Chat et chien

Test	Recombinant	Can f 1	Can f 2
in vitro	E. coli	70%	10%
TC	P. pastoris	52%	32% (tous sont Can f 1+
in vitro	non	75%	25%
TC	non	94%
in vitro	P. pastoris	42%	16%
in vitro	E. coli (CAP)	49%	22% (7/8 sont Can f 1+)

Nb. patients	Prévalence d’allergie (%)	VPP (%)
100	45	93
128	39	93
69	43	80
53	39	100
167	7,2	50
181	32	78

	Sensibilisation
	Chat	Chien	Les deux
rFel d 1	+	-	+
rCan f 1	-	+	+

[1] - Liccardi G, d’Amato G, Russo M, Canonica GW, d’Amato L, de Martino M, et al. Focus on Cat Allergen (Fel d 1): Immunological and Aerodynamic Characteristics, Modality of Airway Sensitization and Avoidance Strategies. Int Arch Allergy Immunol 2003;132:1-12

The increasing frequency of pet ownership (especially cats) in many industrialized countries has raised the level of exposure to the allergens produced by these animals. Moreover, it is likely that modern energy-saving systems and the wide use of upholstered furniture has resulted in closer contact between cats (and their allergens) and humans. Many different methods have been developed to quantify the main cat allergen (Fel d 1) in settled dust and in ambient air. The threshold levels of cat allergen inducing sensitization or triggering respiratory symptoms in sensitized patients have been calculated in settled dust, but airborne amounts of Fel d 1 probably represent a more reliable index of allergen exposure. Noticeably, the amount of Fel d 1 may be relatively high also in confined environments where cats have never been kept. It has been demonstrated that clothes of cat owners are the main source for dispersal of allergens in cat-free environments. This fact may be of relevance, because recent studies have shown that allergic sensitization to cats is more likely to develop in children exposed to moderate levels of this allergen than in children exposed to high amounts of Fel d 1. The ubiquity of cat allergen may justify the common observation that allergen avoidance is often insufficient to reduce the risk of developing allergic sensitization and/or symptom exacerbation in highly susceptible patients. Further efforts are needed to improve the efficacy of Fel d 1 avoidance strategies to try to reduce the risk of allergic sensitization to this allergen.

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[2] - Ichikawa K, Iwasaki E, Baba M, Chapman MD. High prevalence of sensitization to cat allergen among Japanese children with asthma, living without cats. Clin Exp Allergy 1999;29:754-761

BACKGROUND: Cat allergy is common among children with asthma. Many cat- allergic patients in Japan and elsewhere do not keep cats, but nonetheless become sensitized through environmental exposure to cat allergen. OBJECTIVE: To assess the frequency of cat allergy and cat- specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibody responses in young Japanese patients with asthma in relation to self- reported cat exposure and Fel d 1 levels in dust samples. METHODS: Cat dander-specific IgE antibody was measured in sera from asthma patients using the CAP system. IgE and IgG antibody to Fel d 1 was measured by antigen binding radioimmunoassay and by chimeric enzyme immunoassay. Fel d 1 levels in dust samples from a subset of patients' homes were measured by monoclonal antibody-based enzyme immunoassay. RESULTS: Cat- specific IgE (CAP class>/=2) was found in sera from 70% of 44 patients who kept cats and 34% of 394 patients who had never kept cats. The prevalence of sensitization increased progressively to age 6 years (40%: positive), and then increased gradually to age 16 years (approximately 60%: positive) in patients who had never kept cats. There was an excellent correlation between cat CAP values and IgE levels to Fel d 1. The absolute amount of IgE antibody to Fel d 1 ranged from 0.01 to 15.6% of total IgE. Most patients who did not keep cats were exposed to Fel d 1 levels ranging from 0.07-8 microg/g dust. CONCLUSIONS: Sensitization to cat allergen is common among young asthmatic patients in Japan, even among patients who do not keep cats. Use of CAP and the chimeric enzyme-linked immunosorbent assay allows accurate diagnosis of cat allergy and quantification of specific IgE antibody levels

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[3] - Chan-Yeung M, McClean PA, Sandell PR, Slutsky AS, Zamel N. Sensitization to cat without direct exposure to cats. Clin Exp Allergy 1999;29:762-765

BACKGROUND: Allergy to pets, particularly cats, is one of the most important determinants of asthma and asthma-like symptoms in many parts of the world. Cat allergen is found in homes and public places without cats. OBJECTIVE: The purpose of the study is to investigate the prevalence of sensitization to cat on the island of Tristan da Cunha where cats have been eliminated since 1974. METHODS: A cross-sectional survey was conducted in 1993 on all residents on the island including allergy skin testing. Dust samples were collected from 20 homes on the island for measurement of house dust mite and cat allergens. RESULTS: Positive skin test reaction to cat was present in 57 (20.1%) of all islanders and in six (12.8%) of those born in or after 1975, 1 year after cats had been exterminated. Five of these six residents were born within 5 years of extermination of cats; two of these had attended school outside the island. A low level of cat allergen (Fel d 1) was found in only one out of 20 homes even though house dus t mite allergens (Der p 1 or Der f 1) were found in all homes. CONCLUSION: Sensitization to cat allergen occurs on the island of Tristan da Cunha where there is no direct exposure to cats. This is due either to the persistence of the allergen after the removal of the animal or to the allergen being brought in on visitors' clothing

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[4] - Erwin EA, Rönmark E, Wickens K, Perzanowski MS, Barry D, Lundbäck B, et al. Contribution of dust mite and cat specific IgE to total IgE: Relevance to asthma prevalence. J Allergy Clin Immunol 2007;119:359-365

BACKGROUND: The prevalence of asthma is strikingly different in some Westernized countries: approximately 20% in New Zealand and approximately 8% in northern Sweden . OBJECTIVE: We investigated differences in total IgE and in the prevalence of wheezing related to the observation that high exposure to dust mite allergens induces high titers of IgE antibodies . METHODS: Two age-matched, population-based cohorts-1155 children in New Zealand (224 sera) and 3431 children (797 sera) in the Norrbotten area of Sweden-were studied. Sera were assayed for total IgE and specific IgE antibodies to relevant allergens . RESULTS: The mean total IgE among wheezing children was higher in New Zealand than Sweden (218 IU/mL vs 65.2 IU/mL; P < .001). In addition, the prevalence of high titer specific IgE antibody (> or =50 IU/mL) was greater among the wheezing children in New Zealand compared with Sweden (35.7% vs 13.0%; P < .001). Specific IgE antibody to mite in New Zealand was significantly related to high total IgE (> or =200 IU/mL; r = 0.47; P < .001), whereas the IgE antibody response to cat allergens did not make a significant contribution to high total IgE in either country . CONCLUSION: The quantity of IgE antibody produced to dust mite provides a possible explanation for the higher total IgE levels found in children in New Zealand and may help to explain the differences in prevalence and severity of asthma between these 2 countries. CLINICAL IMPLICATIONS: Specific IgE antibody responses to dust mite and cat allergens may contribute differently to total serum IgE and to the prevalence of allergic disease.

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[5] - Takkouche B, González-Barcala FJ, Etminan M, FitzGerald M. Exposure to furry pets and the risk of asthma and allergic rhinitis: a meta-analysis. Allergy 2008;63:857-864

BACKGROUND: Exposure to pets has been implicated as a risk factor for asthma. However, this relation has been difficult to assess in individual studies because of the large potential of selection bias. We sought to examine the association between exposure to furry pets and asthma and allergic rhinitis by means of a meta-analysis . METHODS: We retrieved studies published in any language by searching systematically Medline (1966-March 2007), Embase, LILACS and ISI Proceedings computerized databases, and by examining manually the references of the original articles and reviews retrieved. We included cohort and case-control studies reporting relative risk estimates and confidence intervals of exposure to cats, dogs and unspecified furry animals and subsequent asthma and allergic rhinitis. We excluded cross-sectional studies and those studies that did not measure exposure but rather sensitization to pets . RESULTS: Thirty-two studies were included. For asthma, the pooled relative risk related to dog exposure was 1.14 (95% CI 1.01-1.29), that related to exposure to any furry pet was 1.39 (95% CI 1.00-1.95). Among cohort studies, exposure to cats yielded a relative risk of 0.72 (95% CI 0.55-0.93). For rhinitis, the pooled relative risk of exposure to any furry pet was 0.79 (95% CI 0.68-0.93) . CONCLUSIONS: Exposure to cats exerts a slight preventive effect on asthma, an effect that is more pronounced in cohort studies. On the contrary, exposure to dogs increases slightly the risk of asthma. Exposure to furry pets of undermined type is not conclusive. More studies with exact measurement of exposure are needed to elucidate the role of pet exposures in atopic diseases.

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[6] - de Blay F, Barnig C, Muti D, Schweitzer B, Purohit A. Allergie au chat et au chien. Rev Fr Allergol 2009;49:147-155

L‚allergie au chat et au chien reste à l‚heure actuelle un sujet de discussion. En effet, la connaissance des allergènes responsables des réactions croisées entre le chat et le chien et du rôle du chat et du chien dans l‚apparition de la sensibilisation atopique ou de l‚asthme, n‚est pas encore clairement établie. En ce qui concerne les méthodes de désensibilisation, il existe quelques études limitées rapportant un bénéfice clinique avec l‚allergène du chat. En revanche, en ce qui concerne la désensibilisation aux allergènes de chien, la preuve de l‚efficacité clinique n‚a pas été établie. Il sera nécessaire de mettre en place des études devant démontrer l‚efficacité clinique de la désensibilisation et éventuellement des modifications immunologiques.

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[7] - Platts-Mills TAE. Paradoxical effect of domestic animals on asthma and allergic sensitization. JAMA 2002;288:1012-1014

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[8] - Oryszczyn MP, Annesi-Maesano I, Charpin D, Kauffmann F. Allergy markers in adults in relation to the timing of pet exposure: the EGEA study. Allergy 2003;58:1136-1143

BACKGROUND: Studies suggest that early childhood exposure to pets may protect from the development of atopy, but limited information is available on adults. The association of allergy markers in adulthood with current and childhood exposure to pets was studied considering retrospectively the window of exposure . METHODS: Immunoglobulin E (IgE), skin prick tests (SPT), eosinophils were related to exposure to pets in 187 adult asthmatic cases and 243 controls from the Epidemiological Study on the Genetics and Environment of Asthma, bronchial hyperresponsiveness and atopy (EGEA) study. Analyses were redone after exclusion of subjects who removed pets or experienced symptoms to animals to take into account selection in that retrospective study . RESULTS: In asthmatic cases, current exposure to pets was unrelated to SPT positivity (+), whereas childhood exposure was significantly related to less SPT+ to any allergen, and to cat in particular, with an association restricted to those exposed before 2 years of age [OR = 0.30 (CI 0.12-0.76)]. Considering the relative timing of exposure in relation to asthma onset showed that the protective effect of exposure to pets occurs for pet exposure starting before asthma onset [OR for SPT+ = 0.19 (CI 0.08-0.48)] . CONCLUSION: Results support the hypothesis that exposure to pets in early life, and in particular before asthma onset, may protect against allergen sensitization in adulthood.

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[9] - Lau S, Illi S, Platts-Mills TAE, Riposo D, Nickel R, Grüber C, et al. Longitudinal study on the relationship between cat allergen and endotoxin exposure, sensitization, cat-specific IgG and development of asthma in childhood – report of the German Multicentre Allergy Study (MAS 90). Allergy 2005;60:766-773

BACKGROUND: Controversial data have emerged regarding the question whether cat exposure in childhood favours or decreases the risk of sensitization and allergic airway disease. In a prospective birth-cohort study, we assessed the association between longitudinal cat allergen exposure, sensitization (immunoglobulin E, IgE), IgG antibody (ab) levels to cat and the development of asthma in children up to the age of 10 years . METHODS: Of 1314 newborn infants enrolled in five German cities in 1990, follow-up data at age 10 years were available for 750 children. Assessments included yearly measurements of specific serum IgE to cat and at age 6 and 18 months, 3, 4 and 10 years measurement of cat allergen Fel d 1 in house dust samples. Additionally, Fel d 1-specific IgG ab were determined in 378 serum samples of 207 children. Endotoxin exposure in mattress dust was measured in a subgroup of 153 children at age 10 years. From age 4 years on, International Study of Asthma and Allergy in Childhood (ISAAC) questionnaires were completed yearly in order to assess the prevalence of wheeze and asthma . RESULTS: Serum IgG-levels to cat showed a large variation, however, intraindividually values showed rather constant concentration over a longer time period. The IgG levels at school-age correlated with cat allergen exposure during the first 2 years of life. Specific IgE to cat was clearly associated with wheeze ever, current wheeze and bronchial hyperresponsiveness (BHR), this was also observed for children with specific IgE ab to cat (>0.35 kU/l) plus IgG levels above 125 U/ml. A large percentage of very highly exposed children showed high IgG but no IgE responses to cat, however, not all highly exposed children were found to be protected from sensitization. Children with IgG but without IgE ab to cat showed the lowest prevalence of wheeze ever and current wheeze despite high cat allergen exposure, however, this trend did not achieve significance. While homes of cat owners showed higher Fel d 1 concentrations than homes without cats, homes of cat owners were not found to have higher endotoxin levels in carpet dust samples than homes without cats . CONCLUSIONS: We could confirm that high cat allergen exposure in a cohort with lower community prevalence of cats is associated with higher serum IgG and IgE levels to cat in schoolchildren. Sensitization to cat allergen (IgE) is a risk factor for childhood asthma. While exposure to cat allergen during infancy is associated with sensitization (IgE), only in the very highly exposed children the likelihood of sensitization (IgE) is decreased and high IgG levels to cat without IgE were associated with low risk of wheeze. However, cat-specific IgG ab levels did not protect children with IgE-mediated sensitization from wheeze.

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[10] - Erwin EA, Woodfolk JA, Custis N, Platts-Mills TA. Animal danders. Immunol Allergy Clin North Am 2003;23:469-481

Animals release proteins into their surroundings through secretions, as excretions, or as dander. The quantity of dander that is dispersed by cats, dogs, or humans is sufficient to supply food for dust mites and to supply easily measurable quantities of proteins in dust. Fel d 1, Can f 1, and human IgA or IgG can be found in microgram quantities in dust samples. Allergens also can accumulate from the urine of wild or pet rodents. For cats and dogs, the accumulation of dander particles is not related to the cleanliness of the animals. All animals, including humans, provide a fully adequate supply of organic material for bacterial growth in a carpet, provided conditions are sufficiently humid. The authors' preliminary results in Virginia do not find a significant difference in endotoxin between homes with or without animals. The likely explanation for the nonallergic IgG and IgG4 response to cat, dog, or rat allergens is high exposure to proteins from these animals. If the highest levels of cat allergen in a home can result in immunologic tolerance, it is unlikely that primary avoidance would be successful at reducing exposure. The data showing that 80% of Swedish children with cat allergies never had lived with a cat imply that the concentrations of cat allergen in schools or in houses without a cat are sufficient to cause sensitization. Primary prevention would be possible only on a community basis, which is unlikely to occur. Sensitization to cat, rat, dog, or mouse allergens consistently is associated with asthma. In symptomatic children with positive skin test results, there is a strong case for allergen avoidance and a clear need for controlled trials. Controlled trials of avoidance should include houses without cats and schools. Controlling exposure to cat allergens with the cat in situ requires aggressive measures, such as removing reservoirs, washing the cat, and air cleaning. Many allergic or symptomatic children who live with a cat do not have positive skin test results or positive IgE antibodies to cats. Avoidance measures related to animals should be recommended only for individuals with positive skin test results. Increasing evidence shows that exposure to cats, dogs, rats, and other animals can induce a form of immunologic tolerance without causing allergic disease, and it is important to understand why this change occurs with dander allergens rather than with all allergens. The most probable explanations are related to the form and quantity of airborne allergens.

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[11] - Casset A, Donnay C, de Blay F. Le rôle de l'environnement dans l'asthme: hypothèses et contradicitions. Rev Prat 2005;55:1299-1304

Three hypotheses are described to explain the relation between allergens and environmental co-factors and the onset of atopy: the hygiene hypothesis, the allergenic hypothesis, and the high exposure tolerance inducing a Th2 derived response with blocking IgG4 synthesis. None of these hypotheses have been confirmed. It seems thus difficult to give recommendations for primary prevention of allergic diseases until results of prospective studies allow to consider a more precise behaviour. In contrast, subjects sensitized and exposed to allergens present an increased risk to develop asthma or non specific bronchial hyperreactivity. Therefore, secondary prevention appears as an essential method for treatment of allergic disease, with clinical benefits on symptoms which have recently been demonstrated in a clinical study.

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[12] - Erwin EA, Reefer AJ, Naccara L, Bunyan D, Woodfolk JA, Platts-Mills TAE. Changes in Immune Responses to Cat Allergens during Prolonged Avoidance: Is Tolerance Reversible. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°440

RATIONALE: Students at the University of Virginia were prospectively monitored for changes in sensitization and tolerance to cat during prolonged avoidance of cat allergens METHODS: Over 2 years, 91 students, who had lived with a cat immediately prior to enrollment, were recruited in the fall. At 6-month intervals, questionnaires were administered, skin testing was performed, and serum samples were collected for measurement of cat specific IgE antibody (CAP FEIA, Pharmacia) and IgG antibody to Fel d 1 (radioimmunoprecipitation) In addition, T cell proliferation and cytokine responses to whole allergen and peptides of Fel d 1 were measured in selected subjects RESULTS: Overall, 19% were sensitized (IgE antibody>0.35 IU/ml) and 37% were tolerant (IgE antibody<0.35 IU/ml but IgG antibody>500 units). Among 8 tolerant participants, IgG antibody levels decreased significantly during 20 months of avoidance (paired t test, P=0.008). In addition, small increases in IgE antibody measurements to cat were observed in two students with low levels and three skin test positive subjects with undetectable IgE antibody initially. Strong T cell proliferative responses to peptides of Fel d 1 were observed at both time points in the sensitized subject. One tolerant individual showed increased proliferative responses with avoidance while T cell proliferation remained unchanged in another Production of IL-5 and IL-13 increased in the sensitized subject. By contrast, in the tolerant subjects, IL-10 responses decreased and production of IFN-g increased CONCLUSIONS: IgG antibody responses to Fel d 1 decline during prolonged "avoidance", while IgE antibody responses stay unchanged or can increase

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[13] - Liccardi G, Martin S, Lombardero M, D'Amato M, Barber D, D'Amato G, et al. Cutaneous and serological responses to cat allergen in adults exposed or not to cats. Respir Med 2005;99:535-544

Background: The relationship between pet ownership and the risk of developing respiratory allergic sensitization to pet allergens is still controversial. OBJECTIVE: To determine the degree of cutaneous immediate hypersensitivity and the levels of specific IgE and IgG4 antibodies to cat allergen in cat sensitized patients directly or indirectly exposed to this animal. METHODS: We studied 112 adolescents and adults sensitized to cat allergens (43 with and 69 without a cat at home). There were also 52 control subjects, 27 atopic non-sensitized to cat and 25 non-atopic. The degree of immediate hypersensitivity was assessed by using, in duplicate, skin prick test with four five-fold dilutions of cat hair allergen extract with the content of its major allergen Fel d 1 quantified in micrograms plus positive (10mg/ml histamine chlorhydrate) and negative (saline solution) controls. The resulting wheal areas were analysed by means of Parallel Line Assay. A blood sample was collected from every patient and control subjects for the evaluation of serological cat specific IgE and IgG4 antibodies. RESULTS: Patients with cat at home had a lower cutaneous response than patients without this pet. The difference in the skin sensitivity was estimated in 3.4 times (P<0.01). There was no statistical difference between the levels of cat specific IgE antibodies in the two groups of patients (P=0.065). The levels of Fel d 1 specific IgG4 antibodies showed a statistically significant association with the presence of cat at home, with higher levels in patients owing cat at home than in patients without this pet (P<0.001). CONCLUSION: The results of this study demonstrate that direct cat exposure in adolescents and adults with respiratory allergy is associated with a lower cutaneous response to cat allergenic extract, assessed by SPT and compared with indirect exposure. In patients with cat at home mean levels of specific IgE are statistically comparable whereas the levels of IgG4 are higher in comparison with subjects not exposed to cats. The role of indirect exposure to cat allergens in airways sensitization also in adults is emphasized. Moreover, patients with cat at home show a cutaneous and serological sensitization to cat allergen not higher in comparison with subjects not exposed to cats.

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[14] - Perzanowski MS, Chew GL, Divjan A, Johnson A, Goldstein IF, Garfinkel RS, et al. Cat ownership is a risk factor for the development of anti-cat IgE but not current wheeze at age 5 years in an inner-city cohort. J Allergy Clin Immunol 2008;121:1047-1052

BACKGROUND: Cat ownership is inversely associated with atopy and asthma in some areas of the world, but the relevance of cat ownership to allergic disease in the inner city is less known . OBJECTIVE: We sought to evaluate the relationship between cat ownership and the development of early sensitization and wheeze . METHODS: By using a prospective birth cohort study, Dominican and African American mothers living in New York City underwent repeated questionnaires about their child from birth to age 5 years. Sera collected from children at ages 2 (n = 323), 3 (n = 336), and 5 (n = 242) years were assayed for anti-cat IgE and anti-Fel d 1 IgG antibodies . RESULTS: Cat ownership was a significant risk factor for the development of anti-cat IgE by age 2 years (risk ratio [RR], 6.4; 95% CI, 1.9-22) but not for anti-cat IgE development between the ages of 2 and 5 years (RR, 0.88; 95% CI, 0.24-2.3). Current wheeze was significantly more common among those children with anti-cat IgE at ages 3 (RR, 3.5; 95% CI, 2.1-6.0) and 5 (RR, 3.4; 95% CI, 2.3-4.9) years. Cat ownership was inversely associated with current wheeze at age 5 years among children without anti-cat IgE (RR, 0.26; 95% CI, 0.083-0.81). Among children with anti-cat IgE, a similar trend was observed (RR, 0.57; P = .044, Fisher exact test), although one with borderline statistical significance . CONCLUSIONS: Despite a positive association with sensitization, cat ownership in this inner-city cohort was inversely associated with wheeze, potentially suggesting an IgE-independent protective mechanism in this community.

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[15] - de Blay F, Casset A, Sohy C. Chat, chien : quelles préventions ? Rev Fr Allergol Immunol Clin 2002;42:565-568

Les données de la littérature sur l'effet protecteur de l'exposition précoce aux allergènes de chat et de chien dans l'apparition et la modulation de la sensibilisation et le développement de l'asthme sont à la fois peu abondantes et contradictoires. En effet, les études qui démontrent cet effet bénéfique sont le plus souvent rétrospectives et pratiquement à chaque fois contredites par des études prospectives. Si la présence de grandes quantités de Fel d 1 ou de Can f 1 au début de la vie a un effet protecteur sur l'apparition de la sensibilisation à l'allergène de chat ou de chien, il n'y a aucune certitude pour que ce phénomène perdure pendant plusieurs années. Quant au rôle potentiellement protecteur des IgG4, même s'il est possible d'observer une élévation des taux d'IgG anti-Fel d 1 chez des sujets fortement exposés à l'allergène de chat, la relation de cause à effet entre ces immunoglobulines et l'absence d'asthme n'a pas été démontrée. Ainsi, avant de proposer l'acquisition d' un chat ou d'un chien à une future maman atopique, il convient de lui dire que si un effet protecteur existe, personne n'en connaît la durée. Sachant que ces animaux vivent plus de dix ans, son enfant pourra peut-être un jour développer des IgE vis-à-vis des allergènes de chat ou de chien. Or, il semble démontré tant par l'épidémiologie que par la pratique clinique quotidienne de l'allergologie qu'un sujet sensibilisé et exposé à un allergène augmente fortement ses chances de développer un asthme allergique.

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[16] - Liccardi G, Barber D, Russo M, D’Amato M, D’Amato G. Human Hair: An Unexpected Source of Cat Allergen Exposure. Int Arch Allergy Immunol 2005;137:141-144

BACKGROUND: Cat allergens are ubiquitous because the clothing of cat owners constitutes an important source of distribution of Fel d 1 in cat-free environments. Since Fel d 1 can adhere to a variety of surfaces, we sought to verify if human hair belonging to individuals with or without a cat at home might represent a reservoir and be a possible carrier of cat allergens . METHODS: Seventy-three women (25 with a non-neutered male cat and 25 with a dog at home, and 23 controls without any direct contact with these animals) were recruited. The collection of material from hair was carried out using a modified version of a battery-powdered portable sampler. Particulate material was harvested onto glass fiber filters (25 mm in diameter, with a pore size of 2 microm; AP 20 Millipore, Milan Italy), extracted in phosphate buffer with BSA and then assayed for the evaluation of cat allergen using an ELISA based on anti-Fel d 1 monoclonal antibody . RESULTS: Detectable levels of cat allergen were found in 2 controls, in 2 women with a dog at home and in 13 women with a cat at home, respectively . CONCLUSIONS: In some women with a cat at home, hair constitutes a significant reservoir of Fel d 1. It is likely that these amounts of cat allergen might contribute to allergic sensitization when released in cat-free environments.

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[17] - Gulbahar O, Sin A, Mete N, Kokuludag A, Kirmaz C, Sebik F. Sensitization to cat allergens in non-cat owner patients with respiratory allergy. Ann Allergy Asthma Immunol 2003;90:635-639

BACKGROUND: Cats represent one of the most important sources of indoor allergens. The sensitization rate can reach up to 60% in western countries. Keeping cats indoors is uncommon in big cities in Turkey, but cats living in the streets are common. OBJECTIVE: To investigate the prevalence of sensitization to cats in patients with respiratory allergy from Izmir, Turkey, and its relationship to home cat allergen levels. METHODS: A total of 387 patients (70.8% female; mean age, 34.3 years) with respiratory allergic diseases (rhinitis and/or asthma) were included in this study. Skin prick test to cat was performed. House dust samples were collected from the living room of 25 patients and 14 healthy subjects. The major cat allergen (Fel d 1) levels were measured by Dustscreen. Fel d 1 levels given by the manufacturer were as follows: 0.05, 0.13, 0.40, 1.1, and 6.2 mU/mL. RESULTS: The prevalence of cat sensitivity was 44.7% (n = 173). Only 6 patients (1.6%) had a history of feeding a cat in their houses. Thirty-six (92%) of 39 houses had detectable levels of cat allergen (mean Fel d 1 level, 2.24 +/- 2.69 mU/mL). The mean Fel d 1 levels were 1.58 +/- 2.51 mU/mL in the healthy group, 1.91 +/- 2.61 mU/mL in the asthmatic group, and 3.26 +/- 2.85 mU/mL in the group with allergic rhinitis (P = 0.12). The prevalence of cat sensitivity in patients who had 1.1 mU/mL of Fel d 1 in their homes was 57.1%. This rate was five times lower (11.1%) in patients who had the highest Fel d 1 level (6.2 mU/mL) in their homes. CONCLUSIONS: The prevalence of cat sensitivity in Izmir, where cats are generally not kept within homes, is as high as in western countries. The sampled houses have measurable levels of Fel d 1 even in the absence of indoor cats. High prevalence of cat sensitivity in Izmir is probably due to indirect exposure.

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[18] - Liccardi G, Mazzarello MG, Senna G, Russo M, Barber D, Martin S, et al. The degree of serological sensitization to cat allergen in patients with or without cat at home. Eur Ann Allergy Clin Immunol 2005;37:87-89

BACKGROUND: The relationship between exposure to cat/dog in domestic environments and development of allergic sensitization to pet allergens is still controversial. OBJECTIVE: To test the role of indirect exposure to cat, we aimed to evaluate the amounts of cat specific IgE in sensitized subjects with or without cat at home. METHODS: Three hundred six adult patients with a previous diagnosis of allergic sensitization to cat allergens (by skin prick test) were enrolled for this study. Among 306 men and women sensitized to cat (and other allergens), 150 owned a cat for at least 10 years ( direct exposure) and 156 had never owned this animal (indirect exposure). An internal questionnaire specifically designed for the study was completed by the same allergists during the screening consultation: the results of skin prick tests, personal and clinical data, periods of symptoms and characteristics of cat ownership were carefully recorded. Specific IgE to cat extract was determined by the Pharmacia CAP System FEIA (Pharmacia Diagnostics, Uppsala, Sweden) and Immulite 2000 (Diagnostic Products Corporation, Los Angeles, USA) RESULTS: Although specific IgE to cat allergen were significantly higher in patients with cat at home (F = 42.77; p < 0.001), high amounts of these antibodies were found also in sera of subjects indirectly exposed to cats. DISCUSSION: The results of our study demonstrate that the degree of serological sensitization to cat allergens may be relevant also in subjects without any direct contact with cats (indirect exposure).

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[19] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[20] - Leitermann K, Ohman JL Jr. Cat allergen 1: Biochemical, antigenic, and allergenic properties. J Allergy Clin Immunol 1984;74:147-153

Cat allergen 1, an important agent in human allergic reactions, has been partially purified by affinity chromatography. Heating the purified allergen at 100 degrees C for 30 min resulted in a 28% loss in the antigenicity of the allergen molecule (determined by Laurell rocket assay), although lower temperatures had little effect. Its allergenicity (determined by passive transfer skin test) was diminished slightly after heating to 56 degrees C or 100 degrees C. Reduction with dithiothreitol or 2-mercaptoethanol resulted in greater losses of antigenicity and allergenicity but did not obliterate these properties. Three forms of the affinity-purified allergen (isoallergens) differing slightly in isoelectric point were demonstrated by isoelectric focusing followed by crossed immunoelectrophoresis. The molecular weight of cat allergen 1 under physiologic conditions was 35,000 +/- 2000 as determined by gel filtration in Sephadex G-75. Under the dissociative conditions of sodium dodecyl sulfate-polyacrylamide gel elec trophoresis, with or without prior reduction by dithiothreitol, cat allergen 1 appeared to exist as an antigenically active subunit with a molecular weight of 18,000 +/- 2000. This subunit molecular weight estimate was confirmed by gel filtration in 6M guanidine hydrochloride. The stability of the allergenic and antigenic activity of cat allergen 1 suggests that this activity may be determined partially by the primary sequence of allergenic sites on the molecule. The separation and purification of molecular subunits may allow sequence analysis of these sites

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[21] - Kaiser L, Cirkovic Velickovic T, Adédoyin J, Gronlund H, Thunberg S, Gafvelin G, et al. The crystal structure of a dimeric recombinant major cat allergen Fel d 1 that corresponds to the natural tetrameric protein. Allergy Clin Immunol Int 2005;17(Suppl. 1):579-580

Fel d 1 is the major cat allergen, inducing allergic rhinitis and asthma in sensitized individuals. In its natural form, it has a complex tetrameric structure, composed of two non-covalently linked heterodimers. We have previously determined the 3D structure of a homodimeric recombinant (r) Fel d 1 molecule expressed in E. coli by a direct fusion of chain 2 to chain 1 (rFel d 1(2+1)). The crystal structure revealed a striking similarity to that of uteroglobin, a steroidinducible cytokine-like molecule. However, the crystallization conditions used in that study did not allow the formation of dimers of rFel d 1(2+1), corresponding to the natural tetrameric protein. We here report the structure of a rFel d 1 molecule produced by direct fusion of chain 1 to chain 2 (rFel d 1(1+2)). The immunological properties of rFel d 1(1 +2) were also evaluated and compared to the previously characterised rFel d 1(2+1). Crystals of rFel d 1(1+2) were obtained in 22.5 % PEG 3350, 0.5 M CaCl2. The crystals belong to space group P1 and diffract to 1.6 Å. We believe that the presence of a milder precipitant (PEG 3350) permitted us to keep the dimer of Fel d 1. The dimer is created by side-to-side packing of mainly a-helices five and eight from each monomer, corresponding to chain 2. The dimer interface buries 17 % of the total accessible surface. Two internal and disparate cavities are present in the rFel d 1 (1+2) dimer, that could bind endogenous ligands. We have previously also demonstrated that the soluble rFel d 1(2+1) diplays similar immunological properties to the natural Fel d 1. We have within the present study compared rFel d 1(1+2) to rFel d 1(2+1). Their IgE binding properties (using ELISA) as well as their capacity to activate basophils were very similar. Specific induction of lymphoproliferative responses to rFel d 1 (1+2) was detected both by 3H-thymidine incorporation in PBMCs and in a flowcytometry assisted cell proliferation assay in activated whole blood, showing the specific proliferation of CD3+CD4+ cells in response to rFel d 1 proteins. In conclusion, the recombinant protein obtained by the direct fusion of chain 1 to chain 2 of Fel d 1 possesses immunological and molecular properties that are very similar to the rFel d 1 (2+1) and the natural Fel d 1 allergen. The data derived from the crystal structure of the recombinant dimer will be used for the future design of therapeutical tools for treating allergy to cat.

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[22] - Kaiser L, Grönlund H, Sandalova T, Ljunggren HG, Achour A, Schneider G, et al. Three-Dimensional Structure of Fel d 1, the Major Allergen in Cat. Int Arch Allergy Immunol 2003;132:25-26

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[23] - Kaiser L, Grönlund H, Sandalova T, Ljunggren HG, van Hage-Hamsten M, Achour A, et al. The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family. J Biol Chem 2003;278:37730-37735

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.

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[24] - Kaiser L, Velickovic TC, Badia-Martinez D, Adedoyin J, Thunberg S, Hallen D, et al. Structural Characterization of the Tetrameric form of the Major Cat Allergen Fel d 1. J Mol Biol 2007;370:714-727

Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested fo r uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin

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[25] - Vervloet D, Charpin D, Birnbaum J. Origine des allergènes du chat. Rev Fr Allergol Immunol Clin 1995;35:533-538

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[26] - Ramadour M, Birnbaum J, Magalon C, Lanteaume A, Charpin D, Vervloet D. Cat sex differences in major allergen production (Fel d 1). J Allergy Clin Immunol 1998;101:282-284

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[27] - Zielonka TM, Charpin D, Berbis P, Luciani P, Casanova D, Vervloet D. Effects of castration and testosterone on Fel dI production by sebaceous glands of male cats: I--Immunological assessment. Clin Exp Allergy 1994;24:1169-1173

Fel dI is produced by salivary and sebaceous glands. Hormonal control of sebum production is clearly established. The influence of cat castration and supplementary treatment with testosterone on the production of sebum and Fel dI in cat skin have been researched in this study. On day 1, 12 male cats were anaesthetized and three skin areas carefully shaven. Then the level of lipids on skin surface was measured by means of a photometric method. Finally, the three areas of skin were washed with 5ml of distilled water through a plastic cylinder. Fel dI collected in the washes was measured with a two-site monoclonal antibody based ELISA. On day 2, six cats were castrated, the other six were used as a control group. Two and 4 weeks later, the levels of lipids and Fel dI in skin washes were measured again in all cats. On day 30, the six castrated cats were injected intramuscularly with prolonged-action testosterone. Two weeks later, quantification of lipids and Fel dI in all animals was repeated. Sebum and Fel dI le vels decreased in all castrated animals. Injecting the castrated cats with testosterone led to a significant increase in sebum and Fel dI production. Our findings indicate that Fel dI production is influenced by the production of hormones

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[28] - Mata P, Charpin D, Charpin C, Lucciani P, Vervloet D. Fel d I allergen: skin and or saliva ? Ann Allergy 1992;69:321-322

To determine the relative importance of saliva and sebaceous glands as sources of Fel d I allergen, we compared Fel d I levels at the base and tip of the hair in areas presenting more or less sebaceous glands and areas licked more or less frequently. The amount of Fel d I was significantly higher at the base than the tip of the hair. Further, it was strongly correlated with the density of sebaceous glands. This study demonstrated that the most abundant source of Fel d I allergen is cat skin.

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[29] - Jalil-Colome J, de Andrade AD, Birnbaum J, Casanova D, Mege JL, Lanteaume A, et al. Sex difference in Fel d 1 allergen production. J Allergy Clin Immunol 1996;98:165-168

BACKGROUND: Previous experiments have shown that in vivo Fel d 1 production is under hormonal control. It strongly decreased 1 month after castration of male cats and increased after testosterone injection. OBJECTIVE: These results led us to put forward the hypothesis that Fel d 1 production could be more important in male that in female cats. Five adult male and five adult female cats were studied. METHODS: On day 0 three separate sites of each cat's trunk were shaved, and a 5.72 cm2 area in each site was washed twice (D0a and D0b) with 5 ml of distilled water. Then a collar was attached to the neck to prevent contamination from saliva. Twenty-four hours later on day 1, all areas were washed again once (D1a). Skin washes and fur extracts from the shaved areas were evaluated for Fel d 1 content. The same procedures (apart from collection of fur) were repeated 5 months later. RESULTS: The Fel d 1 level was higher in the first skin wash of male cats compared with that of female cats on each separate site of the trunk in both experiments, initially and 5 months later. Results were significant in the first experiment (median Fel d 1 per milliliter: 69.4 and 28.9 mU, respectively, for the combined three sites; p < 0.05). Fel d 1 production over a 24-hour period was higher in male cats, but the difference did not reach statistical significance. Furthermore, correlation between Fel d 1 levels in washes D0a and in fur was highly significant (p < 0.01). CONCLUSION: These results confirm that Fel d 1 originates from skin. Furthermore, they suggest that Fel d 1 production is higher in male than in female cats

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[30] - Charpin C, Mata P, Charpin D, Lavaut MN, Allasia C, Vervloet D. Fel d I allergen distribution in cat fur and skin. J Allergy Clin Immunol 1991;88:77-82

Immunohistochemical procedures were performed to ascertain Fel d I antigen (Ag) distribution in cat fur and skin biopsy specimens and to analyze Fel d I allergen concentrations in fur. One hundred strands of fur and 24 skin biopsy specimens (6 by 4 by 3 mm) from shaved areas were collected from 11 different cats. Freshly depilated hairs were immunostained by free-floating monoclonal anti-Fel d I, avidin-biotin- peroxidase complex, and either processed for scanning electron microscopic examination or mounted on glass slides for computer- assisted densitometric analysis (SAMBA system). Skin biopsy specimens were promptly frozen and sectioned just before the immunohistochemical processing. Densitometric analysis of fur demonstrated that immunoprecipitate concentrations were tenfold higher at the root than at the tip. However, this finding may be explained by decrease of the thickness of the hair cortex that varied in similar proportions. The Ag accumulated on the strand surface but may focally penetrate into the medulla through the scale-like cortical interstices. In skin biopsy specimens, Fel d I Ag was found in epithelial squamous cells, within the epidermis and hair follicles, on the surface of the epidermis and hair follicles, and in sebaceous gland cells. These data suggest that Fel d I Ag is produced by sebaceous cells and, to a lesser extent, by basal squamous epithelial cells and that it is stored mainly on the surface of the epidermis and fur

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[31] - Carayol N, Birnbaum J, Magnan A, Ramadour M, Lanteaume A, Vervloet D, et al. Fel d 1 production in the cat skin varies according to anatomical sites. Allergy 2000;55:570-573

BACKGROUND: Fel d 1 is the major cat allergen, inducing asthma in sensitized individuals. It is produced by the skin and lies on fur. Recently, it was established that the amount of Fel d 1 on fur varies among anatomical sites. However, it is not known whether the allergen production by skin varies in parallel. The objective was to compare the Fel d 1 production by male cat skin in two anatomical sites, the face and the chest, in order to correlate it with Fel d 1 amounts on fur, and to assess the reaccumulation of Fel d 1 after washing. METHODS: Ten intact male cats were shaved under general anesthesia at both areas, and the fur was collected. The skin was washed and the washing fluid collected for Fel d 1 assays. Fel d 1 levels were measured in microg/g fur and ng/cm2 skin by ELISA before and after washing and 48 h later. RESULTS: In skin washing, the mean Fel d 1 level was significantly higher in the face (1015.2 +/- 821.6 ng/cm2) than the chest (115.2 +/- 66.8 ng/cm2). In the fur, the respective levels were 63.6 +/- 34 and 29.6 +/- 13.6 microg/g. In the skin sample taken after skin washing, the level of Fel d 1 dropped to 25.1 +/- 25.7 ng/cm2 on the face and to 22 +/- 17.4 ng/cm2 on the chest. After 2 days, skin Fel d 1 levels returned to basal values, with higher values on the face than the chest. CONCLUSIONS: This study shows that Fel d 1 levels on the skin are dramatically higher on the facial area than chest. This anatomical variation is concordant with the levels of Fel d 1 found on fur. Washing reduces levels of major allergen on cat skin and fur, but the accumulation on skin is restored within 2 days

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[32] - Nageotte C, Park M, Havstad S, Zoratti E, Ownby D. Duration of airborne Fel d 1 reduction after cat washing. J Allergy Clin Immunol 2006;118:521-522

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[33] - Avner DB, Perzanowski MS, Platts-Mills TA, Woodfolk JA. Evaluation of different techniques for washing cats: quantitation of allergen removed from the cat and the effect on airborne Fel d 1. J Allergy Clin Immunol 1997;100:307-312

BACKGROUND AND OBJECTIVE: The purpose of this study was to examine the quantity and distribution of the major cat allergen, Fel d 1, on cats and to evaluate the efficacy of washing, both in removing allergen from the cat and reducing airborne allergen levels. METHODS: Airborne samples were collected on four glass fiber filters in a 30 m3 room, before and 3 hours after serial washing of eight cats (45-minute sampling at 18 L/min for each filter). Aliquots of hair and bath water were also collected and assayed for Fel d 1 content. RESULTS: Extracting cat hair with tap water or pet shampoo for 3 minutes removed mean levels of 191 and 245 microg of Fel d 1 per gram of hair, respectively; the quantity of allergen on samples of cat hair ranged from 1 microg/gm to more than 1770 microg/gm. The highest concentration of allergen was found on hair from the neck. Estimates of the total Fel d 1 on the cat, based on shaving the whole cat, ranged from 3 to 142 mg (mean = 67 mg). Washing cats reduced airborne allergen 3 hou rs later. Washing three cats at weekly intervals for 5 weeks in a veterinarian's office produced a mean decrease of 44% in airborne Fel d 1 (n = 15, p < 0.02). Washing three cats by immersion for 3 minutes at weekly intervals for a 1-month period produced a mean decrease in airborne allergen of 79% (n = 12, p < 0.001). However, after repeated washing, the airborne levels before the next wash were not consistently decreased. The quantity of Fel d 1 removed by immersion varied from 1 to 35 mg. CONCLUSION: Cats carry large quantities of Fel d 1, only a small proportion of which (approximately 0.002%/hr) becomes airborne. Washing cats by immersion will remove significant allergen from the cat and can reduce the quantity of Fel d 1 becoming airborne. However, the decrease is not maintained at 1 week

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[34] - Cain G, Elderfield AJ, Green R, Smillie FI, Chapman MD, Custovic A, et al. The effect of dry heat on mite, cat, and dog allergens. Allergy 1998;53:1213-1215

BACKGROUND: Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens. METHODS: Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60 degrees, 80 degrees, 100 degrees, 120 degrees, and 140 degrees C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA. RESULTS: For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120 degrees C, allergen levels were reduced to < 1% of control. Der p 2 was more heat stable, requiring 140 degrees C for 30-60 min to achieve > 99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast, Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140 degrees C. CONCLUSIONS: The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens; however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens

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[35] - de Andrade AD, Birnbaum J, Magalon C, Magnol JP, Lanteaume A, Charpin D, et al. Fel d I levels in cat anal glands. Clin Exp Allergy 1996;26:178-180

BACKGROUND: Major cat allergen Fel d I is produced consistently by skin and by sebaceous glands before being spread on the fur. OBJECTIVE: Since cats have tubular anal glands secreting sebum, proteins and lipids, we looked at the possible presence of Fel d I in these secretions and compared the levels found to those already reported in other cat tissues or secretions. METHODS: Thirty-seven cats were studied. Fel d I dosage in the anal sacs' secretions was performed using an enzyme linked immunosorbent assay (ELISA) method and total protein evaluation by the Bradford's method. RESULTS: The geometric mean Fel d I concentration was 41 U/g secretion which represents 3.4% of the total protein levels. This amount is the highest ever reported in cat tissues or secretions. CONCLUSION: The close association of Fel d I protein with skin sebaceous glands and anal sacs both with holocrine function and lipids' secretions in one hand, and the homology of chain I of Fel d I with some steroid-binding proteins in other hand, suggest a possible physiological role for Fel d I in the regulation of lipids on skin and cat fur

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[36] - van Milligen FJ, Vroom TM, Aalberse RC. Presence of Felis domesticus allergen I in the cat's salivary and lacrimal glands. Int Arch Allergy Appl Immunol 1990;92:375-378

Most of the IgE response in persons allergic to cats is directed against the cat allergen Fel d I (Felis domesticus allergen I). Although the presence of Fel d I in saliva has been demonstrated, the source of Fel d I is not completely known. We measured Fel d I concentrations in the cat's lacrimal and salivary glands and in the excretes lacrimal fluid, milk and saliva. The concentration of Fel d I, determined by a radioimmunoassay, in the lacrimal gland was 390-780 mU/g tissue and in the sublingual gland 610 mU/g tissue. In the parotid gland and especially the submandibular gland only low concentrations were found, respectively 70-210 and 40-50 mU/g tissue. In lacrimal fluid 6.8-14 U/ml Fel d I was detected, which is comparable to saliva, 4.2-7.0 U/ml, whereas in cat's milk only 0.33 U/ml Fel d I was found. Immunohistochemical studies with monoclonal antibody directed against Fel d I (anti-Fel d I) showed the presence of Fel d I in the serous cells of the lacrimal gland. Thus, our results demonstrate that in particular the lacrimal gland might be a useful Fel d I source.

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[37] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[38] - Smith W, Butler AJL, Hazell LA, Chapman MD, Pomés A, Nickels DG, et al. Fel d 4, a cat lipocalin allergen. Clin Exp Allergy 2004;34:1732-1738

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals . OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels . METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed . RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog . CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.

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[39] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[40] - Saarne T, Grönlund H, Kull I, Wickman M, van Hage M. Monitoring cat allergy in the BAMSE birth cohort at 4 and 8 years of age by using rFel d 1. Allergy 2007;62(suppl. 83):149-150

Background: Domestic cat is one of the most important sources of indoor allergens causing asthma symptoms. In order to predict and prevent from deterioration of respiratory allergic disease, early detection of sensitization is important. Today, cat dander extract (CDE) is the most commonly used tool in the diagnosis of cat sensitisation. The aims of this study were to investigate if the recombinant form of the major cat allergen Fel d 1 (rFel d 1) could substitute cat dander extract in the diagnosis of cat sensitisation and if children at risk of developing cat allergy could be identified at an earlier stage. Methods: Children (n=144) from the large birth cohort BAMSE in Stockholm, Sweden, were selected on the basis of 4 year and 8 year follow-up questionnaires. The selection criteria were symptoms related to asthma or rhinoconjunctivitis in contact with cat or where such symptoms were suspected. Blood samples were taken from each patient at both 4 and 8 years of age and serum was analysed for allergen-specific IgE to CDE and rFel d 1. IgE antibodies to CDE were measured by the ImmunoCAP˙ System (Phadia AB, Uppsala, Sweden), using the cut-off limit 0.35 kU/L. A quantitative ELISA assay with a cut-off limit of 0.037 kU/L was established for measuring IgE antibodies to rFel d 1. Results: Of those being certain of symptoms to cat at 4 years of age, the same number of children, 25/33 (76%), had detectable levels of IgE to both rFel d 1 and CDE at this age. However, at the same age, 14/42 (33%) of the group suspecting symptoms at 4 years had detectable IgE levels to rFel d 1, but only 9/42 (21%) were detected by CDE. In the group developing symptoms after 4 years of age, 60/69 (87%) were detected with rFel d 1 and 57/69 (83%) with CDE at 8 years. Among these children, 33/69 (48%) had clearly detectable levels of IgE to rFel d 1 already at 4 years of age, while 26/69 (38%) were detected by CDE. A high correlation between the IgE levels measured by the two assays was found. Conclusions: We here show that the single major recombinant cat allergen, rFel d 1, is at least as sensitive as cat dander extract in the diagnosis of cat sensitisation. Furthermore, we show that children at risk of developing cat allergy are sensitised to cat already before the parents‚ awareness of symptoms.

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[41] - O'Neil SE, Heinrich TK, Hales BJ, Piboonpocanum S, Smith W, Thomas WR. The importance of a non-Fel d 1 cat allergen. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°21

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[42] - Reininger R, Varga EM, Zach M, Balic N, Lindemeier AD, Swoboda I, et al. Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1. Clin Exp Allergy 2007;37:116-124

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited . OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1 . METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies . RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds . CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.

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[43] - Boutin Y, Hébert H, Vrancken ER, Mourad W. Allergenicity and cross-reactivity of cat and dog allergenic extracts. Clin Allergy 1988;18:287-293

This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophoretic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized

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[44] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[45] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[46] - Vailes LD, Li Y, Bao Y, de Groot H, Aalberse RC, Chapman MD. Fine specificity of B-cell epitopes on Felis domesticus allergen 1 (Fel d 1): effect of reduction and alkylation or deglycosylation on Fel d 1 structure and antibody binding. J Allergy Clin Immunol 1994;93:22-33

The repertoire of B-cell epitopes on the major cat allergen, Fel d I, was analyzed with monoclonal antibodies (MoAbs) in topographic mapping studies and in immunoassays with antigen derived from other cat (Felidae) species. Four essentially nonoverlapping epitopes on Fel d I, designated Fd1A to D, were defined by use of 15 anti Fel d I MoAbs in cross-inhibition radioimmunoassay. Only MoAbs directed against epitope Fd1B bound to putative Fel d I homologues in hair and dander extracts from seven other feline species (Panthera species, [n = 5], Leptailurus serval, and Leopardus pardalus). Quantitative monosaccharide analysis showed that Fel d I was a glycoprotein, containing high levels of fucose, as well as glucosamine, galactose, and mannose. Binding of MoAbs and human IgG or IgE antibody to native, reduced and alkylated or deglycosylated Fel d I was compared by means of immunoprecipitation and immunoassay, and the effects of these treatments on the structure of Fel d I were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. On reduction and alkylation, Fel d I dissociated into 14 kd and 3.2 kd peptides, and deglycosylation with trifluoromethane sulfonic acid produced a 12 to 14 kd peptide. These procedures resulted in a 100- to 1000-fold loss in murine or human antibody binding activity and caused significant loss of secondary structure, as judged by circular dichroism spectroscopy. Treatment with potassium hydroxide also caused a marked loss in antigenic reactivity. In contrast, enzymatic deglycosylation generated a 9 kd peptide, which showed strong reactivity with murine and human antibodies, comparable to native Fel d I. The results show that MoAbs define a broad repertoire of B-cell epitopes on Fel d I, one of which is expressed by other cat species. These epitopes are conformational and do not appear to involve oligosaccharide residues.

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[47] - Bond JF, Brauer AW, Segal DB, Nault AK, Rogers BL, Kuo MC. Native and recombinant Fel d 1 as probes into the relationship of allergen structure to human IgE immunoreactivity. Mol Immunol 1993;30:1529-1541

To delineate the relationship between the structural conformation and the stability of an allergen and its antigenicity, we have chosen the major allergen from cat dander, Fel dI. From protein sequence analysis data we have examined the structure of the naturally occurring Fel dI and we have found it to exist as an anti-parallel heterodimer. We have used ELISA, RAST, Western blot and histamine release techniques to compare the IgE reactivity of a set of cat allergic patient samples to purified, native Fel dI and the E. coli expressed chains 1 and 2. Results from these studies demonstrate a significant level of IgE reactivity to all forms when examined for direct binding. However, both blot and ELISA competition assays show a much higher reactivity to Fel dI in solution compared to the separate recombinant chains and this is supported by the histamine release data. Although native Fel dI chain 2 contains an N-linked carbohydrate moiety, this does not seem to play a role in the reactivity of IgE to chain 2. Denaturation of Fel dI with alkali conditions leads to a dramatic decrease in IgE reactivity, even though measurable changes to the backbone structure of the protein are minimal. One proposed explanation is that both chains possess a core region determined by their primary structures and that the major IgE epitopes are dependent upon them. The relative reactivity amongst these allergen forms varied with the method of analysis, implying that the conformational requirements for IgE antibody binding are best studied by the application of more than one experimental protocol. Results from these qualitative analyses afford insight into the allergenicity of this exceptionally stable cat pelt protein

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[48] - Kristensen AK, Schou C, Roepstorff P. Determination of isoforms, N-linked glycan structure and disulfide bond linkages of the major cat allergen Fel d1 by a mass spectrometric approach. Biol Chem 1997;378:899-908

The domestic cat (Felis domesticus) is an important source of indoor allergens, the major allergen being Fel d1 (formerly cat allergen 1). Fel d1 is responsible for cat allergy and has also been established to cause cat-induced asthma. The allergen is a 38 kDa dimer composed of two 19 kDa subunits. Each 19 kDa subunit comprises two disulfide linked polypeptide chains, a light alpha-chain and a heavy beta-chain containing an N-linked oligosaccharide. In this study a variety of endoproteinase digestions of the native allergen in combination with HPLC and matrix-assisted laser desorption mass spectrometry was used to determine the position of the disulfide bridges and to demonstrate that the peptide chains are linked in an anti parallel way. Enzymatic digestion of the reduced and alkylated peptides located the N-glycan to Asn33. Moreover, Fel d1 is found to be partially truncated and to exist in several isoforms. Sequential degradation of the glycosylated peptide with specific glycosidases monitored by mass spectrometry, shows that the glycan is a heterogeneous triantennary complex type structure. The heterogeneity is caused by terminal sialic acid and a fucose residue attached to a beta-galactose residue.

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[49] - Duffort O, Carreira J, Nitti G, Polo F, Lombardero M. Studies on the biochemical structure of the major cat allergen Felis domesticus. Mol Immunol 1991;28:301-309

The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.

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[50] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[51] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[52] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[53] - Reininger R, Varga EM, Zach M, Balic N, Lindemeier AD, Swoboda I, et al. Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1. Clin Exp Allergy 2007;37:116-124

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited . OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1 . METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies . RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds . CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.

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[54] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[55] - Blanc A, Donnay C, Metz-Favre C, de Blay F, Pauli G. Sensibilisation concomitante au chat et au chien : cosensibilisation ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2007;47:375-376

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[56] - Boutin Y, Hébert H, Vrancken ER, Mourad W. Allergenicity and cross-reactivity of cat and dog allergenic extracts. Clin Allergy 1988;18:287-293

This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophoretic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized

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[57] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[58] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[59] - Reininger R, Swoboda I, Bohle B, Hauswirth AW, Valent P, Rumpold H, et al. Characterization of recombinant cat albumin. Clin Exp Allergy 2003;33:1695-1702

BACKGROUND: Indoor allergens derived from animals and mites often contribute to exacerbation of skin manifestations in atopic dermatitis (AD) patients . OBJECTIVE: To produce and characterize recombinant cat albumin, a cross-reactive animal allergen . METHODS: A complete cDNA coding for cat albumin was obtained by RT-PCR amplification from cat liver RNA. Recombinant cat albumin was expressed in Escherichia coli as hexahistidine-tagged protein, purified by nickel affinity chromatography and studied for IgE reactivity with sera from cat-allergic patients by ELISA and immunoblotting. Furthermore, CD203c expression of basophils from cat-allergic patients upon exposure to recombinant cat albumin was analysed . RESULTS: Recombinant cat albumin, a cross-reactive animal allergen sharing most IgE epitopes with its natural counterpart, was produced in E. coli. It was recognized preferentially by IgE from AD patients and elicited IgE-dependent basophil activation in sensitized patients . CONCLUSIONS: Recombinant cat albumin may be used as a paradigmatic tool to analyse mechanisms of allergen-triggered exacerbation of AD, for diagnostic and, perhaps for therapeutic purposes.

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[60] - Ichikawa K, Vailes LD, Pomés A, Chapman MD. Identification of a novel cat allergen: cystatin. Int Arch Allergy Immunol 2001;124:55-56

BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan. Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail. AIMS: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed. RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein. Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively. This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin. This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins. Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin. The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL. CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

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[61] - Ichikawa K, Vailes LD, Pomés A, Chapman MD. Molecular cloning, expression and modelling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor. Clin Exp Allergy 2001;31:1279-1286

BACKGROUND: Cats are an important source of indoor allergens. However, only two cat allergens, Fel d 1 and albumin, have been cloned and sequenced. IgE antibodies to Fel d 1 and albumin do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens . OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens . METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding cystatin, was expressed in E. coli. The three dimensional structure of cat cystatin was modelled using the SWISS-MODEL computer program . RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human cystatin A, respectively. The cat cystatin sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed cystatin clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, Can f 1 and Can f 2, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat cystatin. The three-dimensional structure of cat cystatin was modelled on human cystatin structures . CONCLUSION: A newly identified allergen, cystatin (Fel d 3), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

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[62] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[63] - O'Neil SE, Heinrich TK, Hales BJ, Piboonpocanum S, Smith W, Thomas WR. The importance of a non-Fel d 1 cat allergen. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°21

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[64] - Smith W, Butler AJL, Hazell LA, Chapman MD, Pomés A, Nickels DG, et al. Fel d 4, a cat lipocalin allergen. Clin Exp Allergy 2004;34:1732-1738

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals . OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels . METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed . RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog . CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.

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[65] - Smith W, Butler AJL, Hazell LA, Chapman MD, Pomés A, Nickels DG, et al. Fel d 4, a cat lipocalin allergen. Clin Exp Allergy 2004;34:1732-1738

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals . OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels . METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed . RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog . CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.

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[66] - Adédoyin J, Grönlund H, Öman H, Johansson SGO, van Hage M. Cat IgA, representative of new carbohydrate cross-reactive allergens. J Allergy Clin Immunol 2007;119:640-645

BACKGROUND: Allergens from cat are among the most potent elicitors of allergic disease. Four cat allergens have been identified; however, evidence indicates the existence of additional allergens . OBJECTIVE: In this study, we evaluated IgE sensitization to IgA from cat . METHODS: Sera from cat-sensitized patients (n = 81) were analyzed for IgE antibodies to purified cat IgA in the Pharmacia CAP System. Indirect ELISA was performed with cat IgA, cat IgM, and deglycosylated cat IgA. Competitive inhibition ELISA was performed with cat IgA, cat IgM, calf intestine alkaline phosphatase (CIP), and cat serum albumin on solid phase bound cat IgA. IgE reactivity was also evaluated on membrane blotted cat IgA . RESULTS: Thirty-eight percent (31/81) of the cat-sensitized sera were ImmunoCAP-positive to cat IgA. Indirect ELISA demonstrated a high correlation between IgE reactivity to cat IgA and cat IgM (r = 0.94; P < .001). Very low responses were observed to deglycosylated IgA. Strong inhibition of cat IgA was observed in all sera after preincubation with cat IgA and cat IgM. Inhibition was also observed in most sera after preincubation with CIP. Immunoblotting demonstrated that the IgE reactivity was mainly directed to the heavy chain of IgA . CONCLUSION: This study has revealed a new allergen, cat IgA, containing a novel group of cross-reactive epitopes depending on carbohydrates also present on IgM and partially on CIP. CLINICAL IMPLICATIONS: This new group of cross-reactive carbohydrate IgE epitopes should be taken into consideration when diagnosing patients with suspected animal allergy.

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[67] - Adédoyin J, Johansson SG, Gronlund H, van Hage M. Interference in immunoassays by human IgM with specificity for the carbohydrate moiety of animal proteins. J Immunol Methods 2006;310:117-125

In allergen characterization common immunoassays such as ELISA and immunoblotting are often used to evaluate human IgE binding of sera from allergic individuals. In an attempt to find and characterize new cat allergens, heavily stained bands were found at 67 and 90 kDa on blotted cat dander extract. Further characterization confirmed these bands to be cat IgA and IgM heavy chains. Analyses in ELISA of purified cat immunoglobulins linked the human IgE binding to cat IgA and IgM and also revealed complete mutual cross-reactivity between the two isotypes. IgE reactivity was found to be directed to carbohydrates of the immunoglobulins and to IgM from 7 out of 9 animal species, but not to human immunoglobulins. Further investigations revealed that the reaction was not mediated by human IgE but by a factor in the serum cross-linking animal immunoglobulins and alkaline phosphatase. The factor was further studied and shown to be human IgM. This IgM fits into the heterophile antibody classification and is directed against carbohydrates on animal immunoglobulins and on calf intestine alkaline phosphatase.

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[68] - Adédoyin J, Grönlund H, Öman H, Johansson SGO, van Hage M. Cat IgA, representative of new carbohydrate cross-reactive allergens. J Allergy Clin Immunol 2007;119:640-645

BACKGROUND: Allergens from cat are among the most potent elicitors of allergic disease. Four cat allergens have been identified; however, evidence indicates the existence of additional allergens . OBJECTIVE: In this study, we evaluated IgE sensitization to IgA from cat . METHODS: Sera from cat-sensitized patients (n = 81) were analyzed for IgE antibodies to purified cat IgA in the Pharmacia CAP System. Indirect ELISA was performed with cat IgA, cat IgM, and deglycosylated cat IgA. Competitive inhibition ELISA was performed with cat IgA, cat IgM, calf intestine alkaline phosphatase (CIP), and cat serum albumin on solid phase bound cat IgA. IgE reactivity was also evaluated on membrane blotted cat IgA . RESULTS: Thirty-eight percent (31/81) of the cat-sensitized sera were ImmunoCAP-positive to cat IgA. Indirect ELISA demonstrated a high correlation between IgE reactivity to cat IgA and cat IgM (r = 0.94; P < .001). Very low responses were observed to deglycosylated IgA. Strong inhibition of cat IgA was observed in all sera after preincubation with cat IgA and cat IgM. Inhibition was also observed in most sera after preincubation with CIP. Immunoblotting demonstrated that the IgE reactivity was mainly directed to the heavy chain of IgA . CONCLUSION: This study has revealed a new allergen, cat IgA, containing a novel group of cross-reactive epitopes depending on carbohydrates also present on IgM and partially on CIP. CLINICAL IMPLICATIONS: This new group of cross-reactive carbohydrate IgE epitopes should be taken into consideration when diagnosing patients with suspected animal allergy.

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[69] - O'Neil SE, Heinrich TK, Hales BJ, Piboonpocanum S, Smith W, Thomas WR. The importance of a non-Fel d 1 cat allergen. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°21

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[70] - Liccardi G, d'Amato G, Canonica GW, Hrabina M, Piccolo A, d'Amato M, et al. Direct and prolonged exposure to dog does not influence the degree of skin prick test positivity to dog allergen. J Investig Allergol Clin Immunol 2005;15:167-171

Background. The relationship between pet ownership and the risk of developing allergic sensitization to pet allergens is still controversial. We attempted to assess the possible effect of a direct and prolonged exposure to dog allergen on cutaneous immediate hypersensitivity in dog-sensitized patients. Methods. We studied, in a case-control trial, 116 adults with respiratory allergy, sensitized to dog allergens at the standard prick test. Fifty-five had a dog at home for at least 10 years and 61 never owned a dog. The degree of immediate hypersensitivity was assessed quantitatively by skin prick test, performed in quadruplicate with three concentrations of allergenic extract: A (1:20 w/v), B (1:200 w/v) and C (1:2000 w/v). The mean diameter of each wheal was assessed using a visilog image analysis software. Results. No significant difference between the two groups, in the wheal diameters induced by the three concentrations of dog allergen could be demonstrated. Mean wheal diameters were: solution A 4.94 VS 4.63, solution B 3.48 VS 3.26, solution C 2.54 VS 2.47mm. None of the subjects had isoated dog positivity. Conclusion. The results of this study suggest that direct dog exposure in adults with respiratory allergy is not associated to a greater cutaneous responses to dog allergens, as compared to not exposed subjects.

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[71] - Spitzauer S, Schweiger C, Sperr WR, Pandjaitan B, Valent P, Muhl S, et al. Molecular characterization of dog albumin as a cross-reactive allergen. J Allergy Clin Immunol 1994;93:614-627

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.

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[72] - Konieczny A, Morgenstern JP, Bizinkauskas CB, Lilley CH, Brauer AW, Bond JF, et al. The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms. Immunology 1997;92:577-586

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

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[73] - Martinez A, Martinez J, Sanz ML, Bartolome B, Palacios R. Dander is the best epithelial source for dog allergenic extract preparations. Allergy 1994;49:664-667

Four different commercially available raw materials of dog epithelia (dander, hair and skin scraping, skin scraping, and whole skin) were compared by means of biochemical methods such as SDS-PAGE and chromatography with the SMART System, immunochemical methods such as RAST and SDS-PAGE immunoblotting, and cutaneous tests. Dander extract clearly exhibited the highest IgE binding and in vivo allergenic activity. The dog major allergens Can fI and the 19-kDa protein were adequately detected only in dander extract. Thus, it is concluded that dander is the most suitable epithelial source for preparing dog allergenic extracts

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[74] - Ford AW, Kemeny DM. The allergens of dog. II. Identification and partial purification of a major dander allergen. Clin Exp Allergy 1992;22:793-803

A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, using sera from 32 dog-allergic subjects, a number of IgE radio-staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21,000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size-exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21,000 and an isoelectric point of approximately 5.2. An additional protein-staining band could still be seen of MW of 24,000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio-immunoelectrophoresis (CRIE) and that Blands et al. [2] and Lowenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.

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[75] - Ramadour M, Guetat M, Guetat J, El Biaze M, Magnan A, Vervloet D. Dog factor differences in Can f 1 allergen production. Allergy 2005;60:1060-1064

BACKGROUND: The clinical importance of dog allergy is well known, but it is unknown if all types of dogs represent the same risk for allergic patients. The purpose of this work was to evaluate among 288 healthy dogs if the levels of Can f 1 on fur vary between breeds (German Shepherd, Pyrenean Shepherd, Poodle, Cocker spaniel, Spaniel, Griffon, Labrador retriever and Yorkshire terrier), gender, hormonal status, hair length, and according to the presence of seborrhea . METHODS: Each dog was shaved in a limited area and Can f 1 concentrations were measured in mug/g fur by ELISA. The results (geometric mean values and 95% confidence intervals) were analyzed using analysis of variance and with nonparametric tests . RESULTS: A wide variability in Can f 1 levels was found between dog breeds, from Labradors [1.99 (0.03-129.91)] to Yorkshires [16.72 (3.67-76.16)] and Poodles [17.04 (2.79-103.94)] but only the Labrador levels were significantly different from each other breed. Males produced more Can f 1 than females, 11.75 (1.27-108.40) vs 8.89 (0.91-86.39). No difference was found according to hair length or hormonal status. The seborrheic status highly (P = 0.0019) influenced the presence of Can f 1 on hair: 16.66 (1.59-173.96) vs 9.40 (1.03-85.70) . CONCLUSION: Breeds (Labrador retriever), sex and seborrhea seem to influence the levels of Can f 1 on fur.

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[76] - Cain G, Elderfield AJ, Green R, Smillie FI, Chapman MD, Custovic A, et al. The effect of dry heat on mite, cat, and dog allergens. Allergy 1998;53:1213-1215

BACKGROUND: Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens. METHODS: Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60 degrees, 80 degrees, 100 degrees, 120 degrees, and 140 degrees C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA. RESULTS: For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120 degrees C, allergen levels were reduced to < 1% of control. Der p 2 was more heat stable, requiring 140 degrees C for 30-60 min to achieve > 99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast, Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140 degrees C. CONCLUSIONS: The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens; however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens

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[77] - Hodson T, Custovic A, Simpson A, Chapman M, Woodcock A, Green R. Washing the dog reduces dog allergen levels, but the dog needs to be washed twice a week. J Allergy Clin Immunol 1999;103:581-585

BACKGROUND: Many asthmatic patients allergic to dogs refuse to part with their dog, and it is essential to develop techniques for lowering exposure with a dog in the home. OBJECTIVE: This study investigated the effect of dog washing on the subsequent recovery of Can f 1 from dog hair clippings and on the airborne allergen over a 7-day period. METHODS: Dogs, which had not been washed for at least the previous 3 weeks, were washed with a hand-held shower and proprietary shampoo. Hair clippings and dander samples from 25 dogs were collected before and immediately after washing. After these initial studies, 16 dogs had a small tuft of hair clipped from the collar or spinal area before washing and then daily for the next 7 days. Air sampling was performed in 5 homes, and the air samples were collected (airflow rate, 9 L/min) over an 8-hour period per day on 10 consecutive days (3 days of baseline sampling before washing and then 7 consecutive days after washing). Can f 1 level was measured by using 2-site ELISA. R ESULTS: Washing significantly reduced recoverable Can f 1 from clippings (84% reduction: from 73 microg/g to 12 microg/g [geometric mean]; P < .0001) and from dander samples (86% reduction: from 347 microg/g to 50 microg/g [geometric mean]; P < .0001). There was a significant reduction in Can f 1 levels in dog hair over the observed 8-day period (F = 18.4, P < . 0001). By using a multiple comparison test, this observed significance was found to be due to the difference between the baseline levels and those on days 1 and 2 after washing, with no difference in the baseline Can f 1 compared with days 3 to 7. Airborne Can f 1 levels showed a downward trend, which reached statistical significance when the data were grouped into 3 sampling periods as follows: baseline (ie, mean of 3 days before sampling) was compared with days 1 to 4 after washing (41% reduction, 95% CI 13%-60%) and days 5 to 7 after washing (61% reduction, 95% CI 2%-84%; P =.014). CONCLUSIONS: Washing the dog reduces recoverable allergen from dog hair and dander. The dog needs to be washed at least twice a week to maintain the reduction in recoverable Can f 1 from its hair. Washing the dog achieves a modest reduction in the level of airborne Can f 1 in homes with a dog

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[78] - Kamata Y, Miyanoma A, Nakayama E, Miyanoma T, Tajima T, Nishimura K, et al. Characterization of Dog Allergens Can f 1 and Can f 2 --- 2. A Comparison of Can f 1 with Can f 2 Regarding Their Biochemical and Immunological Properties. Int Arch Allergy Immunol 2007;142:301-308

BACKGROUND: The major dog allergens, Can f 1 and Can f 2, are members of the lipocalin protein family. The characterization of both dog allergens is still not complete. Their deduced amino acid sequences indicate the presence of three cysteine residues, probably connected with a disulfide bridge. We compared the biochemical and immunological properties of Can f 1 with those of Can f 2 using gel filtration, electrophoresis, and immunological assays . METHODS: The rCan f 1, rCan f 2 and dog salivary proteins containing natural Can f 1 and Can f 2 were analyzed by HPLC gel filtration. The recombinant Can f 1 (rCan f 1) and rCan f 2 were analyzed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with or without reduction. The binding ability of rabbit IgG purified by protein G affinity chromatography from the antiserum against rCan f 1 and rCan f 2 was examined after a reduction in the recombinant allergens. The immunological cross-reaction between rCan f 1 and rCan f 2 was examined by an enzyme-linked immunosorbent assay (ELISA) using the rabbit IgG against rCan f 1 and rCan f 2. The cross-reaction of human IgE in the serum of a patient with dog allergy between rCan f 1 and rCan f 2 was also analyzed by competitive ELISA . RESULTS: The molecular weights of rCan f 1 and of rCan f 2 were 18 and 21 kDa, respectively, using SDS-PAGE under reducing conditions, but the natural Can f 1 and Can f 2 were separated by HPLC gel filtration into fractions containing proteins of 31 and 34 kDa, respectively. rCan f 1 and rCan f 2 migrated as multiple bands (30-100 kDa) in native PAGE in the presence or absence of a reductant. The molecular weights of natural Can f 1 and of Can f 2 were 20 and 23 kDa, respectively, in SDS-PAGE under reducing conditions. The ability of rabbit IgG to bind to rCan f 1 and rCan f 2 increased after the reduction of the recombinant allergens. The rabbit IgG against rCan f 1 bound to rCan f 2. Cross-reaction of human IgE was observed between rCan f 1 and rCan f 2 . CONCLUSIONS: In the native and recombinant forms, Can f 1 and Can f 2 possessed a dimer structure under natural (non-reduced) condition. The dimers of Can f 1 and of Can f 2 were not built with a disulfide bridge but by non-covalent association. Cleavage of a disulfide bond of rCan f 1 and rCan f 2 increased the ability of binding of rabbit IgG to the allergens. The cross-reactivity of rabbit IgG and human IgE between rCan f 1 and rCan f 2 indicates that the same epitope(s) was present in Can f 1 and Can f 2.

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[79] - Konieczny A, Morgenstern JP, Bizinkauskas CB, Lilley CH, Brauer AW, Bond JF, et al. The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms. Immunology 1997;92:577-586

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

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[80] - Spitzauer S, Schweiger C, Sperr WR, Pandjaitan B, Valent P, Muhl S, et al. Molecular characterization of dog albumin as a cross-reactive allergen. J Allergy Clin Immunol 1994;93:614-627

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.

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[81] - Konieczny A, Morgenstern JP, Bizinkauskas CB, Lilley CH, Brauer AW, Bond JF, et al. The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms. Immunology 1997;92:577-586

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

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[82] - Saarelainen S, Rytkönen-Nissinen M, Rouvinen J, Taivainen A, Auriola S, Kauppinen A, et al. Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity. Clin Exp Allergy 2008;38:374-381

BACKGROUND: Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known . OBJECTIVE: The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL) . METHODS: The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition . RESULTS: ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1 . CONCLUSION: Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

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[83] - Kamata Y, Miyanoma A, Nakayama E, Miyanoma T, Tajima T, Hoshi H. Characterization of Dog Allergens Can f 1 and Can f 2 --- 1. Preparation of Their Recombinant Proteins and Antibodies. Int Arch Allergy Immunol 2007;142:291-300

BACKGROUND: Recombinant dog allergens, rCan f 1 and rCan f 2, and their antibodies are good tools for the characterization of dog allergens in order to develop modern therapeutic and preventive methods for dog allergy . METHODS: In this study, cDNA was synthesized from the mRNA of dog salivary glands and cloned into the pGEX4T vector. rCan f 1 and rCan f 2 containing glutathione S-transferase were prepared by an Escherichia coli expression system. The antibodies against the recombinant allergens were prepared in rabbit. The serum of patients with dog allergy was evaluated by ELISA and immunoblot, using the recombinant allergens, goat anti-human immunoglobulin (Ig) E (epsilon) labeled with biotin, and enzyme-labeled streptavidin. The binding of IgE in the serum of patients with dog allergy to dog saliva as a natural antigen was determined in the presence or absence of dog saliva, rCan f 1 and rCan f 2 as competitors. The anaphylactic potential of rCan f 1 and rCan f 2 was evaluated. The body temperature of the mice sensitized with rCan f 1 and rCan f 2 was monitored after intravenous injection of the allergens. The passive cutaneous anaphylaxis reaction was examined for rCan f 1 and rCan f 2. Dog salivary glands, dog saliva and dog hair/dander extracts were analyzed with antibodies by means of an immunoblot assay. The expression of the mRNA of Can f 1 and Can f 2 was verified in various dog tissues by reverse transcription polymerase chain reaction . RESULTS: The E. coli expression system revealed the yield of rCan f 1 and rCan f 2 in 36 and 30 mg/l of culture. The molecular weights of rCan f 1 and rCan f 2 were 18 and 20 kDa in SDS-PAGE, respectively. rCan f 1 and rCan f 2 were found to bind to specific IgE in the serum of dog allergy patients. The binding of IgE in the patient serum for dog saliva was partially inhibited in the presence of rCan f 1 and rCan f 2. These recombinant allergens showed positive signals in passive cutaneous anaphylaxis reaction and induced anaphylactic shock in the mouse model, resulting in a decrease in body temperature. The polyclonal rabbit antibody for rCan f 1 bound to a protein of 20 kDa in the salivary gland, saliva and hair/dander extracts of dogs. The rabbit antibody for rCan f 2 bound to proteins in the saliva and the hair/dander extracts. The proteins possessed a molecular weight of 22/ 23 kDa. Reverse transcription polymerase chain reaction showed the presence of mRNA expression of Can f 1 and Can f 2 not only in the salivary gland but also in dog skin. A clear expression of Can f 2 mRNA was observed in dog skin . CONCLUSIONS: The recombinant allergens and antibodies for Can f 1 and Can f 2 are available for immunological and biochemical characterization of dog allergens. The molecular weight of the natural Can f 1 and Can f 2 in dog saliva and hair/dander extracts showed a higher molecular weight than that of rCan f 1 and rCan f 2. The significance of dog skin as the tissue producing dog allergens, especially Can f 2, should be considered in further studies.

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[84] - Weber RW. Carya spp. Ann Allergy Asthma Immunol 2004;93(3):A6

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[85] - Konieczny A, Morgenstern JP, Bizinkauskas CB, Lilley CH, Brauer AW, Bond JF, et al. The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms. Immunology 1997;92:577-586

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

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[86] - Schou C, Svendsen UG, Lowenstein H. Purification and characterization of the major dog allergen, Can f l. Clin Exp Allergy 1991;21:321-328

An important dog-hair and dander-specific allergen Ag13 has been purified by means of immunoaffinity chromatography utilizing rabbit antibody specific for Ag13. Purity was judged to be very high as detected by crossed immunoelectrophoresis and SDS-PAGE. The purified allergen was subjected to amino acid analyses. Molecular weight was about 22 kD in HPLC-gel filtration and 25 kD in SDS-PAGE with an additional band at 18 kD. In vitro IgE binding of the allergen was investigated by luminescence immunoassay (LIA) inhibition. Removal of Ag13 from dog hair and dander extract (DHD) removed 50 +/- 1.5% of the IgE binding capacity. The purified allergen inhibited up to 56.5% of the IgE activity to DHD as measured with a pool of serum from dog-allergic patients. Out of 26 dog-allergic patients, 24 had a positive skin-prick test to the allergen. Out of 23 dog-allergic patients, 16 reacted with the allergen in IgE immunoblotting. We suggest that Ag13 be termed Can f I. The allergen will be a marker allergen for environmental dog hair and dander exposure.

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[87] - Saarelainen S, Rytkönen-Nissinen M, Rouvinen J, Taivainen A, Auriola S, Kauppinen A, et al. Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity. Clin Exp Allergy 2008;38:374-381

BACKGROUND: Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known . OBJECTIVE: The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL) . METHODS: The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition . RESULTS: ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1 . CONCLUSION: Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

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[88] - Mattsson L, Lundgren T, Everberg H, Larsson H, Lidholm J. Prostatic kallikrein: A new major dog allergen. J AlIergy Clin Immunol 2009;123:362-368

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[89] - Yman L, Brandt R, Ponterius G. Serum albumin--an important allergen in dog epithelia extracts. Int Arch Allergy Appl Immunol 1973;44:358-368

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[90] - Spitzauer S, Schweiger C, Sperr WR, Pandjaitan B, Valent P, Muhl S, et al. Molecular characterization of dog albumin as a cross-reactive allergen. J Allergy Clin Immunol 1994;93:614-627

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.

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[91] - Saarelainen S, Rytkönen-Nissinen M, Kalkkinen N, Kinnunen T, Nieminen A, Virtanen T. Characterization of Can f 4, a new lipocalin allergen of dog. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°22

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[92] - Saarelainen S, Taivainen A, Rytkönen-Nissinen M, Auriola S, Immonen A, Mäntyjärvi R, et al. Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy. Clin Exp Allergy 2004;34:1576-1582

BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts . OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy . METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting . RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein . CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.

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[93] - Mattsson L, Lundgren T, Everberg H, Larsson H, Lidholm J. Prostatic kallikrein: A new major dog allergen. J AlIergy Clin Immunol 2009;123:362-368

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[94] - Basagaña M, Bartolomé B, Pastor C, Torres F, Alonso R, Vivanco F, et al. Allergy to human seminal fluid: Cross-reactivity with dog dander. J Allergy Clin Immunol 2008;121:233-239

BACKGROUND: Human seminal plasma (HSP) allergy is uncommon, with symptoms ranging from vulvovaginal pruritus to life-threatening anaphylaxis. Although several seminal plasma allergens have been reported and their molecular masses have been estimated to range between 12 and 75 kd, the prostate-specific antigen (PSA) has recently been identified as a causative allergen. Given that in a large number of cases symptoms appeared during or after the first intercourse, a cross-reactivity phenomenon might be implicated . OBJECTIVE: We sought to assess the presence of IgE cross-reactivity among proteins from dog epithelium and HSP and to attempt to identify the allergens involved . METHODS: Forty-one patients with dog epithelium allergy were selected. One of them experienced anaphylaxis in contact with her husband's seminal plasma. Skin prick tests, serum specific IgE measurements, SDS-PAGE immunoblotting, and inhibition tests were performed to study the pattern of IgE-binding proteins and the potential cross-reactivity between HSP and dog epithelium. Mass spectrometry was carried out to identify the protein involved in allergy reactions . RESULTS: Twenty-four percent of the sera from patients with dog epithelium allergy recognized an IgE-binding band of 28 kd in HSP immunoblotting. Mass spectrometry identified this band as the PSA. SDS-PAGE immunoblotting-inhibition showed a complete IgE-binding inhibition when sera from these patients were preincubated with dog dander extract . CONCLUSIONS: IgE cross-reactivity among proteins from dog dander and human PSA is demonstrated.

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[95] - Mattsson L, Lundgren T, Everberg H, Larsson H, Lidholm J. Prostatic kallikrein: A new major dog allergen. J AlIergy Clin Immunol 2009;123:362-368

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[96] - Weidinger S, Mayerhofer A, Raemsch R, Ring J, Köhn FM. Prostate-specific antigen as allergen in human seminal plasma allergy. J Allergy Clin Immunol 2006;117:213-215

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[97] - Ghosh D, Bernstein JA. Characterization of Seminal Plasma Protein Epitopes in Patients With Seminal Plasma Hypersensitivity. J Allergy Clin Immunol 2009;123:S229

BACKGROUND: Human seminal plasma proteins (SPP) cause localized and/or systemic seminal plasma hypersensitivity (SPH). Prostate specific antigen (PSA), a 32 kD serine protease, is reported to be a relevant glycoprotein allergen in SPH. Interestingly, many SPH patients manifest symptoms after initial intercourse suggesting pre-sensitization caused by cross-reactive protein and/or carbohydrate epitope(s). OBJECTIVE: To determine if specific IgE responses to SPP are directed against carbohydrate or protein moieties. METHODS: SPP from the sexual partners of women with SPH were fractionated by FPLC. IgE-reactive bands were identified by immunoblotting using the respective female‚s sera and analyzed by mAb and mass spectrometry. Periodate (NaIO4)-mediated deglycosylation of the membranebound proteins, was used to determine if the relevant epitopes resided in the carbohydrate or protein moieties. A 3D PSA model was constructed by homology modeling to map conserved antigenic patches on the protein surface. RESULTS: Immunoblotting using 10 SPH sera revealed two distinct IgEreactive bands, MW 31 and 10 kD, respectively, identified as PSA (intact and fragmented) using a-PSAmAb and mass spectroscopy. Periodate-treated immunoblots showed little or no reduction in IgE binding to SPP indicating that carbohydrate determinants have minimal importance for eliciting IgE cross-reactivity. 3D modeling demonstrated considerable sequence and structural homology with other serine proteases. Specifically, the region L53-C65 is well conserved and solvent-exposed. CONCLUSIONS: Carbohydrate moieties of SPP allergen(s) do not have a significant role in IgE-binding or IgE-mediated cross-reactivity. A 3D model of PSA identified residues that might explain the structural basis of cross-reactivity between PSA and other serine proteases.

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[98] - Drouet M, Sabbah A, Hassoun S. Thirteen cases of allergy to human seminal plasma. Allergy 1997;52:112-114

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[99] - Brandt R, Yman L. Dog dander allergens. Specificity studies based on the radioallergosorbent technique. Int Arch Allergy Appl Immunol 1980;61:361-370

Cat allergen-specific serum IgE antibodies were detected in 27 (71%) of a group of 38 dog dander-sensitive patients. In 4 (44%) of the cases the dual reaction could be explained by the presence of IgE antibodies to cross-reacting serum proteins. In a larger group the binding of dog dander-specific IgE antibodies could be inhibited by cat epithelium allergens. Two allergenic components of dog dander were separated by chromatography on Sephadex G-75. One of the allergens was closely related to a purified major cat epithelium allergen. The other allergen was apparently dog-specific and showed no cross-reactivity with cat allergens. Both components were present in the dander of dachshund, Airedale terrier, poodle and boxer. The dog dander is the preferable source of dog epithelium allergens provided that the existence of relations to other animals is considered.

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[100] - Reininger R, Varga EM, Zach M, Balic N, Lindemeier AD, Swoboda I, et al. Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1. Clin Exp Allergy 2007;37:116-124

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited . OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1 . METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies . RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds . CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.

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[101] - Blands J, Lowenstein H, Weeke B. Characterization of extract of dog hair and dandruff from six different dog breeds by quantitative immunoelectrophoresis. Identification of allergens by crossed radioimmunoelectrophoresis (CRIE). Acta Allergol 1977;32:147-169

An extract of mixed dog hair and dandruff from six different dog breeds (alsatian, boxer, collie, poodle, and long-haired and short-haired dachshund) was obtained by mild extraction, centrifugation, dialysis and freeze-drying. Extract of hair and dandruff from the individual dog breeds was obtained in the same way, but the material was not freeze-dried. Examination and characterization of the mixed extract by means of crossed immunoelectrophoresis revealed a precipitation pattern composed of 25 antigens, some of which were mutually partially identical, and a high content of dog serum proteins was found. Quantitative and qualitative differences between the individual dog breeds were demonstrated. Partial identity of the antigens of the mixed extract with antigens of serum, antigens of extracts of hair and dandruff from cat, cow, horse and guinea pig, and antigens from extract of house dust was also observed. By means of crossed radioimmunoelectrophoresis, using sera from 21 patients who were RAST-positive to dog hair and dandruff extract, the specific IgE-binding to antigens of the mixed extract was examined. On the basis of these results major and minor allergens were identified. Dog albumin was found to be a very important major allergen, but alpha1-antitrypsin and gamma-globulin were also identified. Furthermore, four non-serum proteins were shown to be allergens. No breed-specific allergens could be identified in the extracts from the individual dog breeds.

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[102] - Ford AW, Alterman L, Kemeny DM. The allergens of dog. I. Identification using crossed radio-immunoelectrophoresis. Clin Exp Allergy 1989;19:183-190

The antigens present in an extract of dog hair and dander were examined by crossed immunoelectrophoresis (CIE) and the IgE-binding allergens by crossed radio-immunoelectrophoresis (CRIE), respectively, using sera from 60 British and Finnish animal-allergic subjects. The extract was comprised of a minimum of 28 antigens, 11 of which were common to dog serum. IgE antibody in the sera of the dog-sensitive patients bound to 21 of the 28 antigens at varying frequencies and intensities. Binding of any intensity occurred most frequently to two serum proteins: antigen 23 (IgG) binding IgE in 88% of cases, and antigen 3 (dog serum albumin, DSA) in 77% of cases. Dander antigen 8 bound in 63% and antigen 1 in 42% of the sera. Strong IgE binding, however, was most commonly associated with dander antigen 8 followed by antigens 1 and 23 (IgG) then 3 (DSA). The ranking of the antigens as allergens was similar for the two populations except that DSA was more important for the British than for the Finnish subjects

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[103] - Turbyville JC, Nelson M, Mikita C. Discordance Between Conventional and Acetone Precipitated (AP) Dog Extract in Skin Prick Testing. J Allergy Clin Immunol 2008;121:S58

RATIONALE: Can f 1 has been identified as the major dog allergen. Conventional dog extract contains approximately 5 ug/mL of Can f 1. AP dog extract is produced with the same material as conventional extract, but concentrated through a process of acetone precipitation, and contains about 150 ug/mL of Can f 1.We sought to determine if this difference in allergen concentration changed the skin test results. METHODS: AP dog was added to the standard aeroallergen panel as part of a clinic project to determine if discrepancies exist between skin test results from conventional and AP dog extracts. We evaluated positive vs. negative skin pricks as well as mean wheal size among positives. A significant positive reaction was defined as > 3 mm increase in wheal size over negative control. RESULTS: 73 patients received skin testing that included AP and conventional dog. 11 patients (15%) had a significant positive skin prick to dog (AP, conventional, or both). Of those testing positive to dog, 3/11 (27%) were positive to both AP and conventional dog, and 8/11 (73%) were positive only to AP dog. Among these 11 patients, the mean wheal size for AP dog was 7.6mmand that for conventional dog was 3.4 mm. No patients were positive to conventional dog and negative to AP dog. CONCLUSIONS: AP dog extract appears to be a more sensitive indicator of dog skin prick reactivity than conventional dog extract. Further studies are needed to determine clinical correlation between AP skin prick results and allergy symptoms to dog.

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[104] - van der Veen MJ, Mulder M, Witteman AM, van Ree R, Aalberse RC, Jansen HM, et al. False-positive skin prick test responses to commercially available dog dander extracts caused by contamination with house dust mite (Dermatophagoides pteronyssinus) allergens. J Allergy Clin Immunol 1996;98:1028-1034

In an outpatient population, a high frequency of positive skin prick test responses to dog dander was found in the absence of detectable IgE to dog dander in the RAST. The majority of these patients were sensitized to house dust mites (Dermatophagoides pteronyssinus) and had no obvious dog-related allergic symptoms. These findings prompted us to investigate whether dog dander skin test preparations are contaminated with house dust mite allergens in amounts sufficient to cause false-positive skin prick test responses in patients sensitized to house dust mites. METHODS: Antigen detection assays with monoclonal and polyclonal antibodies were used to determine concentrations of the major allergen Can f 1 from dog dander and the major allergens Der p 1 and Der p 2 from house dust mites in five commercially available dog dander skin prick test preparations (A to E). RESULTS: Can f 1 concentrations varied for the different extracts (A: 170 micrograms/ml, B: 11.1 micrograms/ml, C: 13.3 micrograms/ml, D: 3.8 micrograms/ml, and E: 59.4 micrograms/ml). Der p 1 was detectable in all extracts (A: 33.4 ng/ml, B:5.1 ng/ml, C:29.6 ng/ml, D: 0.4 ng/ml, and E: 1.9 ng/ml), and Der p 2 was detectable in some of the commercially available dog dander skin prick test preparations tested (A: 31.3 ng/ml, B: 3.0 ng/ml, and C: 7.5 ng/ml). The median house dust mite threshold in the skin prick test was found to be 5.8 ng/ml, of Der p 1 (range, 3.5 to 20.8 ng/ml) in nine patients tested. CONCLUSION: Contamination of commercially available dog dander skin prick test preparations with the major allergens (Der p 1 and Der p 2) of the house dust mite (D. pteronyssinus) was demonstrated. These contaminations cause false-positive responses to skin prick tests with dog dander in patients sensitized to house dust mite.

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[105] - Johansen N, Brink-Andersen U. Discordance analysis of Dog Dander specific IgE assay from two in vitro systems. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1287

Background: As part of the initial In-House Validations, Method Comparison studies were conducted comparing Bayer ADVIA Centaur to Pharmacia CAP FEIA and Magic Lite SQ systems for Dog Dander (e5) specific IgE analysis. 115 patient samples collected from ALK-Abelló sample bank were tested on all three methods. The clinical diagnoses on this population were unknown. Good correlation to Pharmacia CAP method above class 2 was shown. However 17% of the samples were found to be discordant, mostly in class 1 and 2 in the CAP system and negative in the ADVIA Centaur assay. Purpose of study: To investigate the discordant sample population reactivity and the reason for the difference between ADVIA Centaur and CAP FEIA Dog dander sIgE assay. Methods: ADVIA Centaur (AC) and Pharmacia CAP FEIA (CAP) methods were used for sample analysis of sIgE concentration. CRIE and CAP Inhibition sIgE assay was used for the characterization of the allergen assay reagents and the sample allergen reactivity. CAP inhibition analyses were performed using sample inhibition with dog, cat and dust mite extracts also cat major allergen protein (Fel d1) was used. A pre-incubation of sample with the Pharmacia allergen CAP sponge (dog, cat or dust mite) was used identify the different sample allergen reactivity. Results: For the discordant samples the sIgE assays showed higher concentration for cat or dust mite specific IgE than for dog in both AC and CAP system. CRIE on concordant samples show strong reactivity to dog major allergen. The CRIE on discordant samples shows strong reactivity to Cat major allergen. The extract CAP inhibition assays showed an equal or stronger inhibition by cat extract, cat purified major allergen or dust mite allergen extract for discordant samples. Pre-incubation of the sample with the allergen CAP before assay in the CAP system also showed stronger inhibition by cat or dust mite CAP for discordant samples. For concordant samples the inhibition study showed stronger inhibition from dog extract in the dog IgE assay. Conclusion: A fairly good concordance was found between the ADVIA Centaur and CAP FEIA Dog dander sIgE assays. In the Discordance analyses all immuno chemical analyses indicate a cross reactivity and/or cross contamination in the CAP system, as also reported in the literature. The ADVIA Centaur Dog dander sIgE assay is less sensitive to cross reactivity and cross contamination.

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[106] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[107] - Corey JP, Mamikoglu B, Akbar I, Houser SM, Gungor A. ImmunoCAP and HY*TEC enzyme immunoassays in the detection of allergen-specific IgE compared with serial skin end-point titration by receiver operating characteristic analysis. Otolaryngol Head Neck Surg 2000;122:64-70

OBJECTIVES: In this study, we compared 2 different immunoassays, HY*TEC enzyme immunoassay (Hycor Biomedical Inc, Garden Grove, CA) and Pharmacia Upjohn ImmunoCAP (Pharmacia and Upjohn, Kalamazoo, MI) using skin end-point titration as a gold standard by receiver operating characteristic analysis. MATERIALS: One hundred patients were enrolled in this study and were tested with the above 3 modalities. The results were analyzed by receiver operating characteristic analysis, in which the curves give a decision criterion for any diagnostic test relative to its sensitivity and specificity against an accepted gold standard test. Only inhalant allergens (Dermatophagoides pteronyssinus, Dermatophagoides farina, cat dander, giant ragweed, English plantain weed, timothy grass, Alternaria tenuis, Aspergillus fumigatus, meadow fescue, oak tree, white ash tree, and lamb's-quarter weed) were studied. RESULTS: The results obtained with both ImmunoCAP and HY*TEC showed a good correlation with skin end-point titration for D pteronyssinus, D farina, cat dander, giant ragweed, timothy grass, A tenuis, and meadow fescue, with sensitivities ranging from 71% to 88%. However, the sensitivity of both tests to English plantain weed, lamb's-quarter weed, oak tree, and white ash tree was lower (66% down to 39%). Additionally, HY*TEC results were not satisfactory for detecting specific IgE to A fumigatus (30% sensitivity). The overall sensitivity for each test was 65.67% for the HY*TEC and 71.25% for the ImmunoCAP. CONCLUSION: The 2 in vitro testing systems are comparable for most of the allergens, except for A fumigatus, English plantain, lamb's-quarter, and white ash tree. ImmunoCAP and HY*TEC would benefit from improved technology for detecting these specific IgE antibodies. The HY*TEC assay did not give acceptable results for A fumigatus. The overall assay characteristics were good to excellent for each system tested.

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[108] - Kelso JM, Sodhi N, Gosselin VA, Yunginger JW. Diagnostic performance characteristics of the standard Phadebas RAST, modified RAST, and pharmacia CAP system versus skin testing. Ann Allergy 1991;67 :511-514

Results from three in vitro assays for allergen-specific IgE, the standard Phadebas radioallergosorbent test (PhRAST), modified RAST (mRAST), and the new Pharmacia CAP System (CAP) were compared with skin prick testing (SPT) results in 104 patients with allergic rhinitis and/or asthma and 24 nonatopic controls. Five allergens were evaluated: cat, Dermatophagoides pteronyssinus, Alternaria, June grass, and short ragweed. Using SPT results as the reference standard, the PhRAST had the lowest sensitivity (62%) and highest specificity (99%). The CAP achieved higher sensitivity (74%) with comparable specificity (96%) while the mRAST had even higher sensitivity (90%) but lower specificity (87%). The overall frequency of positive results in controls was 0% for PhRAST, 1.7% for CAP, and 3.3% for mRAST. If the threshold for a positive mRAST was raised to greater than or equal to class 2, this assay achieved performance characteristics similar to the CAP. If the results of these in vitro tests are used as the sole guide to the prescription of environmental control and immunotherapy in unselected patients with rhinitis and asthma, the performance characteristics of the CAP make it the preferred test

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[109] - Ewan PW, Coote D. Evaluation of a capsulated hydrophilic carrier polymer (the ImmunoCAP) for measurement of specific IgE antibodies. Allergy 1990;45 :22-29

The Pharmacia CAP System is a new assay for serum specific IgE , utilising a solid phase capable of binding more antigen than conventional systems. The CAP System has been evaluated in 69 consecutive patients referred to one allergy clinic in relation to skin prick test (SPT), radioallergosorbent test (Phadebas RAST) and specific allergy diagnosis for five inhalant allergens, D.pteronyssinus, timothy grass pollen, cat epithelium/dander, Cladosporium and Alternaria. Good correlation was obtained between RAST and CAP for all allergens, e.g. r = 0.974 for D.pteronyssinus and r = 0.964 for grass pollen. When sensitivity and specificity were examined for both CAP and RAST versus SPT, CAP was usually found to be of greater sensitivity than RAST, and of similar or slightly lower specificity. SPT gave more positive reactions than either in vitro test , but CAP gave more positives than RAST. Twenty-two of 336 (6.6%) tests were CAP positive/RAST negative, whereas a negative CAP with a positive RAST occurred in only 2/336 (0.6%) tests. Of patients with any test (SPT or RAST or CAP) for specific IgE positive, up to 20-30% did not have clinical allergy, confirming the importance of the history in interpreting these tests. Our results suggest that, for the allergens tested, the Pharmacia CAP System is more sensitive than the RAST, identifying more positive tests and approximating more closely to the SPT. It offers the additional advantages of speed and efficiency

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[110] - Metz-Favre C, Birba E, de Blay F, Pauli G. Que nous apporte le dosage de l'allergène recombinant rBet v 1 dans la prise en charge de la pollinose printannière ? Rev Fr Allergol Immunol Clin 2004;44:357

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[111] - Gleeson M, Cripps AW, Hensley MJ, Wlodarczyk JH, Henry RL, Clancy RL. A clinical evaluation in children of Pharmacia ImmunoCAP system for inhalant allergens. Clin Exp Allergy 1996;26:697-702

"BACKGROUND: The Pharmacia ImmunoCAP system (CAP) for assaying serum IgE specific antibodies was evaluated in a clinical setting against skin-prick test (SPT) performed using Dome/Hollister-Steir allergen extracts. The five common inhalant allergens D. pteronyssinus, D. farinae, mould mix, grass mix and cat epithelium were tested concurrently by both methods in 167 children aged 7.5-12 years. The specific SPT for D. pteronyssinus and D. farinae were also tested against the CAP house dust mite (HDM) mix . OBJECTIVE: The purpose of the study was to determine the sensitivity and specificity of the Pharmacia ImmunoCAP system for detecting serum IgE specific antibodies to inhalant allergens in a clinical setting, using SPT result as the ""gold standard' . METHODS: The SPTs were performed using Dome/Hollister-Steir allergen extracts. The serum IgE specific antibodies were quantitated using the radioimmunoassay version of the Pharmacia ImmunoCAP system. A history of allergic disease was assessed using a validated questionnaire . RESULTS: SPT gave more positive reactions than CAP with the exception of cat epithelium. The concordance between SPT and CAP results was 91% over all the tests. The concordance with SPT was slightly higher for the specific CAP for D. pteronyssinus and D. farinae (93% and 95% respectively) than for the CAP HDM mix (86% and 90% respectively). There was a higher proportion of positive results for both SPT and CAP in the 115 children defined as having a history of allergic disease. Using SPT defined allergy as the gold standard, the sensitivity of the CAP system was 87% for the two specific house dust mites but was lower for cat epithelium (67%), mould mix (59%) and grass mix (46%). The sensitivity of the CAP system improved for D. pteronyssinus (96%) and the HDM mix (91%) when tested in subjects defined as having a history of allergy associated disease. The specificity of the CAP system showed less variation between allergens and ranged from 90-99% . CONCLUSION: The results of this study of children aged 7.5-12 years demonstrate that, for the inhalant allergens tested, the Pharmacia ImmunoCAP system performs well in the setting of known allergic disease."

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[112] - Williams PB, Dolen WK, Koepke JW, Selner JC. Comparison of skin testing and three in vitro assays for specific IgE in the clinical evaluation of immediate hypersensitivity. Ann Allergy 1992;68:35-45

A new assay, Pharmacia CAP System (PCS), for allergen-specific IgE (sIgE) was evaluated in 198 new patients presenting with respiratory symptoms to an urban allergy practice. An experienced allergist examined each patient and clinically assessed sensitivity to timothy, short ragweed, Alternaria tenuis, cat, or D. farinae. Puncture and selected intracutaneous skin tests (ST) with these inhalant extracts were then performed. The physician again rated the likelihood of clinical sensitivity to each inhalant, and serum was obtained for sIgE measurements by Phadebas RAST, modified RAST, and PCS. Results of the three in vitro tests (IVT) correlated well with each other and generally agreed with physician assessments and ST results. Individual differences for extracts and assay methods were identified. A few patients with negative ST had positive IVT, but more patients with positive ST were negative by IVT. Modified RAST had greater sensitivity but less specificity than the other two IVT. Analysis of receiver operating characteristic curves showed that sensitivity of the three assays when compared at the 95% level of specificity, did not differ. This result suggests that the cutoff criterion for a positive modified RAST result is too low and should be reevaluated. Skin tests remain the most sensitive and specific test available. The Pharmacia CAP System is a clinically useful assay for sIgE and appears to be a clear advancement for IVT technology.

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[113] - Fernandez C, Cardenas R, Martin D, Garcimartin M, Romero S, de la Camara AG, Vives R. Analysis of skin testing and serum-specific immunoglobulin E to predict airway reactivity to cat allergens. Clin Exp Allergy 2007;37:391-399

Background When the clinical history is not conclusive, it may be difficult to make an accurate interpretation of the value of skin tests and serum-specific IgE to cat allergens in asthma cases. Objective To analyse the diagnostic efficiency of skin testing (ST) and serum-specific IgE to cat allergens, based on the results of bronchial-specific challenge with cat epithelium. Methods Sixty-four asthma patients (49 with cat exposure and 15 without) who did not clearly relate their asthma symptoms to cat exposure and had a positive skin prick testing and/or a positive cat dander-specific IgE determination (CAP-system) underwent intradermal skin tests and specific bronchial challenge with cat epithelium. The results were analysed by receiver operating characteristics curves (ROC curves) and logistic regression. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for different cut-off points. Results Twenty-seven patients (42.2%) had a positive bronchial-specific challenge. The area under the ROC curve for serum-specific IgE quantification is 0.85, which makes a good diagnostic tool out of this test. Intradermal ST predicts the outcome of the bronchoprovocation test better than skin prick testing (area under the ROC curve of 0.74 vs. area under the ROC curve of 0.54, respectively). The logistic regression analysis shows that the estimated probability of a positive bronchial challenge is >/=93% if CAP values are >/=17 kU(A)/L, whereas if CAP values are less than 0.35 kU(A)/L the estimated probability of a positive bronchial challenge is 16%. When the intradermal skin test is negative, the estimated probability of a positive bronchoprovocation test is 9%, being the test that better identifies patients with a negative bronchoprovocation test. Conclusions Levels of serum-specific IgE to cat allergens and intradermal ST can be used to diagnose and treat more accurately asthmatic patients sensitized to cat epithelium when there is uncertainty about cat epithelium causality

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[114] - Witteman AM, Stapel SO, Perdok GJ, Sjamsoedin DH, Jansen HM, Aalberse RC, et al. The relationship between RAST and skin test results in patients with asthma or rhinitis: a quantitative study with purified major allergens. J Allergy Clin Immunol 1996;97:16-25

BACKGROUND: Study of the relationship between skin test results and IgE antibody levels is seriously hampered by the use of conventional allergen extracts because the precise amount of relevant allergen for each patient is unknown. OBJECTIVE: This study was designed to investigate skin reactivity with purified major allergens and to assess the relation with serum levels of IgE antibodies and to determine which additional factors contribute to the skin test result. METHODS: We used five purified major allergens (Der p 1, Der p 2, Fel d 1, Lol p 1, and Lol p 5) in skin tests, RASTs, and histamine release tests in 43 multisensitized patients with asthma or rhinitis. RESULTS: The differences in biologic activity of the five major allergens at a given level of specific IgE are within one order of magnitude. A significant residual variation remains in the correlation between skin test results and levels of IgE antibodies, which cannot be explained by imprecision of both tests (Pearson log skin test vs log specific IgE: r = 0.46-0.92). With similar levels of specific IgE, the amount of allergen that is required for a positive skin test result may differ by as much as a factor of 100 between patients. The amount of total IgE in serum contributes significantly to the skin test result. High values of total IgE are accompanied by a lower skin reactivity for allergen. Within individuals, allergens that cause skin test results that deviate from the prediction based on IgE antibody level often show a similar deviation in the histamine release test. This indicates that the type of IgE response (i.e., affinity or epitope recognition pattern) contributes significantly to the skin test result. Skin reactivity for histamine does not significantly influence the skin reactions expressed as allergen threshold. However, increased skin reactions with higher allergen dosages depend on histamine reactivity. CONCLUSION: The major allergens tested show similar biologic activities. In addition to IgE antibody level, total serum IgE and type of IgE antibody response contribute significantly to the skin test threshold for allergens. Even in a system with purified allergens, IgE antibody levels and skin test results are not interchangeable as an indicator of the degree of allergic sensitization

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[115] - Malandain H. The clinical use of specific IgE antibody testing – a further word of caution. Allergy 2004;59:359-360

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[116] - Söderström L, Kober A, Ahlstedt S, de Groot H, Lange CE, Paganelli R, et al. A further evaluation of the clinical use of specific IgE antibody testing in allergic diseases. Allergy 2003;58:921-928

"BACKGROUND: The evaluation and interpretation of the results from blood tests measuring specific immunoglobulin E (IgE) antibody concentration is currently made using the dichotomized result from the test despite a quantitative result is obtained. It has been shown that different levels of IgE antibodies, assessed by blood test and skin prick test, may have a relation to presence of symptoms, implying that there is more information in a quantitative result than in the dichotomous--positive or negative . OBJECTIVE: To investigate the clinical utility of quantification of IgE antibodies in the diagnosis of allergic patients and whether such procedure has any advantage to the presently dichotomously used sensitivity and specificity at a fixed cut-off . METHODS: Data from a previously published study (R. Paganelli, I.J. Ansoteugi, J. Sastre, C.-E. Lange, M.H.W.M. Roovers, H. de Groot, N.B. Lindholm, P.W. Ewan, Allergy, 1998; 53) analysing diagnosis of allergic patients in four different clinics were re-evaluated. In the original study consecutive patients with suspected IgE-mediated allergy had been examined and evaluated according to the clinical routine at each clinic, using case history, physical examination, skin tests and laboratory tests, except the test to be evaluated, and given a ""doctors' allergen-specific diagnosis"" as positive or negative. In the present study the relation between ""doctors' allergen-specific diagnosis"", expressed as pos/neg, and the quantitative levels of specific IgE antibody concentration was analysed using a logistic regression model. This presentation of results was also compared with the more common characteristics of sensitivity and specificity, and also with Receiver-operator characteristics (ROC) curves . RESULTS: The used logistic model described the relationship between allergen-specific diagnosis in each study and the levels of IgE antibodies. The shape of the curve illustrated the physicians' disposition for a positive diagnose in the study, in relation to the specific IgE antibody level. Differences in the shape of the curve was found both between allergens within clinics and between clinics for the same allergen. No association could be demonstrated between prevalence and shape of the curve . CONCLUSIONS: Conventional sensitivity/specificity figures or ROC concepts only use the qualitative statement of whether IgE is present or not. A risk assessment using the quantitative level of IgE antibody to an allergen increases the utility of the information in clinical context compared with a qualitative statement of whether IgE is present or not. The quantification demonstrated the link between specific IgE antibodies and allergic reactions. The use of objective, well performing quantitative tests should help improve diagnostic accuracy and might provide a way for the patient to understand and manage his or her daily situation and risk for reactions."

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[117] - Paganelli R, Ansotegui IJ, Sastre J, Lange CE, Roovers MH, de Groot H, et al. Specific IgE antibodies in the diagnosis of atopic disease. Clinical evaluation of a new in vitro test system, UniCAP, in six European allergy clinics. Allergy 1998;53:763-768

A new immunoassay system utilizing new automatic instrumentation, new software for evaluation of data, and reagents updated for increased speed and accuracy was evaluated. Six clinical studies included 894 consecutive patients. Major symptoms were rhinoconjunctivitis, asthma, atopic dermatitis, and urticaria. The prevalence of inhalant allergy was 54-69%. Phadiatop, detecting atopic sensitization to common inhalant allergens, agreed with clinical diagnosis in 764/836 cases (91.4%). The clinical sensitivity and specificity were 93% and 89%, respectively. The clinical sensitivity and specificity of UniCAP specific IgE derived from 5170 comparisons with clinical diagnosis were 89% and 91%, respectively. Specific IgE measurements in UniCAP and in the Pharmacia CAP System agreed in 266/274 cases (97%). A comparison of the sensitivity and specificity of Pharmacia CAP System RAST in 1987 and with UniCAP specific IgE in 1995 showed equivalent performance without change of efficacy or degradation of IgE antibodies after 8 years. The systems were equivalent also in terms of measured values (r=0.96, slope=1.12), confirming the standardization of allergens and of assay calibration. UniCAP is an efficient laboratory system for routine diagnostic testing of allergy and a valuable tool for basic studies on allergens and antibodies.

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[118] - Williams PB, Ahlstedt S, Barnes JH, Söderström L, Portnoy J. Are our impressions of allergy test performances correct ? Ann Allergy Asthma Immunol 2003;91:26-33

BACKGROUND: The clinical diagnosis is often subjective and susceptible to bias, yet it is the primary standard by which diagnostic tests are judged. Consequently, our opinions regarding various diagnostic tests may not be entirely accurate . OBJECTIVE: To investigate the accuracy of the clinical history compared with concordant skin and quantitative specific IgE (s-IgE) measurements . METHODS: Consecutive, consenting patients (N = 152) at 2 different allergy centers were examined by history and physical examination (HPE) alone to determine their sensitivity to 7 common allergens. Results were classified as positive, negative, or indeterminate. The HPE results were then compared to concordant skin prick testing (SPT) and s-IgE measurements and to quantitative IgE antibody measurements with and without knowledge of the SPT results . RESULTS: Diagnosis by HPE deviated considerably from concordant SPT and s-IgE results. This deviation differed between allergists and allergens, reflecting a positive HPE bias that averaged 22%. Seventy-six percent of the HPE results judged indeterminate were resolved as negative. Using additional information from the quantification of s-IgE antibodies, considerable differences between the sites in the level of s-IgE associated with a positive HPE result with and without SPT results were observed . CONCLUSIONS: Relative to the SPT and quantification of s-IgE antibodies, the diagnosis by HPE alone to common allergens is not consistent. Discrepancies were dependent on both allergen and allergist. The quantitative s-IgE data revealed that allergists use available information from the HPE and SPT differently. Since the HPE is the primary standard used in judging test efficacy (sensitivity and specificity), our current impressions of test performances are not likely to be accurate.

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[119] - Fernandez C, Cardenas R, Martin D, Garcimartin M, Romero S, de la Camara AG, Vives R. Analysis of skin testing and serum-specific immunoglobulin E to predict airway reactivity to cat allergens. Clin Exp Allergy 2007;37:391-399

Background When the clinical history is not conclusive, it may be difficult to make an accurate interpretation of the value of skin tests and serum-specific IgE to cat allergens in asthma cases. Objective To analyse the diagnostic efficiency of skin testing (ST) and serum-specific IgE to cat allergens, based on the results of bronchial-specific challenge with cat epithelium. Methods Sixty-four asthma patients (49 with cat exposure and 15 without) who did not clearly relate their asthma symptoms to cat exposure and had a positive skin prick testing and/or a positive cat dander-specific IgE determination (CAP-system) underwent intradermal skin tests and specific bronchial challenge with cat epithelium. The results were analysed by receiver operating characteristics curves (ROC curves) and logistic regression. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for different cut-off points. Results Twenty-seven patients (42.2%) had a positive bronchial-specific challenge. The area under the ROC curve for serum-specific IgE quantification is 0.85, which makes a good diagnostic tool out of this test. Intradermal ST predicts the outcome of the bronchoprovocation test better than skin prick testing (area under the ROC curve of 0.74 vs. area under the ROC curve of 0.54, respectively). The logistic regression analysis shows that the estimated probability of a positive bronchial challenge is >/=93% if CAP values are >/=17 kU(A)/L, whereas if CAP values are less than 0.35 kU(A)/L the estimated probability of a positive bronchial challenge is 16%. When the intradermal skin test is negative, the estimated probability of a positive bronchoprovocation test is 9%, being the test that better identifies patients with a negative bronchoprovocation test. Conclusions Levels of serum-specific IgE to cat allergens and intradermal ST can be used to diagnose and treat more accurately asthmatic patients sensitized to cat epithelium when there is uncertainty about cat epithelium causality

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[120] - Plebani M, Borghesan F, Basso D, Faggian D. Receiver-operating characteristic (ROC) curves: a fundamental tool for improving the clinical usefulness of in vitro IgE tests. Allergy 1996;51:407-411

In order to establish the most efficient thresholds for serum allergen-specific IgE measured by the Pharmacia CAP System, a "second-generation" in vitro method, we evaluated results from 89 subjects with suspected inhalant allergies, using receiver-operating characteristic (ROC) curve analysis. Sera samples were analyzed by the Pharmacia CAP System for specific IgE, the choice of allergens to be tested being based upon the symptoms and clinical history of each patient. Results were analyzed by ROC analysis for the five most representative allergens, cat dander (e1), Dermatophagoides pteronyssinus (d1), Lolium perenne (g5), wall pellitory (W19), and wormwood (w5). The areas under the ROC curves were found to be satisfactory, ranging from 0.931 (e1) to 0.974 (g5) when we excluded w5, which had a significantly smaller area (0.81). To establish the most efficient threshold for each allergen, we calculated the clinical sensitivity, specificity, efficiency, and negative and positive predictive values. The thresholds giving a higher diagnostic efficiency were as follows: 0.40 kUa/l for d1, 0.55 kUa/l for g5, 0.50 for e1, 0.65 kUa/l for w19, and 1.00 kUa/l for w5. It is concluded that quantitative reporting of specific IgE measurement has numerous advantages, but the choice of the positive threshold seems to be a prerequisite for obtaining the optimal clinical efficiency. It is also suggested that a specific threshold should be adopted for each allergen.

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[121] - Fernandez C, Cardenas R, Martin D, Garcimartin M, Romero S, de la Camara AG, Vives R. Analysis of skin testing and serum-specific immunoglobulin E to predict airway reactivity to cat allergens. Clin Exp Allergy 2007;37:391-399

Background When the clinical history is not conclusive, it may be difficult to make an accurate interpretation of the value of skin tests and serum-specific IgE to cat allergens in asthma cases. Objective To analyse the diagnostic efficiency of skin testing (ST) and serum-specific IgE to cat allergens, based on the results of bronchial-specific challenge with cat epithelium. Methods Sixty-four asthma patients (49 with cat exposure and 15 without) who did not clearly relate their asthma symptoms to cat exposure and had a positive skin prick testing and/or a positive cat dander-specific IgE determination (CAP-system) underwent intradermal skin tests and specific bronchial challenge with cat epithelium. The results were analysed by receiver operating characteristics curves (ROC curves) and logistic regression. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for different cut-off points. Results Twenty-seven patients (42.2%) had a positive bronchial-specific challenge. The area under the ROC curve for serum-specific IgE quantification is 0.85, which makes a good diagnostic tool out of this test. Intradermal ST predicts the outcome of the bronchoprovocation test better than skin prick testing (area under the ROC curve of 0.74 vs. area under the ROC curve of 0.54, respectively). The logistic regression analysis shows that the estimated probability of a positive bronchial challenge is >/=93% if CAP values are >/=17 kU(A)/L, whereas if CAP values are less than 0.35 kU(A)/L the estimated probability of a positive bronchial challenge is 16%. When the intradermal skin test is negative, the estimated probability of a positive bronchoprovocation test is 9%, being the test that better identifies patients with a negative bronchoprovocation test. Conclusions Levels of serum-specific IgE to cat allergens and intradermal ST can be used to diagnose and treat more accurately asthmatic patients sensitized to cat epithelium when there is uncertainty about cat epithelium causality

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[122] - Li TM, Chuang T, Tse S, Li SM, Hovanec-Burns D, El Shami AS. Quantification of allergen-specific IgE (sIgE): Comparison of logistic regression-derived probability curves obtained from two quantitative sIgE methodologies: Pharmacia CAP FEIA and DPC IMMULITE® 2000. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1165

Background: The validity and benefit of probability curves in allergen-specific IgE (sIgE) testing and their applicability to more than a few allergens are still pending. One study (Sampson, 2001) presented probability curves for 4 food allergens and proposed decision points to rule out the need for food challenges in his clinic. Since then, such cutoffs were reported to be dependent on population, age, allergen and disease (Boyano-Martinez, 2001). A recent article (Söderström, 2003) extended the use of probability curves to 8 inhalant allergens. The results were allergen dependent and site specific. None of the studies, however, has addressed the potential impact of IgE specific for cross-reacting carbohydrate determinants (CCD) on these probability curves (Mari, 2002). Method: In previous studies reported by Ollert 2001, patients with suspected IgE-mediated allergy were evaluated according to clinical positive or negative allergen- specific diagnosis. In the present study, we use logistic regression model to compare the probability curves for inhalant allergens using two quantitative sIgE assays: an FEIA (Pharmacia CAP) and DPC's automated chemiluminescent IMMULITE® 2000. The FEIA yields explicit results down to 0.35 kU/L; the "third-generation" IMMULITE 2000 has an analytical sensitivity of 0.1 kU/L. Results: For all the inhalant allergens under study, almost identical overall probability curves were obtained for the FEIA and IMMULITE 2000 methods. For D1, W6, E1, D2, G6 and G3, probability curves obtained with the two methods were not significantly different. The probability curves were allergen dependent. The FEIA and IMMULITE 2000 methods gave similar allergen-dependent profiles, however, and in one case (W6), both methods yielded identical probability curves. Conclusions: The present study demonstrates that the 2 sIgE assays yield comparable probability curves for the inhalant allergens tested. Because the FEIA method is unable to yield explicit results below 0.35 kU/L, the probability curve generated is necessarily truncated at this level. Methods for sIgE measurement that allow for extending probability curves below 0.35 kU/L can also assess assay performance more definitively at the traditional 0.35 kU/L cutoff. Thus the third-generation IMMULITE 2000 method is a better tool for defining probability curves, as the applicability of such curves in allergy testing and the potential impact of IgE to CCD become better understood.

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[123] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[124] - Keating KM, Segal DB, Craig SJ, Nault AK, Semensi V, Wasserman AS, et al. Enhanced immunoreactivity and preferential heterodimer formation of reassociated Fel d I recombinant chains. Mol Immunol 1995;32:287-293

In this study we have addressed the question of whether reassociating the two recombinant protein chains that comprise the major cat dander allergen, Fel d I, would change the overall IgE and allergic patient T cell immunoreactivity compared to the native molecule. To accomplish this, the chains were combined under reducing and denaturing conditions, then allowed to reassociate by dilution and extensive dialysis against a physiological buffer. An initial examination of the reaction products using quantitative capture ELISA demonstrated comparable reactivity to Fel d I. Further analysis, using a pool of cat allergic patient plasma, showed that the products of the reassociation reaction (rFel d I) also possessed an enhanced IgE binding capacity. Depletion ELISA results gave only a 5% difference in reactivity between rFel d I and the native protein versus a 20% difference with the mixture of the two chains. Comparative secondary T cell stimulation assays were subsequently performed using cat allergic patient peripheral blood lymphocytes. Here the results demonstrated no loss of reactivity with the reassociated chains as compared to Fel d I or the two mixed recombinant chains. To biochemically characterize the products of the reassociation reaction we have performed reverse phase HPLC and then analysed the isolated fractions by mass spectrometry. It was clear from these results that like the native Fel d I, the products of the reassociation reaction favored heterodimer formation, with no homodimer being detected. This implies that the reassociated protein chains had preferentially adopted a native-like conformation.

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[125] - Vailes LD, Sun AW, Ichikawa K, Wu Z, Sulahian TH, Chapman MD, et al. High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells. J Allergy Clin Immunol 2002;110:757-762

BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule . OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells . METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry . RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein . CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.

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[126] - Gronlund H, Bergman T, Sandstrom K, Alvelius G, Reininger R, Verdino P, et al. Formation of disulfide-bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli. J Biol Chem 2003;278:40144-40151

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[127] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[128] - Gronlund H, Bergman T, Sandstrom K, Alvelius G, Reininger R, Verdino P, et al. Formation of disulfide-bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli. J Biol Chem 2003;278:40144-40151

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[129] - Vailes LD, Sun AW, Ichikawa K, Wu Z, Sulahian TH, Chapman MD, et al. High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells. J Allergy Clin Immunol 2002;110:757-762

BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule . OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells . METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry . RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein . CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.

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[130] - Smith W, Butler AJL, Hazell LA, Chapman MD, Pomés A, Nickels DG, et al. Fel d 4, a cat lipocalin allergen. Clin Exp Allergy 2004;34:1732-1738

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals . OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels . METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed . RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog . CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.

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[131] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[132] - Roper JM, Berg A, Satinover SM, Ronmark E, Lundback B, Platts-Mills TAE. IgE Antibodies to Mammalian Antigens in a Population-based Cohort of 963 Teenagers Living in Northern Sweden. J Allergy Clin Immunol 2009;123:S22

RATIONALE: In this cold and dry climate, cat and dog sensitization strongly predict asthma risk. By contrast, sensitization to dust mite, cockroach, and fungi are not associated with respiratory symptoms. Using sera from teenagers participating in a prospective study, we evaluated IgE antibodies to mammalian allergens. METHODS: Sera from 963 18 year olds from Northern Sweden were assayed for IgE to cat, dog, and horse using ImmunoCAP. RESULTS: IgE to cat (e1) was present in 219 (23%) sera, and correlated closely with IgE to Fel d 1 (r = 0.91, p < 0.001). The assessment for dog sensitization was influenced by the choice of assay: there were 84 (9%) positive sera to epithelium (e2), compared with 190 (20%) positive sera to dander (e5). The correlation between dog epithelium and cat was higher (r =0.59, p < 0.01) than that of dog dander versus cat (r =0.24, p < 0.01). Additionally, 142 individuals (15%) were positive to horse (e3). While inter- species correlations were significant, 73 individuals (8%) had IgE to only one of the three allergens: 46 sera for cat, 18 for dog, and 9 were only positive for horse. Cross reactivity could be explained by IgE to the carbohydrate galactose-alpha-1,3-galactose, however assay of 150 sera with IgE to mammalian allergens identified only one positive (titer = 0.52 IU/mL). CONCLUSIONS: In this cohort where sensitization to mammalian proteins is strongly associated with asthma, there was extensive correlation between each of the allergens. However, 73 sera were positive to only one mammal.

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[133] - Grönlund H, Adédoyin J, Reininger R, Varga EM, Zach M, Fredriksson M, et al. Higher immunoglobulin E antibody levels to recombinant Fel d 1 in cat-allergic children with asthma compared with rhinoconjunctivitis. Clin Exp Allergy 2008;38:1275-1281

BACKGROUND: Current diagnosis of allergy and asthma to cat is confirmed using cat dander extract (CDE). We have previously engineered a recombinant major cat allergen, rFel d 1, with properties identical to the natural molecule . OBJECTIVE: The aim of the study was to evaluate IgE and IgG4 antibodies to rFel d 1 among sera from cat-allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria . METHODS: Cat-allergic children and adults from Sweden (n=27 and 31, respectively) and Austria (n=41 and 41) with RC and/or asthma were selected. Sera were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by CAP, and IgE to rFel d 1 by ELISA. Healthy subjects and non-cat-allergic patients (n=75) were included as controls . RESULTS: There was a high correlation between IgE responses to rFel d 1 and CDE among the 140 patients (r(s)=0.85, P<0.001); however, measured levels to rFel d 1 were on average 30% higher (P<0.0001). Ninety-eight percent of patients and none of the controls showed IgE to rFel d 1 and there was a threefold increased risk of asthma for half of the children with the highest IgE levels [odds ratio 3.23; 95% confidence interval (CI), 1.19-8.79] by ELISA. IgE responses to rFel d 1 among children with asthma were higher (median 19.4 kU/L) compared with children with RC (median 6.6 kU/L, P<0.05) and adults with asthma (median 3.0 kU/L, P<0.01). Furthermore, children with asthma displayed higher IgG4 levels than the asthmatic adults . CONCLUSION: A single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the current extract-based test. Elevated IgE antibody levels to Fel d 1 are suggested to be a risk factor for asthma in cat-allergic children.

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[134] - Lundgren T, Mattsson L, Marknell DeWitt A, Kihlström A, Kusoffsky E, Nordlund M, et al. Prevalence of specific IgE to rFel d 1, nFel d 2, rFel d 3 and cat dander extract in a cohort of allergic cat-sensitized children. Allergy Clin Immunol Int 2005;17(Suppl. 1):193

Background: Cat dander is an important cause of indoor allergy, eliciting a variety of symptoms such as rhinoconjunctivitis and asthma in affected individuals. The IgE antibody reactivity to cat dander is mainly directed to the major allergen Fel d 1, although reactivities to Fel d 2 (cat serum albumin) in up to 39% of patients and to rFel d 3 in up to 90% of the patients have been reported. The purpose of this study was to evaluate the individual contribution of rFel d 1, nFel d 2 and rFel d 3 to the specific IgE response against cat dander in paediatric patients sensitized to cat dander. Methods: Sera from 97 paediatric subjects (4-15 years, median 4.9) having allergy related symptoms including asthma, rhinoconjunctivitis, eczema and a positive SPT to cat extract, were used in the study. Quantitative determinations of specific IgE against cat dander extract, rFel d 1, nFel d 2 and rFel d 3 were performed using regular and experimental ImmunoCAP tests. Results: Out of the 97 subjects, 80% (78/97) had specific IgE against cat dander extract at a level of 0.35 kUA/L or higher. Specific IgE to rFel d 1 was detected in 86% (83/97) of the subjects whereas specific IgE to nFel d 2 and rFel d 3 was only present in 4% (4/97) and 2% (2/97) respectively. All subjects having specific IgE to nFel d 2 and rFel d 3 were also IgE-positive to rFel d 1. There was a strong correlation between the values of specific IgE to cat dander extract and rFel d 1, with the latter being on average 67% higher. Conclusions: The vast majority of subjects in our study group had specific IgE to rFel d 1 and rFel d 1 even exceeded cat dander extract in diagnostic sensitivity. Very few patients showed IgE reactivity to nFel d 2 or rFel d 3, which is in contrast to previous reports on adult subjects and with different assay methods. Our results suggest that the IgE response to cat dander at young age is essentially restricted to Fel d 1, which is therefore an excellent diagnostic marker of cat sensitization in paediatric patients.

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[135] - Grönlund H, Adédoyin J, Reininger R, Varga EM, Zach M, Fredriksson M, et al. Higher immunoglobulin E antibody levels to recombinant Fel d 1 in cat-allergic children with asthma compared with rhinoconjunctivitis. Clin Exp Allergy 2008;38:1275-1281

BACKGROUND: Current diagnosis of allergy and asthma to cat is confirmed using cat dander extract (CDE). We have previously engineered a recombinant major cat allergen, rFel d 1, with properties identical to the natural molecule . OBJECTIVE: The aim of the study was to evaluate IgE and IgG4 antibodies to rFel d 1 among sera from cat-allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria . METHODS: Cat-allergic children and adults from Sweden (n=27 and 31, respectively) and Austria (n=41 and 41) with RC and/or asthma were selected. Sera were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by CAP, and IgE to rFel d 1 by ELISA. Healthy subjects and non-cat-allergic patients (n=75) were included as controls . RESULTS: There was a high correlation between IgE responses to rFel d 1 and CDE among the 140 patients (r(s)=0.85, P<0.001); however, measured levels to rFel d 1 were on average 30% higher (P<0.0001). Ninety-eight percent of patients and none of the controls showed IgE to rFel d 1 and there was a threefold increased risk of asthma for half of the children with the highest IgE levels [odds ratio 3.23; 95% confidence interval (CI), 1.19-8.79] by ELISA. IgE responses to rFel d 1 among children with asthma were higher (median 19.4 kU/L) compared with children with RC (median 6.6 kU/L, P<0.05) and adults with asthma (median 3.0 kU/L, P<0.01). Furthermore, children with asthma displayed higher IgG4 levels than the asthmatic adults . CONCLUSION: A single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the current extract-based test. Elevated IgE antibody levels to Fel d 1 are suggested to be a risk factor for asthma in cat-allergic children.

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[136] - Saarne T, Grönlund H, Kull I, Wickman M, van Hage M. Monitoring cat allergy in the BAMSE birth cohort at 4 and 8 years of age by using rFel d 1. Allergy 2007;62(suppl. 83):149-150

Background: Domestic cat is one of the most important sources of indoor allergens causing asthma symptoms. In order to predict and prevent from deterioration of respiratory allergic disease, early detection of sensitization is important. Today, cat dander extract (CDE) is the most commonly used tool in the diagnosis of cat sensitisation. The aims of this study were to investigate if the recombinant form of the major cat allergen Fel d 1 (rFel d 1) could substitute cat dander extract in the diagnosis of cat sensitisation and if children at risk of developing cat allergy could be identified at an earlier stage. Methods: Children (n=144) from the large birth cohort BAMSE in Stockholm, Sweden, were selected on the basis of 4 year and 8 year follow-up questionnaires. The selection criteria were symptoms related to asthma or rhinoconjunctivitis in contact with cat or where such symptoms were suspected. Blood samples were taken from each patient at both 4 and 8 years of age and serum was analysed for allergen-specific IgE to CDE and rFel d 1. IgE antibodies to CDE were measured by the ImmunoCAP˙ System (Phadia AB, Uppsala, Sweden), using the cut-off limit 0.35 kU/L. A quantitative ELISA assay with a cut-off limit of 0.037 kU/L was established for measuring IgE antibodies to rFel d 1. Results: Of those being certain of symptoms to cat at 4 years of age, the same number of children, 25/33 (76%), had detectable levels of IgE to both rFel d 1 and CDE at this age. However, at the same age, 14/42 (33%) of the group suspecting symptoms at 4 years had detectable IgE levels to rFel d 1, but only 9/42 (21%) were detected by CDE. In the group developing symptoms after 4 years of age, 60/69 (87%) were detected with rFel d 1 and 57/69 (83%) with CDE at 8 years. Among these children, 33/69 (48%) had clearly detectable levels of IgE to rFel d 1 already at 4 years of age, while 26/69 (38%) were detected by CDE. A high correlation between the IgE levels measured by the two assays was found. Conclusions: We here show that the single major recombinant cat allergen, rFel d 1, is at least as sensitive as cat dander extract in the diagnosis of cat sensitisation. Furthermore, we show that children at risk of developing cat allergy are sensitised to cat already before the parents‚ awareness of symptoms.

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[137] - Jaramillo JC, Sanchez-Gonzalez MJ, Blanca-Lopez N, Jimenez MA, Rodriguez J, Daroca P et al. Usefulness of Inmunoglobulin E Antibody Levels to Recombinant Fel d 1 in Predicting the Cat Specific Bronchial Provocation Test Results. J Allergy Clin Immunol 2009;123:S8

RATIONALE: Recombinant Fel d 1(rFel d 1) could be a good candidate for replacing cat dander extracts in diagnostics for cat allergy.We analyze this topic in asthmatic patients sensitized to cat using the specific bronchial provocation test (BPT) results as reference. METHODS: Thirty-nine asthmatic patients (mean age 31.2 ± 8.4 years) with positive results for skin prick testing (SPT) and/or detectable cat allergens serum specific IgE (CAP -System FEIA) underwent a BPT with cat epithelium. In addition, sera were tested for specific IgE to rFel d 1 (InmunoCAP PHADIA). The cut off point for a positive result of both in vitro tests was 0.35 kUA/l. Statistical analysis of results was carried out by Receiver Operating Characteristics (ROC) curves. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values were also calculated for both in vitro tests. RESULTS: Cat epithelium BPT was positive in 24 subjects. The area under the curve for rFel d 1 was 0.839 (CI 0.70-0.97), and 0.836 (CI 0.70-0.96) (p=0.959) for cat allergen specific IgE. For rFel d 1 91.6% sensitivity, 40% specificity, 70% PPV and 75% NPV were obtained, for cat specific IgE 100% sensitivity, 6.67% specificity, 63% PPVand 100%NPV. CONCLUSIONS: Cat epithelium and rFel d 1 serum-specific IgE are useful tests in predicting the outcome of cat specific BPT. Both tests have similar efficacy; however rFel d1 seems to be more accurate to rule out specific bronchial hyperreactivity.

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[138] - Fernandez C, Cardenas R, Martin D, Garcimartin M, Romero S, de la Camara AG, Vives R. Analysis of skin testing and serum-specific immunoglobulin E to predict airway reactivity to cat allergens. Clin Exp Allergy 2007;37:391-399

Background When the clinical history is not conclusive, it may be difficult to make an accurate interpretation of the value of skin tests and serum-specific IgE to cat allergens in asthma cases. Objective To analyse the diagnostic efficiency of skin testing (ST) and serum-specific IgE to cat allergens, based on the results of bronchial-specific challenge with cat epithelium. Methods Sixty-four asthma patients (49 with cat exposure and 15 without) who did not clearly relate their asthma symptoms to cat exposure and had a positive skin prick testing and/or a positive cat dander-specific IgE determination (CAP-system) underwent intradermal skin tests and specific bronchial challenge with cat epithelium. The results were analysed by receiver operating characteristics curves (ROC curves) and logistic regression. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for different cut-off points. Results Twenty-seven patients (42.2%) had a positive bronchial-specific challenge. The area under the ROC curve for serum-specific IgE quantification is 0.85, which makes a good diagnostic tool out of this test. Intradermal ST predicts the outcome of the bronchoprovocation test better than skin prick testing (area under the ROC curve of 0.74 vs. area under the ROC curve of 0.54, respectively). The logistic regression analysis shows that the estimated probability of a positive bronchial challenge is >/=93% if CAP values are >/=17 kU(A)/L, whereas if CAP values are less than 0.35 kU(A)/L the estimated probability of a positive bronchial challenge is 16%. When the intradermal skin test is negative, the estimated probability of a positive bronchoprovocation test is 9%, being the test that better identifies patients with a negative bronchoprovocation test. Conclusions Levels of serum-specific IgE to cat allergens and intradermal ST can be used to diagnose and treat more accurately asthmatic patients sensitized to cat epithelium when there is uncertainty about cat epithelium causality

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[139] - van Ree R, Van Leeuwen WA, Bulder I, Bond J, Aalberse RC. Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999;104:1223-1230

Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.

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[140] - Hrabak TM, Dice J, Hagan L, Whisman B. Comparison of Protein Antigens Among Commercially Available Dog Extracts. J Allergy Clin Immunol 2007;119(1 suppl):S186-S187

RATIONALE: Lyophilization of a randomly selected, commercially prepared 50ml vial of dog epithelial extract revealed minimal protein content contained in the extract. We formally evaluated multiple, commercially available, dog extracts for protein concentration in addition to the presence of the major allergens Can f 1 and Can f 2. METHODS: 50ml vials of various dog extracts (acetone precipitated, glycerinated, non-glycerinated), some from multiple lots, were obtained directly from the manufacturing labs, dialyzed and lyophilized to remove impurities; Protein Lowery's were subsequently performed on all extracts to determine protein concentration and SDS-PAGE gels evaluated for the presence of Can f 1 and Can f 2 as compared to known standards. RESULTS: Protein concentrations among commercial extracts ranged from 1.45 to 24.25 grams of protein per ml. SDS-PAGE revealed the varied presence of the major allergens, Can f 1 and Can f 2 and only 1 extract apparently contained both allergens. CONCLUSIONS: Considerable variation exists in commercially prepared dog extracts both in protein concentration as well as the presence of the major allergens Can f 1 and Can f 2. Depending on the source of the manufactured dog extract, dog sensitive patients may be missed on routine skin testing, and, those receiving dog immunotherapy may not be obtaining full benefit as the relevant protein allergen may not be present in the vaccine. These results suggest the possible need for a standardized dog extract for diagnosis and treatment of the clinically sensitive, dog allergic patient.

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[141] - Curin M, Reininger R, Swoboda I, Valenta R, Spitzauer S. Skin prick test extracts for dog allergy diagnosis show considerable variations regarding the content of major and minor dog allergens. Allergy 2009;64(Suppl. 90):273

Background: Commercial Skin prick test (SPT) extracts used for the diagnosis of dog allergy are prepared by extracting allergens from natural sources e.g. dog hair and dander. Due to different starting material and extraction methods of different manufacturers it is likely that extracts differ regarding their allergen contents. Methods: The total protein content and composition of dog SPT extracts from five European manufacturers was compared by silverstained SDS-PAGE. Specific antibodies were used to detect major and minor allergens in each extract by immunoblotting. Additionally, sera of patients suffering from dog allergy were used to detect dog allergens in SPT extracts. Results: SPT extracts showed approximately 20-fold variation regarding the total protein content. Signal intensities of major dog allergens Can f 1 and Can f 2 varied approximately 15-fold between the extracts. Moreover, in one of the extracts major allergens Can f 1 and Can f 2 could not be detected at all by immunoblotting. Can f 3, .i.e. dog albumin contents also showed great variability. In one of the dog SPT extracts the presence of human serum albumin was detected with HSA-specific antibodies. Conclusion: The observed variability of commercial dog SPT extracts regarding allergen contents will likely have negative influence on the accuracy of diagnosis of dog allergy. Supported by grant F1804 of the Austrian Science Fund, the Christian Doppler Association and Biomay, Austria.

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[142] - Roper JM, Berg A, Satinover SM, Ronmark E, Lundback B, Platts-Mills TAE. IgE Antibodies to Mammalian Antigens in a Population-based Cohort of 963 Teenagers Living in Northern Sweden. J Allergy Clin Immunol 2009;123:S22

RATIONALE: In this cold and dry climate, cat and dog sensitization strongly predict asthma risk. By contrast, sensitization to dust mite, cockroach, and fungi are not associated with respiratory symptoms. Using sera from teenagers participating in a prospective study, we evaluated IgE antibodies to mammalian allergens. METHODS: Sera from 963 18 year olds from Northern Sweden were assayed for IgE to cat, dog, and horse using ImmunoCAP. RESULTS: IgE to cat (e1) was present in 219 (23%) sera, and correlated closely with IgE to Fel d 1 (r = 0.91, p < 0.001). The assessment for dog sensitization was influenced by the choice of assay: there were 84 (9%) positive sera to epithelium (e2), compared with 190 (20%) positive sera to dander (e5). The correlation between dog epithelium and cat was higher (r =0.59, p < 0.01) than that of dog dander versus cat (r =0.24, p < 0.01). Additionally, 142 individuals (15%) were positive to horse (e3). While inter- species correlations were significant, 73 individuals (8%) had IgE to only one of the three allergens: 46 sera for cat, 18 for dog, and 9 were only positive for horse. Cross reactivity could be explained by IgE to the carbohydrate galactose-alpha-1,3-galactose, however assay of 150 sera with IgE to mammalian allergens identified only one positive (titer = 0.52 IU/mL). CONCLUSIONS: In this cohort where sensitization to mammalian proteins is strongly associated with asthma, there was extensive correlation between each of the allergens. However, 73 sera were positive to only one mammal.

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[143] - Matito A, Blanco S, Gajate P, Martín L, Vives R, Ortega C, et al. ROC Curve Analysis Of Serum-specific IgE Levels To Predict Airway Reactivity To Dog Allergens In Asthmatic Subjects. J Allergy Clin Immunol 2007;119(1 suppl):S82

RATIONALE: Sometimes it is difficult to know if bronchial hyperreactivity in asthmatic subjects sensitized to dog allergens is due precisely to that sensitization to dog allergens. The aim of this work has been to find an optimal cut-off value for serum-specific IgE level predictive of a positive specific bronchial response to dog epithelium in asthmatic patients. METHODS: We performed a bronchial provocation test (BPT) with dog epithelium (following ATS guidelines) in 67 asthmatic patients (mean age 29.4±10.48, female/male 1.68) with a positive skin prick-test (dog dander 100 BU, ALK-Abello) and/or detectable dog allergen serum-specific IgE (CAP-System FEIA). Statistical analyses of results were performed by Receiver Operating Characteristics (ROC) curve. Sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) for several cut-off points were also calculated. RESULTS: Dog epithelium BPT was positive in 30 subjects (44.8%). The area under the ROC curve for CAP values was 0.86. The cut-off 0.35 kUA/L showed 86.6% sensitivity, 72.97% specificity, 72% PPV, and 87% NPV. The cu-off 1.2 kUA/L, 70% sensitivity, 89% specificity, 84% PPV, and 78% NPV. The cut-off 10.7 kUA/L showed 36% sensitivity, 100% specificity, 100% PPV, and 66% NPV. CONCLUSIONS: In asthmatic patients, levels of serum-specific IgE to dog allergens could be useful to predict the outcome of BPT.

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[144] - Söderström L, Kober A, Ahlstedt S, de Groot H, Lange CE, Paganelli R, et al. A further evaluation of the clinical use of specific IgE antibody testing in allergic diseases. Allergy 2003;58:921-928

"BACKGROUND: The evaluation and interpretation of the results from blood tests measuring specific immunoglobulin E (IgE) antibody concentration is currently made using the dichotomized result from the test despite a quantitative result is obtained. It has been shown that different levels of IgE antibodies, assessed by blood test and skin prick test, may have a relation to presence of symptoms, implying that there is more information in a quantitative result than in the dichotomous--positive or negative . OBJECTIVE: To investigate the clinical utility of quantification of IgE antibodies in the diagnosis of allergic patients and whether such procedure has any advantage to the presently dichotomously used sensitivity and specificity at a fixed cut-off . METHODS: Data from a previously published study (R. Paganelli, I.J. Ansoteugi, J. Sastre, C.-E. Lange, M.H.W.M. Roovers, H. de Groot, N.B. Lindholm, P.W. Ewan, Allergy, 1998; 53) analysing diagnosis of allergic patients in four different clinics were re-evaluated. In the original study consecutive patients with suspected IgE-mediated allergy had been examined and evaluated according to the clinical routine at each clinic, using case history, physical examination, skin tests and laboratory tests, except the test to be evaluated, and given a ""doctors' allergen-specific diagnosis"" as positive or negative. In the present study the relation between ""doctors' allergen-specific diagnosis"", expressed as pos/neg, and the quantitative levels of specific IgE antibody concentration was analysed using a logistic regression model. This presentation of results was also compared with the more common characteristics of sensitivity and specificity, and also with Receiver-operator characteristics (ROC) curves . RESULTS: The used logistic model described the relationship between allergen-specific diagnosis in each study and the levels of IgE antibodies. The shape of the curve illustrated the physicians' disposition for a positive diagnose in the study, in relation to the specific IgE antibody level. Differences in the shape of the curve was found both between allergens within clinics and between clinics for the same allergen. No association could be demonstrated between prevalence and shape of the curve . CONCLUSIONS: Conventional sensitivity/specificity figures or ROC concepts only use the qualitative statement of whether IgE is present or not. A risk assessment using the quantitative level of IgE antibody to an allergen increases the utility of the information in clinical context compared with a qualitative statement of whether IgE is present or not. The quantification demonstrated the link between specific IgE antibodies and allergic reactions. The use of objective, well performing quantitative tests should help improve diagnostic accuracy and might provide a way for the patient to understand and manage his or her daily situation and risk for reactions."

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[145] - Williams PB, Ahlstedt S, Barnes JH, Söderström L, Portnoy J. Are our impressions of allergy test performances correct ? Ann Allergy Asthma Immunol 2003;91:26-33

BACKGROUND: The clinical diagnosis is often subjective and susceptible to bias, yet it is the primary standard by which diagnostic tests are judged. Consequently, our opinions regarding various diagnostic tests may not be entirely accurate . OBJECTIVE: To investigate the accuracy of the clinical history compared with concordant skin and quantitative specific IgE (s-IgE) measurements . METHODS: Consecutive, consenting patients (N = 152) at 2 different allergy centers were examined by history and physical examination (HPE) alone to determine their sensitivity to 7 common allergens. Results were classified as positive, negative, or indeterminate. The HPE results were then compared to concordant skin prick testing (SPT) and s-IgE measurements and to quantitative IgE antibody measurements with and without knowledge of the SPT results . RESULTS: Diagnosis by HPE deviated considerably from concordant SPT and s-IgE results. This deviation differed between allergists and allergens, reflecting a positive HPE bias that averaged 22%. Seventy-six percent of the HPE results judged indeterminate were resolved as negative. Using additional information from the quantification of s-IgE antibodies, considerable differences between the sites in the level of s-IgE associated with a positive HPE result with and without SPT results were observed . CONCLUSIONS: Relative to the SPT and quantification of s-IgE antibodies, the diagnosis by HPE alone to common allergens is not consistent. Discrepancies were dependent on both allergen and allergist. The quantitative s-IgE data revealed that allergists use available information from the HPE and SPT differently. Since the HPE is the primary standard used in judging test efficacy (sensitivity and specificity), our current impressions of test performances are not likely to be accurate.

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[146] - Mattsson L, Lundgren T, Everberg H, Larsson H, Lidholm J. Prostatic kallikrein: A new major dog allergen. J AlIergy Clin Immunol 2009;123:362-368

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[147] - Matricardi PM, Bockelbrink A, Beyer K, Keil T, Niggemann B, Grüber C, et al. Primary versus secondary immunoglobulin E sensitization to soy and wheat in the Multi-Centre Allergy Study cohort. Clin Exp Allergy 2008;38:493-500

BACKGROUND: IgE sensitization to soy and wheat is classified as 'primary' when generated by food ingestion and 'secondary' when it as a consequence of primary sensitization to cross-reacting pollen antigens via inhalation. The age-specific relevance of these categories of sensitization throughout childhood is unknown . OBJECTIVE: To monitor the natural course of IgE sensitization against common food allergens in childhood in relation to sensitization against cross-reactive airborne allergens . METHODS: The German Multi-Centre Allergy Study with follow-up from birth to age 13 recruited initially 1314 children. IgE antibody levels against cow's milk, hen's egg, soy, wheat, mites, cat and dog dander, birch and grass pollens were tested. Longitudinal data were analysed from the 273 children with sera obtained at age 2, 5, 7 and 10 years of age . RESULTS: The point prevalence of sensitization (>1.0 kU/L) to milk and egg allergens progressively decreased from about 4% at 2 years to <1% at 10 years. By contrast, the prevalence of IgE to wheat and soy progressively increased with age, from 2% to 7% (soy) and from 2% to 9% (wheat). At 10 years of age, IgE to grass pollen was detected in 97% and 98% of the children reacting against soy and wheat, respectively; IgE to birch pollen was observed in 86% and 82% of the children reacting against soy and wheat, respectively. Early IgE sensitization to soy or wheat preceded that to grass or birch pollen in only 4% and 8% of participants sensitized to soy and wheat, respectively . CONCLUSION: IgE sensitization to soy and wheat is relatively uncommon and mostly primary in early infancy, more frequent and mostly secondary to pollen sensitization at school age. Clinical Implications Awareness should be raised to avoid unnecessary diet restrictions due to the high frequency of clinically irrelevant, secondary sensitization to soy and wheat in schoolchildren with pollinosis.

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[148] - Ribeiro F, Carrapatoso I, Geraldes L, Sousa N, Machado D, Chieira C. Is sensitisation to cat and dog relevant ?. Allergy 2009;64(Suppl. 90):377

Background: Many animals inside our houses are common sources of inhalant allergens that provoke significant allergy symptoms. Objectives: To evaluate cat and dog sensitization in an Immunoallergology outpatient consultation; to compare these sensitizations with other simultaneous sensitizations; evaluating clinical manifestations of allergic disease. Methods: We evaluated retrospectively the results of skin prick tests to aeroallergens (GA2LEN battery) done on patients observed in an Immunoallergology consultation over a 12 month period. Patients with positive tests to cat and/or dog were selected. Clinical manifestations of allergic disease were investigated, as well as their severity and other concomitant sensitizations. The relation between exposure to cat and dog allergens and clinical manifestations was investigated using a questionnaire. Results: From 1145 results evaluated, 230 patients were sensitized to cat and/or dog, and 75 patients (45 female) completed the study. The mean age was 27±12 years old. Clinically, 55 patients had asthma (24 intermittent, 16 mild persistent, 13 moderate persistent and 2 severe persistent); 72 patients had rhinitis (21 mild intermittent, 6 moderate/severe intermittent, 19 mild persistent, 24 moderate/ severe persistent). From out of these 72 patients with rhinitis, 17 had rhinoconjunctivitis and 3 rhinosinusitis. Besides the respiratory symptoms, 7 patients had clinical manifestations of atopic dermatitis, 2 had contact dermatitis, 5 angioedema and 2 urticaria. Fifty-one patients were sensitized to cat and 51 to dog. Simultaneous sensitization to cat and dog was found in 28 patients. Mean wheal size was 4.7mm to cat and 4.2mm to dog, with the mean wheal size being 5.7mm to histamine. Patients had, to a greater or less extent, other sensitizations, namely: mites (59 patients), cockroach (22), weeds (32), trees (20), grasses (17) and moulds (11). Two patients were only sensitized to cat and dog. Most patients did not associate their allergic symptoms with exposure to cat and dog. However, when there was an association, this was more reported in cat sensitization. Conclusion: Isolated sensitization to cat and dog does not seem relevant, usually appearing with other sensitizations and not being given much importance by most patients.

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[149] - Heinzerling L, Frew AJ, Bindslev-Jensen C, Bonini S, Bousquet J, Bresciani M, et al. Standard skin prick testing and sensitization to inhalant allergens across Europe – a survey from the GA2LEN network. Allergy 2005;60:1287-1300

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[150] - Eriksson NE, Holmen A. Skin prick tests with standardized extracts of inhalant allergens in 7099 adult patients with asthma or rhinitis: cross-sensitizations and relationships to age, sex, month of birth and year of testing. J Investig Allergol Clin Immunol 1996;6:36-46

The results of skin prick tests (SPTs) performed between 1981 and 1992 on 7099 adult patients with asthma and/or rhinitis were retrospectively analyzed. Standardized Soluprick extracts of Dermatophagoides pteronyssinus, animals and pollen, and unstandardized extracts of Cladosporium++ and chironomids (red mosquito larvae, Chir), were used. The proportion of atopics (patients with positive SPT results) was 44 percent, decreasing from 61 percent in patients 14-20 years old to 18 percent in patients 61-70 years old. The decrease with age was most pronounced with timothy, cat and horse allergens. In the whole patient group, timothy, cat and birch gave the highest number of positive SPTs. Positive SPTs with dog and Chir were more common in asthmatics than in patients with rhinitis, whereas pollen allergy was more common in patients with rhinitis. Sensitization against D. pteronyssinus, timothy and Chir was more common in men than in women. Of the atopic patients, 65 percent were sensitized against several allergens and 35 percent had a mono-allergy, most frequently to D. pteronyssinus (7.4 percent) and timothy (70 percent). The proportion monoallergies/multi-allergies was higher in older patients than in younger ones. The degree of atopy, expressed as the sum of plusses of the test results with eight allergens for each patient, was higher in younger patients than in older ones. The size of the wheals induced by the positive histamine control increased with age, and the histamine-induced wheals were larger in men than in women and larger in non-atopics than in topics. Strong correlations were found between test results with cat, dog and horse. A greater proportion of the patients with an isolated pollen allergy were born in February-May than was to be expected. The proportion of positive SPTs with mugwort (Artemisia), in relation to positive SPTs with other pollen allergens, decreased from 1981 to 1992. It can be concluded that sensitization to various inhalant allergens is influenced by age, and to a lesser extent by sex, and that pollen sensitization is influenced by the month of birth. During a 12-year period, sensitization to mugwort showed a decrease, as compared to other pollen allergens.

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[151] - Roberts G, Peckitt C, Northstone K, Strachan D, Lack G, Henderson J, et al. Relationship between aeroallergen and food allergen sensitization in childhood. Clin Exp Allergy 2005;35:933-940

BACKGROUND: Previous studies measuring the prevalence of allergen sensitization have been relatively small and used small numbers of allergens. To effectively evaluate children with atopic disease, we need an accurate knowledge of which allergens are important . OBJECTIVE: To measure the prevalence of sensitization within a large unselected birth cohort, to examine the associations between sensitization to different allergens and determine whether atopy can be defined by a small panel of allergens . METHODS: The Avon Longitudinal Study of Parents and Children is a population-based birth cohort of 13,638 singletons surviving to 4 weeks of age. The cohort was skin tested at 7 years of age to house dust mite (Dermatophagoides pteronyssinus), grass pollens, cat, peanuts, mixed tree nuts and egg and one of three other panels: animal danders, foods or aeroallergens. Sensitization was defined as a weal diameter of > or =3 mm. The strength of associations between sensitization to different allergens was tested by calculating the odds ratio adjusted for sensitization to D. pteronyssinus and grass pollen and gender . RESULTS: Valid data were obtained from 6412 singletons. Sensitization was most common to aeroallergens: grass pollens (8.5%), D. pteronyssinus (7.8%), cat (4.9%), D. farinae (3.6%), dog (2.7%), horse (1.4%), rabbit (1.4%). Of the foods tested, the most common sensitization was to peanut (1.4%) and mixed tree nuts (1.0%). More than 95% of subjects with sensitization to any of the 29 allergens tested were sensitized to one of grass, D. pteronyssinus or cat allergen. There were strong associations of multiple sensitizations both within and between different allergen classes (pollens, animals, foods, peanut and tree nuts) . CONCLUSIONS: Seven-year-old children in the UK are primarily sensitized to aeroallergens, but also to peanuts and tree nuts. There are strong associations between sensitization within allergen groups as well as between allergen groups. Further studies are required to observe whether similar associations are seen with clinical allergy to these allergens.

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[152] - Reininger R, Varga EM, Zach M, Balic N, Lindemeier AD, Swoboda I, et al. Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1. Clin Exp Allergy 2007;37:116-124

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited . OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1 . METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies . RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds . CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.

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[153] - Spitzauer S, Pandjaitan B, Muhl S, Ebner C, Kraft D, Valenta R, et al. Major cat and dog allergens share IgE epitopes. J Allergy Clin Immunol 1997;99:100-106

Patients allergic to cats and dogs frequently display IgE reactivity against allergens from different animals, suggesting a cross-sensitization to common allergenic determinants. Although albumins have been recognized as relevant cross-reactive allergens, little is known regarding cross-reactive epitopes of the major cat and dog allergens. OBJECTIVE: In this study, sera from patients allergic to cats and/or dogs were used to investigate the presence of common IgE epitopes among the major cat and dog allergens. METHODS: The IgE reactivity profile of 109 patients who were allergic to allergens from several species of animals was determined with nitrocellulose-blotted cat and dog allergens. Sera from patients who were strongly allergic to the major cat and dog allergens were tested for the presence of cross-reactive IgE antibodies by one-dimensional and two-dimensional immunoblot inhibition experiments and by quantitative measurements obtained with the CAP-FEIA system (Pharmacia). RESULTS: Sixty-eight of 109 patients with animal allergy showed IgE reactivity to cat allergens and dog allergens. Sera from patients with both cat and dog allergy detected allergens of similar molecular weight in nitrocellulose-blotted cat and dog hair/dander extracts. Common, as well as species-restricted, IgE epitopes of the major cat and dog allergens could be demonstrated by IgE inhibition studies. CONCLUSION: Shared IgE epitopes of the major cat and dog allergens may provide an explanation for the clinical observation that allergies to cats and dogs are frequently associated.

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[154] - Cabanas R, Lopez-Serrano MC, Carreira J, Ventas P, Polo F, Caballero MT, et al. Importance of albumin in cross-reactivity among cat, dog and horse allergens. J Investig Allergol Clin Immunol 2000;10:71-77

Different allergenic proteins have been involved in cross-reactivity among animals. Albumins seem to be cross-sensitizing allergenic components. The aim of this study was to assess the importance of albumin as a cross-reactive allergen in patients sensitized to cat, dog and horse. One hundred and seventeen patients sensitized to cat were tested for IgE reactivity using skin prick tests and RAST assays with cat, dog and horse hair/dander extracts and their purified albumin extracts. RAST-inhibition studies were carried out to assess cross-reactivity among cat, dog and horse and among their purified albumins. It was found that 22% of patients exhibited specific IgE to cat albumin; 41% of patients sensitized to cat were also sensitized to dog and horse. Out of these patients, 21% had IgE to three albumins and 17% to two. Reciprocal inhibitions were observed among cat, dog and horse albumins and also among cat, dog and horse hair/dander extracts, using in the latter experiment sera from patients not sensitized to albumins. IgE binding to horse extract was inhibited 30% by its homologous albumin and IgE binding to cat and dog extracts in almost 15% by their respective albumins. It was concluded that albumins from these three animals share some epitopes that account for the cross-reactivity observed in around one-third of patients sensitized to cat, dog and horse. Nevertheless, more than 50% of specific IgE that cross-reacts among these three animals is directed to allergens other than albumin

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[155] - Ohman JL Jr, Bloch KJ, Kendall S, Lowell FC. Allergens of mammalian origin. IV. Evidence for common allergens in cat and dog serum. J Allergy Clin Immunol 1976;57:560-568

IgE antibodies present in serum from 3 patients clinically sensitive to cat and dog were shown to combine with whole cat and dog sera linked to cellulose particles. Employing a modified radioallergosorbent technique (RAST), it was shown that cat and dog sera inhibited the binding of IgE antibodies to insolubilized cat serum and to insolubilized dog serum. These findings suggest that cat and dog sera have common allergens. Other mammalian sera were also tested and found to have little inhibitory activity. Cat and dog sera did not inhibit the binding of IgE antiragweed antibodies to an insolubilized ragweed fraction. Following separation of cat serum by gel filtration on Sephadex G-200 or electrophoresis in acrylamide gel, several fractions were found to inhibit the binding of IgE antibodies to insolubilized cat and dog sera. Whether the heterogeneity observed reflects the presence of single allergens with diverse size and charge properties or multiple allergens remains to be determined.

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[156] - Pauli G. Evolution in the understanding of cross-reactivities of respiratory allergens: the role of recombinant allergens. Int Arch Allergy Immunol 2000;123:183-195

The aim of this review is to show the impact of the use of purified and recombinant allergens to discriminate between co- and cross-sensitization to respiratory allergens. The author describes the evolution of diagnostic tests over the last decades; the tests initially allowed the detection of simultaneously positive cutaneous tests and/or simultaneous positivity of specific IgE to different allergen extracts, but they did not differentiate cross-sensitization from co-sensitization. RAST inhibition studies with crude extracts then established cross-reactivity, but did not identify the cross-reactive allergens involved. Later, immunoblot and CRIE inhibition were able to detect multiple cross-reactive allergens and to assess their physicochemical properties. But it is only since purified and recombinant allergens have been used in the different investigations that identification of cross-reactive allergens has been made possible at a molecular level. This historical approach is illustrated by examples selected from some of the main respiratory allergen sources: tree pollen, grass pollen, weed pollen, acarids, cockroaches and mammalians. For each of these allergen sources, the author gives an updated presentation of major and minor cross-reactive allergen molecules and refers to the last decade's major publications concerning immunochemical investigations carried out in the field of cross-reactive respiratory allergens. Emphasis is placed on the clinical applications for allergic patients: improvement in the accuracy of the diagnosis of sensitization, new concepts of immunotherapy based on genetically engineered hypoallergenic variants of cross-reactive allergens used alone or in combination, evaluation of allergen load with environmental tests using monoclonal antibodies against cross-reactive allergens.

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[157] - Viander M, Valovirta E, Vanto T, Koivikko A. Cross-reactivity of cat and dog allergen extracts: RAST inhibition studies with special reference to the allergenic activity in saliva and urine. Int Arch Allergy Appl Immunol 1983;71:252-260

The commercial cat and dog allergen extracts are traditionally prepared from pelt, fur or dander. However, there is increased evidence of the allergenicity of saliva and urine of the animals. We have investigated 25 asthmatic children with a positive cat and/or dog RAST result. All 20 subjects with a positive cat RAST gave a positive skin prick test result to cat saliva, cat urine and cat hair. Analogously, all 20 subjects with a positive dog RAST had a positive skin reaction to dog saliva, urine and dander. In RAST inhibition experiments with dog and cat allergen discs, dog saliva appeared to be at least as potent as a commercial dog dander and hair extract, while cat saliva was less potent than the respective commercial extract. Both dog and cat salivas were clearly more potent than the respective urine. Significant cross-reactivity was observed between cat hair and dog dander in the RAST inhibition, whereas saliva and urine were shown to be more species-specific. An experimental dog dander preparation had about the same specificity as, and even higher allergenic activity than, that of dog saliva or urine. Our results suggest that saliva actually may be the best source of cat and dog allergen preparations. The importance of urine warrants further investigation.

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[158] - Wüthrich B, Guérin B, Hewitt BE. Cross-allergenicity between extracts of hair from different dog breeds and cat fur. Clin Allergy 1985;15:87-93

Skin tests and RAST determinations with breed-specific dog allergen extracts and a cat allergen preparation were made on forty-four atopic patients divided into three groups. Group 1 were twenty dog-owning atopic patients without clinical signs of dog sensitivity, Group 2 contained twenty-one patients with a clinical history that suggested allergy to dogs, and Group 3 contained ten atopic patients who were sensitive to cats. In neither the in vivo nor the in vitro tests was there any evidence for dog breed specificity, nor was dog albumin found to be a major allergen in the population studied, though a few individuals showed strong RAST activity to albumin. Furthermore, a cat fur extract inhibited the reaction between dog hair and anti-dog serum, and a dog hair extract inhibited the reaction between cat fur extract and anti-cat serum.

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[159] - Spitzauer S, Pandjaitan B, Muhl S, Ebner C, Kraft D, Valenta R, et al. Major cat and dog allergens share IgE epitopes. J Allergy Clin Immunol 1997;99:100-106

Patients allergic to cats and dogs frequently display IgE reactivity against allergens from different animals, suggesting a cross-sensitization to common allergenic determinants. Although albumins have been recognized as relevant cross-reactive allergens, little is known regarding cross-reactive epitopes of the major cat and dog allergens. OBJECTIVE: In this study, sera from patients allergic to cats and/or dogs were used to investigate the presence of common IgE epitopes among the major cat and dog allergens. METHODS: The IgE reactivity profile of 109 patients who were allergic to allergens from several species of animals was determined with nitrocellulose-blotted cat and dog allergens. Sera from patients who were strongly allergic to the major cat and dog allergens were tested for the presence of cross-reactive IgE antibodies by one-dimensional and two-dimensional immunoblot inhibition experiments and by quantitative measurements obtained with the CAP-FEIA system (Pharmacia). RESULTS: Sixty-eight of 109 patients with animal allergy showed IgE reactivity to cat allergens and dog allergens. Sera from patients with both cat and dog allergy detected allergens of similar molecular weight in nitrocellulose-blotted cat and dog hair/dander extracts. Common, as well as species-restricted, IgE epitopes of the major cat and dog allergens could be demonstrated by IgE inhibition studies. CONCLUSION: Shared IgE epitopes of the major cat and dog allergens may provide an explanation for the clinical observation that allergies to cats and dogs are frequently associated.

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[160] - Blanc A, Donnay C, Metz-Favre C, Kopferschmitt-Kubler MC, de Blay F, Pauli G. Rôle de l'albumine sérique en tant qu'allergène croisant entre les mammifères. Rev Fr Allergol Immunol Clin 2006;46:361-362

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[161] - Cabanas R, Lopez-Serrano MC, Carreira J, Ventas P, Polo F, Caballero MT, et al. Importance of albumin in cross-reactivity among cat, dog and horse allergens. J Investig Allergol Clin Immunol 2000;10:71-77

Different allergenic proteins have been involved in cross-reactivity among animals. Albumins seem to be cross-sensitizing allergenic components. The aim of this study was to assess the importance of albumin as a cross-reactive allergen in patients sensitized to cat, dog and horse. One hundred and seventeen patients sensitized to cat were tested for IgE reactivity using skin prick tests and RAST assays with cat, dog and horse hair/dander extracts and their purified albumin extracts. RAST-inhibition studies were carried out to assess cross-reactivity among cat, dog and horse and among their purified albumins. It was found that 22% of patients exhibited specific IgE to cat albumin; 41% of patients sensitized to cat were also sensitized to dog and horse. Out of these patients, 21% had IgE to three albumins and 17% to two. Reciprocal inhibitions were observed among cat, dog and horse albumins and also among cat, dog and horse hair/dander extracts, using in the latter experiment sera from patients not sensitized to albumins. IgE binding to horse extract was inhibited 30% by its homologous albumin and IgE binding to cat and dog extracts in almost 15% by their respective albumins. It was concluded that albumins from these three animals share some epitopes that account for the cross-reactivity observed in around one-third of patients sensitized to cat, dog and horse. Nevertheless, more than 50% of specific IgE that cross-reacts among these three animals is directed to allergens other than albumin

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[162] - Blanc A, Donnay C, Metz-Favre C, de Blay F, Pauli G. Sensibilisation concomitante au chat et au chien : cosensibilisation ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2007;47:375-376

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[163] - Spitzauer S, Pandjaitan B, Muhl S, Ebner C, Kraft D, Valenta R, et al. Major cat and dog allergens share IgE epitopes. J Allergy Clin Immunol 1997;99:100-106

Patients allergic to cats and dogs frequently display IgE reactivity against allergens from different animals, suggesting a cross-sensitization to common allergenic determinants. Although albumins have been recognized as relevant cross-reactive allergens, little is known regarding cross-reactive epitopes of the major cat and dog allergens. OBJECTIVE: In this study, sera from patients allergic to cats and/or dogs were used to investigate the presence of common IgE epitopes among the major cat and dog allergens. METHODS: The IgE reactivity profile of 109 patients who were allergic to allergens from several species of animals was determined with nitrocellulose-blotted cat and dog allergens. Sera from patients who were strongly allergic to the major cat and dog allergens were tested for the presence of cross-reactive IgE antibodies by one-dimensional and two-dimensional immunoblot inhibition experiments and by quantitative measurements obtained with the CAP-FEIA system (Pharmacia). RESULTS: Sixty-eight of 109 patients with animal allergy showed IgE reactivity to cat allergens and dog allergens. Sera from patients with both cat and dog allergy detected allergens of similar molecular weight in nitrocellulose-blotted cat and dog hair/dander extracts. Common, as well as species-restricted, IgE epitopes of the major cat and dog allergens could be demonstrated by IgE inhibition studies. CONCLUSION: Shared IgE epitopes of the major cat and dog allergens may provide an explanation for the clinical observation that allergies to cats and dogs are frequently associated.

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[164] - Reininger R, Varga EM, Zach M, Balic N, Lindemeier AD, Swoboda I, et al. Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1. Clin Exp Allergy 2007;37:116-124

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited . OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1 . METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies . RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds . CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.

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[165] - Smith W, Butler AJL, Hazell LA, Chapman MD, Pomés A, Nickels DG, et al. Fel d 4, a cat lipocalin allergen. Clin Exp Allergy 2004;34:1732-1738

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals . OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels . METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed . RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog . CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.

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[166] - Blanc A, Donnay C, Metz-Favre C, de Blay F, Pauli G. Sensibilisation concomitante au chat et au chien : cosensibilisation ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2007;47:375-376

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Les allergènes du chat

Les allergènes du chien

Tests in vitro et allergie au chat ou au chien

Chat ou chien ? Ou les deux ?

Rubriques

Dans la même rubrique

Les blattes

Les acariens

Le ficus

Phanères de mammifères

Les oiseaux