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Tree nut-induced allergies are typically severe and are usually not outgrown. The IgE immunoglobulin (antibody) class binds to specific regions on the allergen proteins (termed epitopes) leading to allergic symptoms in patients. The relative ratio of linear (sequential, continuous) to conformational (discontinuous, 3-D) epitopes probably varies significantly between allergens and may correlate with patient symptom severity. Many tree nut allergens have been subjected to linear epitope characterization but far less is known about conformational epitopes. To address these issues, we propose to 1) identify and map conformational epitopes on the otherwise well characterized allergens from cashew and almond nuts for comparison to previously mapped linear epitopes from these nut proteins. Aim 2) is to assess the relative contribution of conformational and linear epitopes to the IgE-reactivity of these proteins. Our final aim, 3), is to assess the relative stability of the conformational epitopes when these almond and cashew proteins are subjected to conditions comparable to those that would be encountered during food processing to see if food processing can be used to lessen allergenic potential.
Non-Technical Summary: Tree nut-induced allergies are typically severe and are usually not outgrown. Food allergies occur when a particular class of antibodies, known as IgE, binds to otherwise harmless molecules in certain foods and trigger the cells responsible for causing the allergic symptoms. It is not know why some foods are more prone to causing such reactions. Our research is directed at identifying and describing the specific IgE contact regions on tree nuts proteins that are responsible for tree nut allergies. Specific proteins from almond and cashew will be analyzed to investigate the two main types or reactive sites - those that are dependent on the precise folding of the protein and those that are directed against the uncoiled protein backbone. Antibodies from patients' sera will be used in several assays to investigate the characteristics of the two classes and various food processing methods will be tested to see if problem regions on the proteins can be eliminated. <P> Approach: Allergen epitope mapping will be accomplished by applying molecular genetic mutational analyses of recombinant allergen molecules, and state-of-the-art phage peptide-display mimotope and hydrogen/deuterium exchange (HDX) epitope mapping techniques. To determine the ratio of patients' IgE antibodies recognizing the two forms of epitopes, we will use affinity chromatography to separate the two forms of antibodies and will test them by ELISA and western blot assays. The stability (durability) of the identified epitopes will be assessed by subjected the nut allergen proteins to various chemical and physical denaturing conditions.