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VOLUME 109, NUMBER 1 28 A Sensitive Sandwich EUSA for the Detection of Trace Amount of Cashew Nut in Foods Yanhong Wei*, Shridhar K Sathe*, Suzanne S Teuber§, Kenneth H Roux* *Florida State University, Tallahassee, FL §UC Davis, Davis, CA Cashew is one of several tree nuts that are associated with IgE-mediated hypersensitivity reactions in some patients. Since low levels of cashew protein can provoke an allergic reaction, the sensitive patients must be aware of hidden allergens contained in commercially prepared foods. Toward this end, we have developed a sandwich ELISA to detect cashew major protein (CMP) in food matrices. CME as the name implies, constitutes a major portion (~50%) of those cashew proteins extractable in aqueous solvents. CMP is heat stable and thus is a good marker protein to detect the presence of cashew nut. Polyclonal goat and rabbit antibodies generated against whole cashew extract and CMP, respectively, were first eluted from protein G affinity columns and subsequently adsorbed with protein extracts from a variety of nuts and seeds to minimize crossreactivity. The purified goat and rabbit IgGs were used as capture and secondary antibodies, respectively. Cashew flour and CMP-spiked ground food samples were extracted 1:10 (w:v) in borate-saline buffer and placed in ELISA plates pre-coated with goat antibodies followed by rabbit anti-cashew IgG, alkaline phosphataselabeled goat anti-rabbit IgG, and p-nitrophenyl phosphate substrate. Standard curves were based on reactivity with purified CME The adsorption greatly minimized the inherent cross-reactivity of these reagents for various nuts and seeds such as pistachio, sunflower, and almonds. The assay was optimized to detect as little as 20 ng/ml (0.2 ppm) of CMP and was successfully used to quantify CMP in food matrices such as wheat flour, a raisin bran cereal, and milk chocolate candy. Food samples (100 mg) spiked with 10 and 100 ug of CMP yielded between 64% and 136% of the expected value. The influence of various spices on the detection of CMP was shown to be minimal. This quantitative and specific assay should be of value to the food industry and regulatory agencies for the screening of foods to protect allergic patients from unwanted cashew nut exposure. 9 2 9 Anaphylaxis Induced by Pine Nut iua2 Year Old Girl MD Ibahez*, Manuel Lombardero§, A Jimenez*, L Ferndndez*, MT Laso* *Hospital Nifio Jesfis, Madrid, Spain §ALK-AbelIo, Madrid, Spain Pine nuts are the seeds of pine trees (Pinus radiata) and are consumed as toasted nuts or added to a large amount of sauces and vegetables. We describe a 23-months-old girl who was referred because she had suffered two episodes of angioedema of eyelids, lips, facial and feet, exhibiting moderate respiratory difficulty in the last three months. A third episode immediately happened after socking pine nuts and had angioedema, wheezing and severe dyspnea. This time, the patient needed to be treated in the emergency room with subcutaneous epinephrine and intravenous corticosteroids and antihistamines. After the last attack the parents related the first two reactions with the intake of small amount of pine nuts. The patient tolerated the normal food in her diet, including almond, walnut, hazelnut and other nuts. METHODS: Skin prick tests were performed with common allergens (mites, grass, trees and weed pollens, molds, epithelia and latex) and food (peanut, almond, hazelnut, chestnut, walnut, sunflower seed, egg, milk and fish) of commercial origin (ALK Abello, Madrid, Spain) and all of them were negative. Skin prick tests proved to be positive with commercial pine nuts (wheal of 7x6 mm). Prick by prick test with pine nut was positive (wheal of 12x16 mm). Total serum IgE was 70 KU/1 and Specific IgE (CAP System, Pharmacia, Uppsala, Sweden) against peanut, almond, hazelnut, chestnut, walnut, sunflower seed, pistachio, latex, T. echinococcus and inhalants was negative. Specific IgE (RAST using paper disks sensitised to the corresponding extracts) against pine nut was class 3 and negative for pine (Pinus radiata) pollen. An IgE-Immunoblotting of a pine nut extract carried out by ECL after SDS-PAGE without 2-mercapto ethanol showed a unique protein band with an apparent molecular weight of ~ 17kDa. CONCLUSIONS: We describe a very young girl allergic only to pine nut. She suffered very aggressive attacks after eating this nut. We have demonstrated specific lgE to pine nut by prick, prick-prick and RAST. No specific IgE to other nuts and pine pollen was detected. These data suggest that the allergen or allergens implicated in the patient reaction don't have cross reactivity with nuts or pine pollen. A 17-kDa allergen band was detected by immunodetection. 9qn The Glutelin Fraction of Walnut Kernels (Juglans Regia) ConIJwV rains Unique Allergens Not Visible in the Salt Soluble Fraction Tanya V Leshchinsky*, Suzanne S Teuber*, Shridhar K Sathe§, Kenneth H Roux§, Kar Sze-Tao§ *UC Davis, Davis, CA §Florida State University, Tallahassee, FL Most studies characterizing allergens from nuts and seeds have previously focused on identification and cloning of allergens detected in wateror salt-soluble fractions. Walnut, one of the tree nuts most frequently associated with food-induced allergic reactions, is unusual in that the majority of proteins are poorly soluble and are glutelins as classically defined by solubility. We sought to determine if the glutelin fraction contained allergens that were not previously detected in water or salt extractions. Walnuts were defatted with acetone and the dried flour was then extracted sequentially with 1.0 M NaCI (albumin + globulin fraction), then 70% ethanol (prolamin fraction) and finally, 0.1 M NaOH (glutelin fraction). The fractions were then dialyzed against water and lyophilized. IgE immunoblotting was performed against the fractions using sera from 25 adult patients with lifethreatening systemic reactions to walnuts, 16 adult patients with allergic rhinitis and self-reported mild walnut food reactions involving itching, hives or mild angioedema confined to the mouth and face, and 16 patients with pollinosis, peanut allergy or latex allergy who were not tree nut allergic. Twenty of 25 sera (80%) from severely allergic subjects showed IgE binding to a doublet at approximatelyl2 kDa present in the non-reduced glutelin sample that was unaffected by reduction of the sample with dithiothreitol. No sera from patients with mild walnut allergy and only one sera from a patient with pollinosis and latex allergy had IgE binding to this doublet. This doublet was not represented in the albumin, globulin or prolamin fractions. In conclusion, we have detected a major food allergen in walnut kernel that was previously unknown and may not be represented in commercial walnut extracts available for skin testing or used for in vitro specific IgE assays, unless there are similar proteins (of different molecular weight) soluble in salt solutions. Such possibilities are being explored. 3' Quantitative and Qualitative Variability of Protein Content and I Presence of a 56 kDa Allergen in Different Batches of ColdPressed Soy Oil Younes Errahali, Martine Morisset, Denise Anne Moneret-Vautrin, Gis~le Kanny, Maurice Metche, Jean Pierre Nicolas, Sophie Anne Frdmont University Hospital, Vandoeuvre, France Soy allergy currently affects 6% of atopic patients. To date, protein of coldpressed soy oil have been shown to reveal an allergenic potency. However only a few patients showed a clinical reactivity.The purpose of the study is to investigate possible differences in the protein content of several batches of cold-pressed soy oil and to find out whether any of these proteins are allergenic.The proteins were extracted from 3 different batches of cold-pressed soy oils. After SDS-PAGE separation, a western blot analysis was carried out using the serum from a soy-allergic child and the serum of a soy-sensitized patient (RAST = 18.9 kU/L).The amount of protein detected in oils vary from 0.10 to 1.8~tg/rnl. The SDS-PAGE shows a qualitative differences of the protein profiles in the 3 batches. A 56 kDa IgE-binding protein was observed with both serum and an additional 28 kDa allergen was detected using the serum of one patient. Several proteins have been observed in soy oil and two of these were shown to bind IgE. The variability of the concentration of an allergenic protein in some batches of soy oil could explain why soy oil allergy is rarely identified. Whether this allergen is chnically relevant is under scope. |
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