1a. Objectives (from AD-416)
1. Identify cross-reactive epitopes using the peptide SPOTS membrane and serum IgE from patients allergic to peanut, walnut, and almond. 2. Develop models to predict cross-reactive epitopes among the nut allergens. 3. Synthesize free peptides of the cross-reactive epitopes. 4. Determine clinically relevant cross-reactive epitopes.
1b. Approach (from AD-416)
SPOTS membrane analysis will be used to identify cross-reactive epitopes of nuts. The SPOTS data will be used to enhance the accuracy of prediction models. Free peptides of the cross-reactive epitopes will be synthesized and used to compare dissociation constants to allow the highest affinity peptides to be chosen for skin prick tests. Use SPOTS analysis, prediction tools, and inhibition analysis by basophile-histamine release analysis using basophils isolated from nuts to determine clinically relevant cross-reactive epitopes.
3. Progress Report
1) We detected the amino acid (building blocks of proteins) sequences in other allergenic proteins in Structural Data Base for Allergic Proteins (SDAP) similar to known epitopes (antibody binding sites) of Ara h 1 and Ara h 2, by calculating Property Distance (PD) values. The search result is a list of similar sequences identified in allergenic proteins, presented in decreasing order of similarity (increasing PD) with the query sequence. For example, a SDAP search starting from previously identified epitopes of the peanut allergen Ara h 2, indicated closely related sequences in other nut proteins. One strong IgE epitope of Ara h 2 is similar (low PD value between 5 and 8.65) to regions in two other peanut allergens and several areas of the walnut allergen Jug r 2. This indicated that these two proteins might be cross-reactive, even though they are not similar (E> 0.01) according to a Fast-All (FASTA) protein alignment search. 2) We tested whether the areas in the Jug r 2 that were similar, according to the PD search, would also bind IgE from patient sera, and whether they would be better recognized by sera from patients who were reactive to walnut, peanut, or both. Small peptide strips (6 peptides from Ara h 1, Ara h 2 and Jug r 2, previously found to be strong IgE epitopes), were synthesized and used to characterize sera from the large collection of patient samples. Progress is monitored by on-site location coordinator.