1a. Objectives (from AD-416)
Objective 1: Identify and compare the structural, chemical, functional, and immunological characteristics of peanut with homologous; less allergenic legume (green pea and soy) and tree nut allergens in raw and processed forms towards delineating the clinically-relevant antibody-allergen interactions. Objective 2: Clone, express, and purify the major peanut and select tree nut and legume allergens, and fragments thereof, in recombinant form to further delineate clinically-relevant antibody-allergen interactions. Objective 3: Assess the role of processing-induced chemical or structural modifications on the individual allergens by systematically altering amino acids thought to be important in clinically-symptomatic allergic reactions. Objective 4: Develop computer models and/or determine NMR/crystal structures of native and recombinant peanut and select tree nut and legume allergens in raw and processed forms. Objective 5: Combine the structural information obtained with the empirical knowledge from Objectives 1, 2, and 3 to identify clinically-relevant allergen-antibody interactions in peanut and tree nut allergy. Objective 6: Develop processing technologies for peanut products with reduced allergenic properties. Objective 7: Develop and improve immunoassays for detection of peanut, select tree nut, and soy allergen residues before and after processing (i.e. roasting, baking into cookies, etc.). Objective 8: After establishing standardized protocols for determining threshold doses for peanut, select tree nut, and soy allergens, determine threshold (minimal eliciting) dose of reactivity for processed forms of peanut allergens and develop computational and statistical models to estimate population thresholds.
1b. Approach (from AD-416)
Specifically, peanuts and tree nuts will be subjected to thermal processing (i.e., roasting). New allergens or changes in allergenic properties of existing allergens due to the thermal processing will be identified by immunoassays, using serum (containing IgE antibodies) from peanut and/or tree nut allergic individuals. Proteins found to be immunologically altered by thermal processing will be purified by conventional chromatography and analyzed for alterations in size, structure, digestibility, and binding to various antibodies, including anti-Maillard reaction products and specific anti-allergen antibodies. The specific amino acid residues, thought to be modified during different processing events and to contribute to altered allergenic properties (i.e. IgE binding), will be identified. These amino acids will be identified by cloning and expression of select recombinant major allergens of peanut and tree nuts in E. coli followed by site directed mutagenesis, simulated processing and immunological analysis of previously identified IgE binding sites, specifically, sites thought to be modified by processing. Understanding the molecular basis of processing-induced alterations of allergens will guide development of processing technologies towards reduced allergenicity of nuts. This knowledge will also contribute to the development of better labeling practices and detection tools for industry and regulatory agencies resulting in better protection of consumers.
3. Progress Report
Preliminary results show that structural or folded, human immunoglobulin (IgE, the main mediator of allergic reaction), binding sites (epitopes) of the allergic proteins are more important than linear epitopes; and that enhanced IgE binding to roasted allergens of peanut are most likely due to chemical modifications. These results indicate that targeted processing methods that unfold proteins are likely to reduce the allergenic potential or peanuts and tree nuts. Various processing methods were tested for causing alterations in the allergenic properties of peanuts and tree nuts. The efficacy of ribose on peanut maturity and allergenicity were assessed. Ribose-treated mature and immature peanut extracts were shown to be different in levels of by-products and allergenic potency, suggesting that ribose could be used for assessment of peanut maturity and allergenicity. HP and PUV were tested to determine usefulness to reduce the allergenic potency of almond extracts. Preliminary results showed that HP had no effect, which means that further optimization of the experiment may be; however, PUV showed promising results in reducing the allergenic potency of almond extracts. A similar result was also obtained with shrimp extracts in a collaborative study. Pistachio allergenicity was reduced in humans as shown by skin prick tests following soaking in lemon (acid treatment). Peanut and tree nut allergens (recombinant and native) are in various stages of cloning, expression, and purification towards understanding their structure and allergenicity. For example, production of the 2S albumin proteins (identified as the most potent family of allergens) from walnut, cashew, and soy bean in bacteria is being optimized. Also, major peanut allergen, Ara h 1 was purified in raw, roasted, and recombinant (produced in bacteria) form and examined for immunological, structural, and allergenic properties. Structure and models of allergens are being developed to determine the characteristics most important for allergenicity and potential targets for processing. Chromatographic protocol for separation of D- and L-amino acids (different orientations of a single amino acid) was developed. Difference between raw and roasted samples in D-amino acids was inconclusive. Optimization of the assays is continuing.
1. Use of magnetic beads to separate peanut allergins. Allergy to peanuts can be very severe and is rarely outgrown. ARS scientists at New Orleans, LA, are testing methods to reduce allergenic properties of peanuts. Magnetic beads with different functional groups were used to bind and partially remove allergens from peanut extracts. As a result, the allergenic capacity of the peanut samples was reduced. The research could lead to development of less allergenic peanut-based products and beverages.
2. Association between immunological response and protein structural properties. Methods were developed to unfold purified peanut allergens. These non-native proteins were tested with native (folded) proteins for their ability to bind to IgE, an interaction that is necessary for establishing an immune response. ARS scientists at New Orleans, LA, found that protein folding of the allergens is an important contributor to patient serum IgE binding response. Therefore, if the allergens are unfolded by a processing treatment, e.g., they might be less allergenic, which may allow for a broader use of peanuts in processed foods.
3. Method to detect walnut residue in foods using enzyme-linked immuosorbent assay. Cross contamination of food products with allergenic nuts causes many accidental allergic reactions and losses due to food recalls. ARS scientists at New Orleans, LA, together with collaborators from the University of Nebraska (Lincoln), an assay was developed to detect walnut proteins in food products. Using immunological techniques, we optimized the test for the detection of walnut residue in foods. After additional validated, this method should be useful as a tool for industry and regulators to test for walnut contamination.
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