<OL> <LI> To examine the digestion stability of food allergens including to (a) identify intact allergens and digestion-resistant peptides/fragments after in vitro digestion, and (b) determine and compare the structure of intact allergens and digestion-resistant fragments after in vitro digestion. <LI> To assess the allergenicity of fish and ingredients derived from fish including to (a) evaluate the comparative content and antigenicity of parvalbumin (major fish allergen) from various species of fish, and (b) determine the effects of thermal processing on the structure and antigenicity of parvalbumin. <LI> To evaluate and improve various approaches used to assess the potential allergenicity of novel proteins that may occur in genetically modified and other novel foods. Specifically, this project will provide partial support to allow the development of version 10 of the AllergenOnline database, a publicly available database of known allergen sequences.<LI> To develop and maintain research support databases containing over 15,000 published journal articles on food allergy which provide food industry and worldwide researchers, and the FARRP research team with over critical information on food allergen research.</OL> The outputs of this project will include the publication of critical information on the allergenicity of various fish species and fish-derived ingredients widely used in foods, the publication of critical information on the digestion stability of intact allergens and digestion-resistant fragments and characterization of structural properties that aid in their stability and allergenicity, the release of AllergenOnline version 10 allergen sequence database on the AllergenOnline web site.
Non-Technical Summary: This project has four main aspects related to food allergy. The first objective is to examine the digestive stability of food allergens and identify key structural components that may aid in their stability. Resistance to gastrointestinal digestion is believed to be a key characteristic of many food allergens due to the preservation of the protein structure. Knowledge of common structural characteristics of digestion-resistant food allergens may help to gain insight into the molecular basis of allergenicity of proteins. The second objective is to evaluate the major fish allergen, parvalbumin, by investigating the effects of species, calcium, thermal processing, and Maillard reactions on the antibody binding characteristics. The stability of parvalbumin and its IgE-binding ability under heating conditions will allow us to determine if cooked or heat-processed fish is as hazardous as raw fish. Ultimately, these results will allow better advice to be given to fish-allergic consumers regarding the hazards posed by various species of fish and fish processed in various ways. The third objective is to evaluate and improve methods for the assessment of the allergenicity of novel proteins and to build and curate version 10 of AllergenOnline which is a public database created through the efforts of the Food Allergy Research and Resource Program (FARRP) at the University of Nebraska. AllergenOnline has been very successful in recent years, providing the only peer-reviewed list of allergen sequences. We have a panel of internationally recognized allergy experts who set the criteria and will review the final list of allergens for possible inclusion in version 10 of AllergenOnline. Continuing to refine the criteria and reviewing the data supporting entries greatly enhances the quality and utility of the allergen database to help ensure a minimal risk of unnecessary allergenicity in future food products. The fourth objective is to continue the development and maintenance of research support databases. The Food Allergy Research & Resource Program currently maintains the published literature databases containing nearly 15,000 entries. The information in these databases can be accessed by food industry researchers and worldwide researchers as well as the FARRP research team. Development of an improved web site could enhance scholarly reviews, industry advice and other useful features that are created primarily from the publications databases. <P> Approach: This project will expand the current knowledge related to four major aspects related to food allergy. (1) The digestion stability of known food allergens will be assessed. Protein from raw or roasted seed or tree nuts will be extracted then isolated and purified using ammonia sulfate precipitation and a series of size exclusion, ion exchange, and hydrophobic interaction chromatography utilizing FPLC or HPLC. Protein fractions will be monitored using SDS-PAGE and sequence analysis will be conducted using liquid chromatography and tandem mass spectroscopy (LC/MS/MS). Purified allergens will undergo tryptic digestion at 37C for up to four hours. Digestion stability of these allergens will be monitored using SDS-PAGE, and LC/MS/MS will be used to identify the sequence of resistant proteins and peptides. The secondary structural characteristics and differences of purified native proteins and digestion-resistant proteins and peptides will be assessed using circular dichroism (CD) spectroscopy. (2) The allergenicity of fish and ingredients derived from fish will be assessed by examining antibody binding characteristics of the major fish allergen, parvalbumin, using SDS-PAGE and immunoblotting techniques to investigate if the parvalbumin content and isotypes contribute to the differences in antibody binding. Parvalbumin isotypes will be identified with 2D gel electrophoresis and LC/MS/MS. The effects of thermal processing treatments, calcium concentration, and Maillard reactions on reactivity of parvalbumin-specific IgG and IgE will be evaluated using indirect ELISA methods. (3) Bioinformatics approaches (computer assisted sequence comparison analysis) used to assess the potential allergenicity of novel proteins that may occur in genetically modified and other novel foods will continue to be developed and improved with the release of version 10 of the AllergenOnline database, a publicly available database of known allergen sequences. Proteins identified as allergens in scientific publications will be evaluated by a panel of allergy experts for inclusion in AllergenOnline. Criteria for inclusion of proteins as allergens include: 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. The current version of AllergenOnline will be updated with the anticipated January 2010 release of version 10. (4) The literature on food and other allergens is pivotal to the identification of proteins that should be evaluated for possible inclusion in the AllergenOnline database. Various library databases (e.g. Medline, Toxline, etc.) and Current Contents are screened continuously for publications that merit inclusion into the research support publications database. Each publication is then reviewed and key words are assigned to allow its retrieval during searches on specific topics. The publication is entered into the database which is maintained by the ProCite software package. Literature in this database is used to develop review articles, web site reviews and other materials on food allergens.