Le soja


Aburage	C’est du tofu frit
Concentrats	Au moins 70% de protéines/poids sec. Fabriqués à partir de flocons dégraissés et chauffés. Le procédé d’extraction abaisse le contenu en glucides et corrige le goût de soja (« beany »). Utilisation dans des produits céréaliers (boulangerie, etc..), produits carnés (ex. « pains » de poisson), produits laitiers, etc..
Eau de soja	Synonyme de lait de soja -> voir Tonyu
Farines	Elles sont souvent dégraissées. Moins de 50% de protéines. Utilisées en boulangerie, elles ralentissent le rassissement (ex. 1,5-2% pour le pain blanc).
Huile	La fabrication passe par divers stades de chauffage, notamment au moment du raffinage. La phase de dégommage permet de récupérer les lécithines. Les résidus de fabrication de l’huile de soja sont utilisés en alimentation animale
Isolats	Au moins 90% de protéines. Plusieurs procédés d’obtention (ex. extraction alcaline). Les isolats sont plus solubles que les concentrats, d’où une plus large gamme d’emplois possibles. Utilisations : texturisation (saucisses, surimi, etc), produits laitiers, laits infantiles (ex. hydrolysat = Prégomine), parfois pâtes alimentaires, etc..
Lait de soja	-> voir Tonyu
Lécithines	Les globules graisseux (oil bodies) des graines de soja sont petits : leur rapport surface/volume est grand de sorte que le soja contient une proportion importante de phospholipides. Ceux-ci donnent aux lécithines leur pouvoir émulsifiant. Les lécithines ne sont pas dépourvues de protéines (100 à 500 ppm, mais taux très variables ) et donc sont potentiellement allergisantes. On en trouve aussi dans des cosmétiques
Miso	Mélange de graines cuites (ou flocons), d’eau, de sel et d’agents de fermentation (traditionnellement : Aspergillus oryzae ou A. sojae ; à présent avec des Lactobacillus aussi). Après 6 mois-3 ans de fermentation, le résidu solide est malaxé. Contient 8-14% de sel et 10-14% de protéines (plus ou moins dégradées). Le koji et le moromi sont les noms donnés aux premier et deuxième stade de fabrication du miso.
Natto	Graines cuites à la vapeur puis fermentées en présence de Bacillus natto
Okara	C’est un tempeh obtenu à partir des résidus solides de fabrication du tonyu (lait de soja)
Sauce soja = shoyu	C’est le surnageant du miso. Contient environ 18% de sel.
Tamari	Shoyu plus traditionnel (contient moins de blé)
Tempeh	Sorte de fromage. Les graines de soja détégumentées, bouillies, sont mélangées avec un Rhizopus dont le mycélium envahit la préparation en 24-48h à 30°C. Le tempeh n’est pas salé. Environ 18% de protéines.
Texturé (soja)	Obtenu par divers procédés qui concourent à la formation des fibres protéiques (ex. procédé d’extrusion thermoplastique qui combine haute pression et forte température). Les protéines sont en partie modifiées par formation réseaux macromoléculaires (et de composés de Maillard, à moins de partir d’un isolat). Le soja texturé est ajouté à des produits carnés jusqu’à 30% de poids (viandes hachées, boulettes, etc..). Le soja texturé peut même être moulé pour constituer des substituts de viande (ex. blancs de volaille) …
Tofu	Obtenu par coagulation à chaud du tonyu (lait de soja) puis égouttage, pressage et lavage. Le silken-tofu utilise un coagulant différent (glucono-lactone et non sel minéral) : on obtient une sorte de lait gélifié. Le kori-tofu est une variété deshydratée du tofu.
Tonyu	Ou eau de soja, lait de soja. C’est le liquide obtenu après ébullition de graines concassées. Sert aussi à fabriquer le tofu et le yaourt de soja
Yuba	C’est le film (collecté et séché) qui se forme à la surface du tonyu (lait de soja) chauffé


	Gly m 4	Gly m Bd 30K	Gly m Bd 28K
Lait de soja (tonyu)	+	+	±
Isolat de soja	+	+	+
Soja texturé	+	? *	?
Tofu	+	+	+
Sauce soja	-	- **	-
Miso	-	-	-
Natto	?	-	-

[1] - Franck P. Modifications de l'allergénicité du soja selon les technologies industrielles. Alim'Inter 2003;8:42-46

[2] - Franck P, Moneret-Vautrin DA, Dousset B, Kanny G, Nabet P, Guénard-Bilbaut L, et al. The allergenicity of soybean-based products is modified by food technologies. Int Arch Allergy Immunol 2002;128:212-219

Background: Numerous products based on soybean are available and various food technologies are applied for their production. The allergenicity of natural soybean may be modified by these treatments. Objectives: To compare the allergenicity of native soybean proteins with those of soy milk and texturized protein products. To show additional allergens. Methods: Three commercial products and two infant formulas were studied: Soybean flour, soy milk, texturized soy proteins, two infant formulas; the first containing total proteins and the second containing a soy protein hydrolysate. Sera from 9 patients allergic to soy protein were tested by immunoblotting (IB). IB inhibition was achieved by incubating sera with protein extract from soybean flour. Results: The SDS-PAGE profile of soybean flour protein and soy milk showed a difference in the proportions of the various protein fractions, with a higher concentration of 37-kD protein in flour and 33-kD protein in milk. Infant formula 1 contained proteins with a molecular weight below 28 kD. The texturized extract contained high proportions of 31- to 34- and 38-kD proteins. Immunoblotting revealed a lack of allergenicity in infant formula. Sera recognizing the 38- and 50-kD proteins in texturized soy protein also recognized the 37- and 49-kD proteins in soybean flour and in soy milk, suggesting a protein glycation by texturization processes. The 30- to 34-kD band in texturized proteins was devoid of any allergenicity. This study seems to indicate that the 30-kD allergen (Gly m Bd 30) disappears during the production of texturized soy protein. Conclusion: All technologies applied to soybean-based products induce striking variation in the protein profile and allergenicity. Texturized protein could lack the major allergen Gly m Bd 30. Further studies or texturization might generate modified technologies in order to create hypoallergenic texturized proteins.

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[3] - Crevel R, Sanders I, Dahl L. Evaluation of the allergenicity of residual protein from soy lecithin samples. Allergy Clin Immunol Int 2005;17(Suppl. 1):355-356

Background: Soy lecithins are produced from material removed during the degumming stage of the refining of crude soybean oils. Different lecithins vary widely in composition and characteristics, depending on the extent to which the original material is further processed. Published reports indicate that they also differ greatly in their protein content, and their allergenic reactivity as measured by different in vitro endpoints. The purpose of this study was to evaluate the allergenicity of a range of soy lecithins currently used in the food industry. Methods: The protein component of ten lecithin samples was extracted using acetone/hexane followed by centrifugation and concentration steps, and finally re-suspension in PBS. Total protein was estimated by the Bradford method, and each sample was characterised by SDS-PAGE and immunoblotting analysis, using a rat anti-soy protein IgE antiserum. The functional allergenic activity of the samples was evaluated using the RBL-2H3 mediator release assay. Results: Total protein content ranged from 2.2 to 41.1mg/kg, while SDS-PAGE revealed one or more protein bands in 8 out of 10 samples. In five of the samples those proteins were recognised by rat anti-soy IgE, and matched to bands present in a soy protein extract. Eight out of the 10 extracts produced specific degranulation of RBL-2H3 cells sensitised with rat anti-soy IgE. For each sample, the highest dilution capable of producing a positive response broadly correlated with the total protein content. Conclusions: The study confirms earlier published data indicating that the residual allergenic activity of different soy lecithin samples varies considerably. However the protein contents measured in this study span a much narrower and lower range of values than previously reported. Together with clinical data which indicate that the doses of soy protein required to provoke reactions tend to be higher than for many other commonly allergenic foods, and the fact that soy lecithin is generally used in low concentrations in foods, these findings suggest that the risk of reactions from the presence of undeclared soy lecithin is probably lower than previously implied.

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[4] - Belloque J, Garcia MC, Torre M, Marina ML. Analysis of soybean proteins in meat products: a review. Crit Rev Food Sci Nutr 2002;42:507-532

The use of soyabean proteins as meat extenders has spread significantly due to the interesting nutritional and functional properties that are present in soyabean proteins. Together with these, health and economical reasons are the major causes for the addition of soyabean proteins to meat products. Nevertheless, despite the good properties associated to soyabean proteins, there are many countries in which the addition of these proteins is forbidden or in which the addition of soyabean proteins is allowed up to a certain extent. Thus, the need of analytical methods enabling the detection of added soyabean proteins in meat products is obvious. Microscopic, electrophoretic, immunologic, and chromatographic methods are the most widely used for this purpose. However, the detection of soyabean proteins in meat products presents difficulties related to the composition (meat species, meat quality, soyabean protein source, presence of other non-meat proteins, etc.) and the processing of the meat products, and, although these analytical methods have tried to overcome all these difficulties, there is still not a method enabling quantitative assessment of soyabean proteins in all kinds of meat products.

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[5] - Cordle CT. Soy protein allergy: incidence and relative severity. J Nutr 2004;134:1213S-1219S

Food allergy is a relatively rare and sometimes violent reaction of the immune system to food proteins. The first report characterizing soy allergy appeared in 1934. The Food and Agriculture Organization of the United Nations includes soy in its list of the 8 most significant food allergens. At least 16 potential soy protein allergens have been identified but their relative clinical significance is unknown. Conversely, soy has a long history of successful use in managing cow's milk allergies in infants. To better predict the utility of soy proteins for controlling food allergy, it is important to understand the relative allergenic reactivity of soy compared with other major food proteins. This can be studied using clinical data, animal models, and biochemical approaches; all show diminished reactivity for soy. Clinical studies using in vitro methods and blinded food challenges have generated substantial information. Study populations include high-risk asymptomatic infants and patients with atopic symptoms, positive food challenges, and specific milk allergies. Generally, these studies show lower allergic reactivity for soy proteins vs. other food allergens. Comparisons of food allergen dose-response relationships for triggering allergic symptoms also demonstrate a higher protein concentration threshold for soy (approximately 100 times), indicating lower allergenic reactivity. Extensive investigations of soy immunological reactivity have also been carried out using animal models. Consistent with clinical results, all of these data show substantially diminished immunological reactivity for soy proteins. Biochemical and immunochemical analyses indicate no striking differences between soy and other food proteins that would explain these unexpected differences in allergenic reactivity.

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[6] - Kleine-Tebbe J, Wangorsch A, Vogel L, Crowell DN, Haustein UF, Vieths S. Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1-related PR-10 protein in soybean, SAM22. J Allergy Clin Immunol 2002;110:797-804

BACKGROUND: Anaphylactic reactions to soy products have been attributed to stable class 1 food allergens . OBJECTIVE: IgE- mediated reactions to a soy-containing dietary food product in patients allergic to birch pollen were investigated . METHODS: Detailed case histories were taken from 20 patients. Their sera were analyzed for IgE (UniCAP) specific for birch, grass, mugwort, the recombinant birch allergens rBet v 1 and rBet v2, and soy protein. Extracts from birch pollen, soy isolate, rBet v 1, and the recombinant PR-10 soy protein rSAM22 were coupled to paper disks or nitrocellulose for IgE measurements (enzyme allergosorbent test) or Western blot analysis. Enzyme allergosorbent testing, Western blot inhibition, and histamine release studies were performed with the same allergens . RESULTS: Most patients (17/20) experienced facial, oropharyngeal, and/or systemic allergic symptoms within 20 minutes after ingesting the soy product for the first time. Birch pollen allergy (16/20) was common, along with oral allergy syndrome to apple (12/20) or hazelnut (11/20). IgE levels to birch and Bet v 1 but not to other inhalants were high in 18 of 20 patients. Significant IgE binding to rSAM22 occurred in 17 of 20 patients. Blot experiments with the soy isolate revealed IgE-binding bands at 17 kd (15/20), 22 kd (1/20), and 35 to 38 kd (2/20); the former was inhibited by preincubation of the sera with rBet v 1 or rSAM22. Birch extract and soy isolate, rBet v 1, and rSAM22 induced dose-dependent histamine release in the nanomolar range . CONCLUSION: Immediate-type allergic symptoms in patients with birch pollen allergy after ingestion of soy protein-containing food items can result from cross-reactivity of Bet v 1 -specific IgE to homologous pathogenesis-related proteins, particularly the PR-10 protein SAM22.

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[7] - Adel-Patient K, Ah-Leung S, Creminon C, Nouaille S, Chatel JM, Langella P, et al. Oral administration of recombinant Lactococcus lactis expressing bovine beta-lactoglobulin partially prevents mice from sensitization. Clin Exp Allergy 2005;35:539-546

BACKGROUND: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. OBJECTIVE: We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. METHODS: Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. RESULTS: Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. CONCLUSION: Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

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[8] - Sánchez Alonso A, Méndez Alcalde JD. Allergy to sausages. Alergol Immunol Clin 2000;15:211-215

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[9] - Moneret-Vautrin DA. Modifications of allergenicity linked to food technologies. Allerg Immunol (Paris) 1998;30:9-13

The prevalence of food allergies (FA) has increased over the past fifteen years. The reasons suggested are changes in dietary behaviour and the evolution of food technologies. New cases of FA have been described with chayote, rambutan, arguta, pumpkin seeds, custard apple, and with mycoproteins from Fusarium.... Additives using food proteins are at high risk: caseinates, lysozyme, cochineal red, papaïn, alpha-amylase, lactase etc. Heating can reduce allergenicity or create neo-allergens, as well as storage, inducing the synthesis of allergenic stress or PR proteins. Aeroallergens (miles, moulds) contaminate foods and can induce allergic reactions. Involuntary contamination by peanut proteins on production lines is a problem which is not yet solved. Genetically modified plants are at risk of allergenicity, requiring methodological steps of investigations: the comparison of the amino-acid sequence of the transferred protein with the sequence of known allergens, the evaluation of thermo degradability and of the denaturation by pepsin and trypsin are required, as well as the study with sera from patients allergic to the plant producing the gene. The combination of enzymatic hydrolysis, heating, or the development of genetically modified plants may offer new alternatives towards hypoallergenic foods (57 references).

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[10] - Inomata N, Osuna H, Ikezawa Z. Late-onset anaphylaxis to Bacillus natto-fermented soybeans (natto). J Allergy Clin Immunol 2004;113:998-1000

Natto is a traditional Japanese food produced from fermenting soybeans by the bacteria Bacillus natto (subtilis), and making an appearance in Western cuisine. This study reports an IgE-mediated cutaneous, respiratory, and abdominal symptoms occurring 10 to 12 hours after consuming natto. A 36-year-old man presented with a 2-month history of recurrent generalized urticaria and anaphylaxis. He experienced urticaria >2 to 3 days a week and more recently had at least 3 severe anaphylactic reactions accompanied by dyspnea, chest tightness, abdominal cramps, palpitation, vomiting, dizziness, and headache. He ate dinner containing natto at 6 PM or 8 PM, respectively, and then anaphylactic reactions developed at 6 AM while sleeping. He had never realized that these reactions were elicited by something he had eaten, because he had 2 of these 3 episodes while sleeping. His diet diaries, however, revealed that he had ingested natto 10 to 12 hours before the onset of symptoms in all 3 instances, indicating an association between natto and these 3 anaphylactic events. Total IgE concentration was 710 KJ/mL. Specific IgE antibodies were detected against egg white (a score of 2+), but not against soybean or the other foods. All results of SPTs with multiple commercial foods allergens were negative. Results of the prick-by-prick test with the raw ingredients included in the dinner the patient had eaten 12 hours before an episode were negative for all ingredients except natto Subsequent SPTs with a saline solution containing B natto powder. Prick test to self manufactured natto was positive. Other soy products were negative. Specific IgE to natto was positive. It was suspected that the natto allergens may be produced during fermentation.

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[11] - Inomata N, Osuna H, Kawano K, Yamaguchi J, Yanagimachi M, Matsukura S, et al. Late-onset Anaphylaxis after Ingestion of Bacillus Subtilis-fermented Soybeans (Natto): Clinical Review of 7 Patients. Allergol Int 2007;56:257-261

The objectives of this study was to clarify the clinical and laboratory features and to characterize the allergens in allergy due to fermented soybeans. Seven male patients aged 26 to 42 years with suspected hypersensitivity to fermented soybeans underwent SPTs with fermented soybeans and challenge test with fermented soybeans. All patients reported generalized urticaria and dyspnea; 5, loss of consciousness; 2, collapse; 2, vomiting; and 2, diarrhea after fermented soybean ingestion. The interval between fermented soybean ingestion and onset of symptoms was 5 to 14 hours. All patients were positive on skin prick-prick tests with fermented soybeans. In 2 patients, oral challenge with fermented soybeans was positive 5.5 and 13 hours after ingestion. In ELISA, all 5 patients tested showed elevated IgE levels to the fermented soybean extract. Using 5 patients' sera showed six bands, of which three bands at 38, 28, and 26-kd were bound to sera from 4 patients.

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[12] - Vigan M, Girardin P, Rame JM, Pelletier F. Bronchospasme au tonyu: découvertes déconcertantes à propos d'un cas. Rev Fr Allergol Immunol Clin 2005;45:276-277

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[13] - Klemola T, Vanto T, Juntunen-Backman K, Kalimo K, Korpela R, Varjonen E. Allergy to soy formula and to extensively hydrolyzed whey formula in infants with cow's milk allergy: a prospective, randomized study with a follow-up to the age of 2 years. J Pediatr 2002;140:219-224

OBJECTIVES: We conducted a prospective, randomized study to evaluate the cumulative incidence of allergy or other adverse reactions to soy formula and to extensively hydrolyzed formula up to the age of 2 years in infants with confirmed cow's milk allergy. STUDY DESIGN: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula. If it was suspected that the formula caused symptoms, a double-blind, placebo-controlled challenge (DBPCFC) with the formula was performed. The children were followed to the age of 2 years, and soy-specific immunoglobulin E antibodies were measured at the time of diagnosis and at the ages of 1 and 2 years. RESULTS: An adverse reaction to the formula was confirmed by challenge in 8 patients (10%; 95% confidence interval, 4.4%-18.8%) randomly assigned to soy formula and in 2 patients (2.2%; 95% confidence interval, 0.3% to 7.8%) randomly assigned to extensively hydrolyzed formula. Adverse reactions to soy were similar in IgE-associated and non-IgE-associated cow's milk allergy (11% and 9%, respectively). IgE to soy was detected in only 2 infants with an adverse reaction to soy. Adverse reactions to soy formula were more common in younger (<6 months) than in older (6 to 12 months) infants (5 of 20 vs 3 of 60, respectively, P =.01). CONCLUSIONS: Soy formula was well tolerated by most infants with IgE-associated and non-IgE-associated cow's milk allergy. Development of IgE-associated allergy to soy was rare. Soy formula can be recommended as a first-choice alternative for infants >or=6 months of age with cow's milk allergy.

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[14] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[15] - Maria Y, Leduc V. Complexité du diagnostic de l’allergie alimentaire aux dérivés de soja. Rev Fr Allergol Immunol Clin 2007;47:284

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[16] - Taylor SL, Nordlee JA, Sicherer SH, Sampson HA, Levy MB, Steinman H, et al. Soybean Oil Is Not Allergenic to Soybean-Allergic Individuals. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°296

Rationale Soybean-allergic individuals may be instructed by clinicians to avoid all foods derived from soybeans including soybean oil. However, highly refined soybean oil contains extremely low levels of protein. Previous work suggested that soybean oil was not allergenic, but the number of subjects challenged was small and the protein content of the oil was unknown. These deficiencies are rectified in this study. The study objective was to determine the allergenicity of highly refined soybean oil in soybean- allergic individuals. Method s : Soybean-allergic subjects were selected by convincing history, positive skin test and positive radioallergosorbent test (RAST). Challenge materials consisted of a mixture of 4 soybean oils with the highest protein level from a group of 30 highly refined oils obtained from 30 different worldwide processors. Subjects consumed increasing doses of 1, 5, and 10 grams soybean oil (test material) and canola oil (control material) in a double-blind placebo controlled food challenge. Prepared oatmeal was the challenge vehicle. Result s : Twenty-eight soybean allergic subjects were challenged. No untoward reactions were encountered to either soybean or canola oils. No reaction with this number of subjects indicates with 95% certainty that 89.85% of soybean allergic individuals would not react to this soybean oil. Conclusions : The lack of reactions to commercially available soybean oil supports the previous claim that hot solvent-extracted, bleached and deodorized soybean oil is not allergenic for soy-allergic individuals and avoidance of soybean oil of the type used in this study is unwarranted. The allergenicity of cold-pressed or expeller-pressed soybean oil remains uncertain.

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[17] - Hefle SL, Taylor SL. Allergenicity of edible oils. Food Technol 1999;53:62-68

Allergenicity studies on vegetable oils derived from commonly allergenic foods are described. Aspects considered include: allergenic foods; mechanisms of food allergies; difficulties in avoiding allergenic food proteins due to limited consumer knowledge, inadequate food labelling or poor cleaning practices in food preparation establishments; potentially allergenic oils (peanut (groundnut), soybean, tree nut, sunflower, sesame, cottonseed and (rarely) corn and coconut oils); methods of oil processing (solvent extraction/refining or cold pressing); labelling of oils; lower levels of allergenic proteins in solvent extracted/refined than cold pressed oils and levels required to cause allergic reactions; diagnostic tests for allergenicity (oral challenge, skin testing, radioallergosorbent assay, basophil histamine release, Prausnitz-Kustner test and immunoblotting); and studies on allergenicity of refined and cold pressed soybean, peanut, tree nut, cottonseed, sesame and sunflower oils (providing information concerning experimental techniques employed, shortcomings of the studies, levels of allergenic proteins present in the oils and oil allergenicity). It is concluded that refined oils contain virtually no protein and pose little or no hazard to allergic individuals; crude or cold pressed oils are less commonly available and may contain small amounts of protein and pose some risk

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[18] - Morisset M, Lee T, Codreanu F, Cordebar V, Fremont S, Guenard L, et al. Allergy to an Amino-Acid Formula in Infants: Residual Soy Allergens in Soybean Oil are Incriminated. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°157

RATIONALE: Treatment of allergy to cow milk (CM) proteins comprises avoidance and replacement of CM with various infant formulas. However, clinical manifestations have been associated with CM protein hydrolysates (CMPH). In this situation, in the frame of multiple food allergies (MFA), substitution of CMPH with an amino-acid-based formula (AAF) is recommended METHODS: Seven infants allergic to AAF have been referred for a MFA. In all cases, the breast-feeding was stopped; CMPH and/or soy protein and pork collagen hydrolysates were replaced by Neocate(r) (SHS International), an AAF containing a soy lipid emulsion. As there was no clinical improvement, prick-tests (PT) and atopy patch-test (APT) to soy or Neocate(r) were carried out RESULTS: PIPs were positive in 2/7 patients. APTs to soy and APTs to Neocate(r) were positive respectively for 5/5 and 3/5 infants. In one case, an oral challenge with the soy lipid emulsion was performed and was positive Neocate(r) was replaced by Neocate Advance(r), a soy-free AAF and the symptoms improved within 2-4 weeks in all children Proteins of the Neocate emulsion were extracted. The amount was measured and their molecular mass estimated by SDS-PAGE. A western blot was conducted using the serum of a soy allergic patient . The western blot showed 2 bands (56-66 kDa) CONCLUSIONS: AAFs have represented a great advance. However, the risk of allergy to traces of vegetable proteins in certain oils has been confirmed yet again. Food industries must be made aware of the need to detect allergen traces in foods for infants at high risk of atopy.

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[19] - Müller U, Weber W, Hoffmann A, Franke S, Lange R, Vieths S. Commercial soybean lecithins: a source of hidden allergens ? Eur Food Res Technol 1998;207:341-351

Soybeans are known to be allergenic for adults as well as for infants. Processed products derived from soybeans are used in a wide spectrum of foods, drugs and other industrial products. In particular, soybean lecithins are used as stabilizers and emulsifiers and may not be suspected as possible source of allergens. To test this hypothesis, six commercial soy lecithins were investigated for residual allergenicity and compared with extracts from raw and heat-treated soybeans. They were characterized, the protein content was determined by enzyme-linked immunosorbent assay (ELISA) and allergens were analyzed with specific IgE from patients' sera using the enzyme allergosorbent test (EAST), EAST inhibition and protein blotting followed by immunodetection. For further characterization a polyclonal antiserum directed against soybean extract and a monoclonal antibody (mAb?025) directed against the acidic subunit of the soybean storage protein glycinin were used. The EAST studies revealed that three of six sera from patients with allergy to soybeans contained IgE to four soy lecithins (Topcithin 50, Topcithin 300, Emulfluid FD 12, Epikuron 100 P), the same lecithins which were found to contain residual proteins. Two lecithins with a protein content of less than 20?ppb did not bind IgE. EAST inhibition showed that the allergens from soy lecithin were immunologically more closely related to allergens from heat-treated soybeans than to those from raw soybeans. Protein blotting and immunodetection of the protein extract from the lecithins resulted in various allergen bands between 14?kDa and 94?kDa. A heat-stable allergen of 39?kDa was recognized by the monoclonal antibody and thus identified as a subunit of glycinin. The results obtained were confirmed by a mediator release assay based on a rat basophil leukemia cell line. Lecithins that contained residual proteins caused a specific mediator release, suggesting that these products may induce allergic symptoms. Our results show that soybean lecithins are capable of introducing hidden allergens to processed foods and that the IgE binding potential corresponds to the total protein determined by ELISA. Furthermore, it appears to be possible that by monitoring the protein content soy lecithins can be applied which may be safe for the allergic consumer.

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[20] - Crevel R, Sanders I, Dahl L. Evaluation of the allergenicity of residual protein from soy lecithin samples. Allergy Clin Immunol Int 2005;17(Suppl. 1):355-356

Background: Soy lecithins are produced from material removed during the degumming stage of the refining of crude soybean oils. Different lecithins vary widely in composition and characteristics, depending on the extent to which the original material is further processed. Published reports indicate that they also differ greatly in their protein content, and their allergenic reactivity as measured by different in vitro endpoints. The purpose of this study was to evaluate the allergenicity of a range of soy lecithins currently used in the food industry. Methods: The protein component of ten lecithin samples was extracted using acetone/hexane followed by centrifugation and concentration steps, and finally re-suspension in PBS. Total protein was estimated by the Bradford method, and each sample was characterised by SDS-PAGE and immunoblotting analysis, using a rat anti-soy protein IgE antiserum. The functional allergenic activity of the samples was evaluated using the RBL-2H3 mediator release assay. Results: Total protein content ranged from 2.2 to 41.1mg/kg, while SDS-PAGE revealed one or more protein bands in 8 out of 10 samples. In five of the samples those proteins were recognised by rat anti-soy IgE, and matched to bands present in a soy protein extract. Eight out of the 10 extracts produced specific degranulation of RBL-2H3 cells sensitised with rat anti-soy IgE. For each sample, the highest dilution capable of producing a positive response broadly correlated with the total protein content. Conclusions: The study confirms earlier published data indicating that the residual allergenic activity of different soy lecithin samples varies considerably. However the protein contents measured in this study span a much narrower and lower range of values than previously reported. Together with clinical data which indicate that the doses of soy protein required to provoke reactions tend to be higher than for many other commonly allergenic foods, and the fact that soy lecithin is generally used in low concentrations in foods, these findings suggest that the risk of reactions from the presence of undeclared soy lecithin is probably lower than previously implied.

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[21] - Palm M, Moneret-Vautrin DA, Kanny G, Denery-Papini S, Frémont S. Food allergy to egg and soy lecithins. Allergy 1999;54:1116-1117

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[22] - Hefle SL, Lambrecht DM, Nordlee JA. Soy Sauce Retains Allergenicity Through the Fermentation/ Production Process. J Allergy Clin Immunol 2005;115(2 suppl.):S32

RATIONALE: Soy allergy is one of the most prominent allergies in the worldwide population. The vast majority of soy sauces are produced through the fermentation of soy and wheat. Some soy sauce manufacturers tell finished food product processors (and also soy-and wheat-allergic patients who contact them) that the fermentation process destroys the allergenicity of their soy and wheat fermentation ingredients. This has not been proven to be the case by scientific experimentation, so the risk of reaction from soy sauce ingestion among soy-allergic and wheat-allergic/ celiac patients is unknown METHODS: Ten soy sauces were evaluated using three soy-specific animal IgG-based ELISAS (two of which are commercially available), by soy polymerase chain reaction (PCR) analysis, and also by RAST inhibition using sera from soy-allergic subjects RESULTS: The soy IgG-based ELISA tests and PCR tests showed no detectable residues of soy protein or soy DNA in the ten sauces. However, in RAST inhibition (using soy flour as the solid phase), some soy sauces contained 10-30% residual activity CONCLUSIONS: Soy sauces made by fermentation of soy protein can retain some of their soy allergenicity (10-30% that of soy flour) through the fermentation/production process. Soy IgG-ELISA and PCR analyses do not detect the remaining allergenic residues in soy sauce. Therefore, results from these types of tests should not be used by soy sauce suppliers nor finished food manufacturers to indicate that soy sauce is devoid of allergenic residues and safe for soy-allergic individuals to consume. Soyallergic patients should continue to be counseled to avoid soy sauce

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[23] - Codreanu F. L'activité du réseau Allergo-Vigilance: allergies alimentaires. Alim'Inter 2007;12:239-242

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[24] - Frémont S. Soja. Alim'Inter 2001;6:199-201

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[25] - (allergie au chocolat). Alim'Inter 1998;3(15):9

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[26] - Bessot JC. Allergènes végétaux non polliniques. Rev Fr Allergol Immunol Clin 2003;43:40-52

L'inventaire des allergènes végétaux non polliniques s'est considérablement élargi au cours des 12 dernières années. Ces allergènes, initialement répertoriés dans l'environnement professionnel peuvent aussi être rencontrés dans l'environnement domestique. Certains d'entre eux se comportent à la fois comme des pneumallergènes ou des trophallergènes. Dans cette revue générale, seront envisagés la prévalence, les mécanismes, les aspects cliniques, la démarche diagnostique des allergies aux gommes végétales, aux graines, aux racines, aux feuilles et aux plantes d'intérieur. Le rôle des allergènes du latex, des bois, des farines, des enzymes végétales ne sera pas traité ici. Les gommes végétales (arabique, karaya, guar, psyllium...) provoquent des rhinites ou des asthmes IgE médiés, bien que leurs allergènes soient des polysaccharides. La colophane fait partie des 5 allergènes le plus souvent responsables d'asthmes professionnels en Grande-Bretagne. Parmi les graines, le rôle des graines provenant de céréales, du café, du ricin, du soja sera privilégié. Parmi les racines, certaines plantes médicinales (sanyak, bahna, salsepareille...) ont une importance croissante. Le henné, le tabac, le thé, le lycopode sont aussi des sources d'allergènes. Des allergies IgE dépendantes ont été décrites pour le ficus, mais aussi pour d'autres plantes d'appartement. Certaines étiologies, se limitant actuellement à un ou quelques cas publiés, peuvent annoncer des pathologies allergiques émergentes.

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[27] - Rodrigo MJ, Morell F, Helm RM, Swanson M, Greife A, Anto JM, et al. Identification and partial characterization of the soybean-dust allergens involved in the Barcelona asthma epidemic. J Allergy Clin Immunol 1990;85:778-784

Asthma epidemics in Barcelona, Spain, have been attributed to dust generated by the unloading of soybeans in the harbor. Sera of four different groups of 10 subjects in each group were studied: (1) patients attending an emergency room in Barcelona for an asthma attack on epidemic days, group A, (2) patients attending an emergency room for an attack on nonepidemic days, group B, (3) patients with asthma from other cities, group C, and (4) patients without asthma from Barcelona matched by age and sex with group A, group D. All subjects in group A had IgE to allergens in extracts of various soybean samples. In contrast, only one of the 10 subjects in each of groups B and C and none of those subjects in group D had IgE to uncleaned bean and hull extracts. Radioimmunoassay demonstrated that in sera from patients with asthma during an asthma outbreak reacted primarily to soybean hull and dust extracts. Sodium dodecyl sulfate-polyacrylamide and gel electrophoresis thin-layer isoelectrofocusing demonstrated protein bands of 97.4 to less than 14.4 kd and isoelectric point between 6 and 3.5. By Western blot and thin layer isoelectrofocusing/blotted radioimmunoisoelectrofocusing, IgE of patients with asthma during an asthma outbreak reacted weakly to two protein bands of molecular weight ranging from 42 to 21 kd, strongly to glycoprotein bands with molecular weight less than 14.4 kd, and isoelectric point less than 6, which appeared to be the major allergens

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[28] - Codina R, Oehling Jr AG, Lockey RF. Neoallergens in heated soybean hull. Int Arch Allergy Immunol 1998;117:120-125

BACKGROUND: During the process of harvest, transport and storage, microbial and mold contamination can raise the temperature of soybeans to 75 degreesC or higher. The purposes of this study were (1) to evaluate the allergenicity of fresh and stored soybean hulls and (2) to ascertain whether heat alters the allergenicity of stored soybean hulls. METHODS: Allergen extracts were prepared from (1) stored soybean hulls, (2) fresh soybean hulls and (3) stored soybean hulls heated to 37 degreesC (E1), 55 degreesC (E2) and 80 degreesC (E3) or kept at room temperature (E4) for 16 h. Individual serum from 68 soybean asthmatic (SA) subjects, 30 nonallergic subjects and two serum pools made from 4 SA sera and 4 sera from asthmatics not sensitive to soybean were studied. All sera and serum pools were assayed for content of specific IgE (radioallergosorbent test) and IgG4 (ELISA). The following additional studies were done for extracts E1-E4: (1) SDS-PAGE, (2) SDS-PAGE/Western blot for specific IgE and IgG4 using both serum pools, and (3) study of the effects of heat on inhibiting activity of the extracts prepared from stored soybean hulls using the pool of SA sera. RESULTS: Test results demonstrated a reduced binding of specific IgE and IgG4 to fresh soybean hull extract compared to stored soybean hull extract, and an increased binding for heated extracts (E1-E3) compared to unheated ones (E4). Moreover, there was an increase in potency for IgE and IgG4 bindings for the heated (E1-E3) compared to unheated (E4) extract, as measured by the amount of protein to produce 50% inhibition. Several protein bands with a molecular weight (MW) higher than 20 kD were absent from the SDS-PAGE for E3 but were present in E1, E2 and E4, and a new protein band (MW 15.3 kD) appeared for E3 only. Two new protein bands, with MWs of 15.3 and 10 kD, which bind specific IgE, were present on Western blot and one of the 3 main soybean hull allergens, probably Gly m 2, disappeared in E3. IgG4 Western blot showed similar results, but only the 10 kD protein band was present. CONCLUSION: The results demonstrate that soybean hull allergenicity is affected by heat, and suggest that the heat generated during storage and transport of soybeans could generate 2 new allergen determinants or increases in epitope exposure as a result of conformational changes. The significance of these new IgE and IgG4 binding proteins has yet to be determined.

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[29] - Gómez-Ollés S, Cruz M, Alcántara L, Untoria M, Morell F, Rodrigo M. Assessment of allergenic cross-reactivity between soy low molecular weight proteins and other legumes and cereals. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°286

Background: Asthma epidemics due to soybean dust relevant during unloading of this leguminous in the harbour have been broadly reported. Being Soy hull low molecular weight allergens responsible for asthma attacks, with one 7.5-kDa protein (Gly m I) as the major allergen. The aim of this study was to examine the immunologic cross-reactivity between soybean low molecular weight proteins and proteins from other legumes and cereals extracts. Methods: Inhibition ELISA studies were conducted to assess the presence of cross-reactivity between soybean extract and extracts from several common legumes (green bean, green pea, chickpea and peanut), other legumes and cereals unloaded in the harbour, such as corn, rye, field pea and wheat fodder. Western blot - only with extracts that reach an inhibition >25% in ELISA inhibition experiments- was performed using a pool of sera from 12 allergenic soybean asthma epidemic patients to determine the MW of the band responsible for the cross-reaction. Results: In the IgE ELISA inhibition, non legume or cereal extract tested reach 50% inhibition at the maximum inhibitor concentration of 60 µg/ml. Although, inhibition assays using field pea showed a partial inhibition (approx. 30%) of soybean specific IgE. Immunoblotting revealed IgE binding to mainly a band of low molecular weight (approx. 6 kDa) in soybean extract and field pea extract. Which is not present in the green pea extract. Conclusion: A partial allergenic cross-reactivity exists among proteins of low molecular weight of soybean hull and field pea extracts. Probably due to storage proteins present in dry legumes. So future studies should focus on field pea, in order to identify the protein involve in the cross-reaction and to evaluate the clinical relevance of this in vitro cross-reactivity.

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[30] - Gijzen M, González R, Barber D, Polo F. Levels of airborne Gly m 1 in regions of soybean cultivation. J Allergy Clin Immunol 2003;112:803-805

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[31] - Codina R, Ardusso L, Lockey RF, Crisci CD, Jaén C, Bertoya NH. Identification of the soybean hull allergens involved in sensitization to soybean dust in a rural population from Argentina and N-terminal sequence of a major 50KD allergen. Clin Exp Allergy 2002;32:1059-1063

Background: Sensitization to soybean hull (SH) allergens occurs in subjects from Argentina, a soybean producer country. However, the causative allergens have not been identified. The purposes of this study are to: (i) identify the SH allergens using sera of 29 subjects with asthma and/or allergic rhinitis from Argentina exposed to soybean dust who have a positive (weal with SH/weal with histamine 0.5) skin prick test to SH; and (ii) determine the N-terminal amino acid sequence of a major 50K SH allergen that sensitizes this population. Methods: All sera were assayed for specific IgE (RIA), IgG4 (ELISA), and IgE and IgG4-Western blots. A sera pool from 10 healthy subjects was a negative control. N-terminal amino acid sequencing was performed by the Edman degradation method. Results: Positive specific IgE only was found in 12/29 (41.4%), IgG4 in 3/29 (10.3%), and both IgE and IgG4 in 14/29 (48.3%) sera. IgE-Western blot demonstrates: (i) an allergen, MW 50 K (51.7% binding); (ii) one or two distinct allergens, MW<20.2 K (72.4% binding), depending on the sera; and (iii) 1-5 additional IgE binding proteins, MW>20.2 to <46.9 K (41.4% binding), depending on the sera. IgG4-Western blot demonstrates: (i) a band, MW 70K (31% binding); (ii) a band, MW 50 K (17.2% binding); (iii) one or two additional bands, MW<20.2 K (51.7% binding), depending on the sera; and (iv) a band, MW>20.2 to <28.5 K (20.7% binding). The 50 K allergen N-terminal amino acid sequence of the first 17 amino acids indicates a significant homology with chlorophyll A-B binding protein precursors from tomato, spinach, and petunia. Conclusions: Specific IgE and IgG4 to SH are common in sera from allergic individuals living in rural areas in Argentina. SH contain an IgE binding protein, MW about 50 K, not previously described. Sensitization to this allergen is common in subjects who are repeatedly exposed to soybean dust inhalation.

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[32] - Quirce S, Polo F, Figueredo E, Gonzalez R, Sastre J. Occupational asthma caused by soybean flour in bakers: differences with soybean-induced epidemic asthma. Clin Exp Allergy 2000;30:839-846

BACKGROUND: Soybean dust has been identified as the causative agent of occupational asthma and asthma epidemics. Two main soybean hull allergens responsible for asthma outbreaks, Gly m 1 and Gly m 2, have been identified and purified . OBJECTIVE: The soybean allergens causing occupational asthma in exposed bakers were investigated and compared with those involved in epidemic asthma . METHODS: We report four bakers or confectioners with work-related respiratory symptoms who were exposed to soybean flour used as a baking additive. The causative role of soybean flour was investigated by immunological tests and specific inhalation challenge tests. Soybean flour allergens causing occupational asthma were characterized by immunoblotting. Immunoglobulin (Ig) E-reactivity to Gly m 1 and Gly m 2 was assessed using enzyme-linked immunosorbent assay . RESULTS: Sensitization to soybean flour was demonstrated by skin and serological tests and was confirmed by positive inhalation tests. Bronchial challenge test to soybean flour extract elicited immediate or dual asthmatic responses. Immunoblotting with soybean flour and soybean hull extracts showed IgE-binding mainly to high molecular weight (MW) allergens. There was an important individually different allergic response to inhalant soybean components. None of the patients showed IgE-reactivity against Gly m 1 and only one patient showed IgE-reactivity to the soybean hull allergen Gly m 2 . CONCLUSION: These bakery workers had developed IgE-mediated occupational asthma to soybean flour. The allergens involved in occupational asthma caused by soybean flour are predominantly high MW proteins that are present both in soybean hull and flour, and they are different from the allergens causing asthma outbreaks, which are mainly low MW proteins concentrated in the hull.

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[33] - Bush RK, Schroeckenstein D, Meier-Davis S, Balmes J, Rempel D. Soybean flour asthma: detection of allergens by immunoblotting. J Allergy Clin Immunol 1988;82:251-255

A 43-year-old woman developed asthma 6 years after beginning work in a food-processing plant in which soybean flour was used as a protein extender. Symptoms of sneezing, coughing, and wheezing would begin within minutes of exposure to soybean flour and resolve 2 hours after exposure ceased. Skin tests were positive to a soy extract prepared from the flour. Airway hyperreactivity was confirmed by a positive bronchial challenge to methacholine. Bronchial challenge with soybean flour produced an immediate increase in specific airway resistance from 5.0 to 22.7 L. cm of H2O/L/sec. There was no response to challenge with lactose. The patient's allergic response to soy-flour extract was further characterized by several immunologic methods. IgE binding to soy-flour protein by direct RAST was 5.98 times that of a normal control serum. The soy-flour extract was separated by dodecyl sulfate-polyacrylamide gel electrophoresis. Twenty-four protein bands were detected in the crude soy-flour extract. After immunoblotting and subsequent autoradiography, nine proteins with molecular weights ranging from 54,500 to 14,875 were found. Cross-reactivity studies with other legumes demonstrated apparent immunologic identity between a component in green pea extract and a soybean protein with a molecular weight of 17,000. The clinical significance of this cross-reactivity is not known. We conclude that in this case of occupational asthma to soybean flour, multiple allergens were involved. Immunoblotting may be useful in identifying the allergens involved in occupational asthma.

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[34] - Sandiford CP, Tee RD, Newman Taylor AJ. Identification of crossreacting wheat, rye, barley and soya flour allergens using sera from individuals with wheat-induced asthma. Clin Exp Allergy 1995;25:340-349

We have conducted radio allergosorbent test (RAST), competitive RAST inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using sera from patients with wheat-induced asthma to investigate the immunological relationship between wheat, rye, barley and soya, and to identify common proteins between these flours. RAST showed strong associations between the levels of specific IgE to wheat flour and those of rye and barley flour. Competitive RAST inhibition showed that wheat, rye, barley and soya flours contained crossreacting proteins, in decreasing concentrations. Wheat, rye and barley flours had similar protein profiles on gel electrophoresis. Soya flour contained a number of high molecular weight proteins not present in the other cereals. Western blotting using sera from 21 wheat flour hypersensitive individuals identified a large number of allergens in the different flours. Proteins of 69, 33, 26, 21 and 12 kDa were identified as major wheat flour allergens. Rye flour proteins of 21 and 12 kDa, and barley flour proteins of 69, 52 and 10 kDa were the major allergens identified by serum from wheat hypersensitive individuals. The major common protein of soya and wheat flour had a molecular weight of 21 kDa. The majority of crossreacting allergens identified between the different flours have molecular weights similar to those of known flour enzymes or enzyme inhibitors.

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[35] - Baur X, Pau M, Czuppon A, Fruhmann G. Characterization of soybean allergens causing sensitization of occupationally exposed bakers. Allergy 1996;51:326-330

Fourteen bakers suffering from workplace-related respiratory symptoms and sensitized to soybean were studied. Twelve of them were also allergic to wheat flour, 10 to rye flour, and five to alpha-amylase of Aspergillus oryzae (Asp o 2). IgE estimation by RAST strongly indicated that the trypsin inhibitor and lipoxidase are major allergens of soybean. Various allergenic components could be characterized by immunoblotting after two-dimensional electrophoresis. Our RAST and immunoblotting results show an interindividually different allergic response to inhalative soybean constituents, and that the trypsin inhibitor (20 kDa, pI approximately 4.5) is an important inhalative soybean allergen recognized by IgE antibodies in the sera of 86% of the examined sensitized bakers.

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[36] - Baur X, Pau M, Czuppon A, Fruhmann G. Characterization of soybean allergens causing sensitization of occupationally exposed bakers. Allergy 1996;51:326-330

Fourteen bakers suffering from workplace-related respiratory symptoms and sensitized to soybean were studied. Twelve of them were also allergic to wheat flour, 10 to rye flour, and five to alpha-amylase of Aspergillus oryzae (Asp o 2). IgE estimation by RAST strongly indicated that the trypsin inhibitor and lipoxidase are major allergens of soybean. Various allergenic components could be characterized by immunoblotting after two-dimensional electrophoresis. Our RAST and immunoblotting results show an interindividually different allergic response to inhalative soybean constituents, and that the trypsin inhibitor (20 kDa, pI approximately 4.5) is an important inhalative soybean allergen recognized by IgE antibodies in the sera of 86% of the examined sensitized bakers.

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[37] - Rihs HP, Chen Z, Rueff F, Petersen A, Rozynek P, Heimann H, et al. IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes. J Allergy Clin Immunol 1999;104:1293-1301

Soybean proteins are constituents of a number of food products and represent a panel of potential allergens. Thus far, little is known about the molecular characteristics of soybean allergens. OBJECTIVE: The aim of this study was to identify the soybean profilin by PCR-based complementary (c)DNA cloning and to elucidate its allergenic characteristics. METHODS: Highly degenerate profilin-specific primers were used to identify, by means of PCR, 2 soybean profilin isoforms (GmPRO1 and GmPRO2) by using soybean cDNA as a target. One isoform (GmPRO1) with a length of 394 bp corresponding to 131 amino acid residues was subcloned and expressed in fusion with the maltose-binding protein. Moreover, 3 overlapping recombinant soybean profilin fragments comprising amino acid residues 1-65, 38-88, and 50-131 were also prepared as maltose-binding protein fusion proteins. IgE-binding reactivity of the recombinant proteins and the cross-reactivity of soybean profilin with birch profilin was studied by immunoblotting, enzyme-linked allergosorbent assays (EASTs), and competitive inhibition experiments by using serum samples from 13 soybean-sensitized subjects. RESULTS: Results of immunoblot analysis, EAST, and EAST-inhibition experiments indicate the presence of profilin in soybean extract. The recombinant soybean profilin (rGly m 3) was recognized by IgE in 9 (69%) of the 13 sera tested. Only the full-length rGly m 3 was able to bind with IgE antibodies, whereas the 3 soybean profilin fragments did not show significant binding reactivity, indicating that the IgE binding to rGly m 3 depends on the integrity of a conformational structure, which was not present in the overlapping profilin fragments. The rGly m 3 cross-reacted with birch pollen profilin (Bet v 2), and the IgE binding to Bet v 2 could be inhibited by rGly m 3. CONCLUSIONS: rGly m 3 represents a new soybean allergen with well-characterized primary sequence, and its IgE-binding reactivity is mediated by conformational epitopes.

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[38] - Lin J, Shewry PR, Archer DB, Beyer K, Niggemann B, Haas H, et al. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach. Int Arch Allergy Immunol 2006;141:91-102

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens . METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system . RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system . CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

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[39] - Kleine-Tebbe J, Wangorsch A, Vogel L, Crowell DN, Haustein UF, Vieths S. Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1-related PR-10 protein in soybean, SAM22. J Allergy Clin Immunol 2002;110:797-804

BACKGROUND: Anaphylactic reactions to soy products have been attributed to stable class 1 food allergens . OBJECTIVE: IgE- mediated reactions to a soy-containing dietary food product in patients allergic to birch pollen were investigated . METHODS: Detailed case histories were taken from 20 patients. Their sera were analyzed for IgE (UniCAP) specific for birch, grass, mugwort, the recombinant birch allergens rBet v 1 and rBet v2, and soy protein. Extracts from birch pollen, soy isolate, rBet v 1, and the recombinant PR-10 soy protein rSAM22 were coupled to paper disks or nitrocellulose for IgE measurements (enzyme allergosorbent test) or Western blot analysis. Enzyme allergosorbent testing, Western blot inhibition, and histamine release studies were performed with the same allergens . RESULTS: Most patients (17/20) experienced facial, oropharyngeal, and/or systemic allergic symptoms within 20 minutes after ingesting the soy product for the first time. Birch pollen allergy (16/20) was common, along with oral allergy syndrome to apple (12/20) or hazelnut (11/20). IgE levels to birch and Bet v 1 but not to other inhalants were high in 18 of 20 patients. Significant IgE binding to rSAM22 occurred in 17 of 20 patients. Blot experiments with the soy isolate revealed IgE-binding bands at 17 kd (15/20), 22 kd (1/20), and 35 to 38 kd (2/20); the former was inhibited by preincubation of the sera with rBet v 1 or rSAM22. Birch extract and soy isolate, rBet v 1, and rSAM22 induced dose-dependent histamine release in the nanomolar range . CONCLUSION: Immediate-type allergic symptoms in patients with birch pollen allergy after ingestion of soy protein-containing food items can result from cross-reactivity of Bet v 1 -specific IgE to homologous pathogenesis-related proteins, particularly the PR-10 protein SAM22.

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[40] - Mittag D, Akkerdaas J, Ballmer-Weber BK, Vogel L, Wensing M, Becker WM, et al. Ara h 8, a Bet v 1–homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy. J Allergy Clin Immunol 2004;114:1410-1417

Background We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. Objective : We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1ˆhomologous peanut allergen Ara h 8. Method s : Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. Result s : During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8ˆspecific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. Conclusions : Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.

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[41] - Bisson C, Holzhauser T, Natale M, Giuffrida MG, Fortunato D, Perono Garoffo L, et al. Application of proteomic tools reveals a highly individual IgE response in soybean-allergic patients. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1014

Background: The increased use of soybean products in processed foods and its use in the diet of infants with allergy to cow‚s milk, poses a potential threat to soybean-sensitive food-allergic individuals. Cross-reactivity to other leguminosae and to birch-pollen allergens has been demonstrated. So far, only four soy proteins (Gly m 1-Gly m 4) have been officially accepted as allergens by the IUIS allergen nomenclature subcommittee, of which two, Gly m 1&2, are respiratory allergens. In the framework of an EU project (FAREDAT), this study is aimed at allergen identification in soy using for the first time sera of European patients with confirmed soybean allergy, by a proteomic approach. Methods: Soybean protein extracts were separated by means of 2 dimensional PAGE: different pH gradients (3-10NL, 3-6, 5-8) IPG Strips were used in the first dimension step and 4-12% NuPage gradient gels in the second dimension. Proteins were subsequently transferred to nitrocellulose membranes. Immunolabeling was performed with individual positive DBPCFC subjects (n=5) or patients with a clear cut history of soybean allergy (n=2). IgE reactive proteins were identified by means of mass spectrometry (MALDI/TOF, ESI MS/MS). Results: All the patients showed multiple sensitizations: a, a' and b chain of b- conglycinin, acidic chains of G1 and G2 glycinin, Gly m Bd30K and Gly m 4 were identified as the main IgE reactive proteins. Two patients presented an interesting unique pattern of immunoreactivity. In general, the IgE response between different patients was highly individual. Conclusions: This study confirms that the proteomic approach is a very powerful tool in food allergy studies. A high heterogeneity of the IgE response in soy allergy was found, and our data indicate that other allergens than those included in the official IUIS allergen list are the major allergens in soybean. Further development concerns pre-fractionation of soybean proteins by preparative IEF and subsequent use of micro-range pH gradient IPG Strips in order to identify also the lower abundant-high IgE reactive proteins.

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[42] - Helm RM, Cockrell G, Connaughton C, Sampson HA, Bannon GA, Beilinson V, et al. A soybean G2 glycinin allergen. Int Arch Allergy Immunol 2000;123:205-212

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. METHODS: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. RESULTS: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. CONCLUSIONS: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.

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[43] - Martin-Hernandez C, Benet S, Marvin-Guy LF. Characterization and quantification of proteins in lecithins. J Agric Food Chem 2005;53:8607-8613

Several methods for extraction and quantification of proteins from lecithins were compared. Extraction with hexane-2-propanol-water followed by amino acid analysis is the most suitable method for isolation and quantification of proteins from lecithins. The detection limit of the method is 15 mg protein/kg lecithin, and the quantification limit is 50 mg protein/kg. The relative repeatability limits for samples containing 0-500 and 500-5000 mg protein/kg sample were 12.6 and 7.5%, respectively. The protein recovery ranged between 101 and 123%. The protein content has been determined in different kinds of lecithins. The results were as follows: standard soy lecithins (between 232 and 1338 mg/kg), deoiled soy lecithin (342 mg/kg), phosphatydylcholine-enriched soy lecithins (not detectable and 163 mg/kg), sunflower lecithins (892 and 414 mg/kg), and egg lecithin (50 mg/kg). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of the standard soy and sunflower lecithins are very similar to those of soy flour. The protein profile of the egg lecithin shows several bands with a broad range of molecular masses. The molecular masses of the main proteins of soy lecithins and soy flour have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ranged from 10.5 to 52.2 kDa. Most of the major proteins from soy and sunflower lecithins identified by MALDI-MS and electrospray tandem MS belong to the 11S globulin fraction, which is one of the main fractions of soy and sunflower seeds. In addition, the seed maturation protein P34 from the 7S globulin fraction of soy proteins has also been identified in soy lecithins. This protein has been reported as the most allergenic protein in soybean

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[44] - Bisson C, Holzhauser T, Natale M, Giuffrida MG, Fortunato D, Perono Garoffo L, et al. Application of proteomic tools reveals a highly individual IgE response in soybean-allergic patients. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1014

Background: The increased use of soybean products in processed foods and its use in the diet of infants with allergy to cow‚s milk, poses a potential threat to soybean-sensitive food-allergic individuals. Cross-reactivity to other leguminosae and to birch-pollen allergens has been demonstrated. So far, only four soy proteins (Gly m 1-Gly m 4) have been officially accepted as allergens by the IUIS allergen nomenclature subcommittee, of which two, Gly m 1&2, are respiratory allergens. In the framework of an EU project (FAREDAT), this study is aimed at allergen identification in soy using for the first time sera of European patients with confirmed soybean allergy, by a proteomic approach. Methods: Soybean protein extracts were separated by means of 2 dimensional PAGE: different pH gradients (3-10NL, 3-6, 5-8) IPG Strips were used in the first dimension step and 4-12% NuPage gradient gels in the second dimension. Proteins were subsequently transferred to nitrocellulose membranes. Immunolabeling was performed with individual positive DBPCFC subjects (n=5) or patients with a clear cut history of soybean allergy (n=2). IgE reactive proteins were identified by means of mass spectrometry (MALDI/TOF, ESI MS/MS). Results: All the patients showed multiple sensitizations: a, a' and b chain of b- conglycinin, acidic chains of G1 and G2 glycinin, Gly m Bd30K and Gly m 4 were identified as the main IgE reactive proteins. Two patients presented an interesting unique pattern of immunoreactivity. In general, the IgE response between different patients was highly individual. Conclusions: This study confirms that the proteomic approach is a very powerful tool in food allergy studies. A high heterogeneity of the IgE response in soy allergy was found, and our data indicate that other allergens than those included in the official IUIS allergen list are the major allergens in soybean. Further development concerns pre-fractionation of soybean proteins by preparative IEF and subsequent use of micro-range pH gradient IPG Strips in order to identify also the lower abundant-high IgE reactive proteins.

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[45] - Holzhauser T, Petrovskaya O, Kuehne Y, Wangorsch A, Ballmer-Weber B, Bindslev-Jensen C, et al. Allergenicity of soybean beta-conglycinin versus Gly m Bd 30k in patients with confirmed soybean allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°513

Background: Soybean b-conglycinin and Gly m Bd 30k have been described as IgE-binding proteins, the latter one as putative major soybean allergen. We aimed at characterising both soybean proteins in regard to their relevance in a European patient group with confirmed soybean allergy. Methods: Twenty-five adults were included into the study on the basis of a positive DBPCFC or conclusive history of anaphylaxis to soy. Twenty-two children were included upon positive DBPCFC or open oral challenge. Natural b-conglycinin subunits (a, a', b) were extracted from soybean, recombinant subunits expressed in E.coli, and both purified by continuous-elution electrophoresis. Natural Gly m Bd 30k was enriched by an oleosin fractionation of soybean and the recombinant homologue expressed as His-tagged fusion protein in E.coli and purified by IMAC. Protein identity was proven by N-terminal sequencing and/or peptide mass fingerprinting. IgE-reactivity of the purified soy proteins was investigated by IgE-immunoblotting and/or IgE-ELISA with patients' sera. ELISA- and EAST-inhibition, and mediator release from passively sensitized humanized rat basophilic leukaemia cells was performed with selected patients' sera. Results: b-conglycinin was IgE-reactive in 54 % (12/22) of the children and 20 % (5/25) of the adults. By contrast, enriched natural Gly m Bd 30k bound IgE from less than 50 % of patients‚ sera in immunoblotting, and rGly m Bd 30k did not show any IgE-reactivity. In ELISA-inhibition, IgE-binding to b- conglycinin was fully inhibited by soybean extract and itself. IgE-reactivity to b-conglycinin was almost fully inhibited by peanut extract and purified Ara h 1 in a soybean and peanut allergic patient, however hardly inhibited in a patient allergic to soybean but not to peanut. Similar results were obtained in EAST-inhibition with soybean extract on the solid surface. Specific mediator release confirmed the cross-linking properties of b-conglycinin. Conclusion: In Europe, soybean b-conglycinin is a major allergen for soy-allergic children and an important minor allergen in adults, whereas the relevance of Gly m Bd 30k in IgE-mediated soybean allergy remains unclear and demands further investigation. In some patients cross-reactivity between peanut and soybean is limited.

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[46] - Lin J, Shewry PR, Archer DB, Beyer K, Niggemann B, Haas H, et al. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach. Int Arch Allergy Immunol 2006;141:91-102

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens . METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system . RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system . CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

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[47] - Han YS, Lin J, Bardina L, Bruni FM, Ayuso R, Sampson HA. Identification of Immunoglobulin E-binding Epitopes in Soybean Gly m conglycinin, Gly m glycinin G1/G2 and Two 2S Albumins Using Peptide Microarray Immunoassay. J Allergy Clin Immunol 2008;121:S248

RATIONALE: Soybean, a member of the legume family, is responsible for allergic reactions that are often outgrown. In contrast, allergy to another highly homologous legume, peanut, is rarely outgrown and allergic reactions are more often severe. The identification of IgE epitopes in soybean proteins might explain this clinical discrepancy. METHODS: Commercially synthesized peptides (15-mers, 3 offset) covering the full length of Conglycinin, Glycinin G1/G2 and two 2S albumins of soybean were site-specifically bound to epoxy-derivatized glass slides in quadruplicates. 5 pooled-sera and 3 individual sera of soyallergic patients were assayed. Sera from 5 non-atopic volunteers served as negative controls. Specific IgE-binding was detected using fluorochromelabeled polyclonal antibodies. RESULTS: The majority of epitopes in Glycinin G1/G2 identified by the peptide microarray were consistent with those identified previously by SPOT membrane. In addition, serum pool and 3 individual sera recognized position 210-235 in Glycinin G2 not previously reported as epitopes. IgE binding to Conglycinin was found at regions 316-360 and 508-537. Several regions (2S albumin 1: aa64-84 and 112-125; 2S albumin3: aa31-45) in 2S albumins were recognized by the serum pool and at least 2 individual sera, suggesting that although not previously regarded as allergen, 2S albumins might be considered as such. CONCLUSIONS: Utilizing peptide microarray assays, we successfully mapped IgE-binding epitopes of soybean allergens. A comparison of allergenic epitopes on peanut and soy proteins may provide a molecular explanation for differences in responses to these homologous allergens.

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[48] - Gu X, Beardslee TA, Zeece MG, Sarath G, Markwell JP. Identification of IgE-Binding Proteins in Soy Lecithin. Int Arch Allergy Immunol 2001;126:218-225

Background: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Methods: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. Results: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Conclusions: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

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[49] - Pons L, Chery C, Romano A, Namour F, Artesani MC, Guéant JL. The 18 kDa peanut oleosin is a candidate allergen for IgE-mediated reactions to peanuts. Allergy 2002;57(Suppl. 72):88-93

BACKGROUND: Peanut allergy is one of the five most frequent food allergies in children and in adults. Recently, we purified and evaluated the allergenicity of peanut oleosins, a family of small-sized proteins involved in the formation of peanut oil bodies . METHODS: Allergenicity of the purified native protein and of the recombinant protein was tested by Western blot and by IgE-RIA . RESULTS: We found IgE-binding with oleosin in 3 of 14 sera of patients who had suffered an allergic reaction to peanuts. Two sera reacted weakly against 16-18 kDa proteins corresponding to oleosin monomers, in Western blot. The main reacting bands had a molecular size estimated at approximately 34 kDa, approximately 50 kDa and approximately 68 kDa and could therefore correspond to oleosin oligomers. IgE reactivity was higher in extracts from roasted peanuts. The same phenomenon occurred with crude soybean oil fraction, with two bands of 16.5 and 24 kDa corresponding to monomers, and two bands of 50 kDa and 76 kDa corresponding to dimers and trimers, respectively. The 18 kDa band was observed in the 3 Western blots of a membrane-enriched fraction of recombinant oleosin produced in the Sf9-baculovirus expression system (performed with the 3 patient sera) . CONCLUSIONS: We have characterized a new peanut allergen which belongs to the oleosins, a family of proteins involved in the formation of oil bodies. The protein may be involved in some of the allergic cross-reactions to peanuts and soybeans.

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[50] - Bisson C, Holzhauser T, Natale M, Giuffrida MG, Fortunato D, Perono Garoffo L, et al. Application of proteomic tools reveals a highly individual IgE response in soybean-allergic patients. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1014

Background: The increased use of soybean products in processed foods and its use in the diet of infants with allergy to cow‚s milk, poses a potential threat to soybean-sensitive food-allergic individuals. Cross-reactivity to other leguminosae and to birch-pollen allergens has been demonstrated. So far, only four soy proteins (Gly m 1-Gly m 4) have been officially accepted as allergens by the IUIS allergen nomenclature subcommittee, of which two, Gly m 1&2, are respiratory allergens. In the framework of an EU project (FAREDAT), this study is aimed at allergen identification in soy using for the first time sera of European patients with confirmed soybean allergy, by a proteomic approach. Methods: Soybean protein extracts were separated by means of 2 dimensional PAGE: different pH gradients (3-10NL, 3-6, 5-8) IPG Strips were used in the first dimension step and 4-12% NuPage gradient gels in the second dimension. Proteins were subsequently transferred to nitrocellulose membranes. Immunolabeling was performed with individual positive DBPCFC subjects (n=5) or patients with a clear cut history of soybean allergy (n=2). IgE reactive proteins were identified by means of mass spectrometry (MALDI/TOF, ESI MS/MS). Results: All the patients showed multiple sensitizations: a, a' and b chain of b- conglycinin, acidic chains of G1 and G2 glycinin, Gly m Bd30K and Gly m 4 were identified as the main IgE reactive proteins. Two patients presented an interesting unique pattern of immunoreactivity. In general, the IgE response between different patients was highly individual. Conclusions: This study confirms that the proteomic approach is a very powerful tool in food allergy studies. A high heterogeneity of the IgE response in soy allergy was found, and our data indicate that other allergens than those included in the official IUIS allergen list are the major allergens in soybean. Further development concerns pre-fractionation of soybean proteins by preparative IEF and subsequent use of micro-range pH gradient IPG Strips in order to identify also the lower abundant-high IgE reactive proteins.

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[51] - Ogawa T, Bando N, Tsuji H, Okajima H, Nishikawa K, Sasaoka K. Investigation of the IgE-binding proteins in soybeans by immunoblotting with the sera of the soybean-sensitive patients with atopic dermatitis. J Nutr Sci Vitaminol (Tokyo) 1991;37:555-565

The IgE-binding proteins in soybeans were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractionated soybean proteins probed with the sera of the patients with atopic dermatitis. About 20% of the patients examined were shown to have specific IgE antibodies to soybean proteins. At least 16 soybean proteins with molecular weights ranging from about 70,000 to 14,000 were recognized by the sera of the patients: 10 major IgE-binding components were found in the 7S-globulin fraction, and the others mainly in the 2S-globulin and whey fractions. The IgE antibodies of the patients bound most strongly and frequently to a unique protein with molecular weight of about 30,000 in the 7S-globulin fraction, which appeared to be the major allergen in soybeans and was named as Gly m Bd 30 K. The proteins in the 11S-globulin fraction were scarcely recognized by the patients' sera and assumed to be less allergenic for the patients with atopic dermatitis.

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[52] - Holzhauser T, Petrovskaya O, Kuehne Y, Wangorsch A, Ballmer-Weber B, Bindslev-Jensen C, et al. Allergenicity of soybean beta-conglycinin versus Gly m Bd 30k in patients with confirmed soybean allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°513

Background: Soybean b-conglycinin and Gly m Bd 30k have been described as IgE-binding proteins, the latter one as putative major soybean allergen. We aimed at characterising both soybean proteins in regard to their relevance in a European patient group with confirmed soybean allergy. Methods: Twenty-five adults were included into the study on the basis of a positive DBPCFC or conclusive history of anaphylaxis to soy. Twenty-two children were included upon positive DBPCFC or open oral challenge. Natural b-conglycinin subunits (a, a', b) were extracted from soybean, recombinant subunits expressed in E.coli, and both purified by continuous-elution electrophoresis. Natural Gly m Bd 30k was enriched by an oleosin fractionation of soybean and the recombinant homologue expressed as His-tagged fusion protein in E.coli and purified by IMAC. Protein identity was proven by N-terminal sequencing and/or peptide mass fingerprinting. IgE-reactivity of the purified soy proteins was investigated by IgE-immunoblotting and/or IgE-ELISA with patients' sera. ELISA- and EAST-inhibition, and mediator release from passively sensitized humanized rat basophilic leukaemia cells was performed with selected patients' sera. Results: b-conglycinin was IgE-reactive in 54 % (12/22) of the children and 20 % (5/25) of the adults. By contrast, enriched natural Gly m Bd 30k bound IgE from less than 50 % of patients‚ sera in immunoblotting, and rGly m Bd 30k did not show any IgE-reactivity. In ELISA-inhibition, IgE-binding to b- conglycinin was fully inhibited by soybean extract and itself. IgE-reactivity to b-conglycinin was almost fully inhibited by peanut extract and purified Ara h 1 in a soybean and peanut allergic patient, however hardly inhibited in a patient allergic to soybean but not to peanut. Similar results were obtained in EAST-inhibition with soybean extract on the solid surface. Specific mediator release confirmed the cross-linking properties of b-conglycinin. Conclusion: In Europe, soybean b-conglycinin is a major allergen for soy-allergic children and an important minor allergen in adults, whereas the relevance of Gly m Bd 30k in IgE-mediated soybean allergy remains unclear and demands further investigation. In some patients cross-reactivity between peanut and soybean is limited.

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[53] - Herman EM, Helm RM, Jung R, Kinney AJ. Genetic modification removes an immunodominant allergen from soybean. Plant Physiol 2003;132:36-43

The increasing use of soybean (Glycine max) products in processed foods poses a potential threat to soybean-sensitive food-allergic individuals. In vitro assays on soybean seed proteins with sera from soybean-sensitive individuals have immunoglobulin E reactivity to abundant storage proteins and a few less-abundant seed proteins. One of these low abundance proteins, Gly m Bd 30 K, also referred to as P34, is in fact a major (i.e. immunodominant) soybean allergen. Although a member of the papain protease superfamily, Gly m Bd 30 K has a glycine in the conserved catalytic cysteine position found in all other cysteine proteases. Transgene-induced gene silencing was used to prevent the accumulation of Gly m Bd 30 K protein in soybean seeds. The Gly m Bd 30 K-silenced plants and their seeds lacked any compositional, developmental, structural, or ultrastructural phenotypic differences when compared with control plants. Proteomic analysis of extracts from transgenic seed detected the suppression of Gly m Bd 30 K-related peptides but no other significant changes in polypeptide pattern. The lack of a collateral alteration of any other seed protein in the Gly m Bd 30 K-silenced seeds supports the presumption that the protein does not have a role in seed protein processing and maturation. These data provide evidence for substantial equivalence of composition of transgenic and non-transgenic seed eliminating one of the dominant allergens of soybean seeds.

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[54] - Martin-Hernandez C, Benet S, Marvin-Guy LF. Characterization and quantification of proteins in lecithins. J Agric Food Chem 2005;53:8607-8613

Several methods for extraction and quantification of proteins from lecithins were compared. Extraction with hexane-2-propanol-water followed by amino acid analysis is the most suitable method for isolation and quantification of proteins from lecithins. The detection limit of the method is 15 mg protein/kg lecithin, and the quantification limit is 50 mg protein/kg. The relative repeatability limits for samples containing 0-500 and 500-5000 mg protein/kg sample were 12.6 and 7.5%, respectively. The protein recovery ranged between 101 and 123%. The protein content has been determined in different kinds of lecithins. The results were as follows: standard soy lecithins (between 232 and 1338 mg/kg), deoiled soy lecithin (342 mg/kg), phosphatydylcholine-enriched soy lecithins (not detectable and 163 mg/kg), sunflower lecithins (892 and 414 mg/kg), and egg lecithin (50 mg/kg). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of the standard soy and sunflower lecithins are very similar to those of soy flour. The protein profile of the egg lecithin shows several bands with a broad range of molecular masses. The molecular masses of the main proteins of soy lecithins and soy flour have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ranged from 10.5 to 52.2 kDa. Most of the major proteins from soy and sunflower lecithins identified by MALDI-MS and electrospray tandem MS belong to the 11S globulin fraction, which is one of the main fractions of soy and sunflower seeds. In addition, the seed maturation protein P34 from the 7S globulin fraction of soy proteins has also been identified in soy lecithins. This protein has been reported as the most allergenic protein in soybean

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[55] - Burks AW, Cockrell G, Connaughton C, Guin J, Allen W, Helm RM. Identification of peanut agglutinin and soybean trypsin inhibitor as minor legume allergens. Int Arch Allergy Immunol 1994;105:143-149

Peanuts and soybeans are frequent causes of food hypersensitivity reactions in children. Sera from 12 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut and sera from 5 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to soybean were used to identify and characterize specific legume allergens. Identification of a minor allergen from peanut and a minor allergen from soybean was accomplished using various physicochemical techniques. The peanut fraction, peanut agglutinin, isolated by anion-exchange chromatography and electrolution and confirmed by amino acid sequencing, bound IgE in only 50% of the peanut challenge positive patients. The soybean fraction, soybean trypsin inhibitor, identified by gel filtration and electroelution and confirmed by amino acid sequencing, bound IgE in only 20% of the soy challenge positive patients. The identification of these two known legume proteins as minor allergens should allow further immunologic and structural investigations to compare the major and minor legume allergens.

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[56] - Quirce S. Cross reactivity between enzymes. Alergol Immunol Clin 2000;15:50-52

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[57] - Besler M, Helm RM, Ogawa T. Allergen data collection: soybean (Glycine max). Internet Symp Food Allergens 2000;2(suppl. 3):1-35

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[58] - Gu X, Beardslee TA, Zeece MG, Sarath G, Markwell JP. Identification of IgE-Binding Proteins in Soy Lecithin. Int Arch Allergy Immunol 2001;126:218-225

Background: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Methods: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. Results: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Conclusions: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

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[59] - Errahali Y, Kanny G, Morisset M, Cren-Olive C, Rolando C, Metche M, et al. Sequencing of Soy Oil and Soy Lecithin Allergens. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°507

Rationale It has been shown that soy oil and soy lecithin may contain allergens. The aim of the present study is to characterize, by means of the physico-chemical and immunological methods, the allergenic proteins of soybean seed transferred to soy oil and soy lecithin during industrial refining. Method s : Extraction of proteins was conducted using a buffer solution. The identification of allergens used specific IgE from a soybean allergic patient. Bi-dimensional electrophoresis and sequencing from N and C terminals were used to characterize the allergenic proteins. Result s : Two allergenic proteins present in soybean seed, soy oil and soy lecithin have been isolated and identified: one 56 kDa non glycosylated allergen, whose isoelectric point of 5.4 has been identified as soybean -Amylase (7S). One 20 kDa non glycosylated allergen, whose isoelectric point of 4.6 has been identified as Kunitz Trypsin inhibitor [Glycine max] (2S). Conclusions : We have sequenced two allergenic proteins present as hidden allergens in soy oil and soy lecithin. It is to note that these highly refining-resistant proteins are not the main proteins of soybean seed.

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[60] - Burks AW, Cockrell G, Connaughton C, Guin J, Allen W, Helm RM. Identification of peanut agglutinin and soybean trypsin inhibitor as minor legume allergens. Int Arch Allergy Immunol 1994;105:143-149

Peanuts and soybeans are frequent causes of food hypersensitivity reactions in children. Sera from 12 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut and sera from 5 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to soybean were used to identify and characterize specific legume allergens. Identification of a minor allergen from peanut and a minor allergen from soybean was accomplished using various physicochemical techniques. The peanut fraction, peanut agglutinin, isolated by anion-exchange chromatography and electrolution and confirmed by amino acid sequencing, bound IgE in only 50% of the peanut challenge positive patients. The soybean fraction, soybean trypsin inhibitor, identified by gel filtration and electroelution and confirmed by amino acid sequencing, bound IgE in only 20% of the soy challenge positive patients. The identification of these two known legume proteins as minor allergens should allow further immunologic and structural investigations to compare the major and minor legume allergens.

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[61] - Lin J, Shewry PR, Archer DB, Beyer K, Niggemann B, Haas H, et al. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach. Int Arch Allergy Immunol 2006;141:91-102

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens . METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system . RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system . CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

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[62] - Lin J, Shewry PR, Archer DB, Beyer K, Niggemann B, Haas H, et al. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach. Int Arch Allergy Immunol 2006;141:91-102

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens . METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system . RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system . CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

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[63] - Rosahl S. Lipoxygenases in plants : their role in development and stress response. Z Naturforsch 1996;51:123-138

Lipoxygenases catalyze the hydroperoxidation of polyunsaturated fatty acids and thus the first step in the synthesis of fatty acid metabolites in plants. Products of the LOX pathway have multiple functions as growth regulators, antimicrobial compounds, flavours and odours as well as signal molecules. Based on the effects of LOX products or on the correlation of increases in LOX protein and the onset of specific processes, a physiological function for LOXs has been proposed for growth and development and for the plant response to pathogen infection and wound stress.

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[64] - Baur X, Pau M, Czuppon A, Fruhmann G. Characterization of soybean allergens causing sensitization of occupationally exposed bakers. Allergy 1996;51:326-330

Fourteen bakers suffering from workplace-related respiratory symptoms and sensitized to soybean were studied. Twelve of them were also allergic to wheat flour, 10 to rye flour, and five to alpha-amylase of Aspergillus oryzae (Asp o 2). IgE estimation by RAST strongly indicated that the trypsin inhibitor and lipoxidase are major allergens of soybean. Various allergenic components could be characterized by immunoblotting after two-dimensional electrophoresis. Our RAST and immunoblotting results show an interindividually different allergic response to inhalative soybean constituents, and that the trypsin inhibitor (20 kDa, pI approximately 4.5) is an important inhalative soybean allergen recognized by IgE antibodies in the sera of 86% of the examined sensitized bakers.

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[65] - Frémont S, Sanchez C, Errahali Y, Mouécoucou J, Metche M, Méjean L, et al. Allergénicité alimentaire: importance des interactions protéines-lipides et protéines-polysaccharides. Alim'Inter 2003;8:35-41

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[66] - Errahali Y, Kanny G, Morisset M, Cren-Olive C, Rolando C, Metche M, et al. Sequencing of Soy Oil and Soy Lecithin Allergens. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°507

Rationale It has been shown that soy oil and soy lecithin may contain allergens. The aim of the present study is to characterize, by means of the physico-chemical and immunological methods, the allergenic proteins of soybean seed transferred to soy oil and soy lecithin during industrial refining. Method s : Extraction of proteins was conducted using a buffer solution. The identification of allergens used specific IgE from a soybean allergic patient. Bi-dimensional electrophoresis and sequencing from N and C terminals were used to characterize the allergenic proteins. Result s : Two allergenic proteins present in soybean seed, soy oil and soy lecithin have been isolated and identified: one 56 kDa non glycosylated allergen, whose isoelectric point of 5.4 has been identified as soybean -Amylase (7S). One 20 kDa non glycosylated allergen, whose isoelectric point of 4.6 has been identified as Kunitz Trypsin inhibitor [Glycine max] (2S). Conclusions : We have sequenced two allergenic proteins present as hidden allergens in soy oil and soy lecithin. It is to note that these highly refining-resistant proteins are not the main proteins of soybean seed.

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[67] - Errahali Y, Morisset M, Moneret-Vautrin DA, Kanny G, Metche M, Nicolas JP, et al. Allergen in soy oils. Allergy 2002;57:648-649

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[68] - Herman EM, Helm RM, Jung R, Kinney AJ. Genetic modification removes an immunodominant allergen from soybean. Plant Physiol 2003;132:36-43

The increasing use of soybean (Glycine max) products in processed foods poses a potential threat to soybean-sensitive food-allergic individuals. In vitro assays on soybean seed proteins with sera from soybean-sensitive individuals have immunoglobulin E reactivity to abundant storage proteins and a few less-abundant seed proteins. One of these low abundance proteins, Gly m Bd 30 K, also referred to as P34, is in fact a major (i.e. immunodominant) soybean allergen. Although a member of the papain protease superfamily, Gly m Bd 30 K has a glycine in the conserved catalytic cysteine position found in all other cysteine proteases. Transgene-induced gene silencing was used to prevent the accumulation of Gly m Bd 30 K protein in soybean seeds. The Gly m Bd 30 K-silenced plants and their seeds lacked any compositional, developmental, structural, or ultrastructural phenotypic differences when compared with control plants. Proteomic analysis of extracts from transgenic seed detected the suppression of Gly m Bd 30 K-related peptides but no other significant changes in polypeptide pattern. The lack of a collateral alteration of any other seed protein in the Gly m Bd 30 K-silenced seeds supports the presumption that the protein does not have a role in seed protein processing and maturation. These data provide evidence for substantial equivalence of composition of transgenic and non-transgenic seed eliminating one of the dominant allergens of soybean seeds.

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[69] - Tsuji H, Hiemori M, Kimoto M, Yamashita H, Kobatake R, Adachi M, et al. Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K. Biochim Biophys Acta 2001;1518:178-182

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.

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[70] - Hiemori M, Bando N, Ogawa T, Shimada H, Tsuji H, Yamanishi R, et al. Occurrence of IgE antibody-recognizing N-linked glycan moiety of a soybean allergen, Gly m Bd 28K. Int Arch Allergy Immunol 2000;122:238-245

It has been reported that N-linked glycan moieties of glycoproteins function as IgE-reactive determinants. Gly m Bd 28K, a soybean allergen, was a glycoprotein with glycan moieties, which are supposed to be the Man(3)GlcNAc(2) backbone with the beta1-->2 xylose and alpha1-->3 fucose branches. The purpose of the present study was to examine the IgE-binding ability of the glycan moiety of Gly m Bd 28K in the binding reaction with patients' sera. METHODS: A peptide containing the glycan moiety was prepared from Gly m Bd 28K by digestion with lysyl endopeptidase. The binding site of the glycan moiety was determined by amino acid sequence analyses. The glycan moiety of the allergen was characterized using anti-horseradish peroxidase antibody (anti-HRP) recognizing the N-linked glycan moieties of glycoproteins. The binding of patients' IgE antibodies with their glycan moiety was examined by an immunostaining technique using the glycopeptide and its deglycosylated peptide derived from Gly m Bd 28K. RESULTS: The binding site of the glycan moiety in Gly m Bd 28K was shown to be its Asn20 residue. Gly m Bd 28K did react with anti-HRP and the sera of soybean-sensitive patients, but the binding of IgE antibodies was inhibited by the preincubation with anti-HRP. Moreover, the glycopeptide also reacted with the sera of soybean-sensitive patients, but its deglycosylated peptide did not react with any IgE antibodies of patients' sera. CONCLUSIONS: The specific IgE antibodies recognizing the N-linked glycan moieties of Gly m Bd 28K and other glycoproteins with homologous glycan moieties occur in the sera of soybean-sensitive patients. It was indicated that the N-linked glycan moieties such as that of Gly m Bd 28K may be one of the common IgE-reactive determinants distributed in various plant food proteins.

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[71] - Xiang P, Haas EJ, Zeece MG, Markwell J, Sarath G. C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen. Planta 2004;220:53-63

Gly m Bd 28 K is a major soybean ( Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the beta subunit of soybean beta-conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first beta-sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.

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[72] - Hiemori M, Ito H, Kimoto M, Yamashita H, Nishizawa K, Maruyama N, et al. Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen. Biochim Biophys Acta 2004;1675:174-183

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.

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[73] - Bisson C, Holzhauser T, Natale M, Giuffrida MG, Fortunato D, Perono Garoffo L, et al. Application of proteomic tools reveals a highly individual IgE response in soybean-allergic patients. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1014

Background: The increased use of soybean products in processed foods and its use in the diet of infants with allergy to cow‚s milk, poses a potential threat to soybean-sensitive food-allergic individuals. Cross-reactivity to other leguminosae and to birch-pollen allergens has been demonstrated. So far, only four soy proteins (Gly m 1-Gly m 4) have been officially accepted as allergens by the IUIS allergen nomenclature subcommittee, of which two, Gly m 1&2, are respiratory allergens. In the framework of an EU project (FAREDAT), this study is aimed at allergen identification in soy using for the first time sera of European patients with confirmed soybean allergy, by a proteomic approach. Methods: Soybean protein extracts were separated by means of 2 dimensional PAGE: different pH gradients (3-10NL, 3-6, 5-8) IPG Strips were used in the first dimension step and 4-12% NuPage gradient gels in the second dimension. Proteins were subsequently transferred to nitrocellulose membranes. Immunolabeling was performed with individual positive DBPCFC subjects (n=5) or patients with a clear cut history of soybean allergy (n=2). IgE reactive proteins were identified by means of mass spectrometry (MALDI/TOF, ESI MS/MS). Results: All the patients showed multiple sensitizations: a, a' and b chain of b- conglycinin, acidic chains of G1 and G2 glycinin, Gly m Bd30K and Gly m 4 were identified as the main IgE reactive proteins. Two patients presented an interesting unique pattern of immunoreactivity. In general, the IgE response between different patients was highly individual. Conclusions: This study confirms that the proteomic approach is a very powerful tool in food allergy studies. A high heterogeneity of the IgE response in soy allergy was found, and our data indicate that other allergens than those included in the official IUIS allergen list are the major allergens in soybean. Further development concerns pre-fractionation of soybean proteins by preparative IEF and subsequent use of micro-range pH gradient IPG Strips in order to identify also the lower abundant-high IgE reactive proteins.

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[74] - Xiang P, Baird LM, Jung R, Zeece MG, Markwell J, Sarath G. P39, a Novel Soybean Protein Allergen, Belongs to a Plant-Specific Protein Family and Is Present in Protein Storage Vacuoles. J Agric Food Chem 2008;56:2266-2272

Soybean lecithins are seeing increasing use in industry as an emulsifier and food additive. They are also a growing source of human food allergies, which arise principally from the proteins fractionating with the lecithin fraction during manufacture. A previous study (Gu, X.; Beardslee, T.; Zeece, M.; Sarath, G.; Markwwell, J. Int Arch. Allergy Immunol. 2001, 126, 218-225) identified several allergenic proteins in soybean lecithins and a soybean IgE-binding protein termed P39 was discovered. However, very little was known about this protein except that it was coded by the soybean genome. This paper investigates key biological and immunological properties of this potential soybean lecithin allergen. P39 is encoded by a multigene family in soybeans and in several other higher plants. The soybean P39-1 protein and its essentially indistinguishable homologue, P39-2, have been cloned and studied. These proteins and their homologues belong to a family of plant-specific proteins of unknown function. In soybeans, P39-1 is seed specific, and its transcript levels are highest in developing seeds and decline during seed maturation. In contrast, P39 protein was detectable only in the fully mature, dry seed. Subcellular fractionation revealed that P39 protein was strongly associated with oil bodies; however, immunolocalization indicated P39 was distributed in the matrix of the protein storage vacuoles, suggesting that association with oil bodies was an artifact arising from the extraction procedure. By the use of recombinant techniques it has also been documented that IgE-binding epitopes are present on several different portions of the P39-1 polypeptide

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[75] - Gu X, Beardslee TA, Zeece MG, Sarath G, Markwell JP. Identification of IgE-Binding Proteins in Soy Lecithin. Int Arch Allergy Immunol 2001;126:218-225

Background: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Methods: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. Results: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Conclusions: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

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[76] - Müller U, Weber W, Hoffmann A, Franke S, Lange R, Vieths S. Commercial soybean lecithins: a source of hidden allergens ? Eur Food Res Technol 1998;207:341-351

Soybeans are known to be allergenic for adults as well as for infants. Processed products derived from soybeans are used in a wide spectrum of foods, drugs and other industrial products. In particular, soybean lecithins are used as stabilizers and emulsifiers and may not be suspected as possible source of allergens. To test this hypothesis, six commercial soy lecithins were investigated for residual allergenicity and compared with extracts from raw and heat-treated soybeans. They were characterized, the protein content was determined by enzyme-linked immunosorbent assay (ELISA) and allergens were analyzed with specific IgE from patients' sera using the enzyme allergosorbent test (EAST), EAST inhibition and protein blotting followed by immunodetection. For further characterization a polyclonal antiserum directed against soybean extract and a monoclonal antibody (mAb?025) directed against the acidic subunit of the soybean storage protein glycinin were used. The EAST studies revealed that three of six sera from patients with allergy to soybeans contained IgE to four soy lecithins (Topcithin 50, Topcithin 300, Emulfluid FD 12, Epikuron 100 P), the same lecithins which were found to contain residual proteins. Two lecithins with a protein content of less than 20?ppb did not bind IgE. EAST inhibition showed that the allergens from soy lecithin were immunologically more closely related to allergens from heat-treated soybeans than to those from raw soybeans. Protein blotting and immunodetection of the protein extract from the lecithins resulted in various allergen bands between 14?kDa and 94?kDa. A heat-stable allergen of 39?kDa was recognized by the monoclonal antibody and thus identified as a subunit of glycinin. The results obtained were confirmed by a mediator release assay based on a rat basophil leukemia cell line. Lecithins that contained residual proteins caused a specific mediator release, suggesting that these products may induce allergic symptoms. Our results show that soybean lecithins are capable of introducing hidden allergens to processed foods and that the IgE binding potential corresponds to the total protein determined by ELISA. Furthermore, it appears to be possible that by monitoring the protein content soy lecithins can be applied which may be safe for the allergic consumer.

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[77] - Gijzen M, Miller SS, Kuflu K, Buzzell RI, Miki BL. Hydrophobic protein synthesized in the pod endocarp adheres to the seed surface. J Plant Physiol 1999;120:951-959

Abstract Soybean (Glycine max [L.] Merr.) hydrophobic protein (HPS) is an abundant seed constituent and a potentially hazardous allergen that causes asthma in persons allergic to soybean dust. By analyzing surface extracts of soybean seeds with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal microsequencing, we determined that large amounts of HPS are deposited on the seed surface. The quantity of HPS present varies among soybean cultivars and is more prevalent on dull-seeded phenotypes. We have also isolated cDNA clones encoding HPS and determined that the preprotein is translated with a membrane-spanning signal sequence and a short hydrophilic domain. Southern analysis indicated that multiple copies of the HPS gene are present in the soybean genome, and that the HPS gene structure is polymorphic among cultivars that differ in seed coat luster. The pattern of HPS gene expression, determined by in situ hybridization and RNA analysis, shows that HPS is synthesized in the endocarp of the inn er ovary wall and is deposited on the seed surface during development. This study demonstrates that a seed dust allergen is associated with the seed luster phenotype in soybean and that compositional properties of the seed surface may be altered by manipulating gene expression in the ovary wall

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[78] - Gonzalez R, Polo F, Zapatero L, Caravaca F, Carreira J. Purification and characterization of major inhalant allergens from soybean hulls. Clin Exp Allergy 1992;22:748-755

Proteins responsible for respiratory allergy to soybean have been purified from an extract of soybean hulls. The purification procedure combined size exclusion and reverse-phase HPLC. Two pure glycoproteins (S1 and S2) exhibiting IgE-binding ability, as demonstrated by immunoblotting and ELISA techniques, were obtained. Both proteins displayed low molecular weight values on SDS-PAGE (S1, 7.0 kD; S2 7.5 kD). Protein S1 showed charge microheterogeneity, rendering two bands at pH 6.1-6.2 on IEF, whereas S2 showed a single band at pH 6.8. Amino acid composition analyses revealed a strong homology between S1 and S2 and, as a characteristic feature, a high percentage of hydrophobic residues, mainly leucine and isoleucine. Concerning the allergenic activity, both proteins were recognized by the specific IgE from 95% of patients who suffered asthma attacks during the asthma outbreaks of 1987 and 1988 in Cartagena (Spain), caused by soybean dust. Besides, proteins S1 and S2 were able to, separately, inhibit up to 75% the binding of specific IgE to the whole extract. Moreover, purified proteins totally crossreacted, even though protein S2 seemed to be slightly more active in all the immunochemical techniques employed. Results presented allow us to conclude that both proteins are isoallergens and to name them as Gly m IA (protein S2) and Gly m IB (protein S1), according to the IUIS-allergen nomenclature system.

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[79] - Codina R, Lockey RF, Fernandez-Caldas E, Rama R. Purification and characterization of a soybean hull allergen responsible for the Barcelona asthma outbreaks. II. Purification and sequencing of the Gly m 2 allergen. Clin Exp Allergy 1997;27:424-430

BACKGROUND: A low MW allergen from soybean hull, Gly m 1, with two isoallergens, Gly m 1 A and Gly m 1 B, was associated with the asthma outbreaks that occurred in Cartagena, Spain. Using sera of asthmatic epidemic patients (AEP) from Barcelona, three main soybean hull allergens, two of them with MWs and pIs identical to those reported for Gly m 1 A and Gly m 1 B, were identified. OBJECTIVE: The purpose of this study was to purify and to study the N-terminal amino acid sequence of the third allergen, which has a MW of 8 kDa. METHOD: The purification procedure combined the double dialysis method and preparative isoelectofocusing (IEF). Specific IgE determination to the fractions obtained demonstrated three peaks, one of them corresponding to the 8 kDa allergen. The pooled fractions containing this allergen were studied by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-PAGE/Western blot and IEF/Western blot. Only a band with a MW of 8 kDa and a pI of 6 was obtained. Its allergenic activity was measured and it was demonstrated that the allergenicity of soybean hull correlates with the presence of the 8 kDa allergen. The N-terminal amino acid sequence of the first 20 amino acids, which was registered at the PIR Data Submission as the N-terminal partial sequence of Gly m 2, was determined according the Edman degradation method. RESULTS: Gly m 2 N-terminal amino acid sequence lacks homology with that reported for the allergen Gly m 1 but has a homology of 71% with a storage protein from cotyledon of Vigna radiata (cow pea) and 64% with a "disease response protein' from Pisum sativum (green pea). These results suggest that Gly m 2 in soybeans could protect against diseases which affect soybean plants. CONCLUSION: This study demonstrates the existence of another soybean hull allergen, Gly m 2, partially responsible for the soybean asthma outbreaks that occurred in Barcelona, Spain.

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[80] - Codina R, Oehling Jr AG, Lockey RF. Neoallergens in heated soybean hull. Int Arch Allergy Immunol 1998;117:120-125

BACKGROUND: During the process of harvest, transport and storage, microbial and mold contamination can raise the temperature of soybeans to 75 degreesC or higher. The purposes of this study were (1) to evaluate the allergenicity of fresh and stored soybean hulls and (2) to ascertain whether heat alters the allergenicity of stored soybean hulls. METHODS: Allergen extracts were prepared from (1) stored soybean hulls, (2) fresh soybean hulls and (3) stored soybean hulls heated to 37 degreesC (E1), 55 degreesC (E2) and 80 degreesC (E3) or kept at room temperature (E4) for 16 h. Individual serum from 68 soybean asthmatic (SA) subjects, 30 nonallergic subjects and two serum pools made from 4 SA sera and 4 sera from asthmatics not sensitive to soybean were studied. All sera and serum pools were assayed for content of specific IgE (radioallergosorbent test) and IgG4 (ELISA). The following additional studies were done for extracts E1-E4: (1) SDS-PAGE, (2) SDS-PAGE/Western blot for specific IgE and IgG4 using both serum pools, and (3) study of the effects of heat on inhibiting activity of the extracts prepared from stored soybean hulls using the pool of SA sera. RESULTS: Test results demonstrated a reduced binding of specific IgE and IgG4 to fresh soybean hull extract compared to stored soybean hull extract, and an increased binding for heated extracts (E1-E3) compared to unheated ones (E4). Moreover, there was an increase in potency for IgE and IgG4 bindings for the heated (E1-E3) compared to unheated (E4) extract, as measured by the amount of protein to produce 50% inhibition. Several protein bands with a molecular weight (MW) higher than 20 kD were absent from the SDS-PAGE for E3 but were present in E1, E2 and E4, and a new protein band (MW 15.3 kD) appeared for E3 only. Two new protein bands, with MWs of 15.3 and 10 kD, which bind specific IgE, were present on Western blot and one of the 3 main soybean hull allergens, probably Gly m 2, disappeared in E3. IgG4 Western blot showed similar results, but only the 10 kD protein band was present. CONCLUSION: The results demonstrate that soybean hull allergenicity is affected by heat, and suggest that the heat generated during storage and transport of soybeans could generate 2 new allergen determinants or increases in epitope exposure as a result of conformational changes. The significance of these new IgE and IgG4 binding proteins has yet to be determined.

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[81] - Bisson C, Holzhauser T, Natale M, Giuffrida MG, Fortunato D, Perono Garoffo L, et al. Application of proteomic tools reveals a highly individual IgE response in soybean-allergic patients. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1014

Background: The increased use of soybean products in processed foods and its use in the diet of infants with allergy to cow‚s milk, poses a potential threat to soybean-sensitive food-allergic individuals. Cross-reactivity to other leguminosae and to birch-pollen allergens has been demonstrated. So far, only four soy proteins (Gly m 1-Gly m 4) have been officially accepted as allergens by the IUIS allergen nomenclature subcommittee, of which two, Gly m 1&2, are respiratory allergens. In the framework of an EU project (FAREDAT), this study is aimed at allergen identification in soy using for the first time sera of European patients with confirmed soybean allergy, by a proteomic approach. Methods: Soybean protein extracts were separated by means of 2 dimensional PAGE: different pH gradients (3-10NL, 3-6, 5-8) IPG Strips were used in the first dimension step and 4-12% NuPage gradient gels in the second dimension. Proteins were subsequently transferred to nitrocellulose membranes. Immunolabeling was performed with individual positive DBPCFC subjects (n=5) or patients with a clear cut history of soybean allergy (n=2). IgE reactive proteins were identified by means of mass spectrometry (MALDI/TOF, ESI MS/MS). Results: All the patients showed multiple sensitizations: a, a' and b chain of b- conglycinin, acidic chains of G1 and G2 glycinin, Gly m Bd30K and Gly m 4 were identified as the main IgE reactive proteins. Two patients presented an interesting unique pattern of immunoreactivity. In general, the IgE response between different patients was highly individual. Conclusions: This study confirms that the proteomic approach is a very powerful tool in food allergy studies. A high heterogeneity of the IgE response in soy allergy was found, and our data indicate that other allergens than those included in the official IUIS allergen list are the major allergens in soybean. Further development concerns pre-fractionation of soybean proteins by preparative IEF and subsequent use of micro-range pH gradient IPG Strips in order to identify also the lower abundant-high IgE reactive proteins.

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[82] - Müller U, Weber W, Hoffmann A, Franke S, Lange R, Vieths S. Commercial soybean lecithins: a source of hidden allergens ? Eur Food Res Technol 1998;207:341-351

Soybeans are known to be allergenic for adults as well as for infants. Processed products derived from soybeans are used in a wide spectrum of foods, drugs and other industrial products. In particular, soybean lecithins are used as stabilizers and emulsifiers and may not be suspected as possible source of allergens. To test this hypothesis, six commercial soy lecithins were investigated for residual allergenicity and compared with extracts from raw and heat-treated soybeans. They were characterized, the protein content was determined by enzyme-linked immunosorbent assay (ELISA) and allergens were analyzed with specific IgE from patients' sera using the enzyme allergosorbent test (EAST), EAST inhibition and protein blotting followed by immunodetection. For further characterization a polyclonal antiserum directed against soybean extract and a monoclonal antibody (mAb?025) directed against the acidic subunit of the soybean storage protein glycinin were used. The EAST studies revealed that three of six sera from patients with allergy to soybeans contained IgE to four soy lecithins (Topcithin 50, Topcithin 300, Emulfluid FD 12, Epikuron 100 P), the same lecithins which were found to contain residual proteins. Two lecithins with a protein content of less than 20?ppb did not bind IgE. EAST inhibition showed that the allergens from soy lecithin were immunologically more closely related to allergens from heat-treated soybeans than to those from raw soybeans. Protein blotting and immunodetection of the protein extract from the lecithins resulted in various allergen bands between 14?kDa and 94?kDa. A heat-stable allergen of 39?kDa was recognized by the monoclonal antibody and thus identified as a subunit of glycinin. The results obtained were confirmed by a mediator release assay based on a rat basophil leukemia cell line. Lecithins that contained residual proteins caused a specific mediator release, suggesting that these products may induce allergic symptoms. Our results show that soybean lecithins are capable of introducing hidden allergens to processed foods and that the IgE binding potential corresponds to the total protein determined by ELISA. Furthermore, it appears to be possible that by monitoring the protein content soy lecithins can be applied which may be safe for the allergic consumer.

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[83] - Martin-Hernandez C, Benet S, Marvin-Guy LF. Characterization and quantification of proteins in lecithins. J Agric Food Chem 2005;53:8607-8613

Several methods for extraction and quantification of proteins from lecithins were compared. Extraction with hexane-2-propanol-water followed by amino acid analysis is the most suitable method for isolation and quantification of proteins from lecithins. The detection limit of the method is 15 mg protein/kg lecithin, and the quantification limit is 50 mg protein/kg. The relative repeatability limits for samples containing 0-500 and 500-5000 mg protein/kg sample were 12.6 and 7.5%, respectively. The protein recovery ranged between 101 and 123%. The protein content has been determined in different kinds of lecithins. The results were as follows: standard soy lecithins (between 232 and 1338 mg/kg), deoiled soy lecithin (342 mg/kg), phosphatydylcholine-enriched soy lecithins (not detectable and 163 mg/kg), sunflower lecithins (892 and 414 mg/kg), and egg lecithin (50 mg/kg). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of the standard soy and sunflower lecithins are very similar to those of soy flour. The protein profile of the egg lecithin shows several bands with a broad range of molecular masses. The molecular masses of the main proteins of soy lecithins and soy flour have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ranged from 10.5 to 52.2 kDa. Most of the major proteins from soy and sunflower lecithins identified by MALDI-MS and electrospray tandem MS belong to the 11S globulin fraction, which is one of the main fractions of soy and sunflower seeds. In addition, the seed maturation protein P34 from the 7S globulin fraction of soy proteins has also been identified in soy lecithins. This protein has been reported as the most allergenic protein in soybean

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[84] - Awazuhara H, Kawai H, Baba M, Matsui , Komiyama A. Antigenicity of the proteins in soy lecithin and soy oil in soybean allergy. Clin Exp Allergy 1998;28:1559-1564

BACKGROUND: Soy lecithin and soy oil are usually produced from the hexane extract of soybean. Some of the soybean proteins are included in the extract and are therefore present in small amounts in both soy lecithin and soy oil. The antigenicity of the proteins present in defatted soybean has been studied with respect to soybean allergy, but the antigenicity of those found in the extract is yet to be investigated. OBJECTIVE: The antigenicity of soy lecithin and soy oil proteins with regard to soybean allergy were investigated. METHODS: The proteins present in soy lecithin and soy oil were determined according to already established method and analysed by SDS-PAGE. The IgE- and IgG4-binding abilities of the soy lecithin proteins were investigated by immunoblotting with sera from 30 soybean-sensitive patients, including seven with a positive challenge test. Immunoblotting of soy oil proteins was performed with the sera from some of these patients. RESULTS: In 100 g of sample, the soy lecithin and soy oil contained 2.8 mg and 1.4-4.0 microg of proteins, respectively. The results of SDS-PAGE demonstrated the presence of only three proteins, with molecular weights of about 58-67 kDa in soy oil, and suggested that soy lecithin also contains these proteins. The soy lecithin also contained many proteins besides these. In the soy lecithin, the detection rate of only one protein, with a molecular weight of 31 kDa, by the serum IgE of patients was significantly different compared with controls (detection rate: 40%). The proteins with molecular weights of 58-67 kDa rarely bound to serum IgE. Only one of the patients who presented a positive challenge test had IgE antibodies to soy lecithin proteins. IgG4-binding proteins were found only rarely in soy lecithin. Neither the IgE nor the IgG4 present in the patients' sera reacted to any soy oil protein. CONCLUSION: Proteins present in soy lecithin and soy oil have little antigenicity with regard to soybean allergy

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[85] - Paschke A, Zunker K, Wigotzki M, Steinhart H. Determination of the IgE-binding activity of soy lecithin and refined and non-refined soybean oils. J Chromatogr B Biomed Appl 2001;756:249-254

In the present study refined and non-refined soybean oils as well as soy lecithins were investigated for residual allergenicity and compared with extracts from native soybeans. By means of immunoblotting and EAST inhibition experiments no IgE-binding activity was detectable in refined soybean oils, which is probably due to thermal treatment during the refining. The investigated non-refined oils and soy lecithins showed a residual IgE-binding activity. In addition in the lecithin extracts a new IgE-binding structure with a molecular mass of approximately 16 kDa was detectable.

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[86] - Moreno FJ. Gastrointestinal digestion of food allergens: Effect on their allergenicity. Biomed Pharmacother 2007;61:50-60

This paper reviews the in vitro digestion models developed to assess the stability digestion of food allergens, as well as the factors derived from the methodology and food structure that may affect the assay results. The adequacy of using the digestion stability of food allergens as a criterion for assessing potential allergenicity is also discussed. Data based on the traditional pepsin digestibility test in simulated gastric fluid are discussed in detail, with special attention to the influence of the pH and pepsin: allergen ratio in the pepsinolysis rate. This review points out the importance of using physiologically relevant in vitro digestion systems for evaluating digestibility of allergens. This would imply the sequential use of digestive enzymes in physiological concentrations, simulation of the stomach/small intestine environment (multi-phase models) with addition of surfactants such as phospholipids or bile salts, as well as the consideration of the gastrointestinal transit and the effect of the food matrices on the allergen digestion and subsequent absorption through the intestinal mucosa. In vitro gastrointestinal digestion protocols should be preferably combined with immunological assays in order to elucidate the role of large digestion-resistant fragments and the influence of the food matrix on the stimulation of the immune system.

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[87] - Untersmayr E, Bakos N, Schöll I, Kundi M, Roth-Walter F, Szalai K, et al. Anti-ulcer drugs promote IgE formation toward dietary antigens in adult patients. FASEB J 2005;19:656-658

Recently, we have demonstrated that anti-ulcer drugs, such as H2-receptor blockers and proton pump inhibitors, promote the development of immediate type food allergy toward digestion-labile proteins in mice. The aim of this study was to examine the allergological relevance of these findings in humans. In an observational cohort study, we screened 152 adult patients from a gastroenterological outpatient clinic with negative case histories for atopy or allergy, who were medicated with H2-receptor blockers or proton pump inhibitors for 3 months. IgE reactivities to food allergens before and after 3 months of anti-acid treatment were compared serologically. Ten percent of the patients showed a boost of preexisting IgE antibodies and 15% de novo IgE formation toward numerous digestion-labile dietary compounds, like milk, potato, celery, carrots, apple, orange, wheat, and rye flour. Thus, the relative risk to develop food-specific IgE after anti-acid therapy was 10.5 (95% confidence interval: 1.44-76.48). The long-term effect was evaluated 5 months after therapy. Food-specific IgE could still be measured in 6% of the patients, as well as significantly elevated serum concentrations of ST2, a Th2-specific marker. An unspecific boost during the pollen season could be excluded, as 50 untreated control patients revealed no changes in their IgE pattern. In line with our previous animal experiments, our data strongly suggest that anti-ulcer treatment primes the development of IgE toward dietary compounds in long-term acid-suppressed patients.

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[88] - Astwood JD, Leach JN, Fuchs RL. Stability of food allergens to digestion in vitro. Nat Biotechnol 1996;14:1269-1273

One of the concerns regarding the development of genetically modified foods is the introduction of allergenic molecules, predominantly proteins. Prospective testing for allergenic proteins from sources with no prior history of causing allergy is hampered by the absence of suitable techniques and models. Stability to digestion was tested as a candidate physicochemical property for use in distinguishing allergenic proteins from non-allergenic proteins. A simple model of gastric digestion was tested using some major food allergens (peanut Ara h2 and lectin, soybean [beta]-conglycinin subunits, SKTI and Gly m BD 30K, mustard Bra J IE, milk casein and [beta]-lactoglobulin, bovine serum albumin, and several egg proteins). Soybean [beta]-conglycinin was stable for 60 min; in comparison, a non-allergenic protein (spinach RUBISCO) was digested within 15 s. Data support the hypothesis that food allergens must be sufficiently stable to reach the intestinal mucosa where absorption and sensitization can occur. It is concluded that stability to digestion is an important parameter which distinguishes food allergens from non-allergens

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[89] - Roychaudhuri R, Sarath G, Zeece M, Markwell J. Stability of the allergenic soybean Kunitz trypsin inhibitor. Biochim Biophys Acta 2004;1699:207-212

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.

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[90] - Lin J, Shewry PR, Archer DB, Beyer K, Niggemann B, Haas H, et al. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach. Int Arch Allergy Immunol 2006;141:91-102

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens . METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system . RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system . CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

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[91] - Fu TJ, Abbott UR, Hatzos C. Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study. J Agric Food Chem 2002;50:7154-7160

Information on the comparative digestibility of food allergens and nonallergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. In this work, we compared the digestive stability of a number of food allergens and proteins of unproven allergenicity and examined whether allergens possess a higher stability than nonallergenic proteins of similar cellular functions, and whether there is a correlation between protein digestibility and allergenicity. The stability of groups of storage proteins, plant lectins, contractile proteins, and enzymes, both allergens and proteins with unproven allergenicity, in a standard simulated gastric fluid and a standard simulated intestinal fluid was measured. Food allergens were not necessarily more resistant to digestion than nonallergenic proteins. There was not a clear relationship between digestibility measured in vitro and protein allergenicity.

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[92] - Nielsen SS, Deshpande SS, Hermodson MA, Scott MP. Comparative digestibility of legume storage proteins. J Agric Food Chem 1988;36:896-902

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[93] - Astwood JD, Leach JN, Fuchs RL. Stability of food allergens to digestion in vitro. Nat Biotechnol 1996;14:1269-1273

One of the concerns regarding the development of genetically modified foods is the introduction of allergenic molecules, predominantly proteins. Prospective testing for allergenic proteins from sources with no prior history of causing allergy is hampered by the absence of suitable techniques and models. Stability to digestion was tested as a candidate physicochemical property for use in distinguishing allergenic proteins from non-allergenic proteins. A simple model of gastric digestion was tested using some major food allergens (peanut Ara h2 and lectin, soybean [beta]-conglycinin subunits, SKTI and Gly m BD 30K, mustard Bra J IE, milk casein and [beta]-lactoglobulin, bovine serum albumin, and several egg proteins). Soybean [beta]-conglycinin was stable for 60 min; in comparison, a non-allergenic protein (spinach RUBISCO) was digested within 15 s. Data support the hypothesis that food allergens must be sufficiently stable to reach the intestinal mucosa where absorption and sensitization can occur. It is concluded that stability to digestion is an important parameter which distinguishes food allergens from non-allergens

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[94] - Fu TJ, Abbott UR, Hatzos C. Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study. J Agric Food Chem 2002;50:7154-7160

Information on the comparative digestibility of food allergens and nonallergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. In this work, we compared the digestive stability of a number of food allergens and proteins of unproven allergenicity and examined whether allergens possess a higher stability than nonallergenic proteins of similar cellular functions, and whether there is a correlation between protein digestibility and allergenicity. The stability of groups of storage proteins, plant lectins, contractile proteins, and enzymes, both allergens and proteins with unproven allergenicity, in a standard simulated gastric fluid and a standard simulated intestinal fluid was measured. Food allergens were not necessarily more resistant to digestion than nonallergenic proteins. There was not a clear relationship between digestibility measured in vitro and protein allergenicity.

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[95] - Lin J, Shewry PR, Archer DB, Beyer K, Niggemann B, Haas H, et al. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach. Int Arch Allergy Immunol 2006;141:91-102

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens . METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system . RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system . CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

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[96] - van Boxtel EL, van den Broek LA, Koppelman SJ, Gruppen H. Legumin allergens from peanuts and soybeans: Effects of denaturation and aggregation on allergenicity. Mol Nutr Food Res 2008;52:674-682

Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92 degrees C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed.

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[97] - Astwood JD, Leach JN, Fuchs RL. Stability of food allergens to digestion in vitro. Nat Biotechnol 1996;14:1269-1273

One of the concerns regarding the development of genetically modified foods is the introduction of allergenic molecules, predominantly proteins. Prospective testing for allergenic proteins from sources with no prior history of causing allergy is hampered by the absence of suitable techniques and models. Stability to digestion was tested as a candidate physicochemical property for use in distinguishing allergenic proteins from non-allergenic proteins. A simple model of gastric digestion was tested using some major food allergens (peanut Ara h2 and lectin, soybean [beta]-conglycinin subunits, SKTI and Gly m BD 30K, mustard Bra J IE, milk casein and [beta]-lactoglobulin, bovine serum albumin, and several egg proteins). Soybean [beta]-conglycinin was stable for 60 min; in comparison, a non-allergenic protein (spinach RUBISCO) was digested within 15 s. Data support the hypothesis that food allergens must be sufficiently stable to reach the intestinal mucosa where absorption and sensitization can occur. It is concluded that stability to digestion is an important parameter which distinguishes food allergens from non-allergens

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[98] - Mills EN, Marigheto NA, Wellner N, Fairhurst SA, Jenkins JA, Mann R, et al. Thermally induced structural changes in glycinin, the 11S globulin of soya bean (Glycine max)--an in situ spectroscopic study. Biochim Biophys Acta 2003;1648:105-114

The thermal denaturation behaviour of glycinin solutions has been studied in situ as a function of ionic strength using various spectroscopic methods. Changes in secondary structure occurred at temperatures above 60 degrees C, well before the onset of gelation. Even after heating to 95 degrees C, much of the native beta-sheet structure of glycinin was retained, as indicated by the amide I peak maximum at 1635 cm(-1) in the Fourier transformed infrared (FT-IR) spectrum. This was accompanied by an increase in the 1625 cm(-1) band, indicative of the formation of intermolecular beta-sheet associated with protein aggregation. Nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of highly mobile regions in glycinin comprising predominantly of Gln and Glu residues, corresponding to mobile regions previously identified by crystallographic studies. There was also evidence of a hydrogen-bonded structure within this mobile region, which may correspond to an alpha-helical region from Pro(256) to (or just before) Pro(269) in proglycinin. This structure disappeared at 95 degrees C, when heat-set gel formation occurred, as indicated by a sudden broadening and weakening of the NMR signal. Otherwise the NMR spectrum changed little during heating, emphasising the remarkable thermal stability of glycinin. It is proposed that during heating the core beta-barrel structure remains intact, but that the interface between the beta-domains melts, revealing hydrophobic faces which may then form new structures in a gel-network. As Cys(45), which forms the disulfide with Cys(12) linking the acidic and basic polypeptides, is found in this interface, such a rearrangement of the individual beta-domains could be accompanied by cleavage of this disulfide bond, as is observed experimentally. Such information contributes to our understanding the aggregative behaviour of proteins, and hence develops knowledge-based strategies for controlling and manipulating it.

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[99] - Mills EN, Huang L, Noel TR, Gunning AP, Morris VJ. Formation of thermally induced aggregates of the soya globulin beta-conglycinin. Biochim Biophys Acta 2001;1547:339-350

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

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[100] - Besler M, Helm RM, Ogawa T. Allergen data collection: soybean (Glycine max). Internet Symp Food Allergens 2000;2(suppl. 3):1-35

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[101] - Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A, et al. Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004;113:148-154

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[102] - Roychaudhuri R, Sarath G, Zeece M, Markwell J. Stability of the allergenic soybean Kunitz trypsin inhibitor. Biochim Biophys Acta 2004;1699:207-212

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.

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[103] - Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A, et al. Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004;113:148-154

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[104] - Wilson S, Blaschek K, de Mejia E. Allergenic proteins in soybean: processing and reduction of P34 allergenicity. Nutr Rev 2005;63:47-58

Soybean ranks among the "big 8" of the most allergenic foods, and with increasing consumption of soybean products, the incidence of soy-caused allergies is expected to escalate. Soybean and its derivatives have become ubiquitous in vegetarian and many meat-based food products, and as a result, dietary avoidance has become difficult. However, soybeans can be manipulated in a variety of ways to alter their allergenicity. Several studies have focused on reducing the allergenicity of soybeans by changing the structure of the immunodominant allergen P34 using food processing, agronomic, or genetic manipulation techniques. A review of the literature pertaining to these studies is presented here.

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[105] - Bando N, Tsuji H, Hiemori M, Yoshizumi K, Yamanishi R, Kimoto M, et al. Quantitative analysis of Gly m Bd 28K in soybean products by a sandwich enzyme-linked immunosorbent assay. J Nutr Sci Vitaminol (Tokyo) 1998;44:655-664

A sandwich enzyme-linked immunosorbent assay for the soybean allergen, which consists of a monoclonal antibody (D4) as the fixing (first) antibody and another peroxidase-conjugated monoclonal antibody (C5) as the second, has been developed. Both D4 and C5 monoclonal antibodies strongly bound to the guanidine/HCl-denatured allergen, Gly m Bd 28K. Therefore the samples used in the present experiment were extracted with sodium phosphate buffer (pH 8.0) containing 6 M guanidine and 10 mM 2-mercaptoethanol, then completely dialyzed against phosphate-buffered saline (PBS). The dialyzed samples were subjected to the assay. Various soybean products were observed to contain the allergen at high concentrations, such as soybean protein isolate (SPI), tofu, kori-dofu, and yuba, but its content in soy milk and abura-age were found to be low. In fermented products such as natto, soy sauce, and miso, and even in the processed foods with soybean protein isolate (SPI), the allergen was not detected. These results were also confirmed by an immunoblotting technique with D4.

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[106] - Franck P, Moneret-Vautrin DA, Dousset B, Kanny G, Nabet P, Guénard-Bilbaut L, et al. The allergenicity of soybean-based products is modified by food technologies. Int Arch Allergy Immunol 2002;128:212-219

Background: Numerous products based on soybean are available and various food technologies are applied for their production. The allergenicity of natural soybean may be modified by these treatments. Objectives: To compare the allergenicity of native soybean proteins with those of soy milk and texturized protein products. To show additional allergens. Methods: Three commercial products and two infant formulas were studied: Soybean flour, soy milk, texturized soy proteins, two infant formulas; the first containing total proteins and the second containing a soy protein hydrolysate. Sera from 9 patients allergic to soy protein were tested by immunoblotting (IB). IB inhibition was achieved by incubating sera with protein extract from soybean flour. Results: The SDS-PAGE profile of soybean flour protein and soy milk showed a difference in the proportions of the various protein fractions, with a higher concentration of 37-kD protein in flour and 33-kD protein in milk. Infant formula 1 contained proteins with a molecular weight below 28 kD. The texturized extract contained high proportions of 31- to 34- and 38-kD proteins. Immunoblotting revealed a lack of allergenicity in infant formula. Sera recognizing the 38- and 50-kD proteins in texturized soy protein also recognized the 37- and 49-kD proteins in soybean flour and in soy milk, suggesting a protein glycation by texturization processes. The 30- to 34-kD band in texturized proteins was devoid of any allergenicity. This study seems to indicate that the 30-kD allergen (Gly m Bd 30) disappears during the production of texturized soy protein. Conclusion: All technologies applied to soybean-based products induce striking variation in the protein profile and allergenicity. Texturized protein could lack the major allergen Gly m Bd 30. Further studies or texturization might generate modified technologies in order to create hypoallergenic texturized proteins.

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[107] - Shreffler WG, Sampson HA, Sicherer SH. Hypoallergenicity of soy sauce. J Allergy Clin Immunol 2001;107(3 Suppl.):S140

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[108] - Franck P, Moneret-Vautrin DA, Dousset B, Kanny G, Nabet P, Guénard-Bilbaut L, et al. The allergenicity of soybean-based products is modified by food technologies. Int Arch Allergy Immunol 2002;128:212-219

Background: Numerous products based on soybean are available and various food technologies are applied for their production. The allergenicity of natural soybean may be modified by these treatments. Objectives: To compare the allergenicity of native soybean proteins with those of soy milk and texturized protein products. To show additional allergens. Methods: Three commercial products and two infant formulas were studied: Soybean flour, soy milk, texturized soy proteins, two infant formulas; the first containing total proteins and the second containing a soy protein hydrolysate. Sera from 9 patients allergic to soy protein were tested by immunoblotting (IB). IB inhibition was achieved by incubating sera with protein extract from soybean flour. Results: The SDS-PAGE profile of soybean flour protein and soy milk showed a difference in the proportions of the various protein fractions, with a higher concentration of 37-kD protein in flour and 33-kD protein in milk. Infant formula 1 contained proteins with a molecular weight below 28 kD. The texturized extract contained high proportions of 31- to 34- and 38-kD proteins. Immunoblotting revealed a lack of allergenicity in infant formula. Sera recognizing the 38- and 50-kD proteins in texturized soy protein also recognized the 37- and 49-kD proteins in soybean flour and in soy milk, suggesting a protein glycation by texturization processes. The 30- to 34-kD band in texturized proteins was devoid of any allergenicity. This study seems to indicate that the 30-kD allergen (Gly m Bd 30) disappears during the production of texturized soy protein. Conclusion: All technologies applied to soybean-based products induce striking variation in the protein profile and allergenicity. Texturized protein could lack the major allergen Gly m Bd 30. Further studies or texturization might generate modified technologies in order to create hypoallergenic texturized proteins.

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[109] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[110] - Vigan M, Girardin P, Rame JM, Pelletier F. Bronchospasme au tonyu: découvertes déconcertantes à propos d'un cas. Rev Fr Allergol Immunol Clin 2005;45:276-277

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[111] - Klemola T, Vanto T, Juntunen-Backman K, Kalimo K, Korpela R, Varjonen E. Allergy to soy formula and to extensively hydrolyzed whey formula in infants with cow's milk allergy: a prospective, randomized study with a follow-up to the age of 2 years. J Pediatr 2002;140:219-224

OBJECTIVES: We conducted a prospective, randomized study to evaluate the cumulative incidence of allergy or other adverse reactions to soy formula and to extensively hydrolyzed formula up to the age of 2 years in infants with confirmed cow's milk allergy. STUDY DESIGN: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula. If it was suspected that the formula caused symptoms, a double-blind, placebo-controlled challenge (DBPCFC) with the formula was performed. The children were followed to the age of 2 years, and soy-specific immunoglobulin E antibodies were measured at the time of diagnosis and at the ages of 1 and 2 years. RESULTS: An adverse reaction to the formula was confirmed by challenge in 8 patients (10%; 95% confidence interval, 4.4%-18.8%) randomly assigned to soy formula and in 2 patients (2.2%; 95% confidence interval, 0.3% to 7.8%) randomly assigned to extensively hydrolyzed formula. Adverse reactions to soy were similar in IgE-associated and non-IgE-associated cow's milk allergy (11% and 9%, respectively). IgE to soy was detected in only 2 infants with an adverse reaction to soy. Adverse reactions to soy formula were more common in younger (<6 months) than in older (6 to 12 months) infants (5 of 20 vs 3 of 60, respectively, P =.01). CONCLUSIONS: Soy formula was well tolerated by most infants with IgE-associated and non-IgE-associated cow's milk allergy. Development of IgE-associated allergy to soy was rare. Soy formula can be recommended as a first-choice alternative for infants >or=6 months of age with cow's milk allergy.

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[112] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[113] - Maria Y, Leduc V. Complexité du diagnostic de l’allergie alimentaire aux dérivés de soja. Rev Fr Allergol Immunol Clin 2007;47:284

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[114] - Hefle SL. Impact of processing on food allergens. Adv Exp Med Biol 1999;459:107-119

In general, allergenic foods are resistant to processes commonly used in food manufacturing. Nearly all the causative proteins (allergens) retain their allergenicity after treatment by heat and/or proteolysis. Notable exceptions exist; for example, the allergenicity of many fresh fruits and vegetables is decreased or removed by relatively mild processes such as gentle heating or mashing. The use of proteolytic enzymes to remove allergenicity is successfully used in the production of hypoallergenic infant formulas, but this approach with other allergenic foods has resulted in only limited success. Processing effects can result in decreased or complete removal of allergenic qualities of a food, such as the removal of proteins in oilseed processing, which renders the oils hypoallergenic and safe for consumption by allergic individuals. This discussion will address the different allergenic foods and processes which can affect or decrease their allergenicity. [References: 101]

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[115] - Liu K, Hsieh FH. Protein-Protein Interactions during High-Moisture Extrusion for Fibrous Meat Analogues and Comparison of Protein Solubility Methods Using Different Solvent Systems. J Agric Food Chem 2008;56:2681-2687

Soy protein, mixed with gluten and starch, was extruded into fibrous meat analogues under high-moisture and high-temperature conditions. The protein solubility of samples collected at different extruder zones and extrudates made with different moistures was determined by 11 extraction solutions consisting of 6 selective reagents and their combinations: phosphate salts, urea, DTT, thiourea, Triton X-100, and CHAPS. Protein solubility by most extractants showed decreasing patterns as the material passed through the extruder, but the solution containing all 6 reagents, known as isoelectric focus (IEF) buffer, solubilized the highest levels and equal amounts of proteins in all samples, indicating that there are no other covalent bonds involved besides disulfide bonds. With regard to relative importance between disulfide bonds and non-covalent interactions, different conclusions could be made from protein solubility patterns, depending on the type of extracting systems and a baseline used for comparison. The observation points out pitfalls and limitation of current protein solubility methodology and explains why controversy exists in the literature. Using the IEF buffer system with omission of one or more selective reagents is considered to be the right methodology to conduct protein solubility study and thus recommended. Results obtained with this system indicate that disulfide bonding plays a more important role than non-covalent bonds in not only holding the rigid structure of extrudates but also forming fibrous texture. The sharpest decrease in protein solubility occurred when the mix passed through the intermediate section of the extruder barrel, indicating formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die, the chemical nature of the product did not change significantly.

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[116] - Franck P, Moneret-Vautrin DA, Dousset B, Kanny G, Nabet P, Guénard-Bilbaut L, et al. The allergenicity of soybean-based products is modified by food technologies. Int Arch Allergy Immunol 2002;128:212-219

Background: Numerous products based on soybean are available and various food technologies are applied for their production. The allergenicity of natural soybean may be modified by these treatments. Objectives: To compare the allergenicity of native soybean proteins with those of soy milk and texturized protein products. To show additional allergens. Methods: Three commercial products and two infant formulas were studied: Soybean flour, soy milk, texturized soy proteins, two infant formulas; the first containing total proteins and the second containing a soy protein hydrolysate. Sera from 9 patients allergic to soy protein were tested by immunoblotting (IB). IB inhibition was achieved by incubating sera with protein extract from soybean flour. Results: The SDS-PAGE profile of soybean flour protein and soy milk showed a difference in the proportions of the various protein fractions, with a higher concentration of 37-kD protein in flour and 33-kD protein in milk. Infant formula 1 contained proteins with a molecular weight below 28 kD. The texturized extract contained high proportions of 31- to 34- and 38-kD proteins. Immunoblotting revealed a lack of allergenicity in infant formula. Sera recognizing the 38- and 50-kD proteins in texturized soy protein also recognized the 37- and 49-kD proteins in soybean flour and in soy milk, suggesting a protein glycation by texturization processes. The 30- to 34-kD band in texturized proteins was devoid of any allergenicity. This study seems to indicate that the 30-kD allergen (Gly m Bd 30) disappears during the production of texturized soy protein. Conclusion: All technologies applied to soybean-based products induce striking variation in the protein profile and allergenicity. Texturized protein could lack the major allergen Gly m Bd 30. Further studies or texturization might generate modified technologies in order to create hypoallergenic texturized proteins.

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[117] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[118] - Frias J, Song YS, Martínez-Villaluenga C, De Mejia EG, Vidal-Valverde C. Immunoreactivity and Amino Acid Content of Fermented Soybean Products. J Agric Food Chem 2008;56:99-105

Food allergy has become a public health problem that continues to challenge both the public and the food industry. The objective of this research was the detection and quantification of the major human allergenic soy proteins and to study the reduction in immunoreactivity and improvement of amino acid content after fermentation of soybean flour. Fermentation was carried out in the solid state of cracked seeds inoculated with Aspergillus oryzae, Rhizopus oryzae, and Bacillus subtilis and in the liquid state of milled soybean flours fermented naturally by microorganisms present only in the seeds or by inoculation with Lactobacillus plantarum. ELISA and Western blot were used to quantify IgE antibody response, and HPLC was used to identify and quantify total amino acids. L. plantarum fermented soy flour showed the highest reduction in IgE immunoreactivity (96-99%) depending upon the sensitivity of the plasma used. Among the solid fermented products, the lowest reduction in immunoreactivity was obtained when mold strains, R. oryzae and A. oryzae, were used (66 and 68%, respectively, for human plasma 97.5 kUA/L). Among the solid fermented products, those inoculated with B. subtilis yielded a 81 and 86% reduction in immunoreactivity against both human plasma 97.5 IgE kUA/L and human pooled plasma samples, respectively. When soybean was subjected to liquid fermentation, most of the total amino acids increased significantly ( p ↩

[119] - Hefle SL, Lambrecht DM, Nordlee JA. Soy Sauce Retains Allergenicity Through the Fermentation/ Production Process. J Allergy Clin Immunol 2005;115(2 suppl.):S32

RATIONALE: Soy allergy is one of the most prominent allergies in the worldwide population. The vast majority of soy sauces are produced through the fermentation of soy and wheat. Some soy sauce manufacturers tell finished food product processors (and also soy-and wheat-allergic patients who contact them) that the fermentation process destroys the allergenicity of their soy and wheat fermentation ingredients. This has not been proven to be the case by scientific experimentation, so the risk of reaction from soy sauce ingestion among soy-allergic and wheat-allergic/ celiac patients is unknown METHODS: Ten soy sauces were evaluated using three soy-specific animal IgG-based ELISAS (two of which are commercially available), by soy polymerase chain reaction (PCR) analysis, and also by RAST inhibition using sera from soy-allergic subjects RESULTS: The soy IgG-based ELISA tests and PCR tests showed no detectable residues of soy protein or soy DNA in the ten sauces. However, in RAST inhibition (using soy flour as the solid phase), some soy sauces contained 10-30% residual activity CONCLUSIONS: Soy sauces made by fermentation of soy protein can retain some of their soy allergenicity (10-30% that of soy flour) through the fermentation/production process. Soy IgG-ELISA and PCR analyses do not detect the remaining allergenic residues in soy sauce. Therefore, results from these types of tests should not be used by soy sauce suppliers nor finished food manufacturers to indicate that soy sauce is devoid of allergenic residues and safe for soy-allergic individuals to consume. Soyallergic patients should continue to be counseled to avoid soy sauce

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[120] - Shreffler WG, Sampson HA, Sicherer SH. Hypoallergenicity of soy sauce. J Allergy Clin Immunol 2001;107(3 Suppl.):S140

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[121] - Palm M, Moneret-Vautrin DA, Kanny G, Denery-Papini S, Frémont S. Food allergy to egg and soy lecithins. Allergy 1999;54:1116-1117

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[122] - Paschke A, Zunker K, Wigotzki M, Steinhart H. Determination of the IgE-binding activity of soy lecithin and refined and non-refined soybean oils. J Chromatogr B Biomed Appl 2001;756:249-254

In the present study refined and non-refined soybean oils as well as soy lecithins were investigated for residual allergenicity and compared with extracts from native soybeans. By means of immunoblotting and EAST inhibition experiments no IgE-binding activity was detectable in refined soybean oils, which is probably due to thermal treatment during the refining. The investigated non-refined oils and soy lecithins showed a residual IgE-binding activity. In addition in the lecithin extracts a new IgE-binding structure with a molecular mass of approximately 16 kDa was detectable.

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[123] - Taylor SL, Nordlee JA, Sicherer SH, Sampson HA, Levy MB, Steinman H, et al. Soybean Oil Is Not Allergenic to Soybean-Allergic Individuals. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°296

Rationale Soybean-allergic individuals may be instructed by clinicians to avoid all foods derived from soybeans including soybean oil. However, highly refined soybean oil contains extremely low levels of protein. Previous work suggested that soybean oil was not allergenic, but the number of subjects challenged was small and the protein content of the oil was unknown. These deficiencies are rectified in this study. The study objective was to determine the allergenicity of highly refined soybean oil in soybean- allergic individuals. Method s : Soybean-allergic subjects were selected by convincing history, positive skin test and positive radioallergosorbent test (RAST). Challenge materials consisted of a mixture of 4 soybean oils with the highest protein level from a group of 30 highly refined oils obtained from 30 different worldwide processors. Subjects consumed increasing doses of 1, 5, and 10 grams soybean oil (test material) and canola oil (control material) in a double-blind placebo controlled food challenge. Prepared oatmeal was the challenge vehicle. Result s : Twenty-eight soybean allergic subjects were challenged. No untoward reactions were encountered to either soybean or canola oils. No reaction with this number of subjects indicates with 95% certainty that 89.85% of soybean allergic individuals would not react to this soybean oil. Conclusions : The lack of reactions to commercially available soybean oil supports the previous claim that hot solvent-extracted, bleached and deodorized soybean oil is not allergenic for soy-allergic individuals and avoidance of soybean oil of the type used in this study is unwarranted. The allergenicity of cold-pressed or expeller-pressed soybean oil remains uncertain.

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[124] - Hefle SL, Taylor SL. Allergenicity of edible oils. Food Technol 1999;53:62-68

Allergenicity studies on vegetable oils derived from commonly allergenic foods are described. Aspects considered include: allergenic foods; mechanisms of food allergies; difficulties in avoiding allergenic food proteins due to limited consumer knowledge, inadequate food labelling or poor cleaning practices in food preparation establishments; potentially allergenic oils (peanut (groundnut), soybean, tree nut, sunflower, sesame, cottonseed and (rarely) corn and coconut oils); methods of oil processing (solvent extraction/refining or cold pressing); labelling of oils; lower levels of allergenic proteins in solvent extracted/refined than cold pressed oils and levels required to cause allergic reactions; diagnostic tests for allergenicity (oral challenge, skin testing, radioallergosorbent assay, basophil histamine release, Prausnitz-Kustner test and immunoblotting); and studies on allergenicity of refined and cold pressed soybean, peanut, tree nut, cottonseed, sesame and sunflower oils (providing information concerning experimental techniques employed, shortcomings of the studies, levels of allergenic proteins present in the oils and oil allergenicity). It is concluded that refined oils contain virtually no protein and pose little or no hazard to allergic individuals; crude or cold pressed oils are less commonly available and may contain small amounts of protein and pose some risk

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[125] - Morisset M, Lee T, Codreanu F, Cordebar V, Fremont S, Guenard L, et al. Allergy to an Amino-Acid Formula in Infants: Residual Soy Allergens in Soybean Oil are Incriminated. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°157

RATIONALE: Treatment of allergy to cow milk (CM) proteins comprises avoidance and replacement of CM with various infant formulas. However, clinical manifestations have been associated with CM protein hydrolysates (CMPH). In this situation, in the frame of multiple food allergies (MFA), substitution of CMPH with an amino-acid-based formula (AAF) is recommended METHODS: Seven infants allergic to AAF have been referred for a MFA. In all cases, the breast-feeding was stopped; CMPH and/or soy protein and pork collagen hydrolysates were replaced by Neocate(r) (SHS International), an AAF containing a soy lipid emulsion. As there was no clinical improvement, prick-tests (PT) and atopy patch-test (APT) to soy or Neocate(r) were carried out RESULTS: PIPs were positive in 2/7 patients. APTs to soy and APTs to Neocate(r) were positive respectively for 5/5 and 3/5 infants. In one case, an oral challenge with the soy lipid emulsion was performed and was positive Neocate(r) was replaced by Neocate Advance(r), a soy-free AAF and the symptoms improved within 2-4 weeks in all children Proteins of the Neocate emulsion were extracted. The amount was measured and their molecular mass estimated by SDS-PAGE. A western blot was conducted using the serum of a soy allergic patient . The western blot showed 2 bands (56-66 kDa) CONCLUSIONS: AAFs have represented a great advance. However, the risk of allergy to traces of vegetable proteins in certain oils has been confirmed yet again. Food industries must be made aware of the need to detect allergen traces in foods for infants at high risk of atopy.

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[126] - Rozenfeld P, Docena GH, Añon MC, Fossati CA. Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk. Clin Exp Immunol 2002;130:49-58

Soy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific monoclonal antibodies were used in immunoblotting and competitive ELISA studies to identify a 30-kD component from soybean that cross-reacts with cow's milk caseins. Its IgE binding capacity was tested by EAST, employing sera from cow's milk allergic patients, not previously exposed to soy proteins. The 30 kD protein was isolated and partially sequenced. It is constituted by two polypeptides (A5 and B3) linked by a disulphide bond. The protein's capacity to bind to the different antibodies relies on the B3 poly-peptide. These results indicate that soy-based formula, which contains the A5-B3 glycinin molecule, could be involved in allergic reactions observed in cow's milk allergic patients exposed to soy-containing foods.

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[127] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[128] - Maria Y, Leduc V. Complexité du diagnostic de l’allergie alimentaire aux dérivés de soja. Rev Fr Allergol Immunol Clin 2007;47:284

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[129] - Ballmer-Weber BK, Holzhauser T, Scibilia J, Mittag D, Zisa G, Ortolani C, et al. Clinical characteristics of soybean allergy in Europe: A double-blind, placebo-controlled food challenge study. J Allergy Clin Immunol 2007;119:1489-1496

BACKGROUND: Soybean is a relevant allergenic food, but little is known about individual threshold doses in soy allergy . OBJECTIVE: We sought to determine the clinical characteristics of soy allergy in Europe, including a dose-response curve . METHODS: Patients with a history of soy allergy underwent a titrated, double-blind, placebo-controlled food challenge. A statistical model was used to calculate the risk of allergic consumers to experience an allergic reaction to soy. Sera were analyzed for specific IgE to soy, peanut, Bet v 1, and Gly m 4 . RESULTS: All patients but one responded primarily with subjective symptoms to the challenge followed by objective symptoms in 11 subjects, ranging from rhinitis up to a decrease in blood pressure. Cumulative threshold doses for allergic reactions ranged from 10 mg to 50 g for subjective symptoms and from 454 mg to 50 g for objective symptoms. The pattern of IgE reactivity against proteins with molecular weights of between approximately 10 and 70 kd was highly individual among the patients and did not correlate with the severity of symptoms . CONCLUSIONS: When data are fitted by using a normal distribution statistical model, they predict that 1% of patients with soy allergy would react subjectively and objectively with 0.21 and 37.2 mg of soy protein, respectively. CLINICAL IMPLICATIONS: Both the clinical and immunologic basis of soy allergy in Europe are highly complex, which affects the diagnosis of soy allergy and the advice given to patients with soy allergy in regard to risk management.

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[130] - Wilson IBH, Zeleny R, Kolarich D, Staudacher E, Stroop CJM, Kamerling JP, et al. Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha-1,3-linked fucose and xylose substitutions. Glycobiology 2001;11:261-274

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.

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[131] - Kimura Y, Ohno A, Takagi S. Structural analysis of N-glycans of storage glycoproteins in soybean (Glycine max. L) seed. Biosci Biotechnol Biochem 1997;61:1866-1871

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz 1H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man3Xyl1GlcNac2, Man3Fuc1Xyl1GlcNac2; 3.4%).

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[132] - Hiemori M, Bando N, Ogawa T, Shimada H, Tsuji H, Yamanishi R, et al. Occurrence of IgE antibody-recognizing N-linked glycan moiety of a soybean allergen, Gly m Bd 28K. Int Arch Allergy Immunol 2000;122:238-245

It has been reported that N-linked glycan moieties of glycoproteins function as IgE-reactive determinants. Gly m Bd 28K, a soybean allergen, was a glycoprotein with glycan moieties, which are supposed to be the Man(3)GlcNAc(2) backbone with the beta1-->2 xylose and alpha1-->3 fucose branches. The purpose of the present study was to examine the IgE-binding ability of the glycan moiety of Gly m Bd 28K in the binding reaction with patients' sera. METHODS: A peptide containing the glycan moiety was prepared from Gly m Bd 28K by digestion with lysyl endopeptidase. The binding site of the glycan moiety was determined by amino acid sequence analyses. The glycan moiety of the allergen was characterized using anti-horseradish peroxidase antibody (anti-HRP) recognizing the N-linked glycan moieties of glycoproteins. The binding of patients' IgE antibodies with their glycan moiety was examined by an immunostaining technique using the glycopeptide and its deglycosylated peptide derived from Gly m Bd 28K. RESULTS: The binding site of the glycan moiety in Gly m Bd 28K was shown to be its Asn20 residue. Gly m Bd 28K did react with anti-HRP and the sera of soybean-sensitive patients, but the binding of IgE antibodies was inhibited by the preincubation with anti-HRP. Moreover, the glycopeptide also reacted with the sera of soybean-sensitive patients, but its deglycosylated peptide did not react with any IgE antibodies of patients' sera. CONCLUSIONS: The specific IgE antibodies recognizing the N-linked glycan moieties of Gly m Bd 28K and other glycoproteins with homologous glycan moieties occur in the sera of soybean-sensitive patients. It was indicated that the N-linked glycan moieties such as that of Gly m Bd 28K may be one of the common IgE-reactive determinants distributed in various plant food proteins.

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[133] - Holzhauser T, Petrovskaya O, Kuehne Y, Wangorsch A, Ballmer-Weber B, Bindslev-Jensen C, et al. Allergenicity of soybean beta-conglycinin versus Gly m Bd 30k in patients with confirmed soybean allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°513

Background: Soybean b-conglycinin and Gly m Bd 30k have been described as IgE-binding proteins, the latter one as putative major soybean allergen. We aimed at characterising both soybean proteins in regard to their relevance in a European patient group with confirmed soybean allergy. Methods: Twenty-five adults were included into the study on the basis of a positive DBPCFC or conclusive history of anaphylaxis to soy. Twenty-two children were included upon positive DBPCFC or open oral challenge. Natural b-conglycinin subunits (a, a', b) were extracted from soybean, recombinant subunits expressed in E.coli, and both purified by continuous-elution electrophoresis. Natural Gly m Bd 30k was enriched by an oleosin fractionation of soybean and the recombinant homologue expressed as His-tagged fusion protein in E.coli and purified by IMAC. Protein identity was proven by N-terminal sequencing and/or peptide mass fingerprinting. IgE-reactivity of the purified soy proteins was investigated by IgE-immunoblotting and/or IgE-ELISA with patients' sera. ELISA- and EAST-inhibition, and mediator release from passively sensitized humanized rat basophilic leukaemia cells was performed with selected patients' sera. Results: b-conglycinin was IgE-reactive in 54 % (12/22) of the children and 20 % (5/25) of the adults. By contrast, enriched natural Gly m Bd 30k bound IgE from less than 50 % of patients‚ sera in immunoblotting, and rGly m Bd 30k did not show any IgE-reactivity. In ELISA-inhibition, IgE-binding to b- conglycinin was fully inhibited by soybean extract and itself. IgE-reactivity to b-conglycinin was almost fully inhibited by peanut extract and purified Ara h 1 in a soybean and peanut allergic patient, however hardly inhibited in a patient allergic to soybean but not to peanut. Similar results were obtained in EAST-inhibition with soybean extract on the solid surface. Specific mediator release confirmed the cross-linking properties of b-conglycinin. Conclusion: In Europe, soybean b-conglycinin is a major allergen for soy-allergic children and an important minor allergen in adults, whereas the relevance of Gly m Bd 30k in IgE-mediated soybean allergy remains unclear and demands further investigation. In some patients cross-reactivity between peanut and soybean is limited.

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[134] - Ofori-Anti AO, Pramod SN, Goodman RE. In vitro IgE Binding to Cross-Reactive Carbohydrate Determinants or Peptide Epitopes in Legume Extracts ? J Allergy Clin Immunol 2008;121:S241

RATIONALE: Many studies have reported in vitro IgE cross-reactivity among seed proteins of various legumes, often without clinical histories of reactions. Few have considered the role of cross-reactive carbohydrate determinants (CCD). IgE from some subjects bound proteins in easily recognized legumes, without complaint, suggesting IgE to CCD. METHODS: Sera from individuals reporting food allergy to peanut and/ or soybean were tested for specific IgE to extracts of peanut, soybean, lupin, chickpea, lentil, beans (kidney, navy, lima and fava), blackgram, pigeonpea and cowpea (blackeyed pea), rice and wheat using direct binding immunoblots and ELISA. Inhibition was performed with native legume (peanut and navy or kidney bean) and wheat extracts, bromelain, horseradish peroxidase (HRP) and extensively digested (proteinase K and pepsin) proteins. RESULTS: IgE from two of five subjects bound specifically to a few proteins in some legumes, but inhibition with CCD (HRP, bromelain and digested wheat and legume extracts) was complete, while for others binding was peptide specific, or due to a mixture of peptide and CCD. HRP is expensive, and bromelain is a crude extract. Inhibition with irrelevant and relevant protease digested extracts provides a better alternative although Proteinase K digestion is insufficient to eliminate peptide inhibition. CONCLUSIONS: Evaluating food allergic subjects by in vitro IgE testing may be helpful, but has the potential to identify species or proteins due to IgE binding to CCD, which are not likely to cause allergic symptoms. Specific inhibition with irrelevant source extracts that have common CCD, with highly digested relevant extracts or HRP improves the accuracy of analysis.

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[135] - Chen L, Ofori-Anti AO, Goodman RE. Detection of Legume Proteins Cross-reactivity by Immunoblot Using Human Plasma of Individuals with Food Allergies to Peanut and/or Soybean. J Allergy Clin Immunol 2007;119(1 suppl):S193

RATIONALE: Peanut and soybean are well recognized allergenic food crops. Allergy to other legumes is becoming more widely reported and is thought to be partly due to cross-reactivity of structurally similar storage proteins. We have initiated IgE-binding studies to evaluate the extent of cross-reactivity, which may be due to similarities in protein or glycan structure. METHODS: Extracts of 11 species of commonly consumed legumes were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. Proteins were stained with colloidal blue or glycoprotein stain, or blotted onto membranes for IgE-binding using plasma of subjects with food allergies to peanut and/or soybean. Inhibition of IgE-binding was conducted by preincubation of inhibitors, legume extracts and horseradish peroxidase (glycan inhibitor), in plasma dilutions during immunoblotting. RESULTS: Protein profiles in stained gels were different under reducing and non-reducing conditions. Glycoproteins were present in all legume samples. Most plasma samples exhibited positive IgE-binding to major proteins in a few legume extracts, with the molecular weights ranging from 10 to 75 kDa. IgE-binding of some plasma to legume proteins was inhibited by peanut protein extract. Most of the IgE-binding was not totally inhibited by any legume extract. Some plasma showed strong IgE-binding to HRP, a glycoprotein containing cross-reactive carbohydrate determinants (CCD), and is thought to be clinical irrelevant. CONCLUSIONS: These results suggest that cross-reactivity may be present among various legume species. However, these preliminary results demonstrate that careful experimental design and controls are required to differentiate the specificity of IgE-binding, which in some cases is due to CCD.

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[136] - 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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[137] - Bardare M, Magnolfi C, Zani G. Soy sensitivity: personal observation on 71 children with food intolerance. Allerg Immunol (Paris) 1988;20:63-66

The controversies on the use of soy milk as a substitute in cow's milk intolerance prompted us to study: the incidence of soy sensitivity in a pediatric population (71 children, mean age 5.9 years, 45 boys and 26 girls) with food intolerance: the influence of a prior soy milk feeding on soy sensitivity: the relationship between soy, cow's milk and seed allergy. The patients were subdivided in two groups, one of atopic patients (50 subjects, 28 of which previously fed soy) and the other of non atopic patients (21 subjects, 12 of which previously fed soy). In the atopic group prick and RAST gave positive results to soy in 46% of case, with no difference between subjects fed soy and not. There was a relationship between any and peanut RAST in 82%; between soy and pea in 70%; between soy and cow's milk in 27% of cases. Soy milk challenge was positive in 10 out of 58 children (6 atopic and 4 non atopic); 4 our of 21 atopic patients with a cow's milk intolerance had a positive soy milk challenge: 3 of 10 non atopic patients with cow's milk intolerance were reactive to soy too. 77% of atopic and 90% of non atopic children were responsive both to seeds and soy. It can be concluded that soy sensitivity is rather rare in patients with food intolerance (17.2% of cases) and is not correlated with cow's milk intolerance while is significantly correlated with seeds allergy; there is no difference between atopic and non atopic subjects and between patients previously fed soy and never fed soy.

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[138] - Eigenmann PA, Sicherer SH, Borkowski TA, Cohen BA, Sampson HA. Prevalence of IgE-Mediated Food Allergy Among Children With Atopic Dermatitis. Pediatrics 1998;101(3):e8

OBJECTIVE: There is a growing body of clinical and laboratory evidence to support the notion that food allergy plays a role in the pathogenesis of atopic dermatitis (AD). However, the incidence of IgE-mediated food allergy in children with AD is not well established. DESIGN: A prospective study to determine the prevalence of IgE-mediated food hypersensitivity among patients referred to a university-based dermatologist for evaluation of AD. SETTING: University hospital pediatric dermatology clinic. PATIENTS: A total of 63 patients with AD were recruited (35 male; 32 white, 24 African-American, 7 Asian) . METHODS: Patients were assigned an AD symptom score (SCORAD) and were screened for food-specific serum IgE antibodies to six foods (milk, egg, wheat, soy, peanut, fish) known to be the most allergenic in children. The levels of food-specific serum IgE were determined by the CAP System fluoroscein-enzyme immunoassay (CAP); patients with a value >/=0.7 kIUa/L were invited for an additional allergy evaluation. Those with CAP values below the cutoff were considered not food allergic. Patients were considered to be allergic if they met one of the following criteria for at least one food: 1) reaction on food challenge; 2) CAP value more than the 95% confidence interval predictive for a reaction; 3) convincing history of an acute significant (hives, respiratory symptoms) reaction after the isolated ingestion of a food to which there was a positive CAP or prick skin test . RESULTS: A total of 63 patients (median age, 2.8 years; median SCORAD, 41.1) were recruited; 22 had negative CAP values (without a significant difference in age or SCORAD score, compared with the 41 with positive specific IgE values). Further allergy evaluation was offered to the 41 remaining patients; 10 were lost to follow-up and 31 were evaluated further. Of these, 19 underwent a total of 50 food challenges (36 double-blind, placebo-controlled, and 14 open), with 11 patients experiencing 18 positive challenges (94% with skin reactions). Additionally, 6 patients had a convincing history with a predictive level of IgE; 5 had a convincing history with positive, indeterminate levels of IgE; and 1 had predictive levels of IgE (to egg and peanut) without a history of an acute reaction. Overall, 23/63 (37%; 95% confidence interval, 25% to 50%) had clinically significant IgE-mediated food hypersensitivity without a significant difference in age or symptom score between those with or without food allergy . CONCLUSIONS: Approximately one third of children with refractory, moderate-severe AD have IgE-mediated clinical reactivity to food proteins. The prevalence of food allergy in this population is significantly higher than that in the general population, and an evaluation for food allergy should be considered in these patients.

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[139] - Giampietro PG, Ragno V, Daniele S, Cantani A, Ferrara M, Businco L. Soy hypersensitivity in children with food allergy. Ann Allergy 1992;69:143-146

To evaluate humoral (IgE antibodies) and clinical (positive challenge test) soy hypersensitivity prevalence, we studied 317 children (271 boys and 100 girls) with a median age of 5 months (range 1-120) who visited the Division of Allergy and Clinical Immunology of the Pediatric Department of the University of Roma "La Sapienza" because of histories and symptoms suggestive of food allergy. Atopic dermatitis (AD) was present in 247/317 children (78%), diarrhea in 19 (6%), urticaria in 22 (7%), and rhinitis and/or asthma in 29 (9%). All children underwent diagnostic procedures including family and personal history, physical examination, PRIST, and RAST to cows milk (CM), egg, wheat, soy, and Dermatophagoides pteronyssinus (Dpt). Open challenge tests to soy were performed in the hospital under observation and with emergency equipment at hand. The prevalence of humoral sensitization to CM was 54%, to egg 46%, to Dpt 35%, to wheat 24%, and to soy 22%. Only five children had IgE only to soy; six to soy and egg; and 58 to soy, CM, and egg. Only ten children (3%) had positive challenge to soy and only five of them had IgE to soy. RAST had a sensitivity of 0.69, a specificity of 0.83, a negative predictive value of 0.77, and a positive predictive value of only 0.06.

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[140] - Bjerremann Jensen L, Andersen M, Skov P, Poulsen L, Bindslev-Jensen C. Investigating the common allergic reactivity within the legume botanical family using skin prick test (SPT), specific immunoglobulin E (IgE) and histamine release (HR). EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°578

Background: The aim of the study was to investigate possible common allergic reactions between peanut and different foods from the legume family. Methods: The legumes investigated were peanut, soybean, lupine, and fresh as well as blanched green pea. Patients were included on the basis of confirmed food allergy to peanut according to EAACI guidelines. SPTs were performed by prick-prick with legumes using as cutoff a wheal with a diameter >3 mm. IgE were determined using the CAP system (Pharmacia, Sweden) with a cutoff of 0.35 kUA/L. HR was performed by direct stimulation of patient basophils with legume extracts and the released histamine was measured spectrofluorometrically (RefLab ApS, Denmark). Results: 72 peanut allergic patients were included. The sensitivity for SPT, IgE and HR was 92-97% for peanut. Testing the other legumes with SPT and HR produced most positives for lupine (45%, 80%) and fresh pea (41%, 91%) followed by soybean (28%, 19%) and blanched pea (16%, 5%), whereas soybean produced most positives in IgE (58%) followed by pea (48%) and lupine (47%). Pairwise comparison between the three diagnostic tests in general gave the highest concordance between SPT and HR. A subpopulation of the patients was challenged with the legumes. The most common reported symptom for reactions related to the various foods was the oral allergy syndrome (OAS). 22 patients were challenged with soybean, and of these 2 were positive. 1 of 6 challenges was positive with lupine whereas 0 of 12 was positive with fresh pea. This suggests that clinical coreactivity between peanut and other legumes is substantially lower than indicated by diagnostic tests. Conclusion: Patients with peanut allergy might in a few cases be clinically allergic to other members of the legume family. However, application of SPT, IgE or HR tests results in an overestimation of the positive reactions.

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[141] - Sicherer SH, Morrow EH, Sampson HA. Dose-response in double-blind, placebo-controlled oral food challenges in children with atopic dermatitis. J Allergy Clin Immunol 2000;105:582-586

"BACKGROUND: Double-blind, placebo-controlled oral food challenges (DBPCFCs) are considered the ""gold standard"" for diagnosing food hypersensitivity, but the dose that elicits positive challenges, or determinants that may predict dose-response relationships, have not been reported . OBJECTIVE: Our purpose was to determine the quantity of food that elicits reactions during DBPCFCs and to evaluate parameters that may predict the provocative dose and severity of reaction . METHODS: We reviewed challenge data for all positive challenges to 6 common allergenic foods in children with atopic dermatitis evaluated for food allergy over a 13-year period. Challenge food was generally administered in 6 doses at 10- to 15-minute intervals beginning with 400 to 500 mg and completing with a total of 8 to 10 g of food. An open feeding of a larger portion followed negative challenges. At the physician's discretion, a lower starting dose was occasionally used (100 mg, 250 mg). Food-specific IgE antibody concentrations (radioallergosorbent test [RAST]) were determined on stored sera of 20% of the challenges selected randomly and 99.6% had prick skin tests (PSTs) performed to the challenged food . RESULTS: A total of 196 children (45% male; median age 5 y 9 mo; atopic dermatitis 98%, asthma 62%) had 513 positive challenges distributed as follows: egg 267, milk 117, soy 53, wheat 40, peanut 24, fish 12. The percentage of children reacting at the first dose (500 mg or less) was as follows: egg 49%, milk 55%, soy 28%, wheat 25%, peanut 26%, and fish 17%. Twenty-six milk challenges and 22 egg challenges were positive at a first dose of 250 mg; 3 milk challenges and 7 egg challenges were positive at a first dose of 100 mg. Eleven percent of the reactions that occurred on the first dose were severe. The percentage reacting after the final dose of the DBPCFC (or during open challenge) were egg 11%, milk 12%, soy 19%, wheat 12.5%, peanut 8.7%, and fish 25%. There was not a strong correlation between PST absolute wheal size or score (adjusted for histamine controls) and dose at reaction or severity of reaction (R(s) range -0.22 to 0.39 for particular foods). Serum concentration of food-specific IgE did not correlate well with the dose causing a reaction or with severity (R(s) range -0.40 to 0.55 for particular foods) . CONCLUSIONS: This food-allergic population may react to as little as 100 mg of food, possibly less, and the dose causing a reaction and the severity of reaction is not predicted by PST or RAST. Lower doses (100 mg or less) should be investigated for their appropriateness in initiating DBPCFCs."

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[142] - Sampson HA, Albergo R. Comparison of results of skin tests, RAST, and double-blind, placebo-controlled food challenges in children with atopic dermatitis. J Allergy Clin Immunol 1984;74:26-33

Forty children with atopic dermatitis were evaluated for clinical evidence of hypersensitivity to foods by double-blind, placebo-controlled food challenges. Twenty-four children (60%) experienced 33 positive challenges, manifested by cutaneous symptoms in 31 (94%), gastrointestinal symptoms in 14 (42%), nasal symptoms in nine (27%), and respiratory in six (18%). Results of prick skin tests (STs) and RASTs to eight food antigens frequently eliciting hypersensitivity reactions were compared with those from food challenges to determine the diagnostic accuracy in children with atopic dermatitis. Defining a positive ST as a wheal 3 mm larger than the negative control wheal and a positive RAST as a Phadebas RAST score of 3 or 4, the sensitivity, specificity, and predictive accuracies of these tests were found to be comparable except in the case of wheat antigen where the ST was clearly superior to the RAST. Accepting a RAST score of 2 or more as a positive slightly improved sensitivity in some cases but dramatically decreased specificity. Combining results of STs and RASTs did not improve significantly the diagnostic accuracy over results of the tests used individually. These studies demonstrate no advantage of RAST alone or in combination with prick skin testing over prick skin testing alone in the evaluation of food hypersensitivity in children with atopic dermatitis. Furthermore, skin testing should be considered a good test for excluding immediate food hypersensitivity but only a suggestive positive indicator of hypersensitivity due to the high rate of clinically insignificant positive STs.

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[143] - Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001;107:891-896

BACKGROUND: The double-blind, placebo-controlled food challenge is considered the gold standard for diagnosing food allergy. However, in a retrospective analysis of children and adolescents with atopic dermatitis and food allergy, discrete food-specific IgE concentrations were established that could predict clinical reactivity to egg, milk, peanut, and fish with greater than 95% certainty. OBJECTIVE: The purpose of this investigation was to determine the utility of these 95% predictive decision points in a prospective evaluation of food allergy. METHODS: Sera from 100 consecutive children and adolescents referred for evaluation of food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by using the Pharmacia CAP System FEIA. Food-specific IgE values were compared with history and the results of skin prick tests and food challenges to determine the efficacy of previously established 95% predictive decision points in identifying patients with increased probability of reacting during a specific food challenge. RESULTS: One hundred children (62% male; median age, 3.8 years; range, 0.4-14.3 years) were evaluated for food allergy. The diagnosis of food allergy was established by means of history or oral food challenge. On the basis of the previously established 95% predictive decision points for egg, milk, peanut, and fish allergy, greater than 95% of food allergies diagnosed in this prospective study were correctly identified by quantifying serum food-specific IgE concentrations. CONCLUSION: In a prospective study of children and adolescents referred for evaluation of food allergy, previously established 95% predictive decision points of food-specific IgE antibody concentrations for 4 major food allergens were effective in predicting clinical reactivity. Quantification of food-specific IgE is a useful test for diagnosing symptomatic allergy to egg, milk, peanut, and fish in the pediatric population and could eliminate the need to perform double-blind, placebo-controlled food challenges in a significant number of children.

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[144] - Sampson HA, Ho DG. Relationship between food-specific IgE concentrations and the risk of positive food challenges in children and adolescents. J Allergy Clin Immunol 1997;100:444-451

"BACKGROUND: The double-blind, placebo-controlled food challenge (DBPCFC) is the ""gold standard"" for diagnosis of food hypersensitivity. Skin prick tests and RASTs are sensitive indicators of food-specific IgE antibodies but poor predictors of clinical reactivity. Previous studies suggested that high concentrations of food-specific IgE antibody were predictive of food-induced clinical symptoms. Because the CAP System FEIA (Pharmacia Diagnostics, Uppsala, Sweden) provides a quantitative assessment of allergen-specific IgE antibody, this study was undertaken to determine the potential utility of the CAP System FEIA in diagnosis of IgE-mediated food hypersensitivity . METHODS: Sera from 196 patients with food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by CAP System FEIA. Sera were randomly selected from 300 stored samples of children and adolescents who had been evaluated by history, skin prick tests, and DBPCFCs. The study population was highly atopic; all patients had atopic dermatitis, and approximately 50% had asthma and allergic rhinitis at the time of initial evaluation. The performance characteristics of the CAP System FEIA were compared with those of skin prick tests and the outcome of DBPCFCs or ""convincing"" histories of anaphylactic reactions . RESULTS: The prevalence of specific food allergies in the study population varied from 22% for wheat to 73% for egg. Allergy to egg, milk, peanut, and soy accounted for 87% of confirmed reactions. The performance characteristics of skin prick tests and CAP System FEIA (egg, milk, peanut, fish) were comparable, with excellent sensitivity and negative predictive accuracy but poor specificity and positive predictive accuracy. The performance characteristics of the CAP System FEIA for soy and wheat were poor. For egg, milk, peanut, and fish allergy, diagnostic levels of IgE, which could predict clinical reactivity in this population with greater than 95% certainty, were identified: egg, 6 kilounits of allergen-specific IgE per liter (kU[A]/L); milk, 32 kU(A)/L; peanut, 15 kU(A)/L; and fish, 20 kU(A)/L . CONCLUSIONS: When compared with the outcome of DBPCFCs, results of CAP System FEIA are generally comparable to those of skin prick tests in predicting symptomatic food hypersensitivity. Furthermore, by measuring the concentrations of food-specific IgE antibodies with the CAP System FEIA, it is possible to identify a subset of patients who are highly likely (>95%) to experience clinical reactions to egg, milk, peanut, or fish. This could eliminate the need to perform DBPCFCs in a significant number of patients suspected of having IgE-mediated food allergy."

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[145] - Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001;107:891-896

BACKGROUND: The double-blind, placebo-controlled food challenge is considered the gold standard for diagnosing food allergy. However, in a retrospective analysis of children and adolescents with atopic dermatitis and food allergy, discrete food-specific IgE concentrations were established that could predict clinical reactivity to egg, milk, peanut, and fish with greater than 95% certainty. OBJECTIVE: The purpose of this investigation was to determine the utility of these 95% predictive decision points in a prospective evaluation of food allergy. METHODS: Sera from 100 consecutive children and adolescents referred for evaluation of food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by using the Pharmacia CAP System FEIA. Food-specific IgE values were compared with history and the results of skin prick tests and food challenges to determine the efficacy of previously established 95% predictive decision points in identifying patients with increased probability of reacting during a specific food challenge. RESULTS: One hundred children (62% male; median age, 3.8 years; range, 0.4-14.3 years) were evaluated for food allergy. The diagnosis of food allergy was established by means of history or oral food challenge. On the basis of the previously established 95% predictive decision points for egg, milk, peanut, and fish allergy, greater than 95% of food allergies diagnosed in this prospective study were correctly identified by quantifying serum food-specific IgE concentrations. CONCLUSION: In a prospective study of children and adolescents referred for evaluation of food allergy, previously established 95% predictive decision points of food-specific IgE antibody concentrations for 4 major food allergens were effective in predicting clinical reactivity. Quantification of food-specific IgE is a useful test for diagnosing symptomatic allergy to egg, milk, peanut, and fish in the pediatric population and could eliminate the need to perform double-blind, placebo-controlled food challenges in a significant number of children.

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[146] - Östblom E, Lilja G, Ahlstedt S, van Hage M, Wickman M. Patterns of quantitative food-specific IgE-antibodies and reported food hypersensitivity in 4-year-old children. Allergy 2008;63:418-424

BACKGROUND: Diagnosis of food hypersensitivity (FHS) is difficult and interpretation of food allergy tests is complicated . OBJECTIVE: To investigate the probability of reported FHS in relation to levels of food-specific IgE-antibodies (AB) in a population-based setting of 4-year-old children (n = 2336) . METHODS: Information on FHS was obtained from a questionnaire and specific IgE-AB to milk, egg, fish, peanut, soy and wheat were analysed . RESULTS: Thirty-one per cent of the children with reported FHS (n = 284) were sensitized (> or =0.35 kU(A)/l) to at least one of the tested foods compared with 11% of children without FHS (n = 2052). Furthermore, the probability of reported symptoms to milk, egg and fish increased with increasing levels of food-specific IgE-AB to the same food allergens. A similar trend was seen for peanut and wheat, but not for soy. Increasing levels of specific IgE-AB to milk or egg were also associated with an increasing risk of reported symptoms caused by other foods . CONCLUSIONS: Quantitative measurements of IgE-AB to milk, egg and fish are useful to evaluate IgE-associated FHS in preschool children also in a population based sample. Such measurements appear to be of limited value for soy bean and wheat, in particular as a screening method.

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[147] - Celik-Bilgili S, Mehl A, Verstege A, Staden U, Nocon M, Beyer K, et al. The predictive value of specific immunoglobulin E levels in serum for the outcome of oral food challenges. Clin Exp Allergy 2005;35:268-273

Summary Background Specific serum IgE is considered as one of the important diagnostic measures in the diagnostic work-up of food allergy. Objective To evaluate the role of specific serum IgE in predicting the outcome of oral food challenges, and to determine threshold concentrations of specific serum IgE that could render double-blind, placebo-controlled food challenges unnecessary. Methods In 501 children (median age 13 months), 992 controlled oral challenges were performed with cow's milk (CM), hen's egg (HE), wheat and soy. 440/501 (88%) children suffered from atopic dermatitis. For all children, specific IgE concentrations in serum were determined. Sensitivity, specificity, positive and negative predictive values, receiver operator characteristics-curves as well as predictive decision points were calculated. Results Four hundred and forty-five out of 992 oral food challenges with allergens were assessed as positive. Sensitivity of specific serum IgE was 97% for HE, 83% for CM, 69% for soy, and 79% for wheat. Specificity was 51% for HE, 53% for CM, 50% for soy, and 38% for wheat. Calculating 90%, 95% and 99% predicted probabilities using logistic regression revealed predictive decision points of 6.3, 12.6, and 59.2 kU/L for HE, respectively. Subdividing our children in those of below or above 1 year of age resulted in a markedly different predicted probability for HE. For CM, only the 90% predicted probability (88.8 kU/L) could be calculated. No decision points could be determined for CM, wheat and soy. Conclusion In general, specific serum IgE levels showed a correlation with the outcome of positive oral food challenges for CM and HE. Meaningful predictive decision points can be calculated for HE, which may help to avoid oral food challenges in some cases. However, data need to be ascertained for each allergen separately. Furthermore, the age of the patient population under investigation must also be taken into account.

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[148] - Mehl A, Verstege A, Staden U, Kulig M, Nocon M, Beyer K, et al. Utility of the ratio of food-specific IgE/total IgE in predicting symptomatic food allergy in children. Allergy 2005;60:1034-1039

BACKGROUND: Double-blind, placebo-controlled food challenges are time-consuming, expensive and not without risk to patients. Therefore, an in vitro test that could accurately diagnose food allergy would be of great value . OBJECTIVE: To evaluate the utility of the ratio of specific immunoglobulin E (IgE)/total IgE compared with specific IgE (sIgE) alone in predicting symptomatic food allergy . METHODS: We retrospectively analysed 992 controlled oral food challenges performed in 501 children (median age 13 months). The ratio of sIgE/total IgE was calculated and tested for correlation with the outcome of food challenges. Receiver operator characteristics (ROC)-curves were performed; predicted probabilities and predictive decision points were calculated . RESULTS: A significant correlation was found between the ratio and the outcome of food challenges for cow's milk (CM), hen's egg (HE), and wheat, but not for soy. The ROC and predicted probability curves as well as sensitivity and specificity of the decision points of the ratio were similar to those of sIgE levels for CM, HE and wheat . CONCLUSION: In view of the greater effort needed to determine the ratio, without benefit compared with the sIgE alone, the calculation of the ratio of sIgE/total IgE for diagnosing symptomatic food allergy offers no advantage for CM, HE, wheat or soy. For the majority of cases controlled oral food challenges still remain the method of choice.

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[149] - Komata T, Imai T, Tomikawa M, Tachimoto H, Shukuya A, Ebisawa M. Usefulness of egg white, milk, wheat and soy specific IgE antibody titers in the diagnosis of food allergy. Allergy Clin Immunol Int 2005;17(Suppl. 1):331-332

Background: The relationship between food-specific IgE titers and the outcome of food challenges is established through threshold values and probability curves. One major difference between the different suggested threshold values is the age of the used populations. OBJECTIVE: The purpose of the study was to determine whether antigen specific IgE values could become the index of food allergy diagnosis in Japan and to determine the effect of different ages. METHODS: Children whose specific IgE against major food antigens (egg white, cow's milk, wheat and soy bean) were measured within six months after their first visit, were included in the study. The diagnosis of food allergy was made at the point of blood drawn, by firm episodes after the ingestion of those foods, or the results of food challenge tests. Specific IgE to the relevant antigen were measured using the Pharmacia CAP-System FEIA®. The total numbers of examination were; egg whites; 1025 subjects, milk; 458 subjects, wheat; 437 subjects, and soy bean; 422 subjects. The children were poststratfied into 6 age groups, < 0.5 year, 0.5-1 year, 1 year, 2 year, 3 year and 4 years of age. The specific IgE antibody concentrations were related ro the oucome using a logistic regression model. The estimated models were expressed as probabilities. Tests and confidence intervals were according to Wald, using a p value of 0.05 as significant. Computerised statistical analysis was performed using SAS System V8.01 Results: For each antigen a significant relationship between the probability of reacting to food and the specific IgE antibody concentrations could be found. When the population was post stratified for ages, different shapes of the probability curves were found. For egg white four significantly different relationships were found, indicating that for children of 1 year or younger even a very low concentration of specific IgE antibodies was associated with a rather high probability for reaction, the younger the child was the higher probability. The same pattern was found also for milk and wheat. CONCLUSIONS: Our results show that the age and the kinds of antigen are major factors when relating the specific IgE antibody concentration to the probability of reacting to food. They also make an explanation to the previously published different threshold values, indicating that age may explain the difference in threshold values.

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[150] - Roehr CC, Reibel S, Ziegert M, Sommerfeld C, Wahn U, Niggemann B. Atopy patch tests, together with determination of IgE levels, reduce the need for need for oral food challenges in children with atopic dermatitis. J Allergy Clin Immunol 2001;107:548-553

BACKGROUND: Atopic dermatitis is commonly associated with food allergy. In addition to skin prick tests (SPTs) and measurements of specific IgE levels, the atopy patch test (APT) has recently been introduced into the diagnostic procedure for food allergy . OBJECTIVE: Our aim was to evaluate whether a combination of allergologic tests could improve the prognostic value of the individual tests for positive food challenge results. We hypothesized that the combination of a positive APT result plus proof of specific IgE, a positive SPT result, or both would render double-blind, placebo-controlled, food challenges unnecessary . METHODS: One hundred seventy-three double-blind, placebo-controlled, food challenges were performed in 98 children (median age, 13 months) with atopic dermatitis. All children were subjected to SPTs, APTs, and determination of specific IgE. Sensitivity, specificity, and positive and negative predictive values were calculated . RESULTS: Ninety-five (55%) of 173 oral provocations were assessed as positive. For evaluating suspected cow's milk (CM) allergy, the APT was the best single predictive test (positive predictive value [PPV], 95%), and the combination of a positive APT result with evidence of specific IgE or an APT result together with a positive skin prick test response optimized the PPV to 100%. For hen's egg (HE) allergy, the APT was also the best single predictive test (PPV, 94%). The combination of 2 or more tests did not exceed the APT's predictive value. In both CM and HE challenges, the predictability of oral challenges depended on the level of specific IgE. For wheat allergy, the APT proved to be the most reliable test, and the PPV of 94% could not be improved by a combination with other allergologic tests . CONCLUSION: The combination of positive APT results and measurement of levels of specific IgE (CM, > or = 0.35 kU/L; HE, > or = 17.5 kU/L) makes double-blind, placebo-controlled, food challenges superfluous for suspected CM and HE allergy.

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[151] - Perry TT, Matsui EC, Conover-Walker MK, Wood RA. The relationship of allergen-specific IgE levels and oral food challenge outcome. J Allergy Clin Immunol 2004;114:144-149

Background Oral food challenges remain the gold standard for the diagnosis of food allergy. However, clear clinical and laboratory guidelines have not been firmly established to determine when oral challenges should be performed. Objective : We sought to determine the value of food-specific IgE levels in predicting challenge outcome. Method s : A retrospective chart review of 604 food challenges in 391 children was performed. All children had food-specific IgE levels measured by means of CAP-RAST before challenge. Data were analyzed to determine the relationship between food-specific IgE levels and challenge outcome, as well as the relationship between other clinical parameters and challenge outcome. Result s : Forty-five percent of milk challenges were passed compared with 57% for egg, 59% for peanut, 67% for wheat, and 72% for soy. Specific IgE levels were higher among patients who failed challenges than among those who passed (P .03 for each food). When seeking a specific IgE level at which a 50% pass rate could be expected, a cutoff level of 2 kUA/L was determined for milk, egg, and peanut. Data were less clear for wheat and soy. Coexistent eczema or asthma was associated with failed egg challenges, but other atopic disease was otherwise not associated with challenge outcome. Conclusions : Allergen-specific IgE concentrations to milk, egg, and peanut and, to a lesser extent, wheat and soy serve as useful predictors of challenge outcome and should be considered when selecting patients for oral challenge to these foods.

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[152] - Sampson HA, Ho DG. Relationship between food-specific IgE concentrations and the risk of positive food challenges in children and adolescents. J Allergy Clin Immunol 1997;100:444-451

"BACKGROUND: The double-blind, placebo-controlled food challenge (DBPCFC) is the ""gold standard"" for diagnosis of food hypersensitivity. Skin prick tests and RASTs are sensitive indicators of food-specific IgE antibodies but poor predictors of clinical reactivity. Previous studies suggested that high concentrations of food-specific IgE antibody were predictive of food-induced clinical symptoms. Because the CAP System FEIA (Pharmacia Diagnostics, Uppsala, Sweden) provides a quantitative assessment of allergen-specific IgE antibody, this study was undertaken to determine the potential utility of the CAP System FEIA in diagnosis of IgE-mediated food hypersensitivity . METHODS: Sera from 196 patients with food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by CAP System FEIA. Sera were randomly selected from 300 stored samples of children and adolescents who had been evaluated by history, skin prick tests, and DBPCFCs. The study population was highly atopic; all patients had atopic dermatitis, and approximately 50% had asthma and allergic rhinitis at the time of initial evaluation. The performance characteristics of the CAP System FEIA were compared with those of skin prick tests and the outcome of DBPCFCs or ""convincing"" histories of anaphylactic reactions . RESULTS: The prevalence of specific food allergies in the study population varied from 22% for wheat to 73% for egg. Allergy to egg, milk, peanut, and soy accounted for 87% of confirmed reactions. The performance characteristics of skin prick tests and CAP System FEIA (egg, milk, peanut, fish) were comparable, with excellent sensitivity and negative predictive accuracy but poor specificity and positive predictive accuracy. The performance characteristics of the CAP System FEIA for soy and wheat were poor. For egg, milk, peanut, and fish allergy, diagnostic levels of IgE, which could predict clinical reactivity in this population with greater than 95% certainty, were identified: egg, 6 kilounits of allergen-specific IgE per liter (kU[A]/L); milk, 32 kU(A)/L; peanut, 15 kU(A)/L; and fish, 20 kU(A)/L . CONCLUSIONS: When compared with the outcome of DBPCFCs, results of CAP System FEIA are generally comparable to those of skin prick tests in predicting symptomatic food hypersensitivity. Furthermore, by measuring the concentrations of food-specific IgE antibodies with the CAP System FEIA, it is possible to identify a subset of patients who are highly likely (>95%) to experience clinical reactions to egg, milk, peanut, or fish. This could eliminate the need to perform DBPCFCs in a significant number of patients suspected of having IgE-mediated food allergy."

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[153] - Celik-Bilgili S, Mehl A, Verstege A, Staden U, Nocon M, Beyer K, et al. The predictive value of specific immunoglobulin E levels in serum for the outcome of oral food challenges. Clin Exp Allergy 2005;35:268-273

Summary Background Specific serum IgE is considered as one of the important diagnostic measures in the diagnostic work-up of food allergy. Objective To evaluate the role of specific serum IgE in predicting the outcome of oral food challenges, and to determine threshold concentrations of specific serum IgE that could render double-blind, placebo-controlled food challenges unnecessary. Methods In 501 children (median age 13 months), 992 controlled oral challenges were performed with cow's milk (CM), hen's egg (HE), wheat and soy. 440/501 (88%) children suffered from atopic dermatitis. For all children, specific IgE concentrations in serum were determined. Sensitivity, specificity, positive and negative predictive values, receiver operator characteristics-curves as well as predictive decision points were calculated. Results Four hundred and forty-five out of 992 oral food challenges with allergens were assessed as positive. Sensitivity of specific serum IgE was 97% for HE, 83% for CM, 69% for soy, and 79% for wheat. Specificity was 51% for HE, 53% for CM, 50% for soy, and 38% for wheat. Calculating 90%, 95% and 99% predicted probabilities using logistic regression revealed predictive decision points of 6.3, 12.6, and 59.2 kU/L for HE, respectively. Subdividing our children in those of below or above 1 year of age resulted in a markedly different predicted probability for HE. For CM, only the 90% predicted probability (88.8 kU/L) could be calculated. No decision points could be determined for CM, wheat and soy. Conclusion In general, specific serum IgE levels showed a correlation with the outcome of positive oral food challenges for CM and HE. Meaningful predictive decision points can be calculated for HE, which may help to avoid oral food challenges in some cases. However, data need to be ascertained for each allergen separately. Furthermore, the age of the patient population under investigation must also be taken into account.

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[154] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[155] - Treudler R, Süß A, Werner M, Thiery J, Simon JC. Severe allergic reactions to soybean products: Special risk for patients with birch pollen allergy. Allergy 2007;62(suppl. 83):367

Background: Soy bean products have become of increasing popularity because they are expected to have positive effects on human‚s health. Unfortunately, during the last years we had to treat rising numbers of patients with symptoms of severe intolerance reactions (anaphylaxis) to soy bean products. We aimed at investigating testing procedures for soy bean allergy and if birch pollen allergy is a special risk factor for these reactions. Methods: Group 1: Ten patients with history of anaphylaxis to soy bean products had skin prick test with standard allergens, the suspicious soy bean product and were investigated for specific IgE to Birch, Bet v1, Gly m4 and soy bean. Group 2: 50 consecutive patients presenting for pollinosis were investigated by skin prick test (SPT) with standard allergens and with a commercially available soy bean prick test preparation. Group 3: In third step, all patients with known type-1 allergy to birch pollen (SPT) were investigated for former soy bean comsumption. Regardless of the comsumption, all had SPT with a soy bean milk product and we looked for total IgE, specific IgE to soy bean, birch, Bet v1, and Gly m4. Results: Group 1: All patients had a combined sensitization to birch, soy bean and Gly m4. Interestingly, testing for soy bean was reactive in all of them only with the suspicious product. Group 2: No patient showed any reaction to the commercially available soy bean test solution, 19 of them were reactive to birch. Group 3: 13/20 patients with birch pollen allergy had a positive SPT to the soy bean milk product. Only 4 of them remembered to have had comsumed soy bean products. All patients with positive SPT to soy bean also had sIgE against Gly m4. Comment: Cross reactions to soy bean allergens seem to be frequent in patients with birch pollen allergy. Gly m4 was identified as a relevant cross reacting allergen. The commercially available test preparations for skin or in vitro tests are not helpful in detecting sensitizations to soy beans, probably because the heatlabile proteins, among them Gly m4, are destroyed. Investigations of larger groups of patients with birch pollen allergy are ongoing in our department. We recommend to advise all patients with birch pollen allergy to be reluctant with consumption of soy protein rich products.

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[156] - Mattsson L, Marknell DeWitt Å, Gubesch M, Ballmer-Weber B, Kofler H, Würtenberger-Wagner C, et al. Recombinant Gly m 4, a useful reagent in the investigation of birch pollen associated soybean allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1067

Background: Food allergy to soybean-derived products may be mediated by the allergen Gly m 4. This type of soybean allergy affects a fraction of birch pollen allergic individuals and is due to cross-reactivity between Gly m 4 and the major birch pollen allergen Bet v 1. Gly m 4 is a low-abundance protein in natural soybean extract which is therefore considered inadequate for detection of specific IgE in this particular category of patients. The aim of this study was to examine the utility of rGly m 4 ImmunoCAP in the laboratory investigation of cases of suspected soybean allergy with a low or negative specific IgE test result for soybean. Methods: Serum samples of 51 subjects from Germany (n=25), Switzerland (n=15) and Austria (n=11) with a convincing case history of soybean allergy (n=36) and a positive double-blind placebo-controlled food challenge (n=15), a concomitant allergy to birch pollen and a low or undetectable IgE response to soybean extract were analysed for IgE binding to rGly m 4 and rBet v 1 using ImmunoCAP. For comparison, sera of a group of birch pollen sensitised subjects (n=85) without known soybean allergy were analysed for IgE binding to the same allergens. Results: 42 of the 51 (82%) soybean allergic subjects studied tested negative for specific IgE to natural soybean extract and the 9 positive results obtained were all below 2.3 kUA/L. In contrast, sera of 45 of the 51 subjects (88%) displayed specific IgE binding to rGly m 4, with levels ranging from 0.43 to 58.1 kUA/L. All but 2 showed IgE reactivity to rBet v 1 and the values obtained were in all cases higher than those to rGly m 4 (median 3.7 times higher). All of the 85 birch pollen allergic subjects had serum IgE to rBet v 1 and 58 (68%) showed detectable IgE binding to rGly m 4. The difference in IgE binding intensity to rBet v 1 and rGly m 4 tended to be greater among these subjects (median 7.4 times higher). Conclusions: The vast majority of subjects with combined birch pollen and soybean allergy studied here were found to be sensitised to rGly m 4, despite having absent or low specific IgE levels to soybean extract. Among the Bet v 1 reactive birch pollen allergic subjects without known soybean allergy, approximately 2/3 showed IgE binding to rGly m 4. The use of rGly m 4 enables detection of relevant sensitisation in subjects with birch pollen related soybean allergy and is therefore a useful tool in the investigation of such patients.

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[157] - Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A, et al. Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004;113:148-154

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[158] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[159] - Mattsson L, Marknell DeWitt Å, Gubesch M, Ballmer-Weber B, Kofler H, Würtenberger-Wagner C, et al. Recombinant Gly m 4, a useful reagent in the investigation of birch pollen associated soybean allergy. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1067

Background: Food allergy to soybean-derived products may be mediated by the allergen Gly m 4. This type of soybean allergy affects a fraction of birch pollen allergic individuals and is due to cross-reactivity between Gly m 4 and the major birch pollen allergen Bet v 1. Gly m 4 is a low-abundance protein in natural soybean extract which is therefore considered inadequate for detection of specific IgE in this particular category of patients. The aim of this study was to examine the utility of rGly m 4 ImmunoCAP in the laboratory investigation of cases of suspected soybean allergy with a low or negative specific IgE test result for soybean. Methods: Serum samples of 51 subjects from Germany (n=25), Switzerland (n=15) and Austria (n=11) with a convincing case history of soybean allergy (n=36) and a positive double-blind placebo-controlled food challenge (n=15), a concomitant allergy to birch pollen and a low or undetectable IgE response to soybean extract were analysed for IgE binding to rGly m 4 and rBet v 1 using ImmunoCAP. For comparison, sera of a group of birch pollen sensitised subjects (n=85) without known soybean allergy were analysed for IgE binding to the same allergens. Results: 42 of the 51 (82%) soybean allergic subjects studied tested negative for specific IgE to natural soybean extract and the 9 positive results obtained were all below 2.3 kUA/L. In contrast, sera of 45 of the 51 subjects (88%) displayed specific IgE binding to rGly m 4, with levels ranging from 0.43 to 58.1 kUA/L. All but 2 showed IgE reactivity to rBet v 1 and the values obtained were in all cases higher than those to rGly m 4 (median 3.7 times higher). All of the 85 birch pollen allergic subjects had serum IgE to rBet v 1 and 58 (68%) showed detectable IgE binding to rGly m 4. The difference in IgE binding intensity to rBet v 1 and rGly m 4 tended to be greater among these subjects (median 7.4 times higher). Conclusions: The vast majority of subjects with combined birch pollen and soybean allergy studied here were found to be sensitised to rGly m 4, despite having absent or low specific IgE levels to soybean extract. Among the Bet v 1 reactive birch pollen allergic subjects without known soybean allergy, approximately 2/3 showed IgE binding to rGly m 4. The use of rGly m 4 enables detection of relevant sensitisation in subjects with birch pollen related soybean allergy and is therefore a useful tool in the investigation of such patients.

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Imprimer la bibliographie

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Soja et allergie respiratoire

Les allergènes du soja

Soja : stabilité des allergènes

Diagnostic d’une allergie au soja

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