Alliance for food Protection, NE

Non-Technical Summary: This project has two major aspects related to food allergy. The first objective is to develop detection methods for residues of two commonly allergenic foods - buckwheat and pistachio. This group has previously develop detection methods for peanut, milk, egg, other tree nuts, soybeans, shrimp, clam, mustard, and lupine so these two additional methods help to complete the set needed by the food industry. The industry can uses to determine if its cleaning practices are sufficient or whether the use of shared equipment presents potential hazards to allergic consumers. The new buckwheat and pistachio assays will give food processors sensitive and specific techniques to detect inadvertent contamination of their raw materials and finished products with residues of either of these allergenic foods. Ultimately, the antibodies used in the pistachio detection method may be useful in the development of a multi-plex tree nut ELISA as we already have antibodies for walnut, almond, hazelnut, pecan, and cashew (but Brazil nut, macadamia nut, and pine nut are also missing). The second objective relates to the refinement of methods for the assessment of the potential allergenicity of genetically modified or other novel foods. These methods are needed to assess the potential risk and prevent the unintended introduction of a major allergen or protein that would likely cause allergic cross-reactions in some consumers. Bioinformatics approaches (computer assisted sequence comparison analysis) have been developed as one of the most important tools to identify potentially allergenic proteins. AllergenOnline is a public database created through the efforts of the Food Allergy Research and Resource Program (FARRP) at the University of Nebraska. The database provides a publicly available, searchable list of protein sequences of known allergens and bioinformatics strategies useful in comparing these sequences to those of novel proteins. FARRP will continue to screen the literature to identify new allergens so that the list can be annually updated. This database is used by academic and government scientists developing genetically engineered and other novel foods, by regulatory officials in many countries, and by the agricultural biotechnology industry in a key step in the overall allergenicity assessment process. As part of this project, we will assess the validity of the search criteria of 35% identity over any 80 amino acid segment as a predictor of allergenicity through serum testing of legume allergic subjects, using allergic sera. IgE-binding proteins will be separated, identified, and compared to known allergens in the database. <P> Approach: Antisera will be made against buckwheat and pistachio using standard procedures in rabbits, goats, and sheep. Two formats, a competition ELISA and a double antibody, sandwich-type ELISA will be tried. Sandwich-type ELISAs have been developed successfully for the detection of undeclared peanut, casein, egg, almond, walnut, hazelnut, cashew, sesame seed, shrimp, clam, lupine, and mustard proteins in food products in our laboratory. The sensitivity of the ELISAs will be determined using standard curves prepared with protein extracts from the specific foods with targeted detection limit of 1-2 ppm in various food systems. Regression analysis will be performed in the interpretation of the ELISA results. Once an assay with suitable sensitivity has been developed, the recovery of buckwheat spiked into wheat flour will be assessed, while the recovery of pistachio spiked into a cake mix will be assessed to determine if the presence of other food components will affect the sensitivity of the assay. The specificity of the ELISAs will be assessed by subjecting numerous components and ingredients of food and food products to rigorous testing in the ELISAs to evaluate any cross-reactions or non-specific interference using a broad assortment of foods and food ingredients. Since food processing operations (heat, acidity, etc.) can affect the extractability of food proteins or their detectability in immunoassays, buckwheat and pistachio, respectively, will be incorporated into foods at various levels, processed according to typical industry standards, and analyzed by the ELISAs to determine if processing will affect the utility of the ELISAs. The structural criteria to identify novel proteins as potential allergens will be assessed. The validity of the search criteria of 35% identity over any 80 amino acid segment will be evaluated for predictive value by serum testing of legume allergic subjects, using sera from the US, EU and India. IgE binding proteins will be identified. Cross-reactive proteins identified by either overall sequence homology or by the 35% identity over any 80 amino acid segment criteria will be compared among legumes based on IgE binding. Cross-reactive proteins from legumes will be evaluated for sequence identity by separating proteins using 2-dimensional gel electrophoresis and sending them for peptide sequencing by Liquid Chromatography and Maldi MS/MS. Further analysis will be performed by matching sequences to the NCBI website and comparing to known allergens. The current version of AllergenOnline allergen sequence database will be updated with the anticipated January 2009 release of version 9.0. The criteria for the inclusion of proteins as allergens in the database are: Protein (gene) isolated from a documented allergenic source, study subjects with described allergic symptoms consistent with exposure, specific IgE testing included controls and characterized test materials. Details of relevant allergenicity assessment data will also be retained in an archive e.g., the number of allergic subjects that tested positive to the protein, type of testing (qualitative or quantitative IgE binding, challenge tests).