Determination of Allergenic Cross-Reactivity between 11S-Globulins (Seed Storage Proteins) in Peanut, Tree-Nut and Sesame Allergic Patients Using Peptide Microarray Immunoassay

Abstract

RATIONALE: The 11S-globulins of peanut and tree-nuts are highly homologous. Structural similarity among proteins is believed to be the molecular basis for immunological and clinical cross-sensitization, which is common in peanut and tree-nut allergic patients.METHODS: Commercially synthesized peptides covering the 11S-globulin acidic subunit of peanut and the acidic and basic subunits of hazelnut, Brazil nut, cashew, walnut and sesame seed were site-specifically bound to epoxy-derivatized glass slides at two locations in triplicate. Peptides were 20 residues in length with an offset of four. A total of 102 sera from patients allergic to at least one tree-nut or peanut were assayed. Sera from 10 non-atopic volunteers and 13 atopic but not peanut/tree-nut allergic individuals served as negative controls. Specific IgE- and IgG4- binding was detected using fluorochrome-labeled monoclonal secondary antibodies.RESULTS: We mapped IgE and IgG4 epitopes of multiple 11S allergens in parallel by microarray immunoassay. Seven cross-reactive antigenic regions were defined by shared recognition between at least two of the six homologues. Unexpectedly, these regions did not coincide with regions of highest identity. Extrapolating from the soy 11S structure, the majority of these sites are expected to be on the surface of the native protein.CONCLUSIONS: Clinical cross-reactivity between peanut and nut allergic individuals is not explained by primary sequence homology of 11S-globulins, as determined by peptide microarray analysis. RATIONALE: The 11S-globulins of peanut and tree-nuts are highly homologous. Structural similarity among proteins is believed to be the molecular basis for immunological and clinical cross-sensitization, which is common in peanut and tree-nut allergic patients. METHODS: Commercially synthesized peptides covering the 11S-globulin acidic subunit of peanut and the acidic and basic subunits of hazelnut, Brazil nut, cashew, walnut and sesame seed were site-specifically bound to epoxy-derivatized glass slides at two locations in triplicate. Peptides were 20 residues in length with an offset of four. A total of 102 sera from patients allergic to at least one tree-nut or peanut were assayed. Sera from 10 non-atopic volunteers and 13 atopic but not peanut/tree-nut allergic individuals served as negative controls. Specific IgE- and IgG4- binding was detected using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: We mapped IgE and IgG4 epitopes of multiple 11S allergens in parallel by microarray immunoassay. Seven cross-reactive antigenic regions were defined by shared recognition between at least two of the six homologues. Unexpectedly, these regions did not coincide with regions of highest identity. Extrapolating from the soy 11S structure, the majority of these sites are expected to be on the surface of the native protein. CONCLUSIONS: Clinical cross-reactivity between peanut and nut allergic individuals is not explained by primary sequence homology of 11S-globulins, as determined by peptide microarray analysis.