Identification of IgE sequential epitopes of lentil (Len c 1) by means of peptide microarray immunoassay

Background: Lentils are often responsible for allergic reactions to legumes in Mediterranean children. Although the primary sequence of the major allergen Len c 1 is known, the location of the IgE-binding epitopes remains undefined. Objective: We sought to identify IgE-binding epitopes of Len c 1 and relate epitope binding to clinical characteristics. Methods: One hundred thirty-five peptides corresponding to the primary sequence of Len c 1 were probed with sera from 33 patients with lentil allergy and 15 nonatopic control subjects by means of microarray immunoassay. Lentil-specific IgE levels, skin prick test responses, and clinical reactions to lentil were determined. Epitopes were defined as overlapping signal above interslide and intraslide cutoffs and confirmed by using inhibition assays with a peptide from the respective region. Hierarchic clustering of microarray data was used to correlate binding patterns with clinical findings. Results: The patients with lentil allergy specifically recognized IgE-binding epitopes located in the C-terminal region between peptides 107 and 135. Inhibition experiments confirmed the specificity of IgE binding in this region, identifying different epitopes. Linkage of cluster results with clinical data and lentil-specific IgE levels displayed a positive correlation between lentil-specific IgE levels, epitope recognition, and respiratory symptoms. Modeling based on the 3-dimensional structure of a homologous soy vicilin suggests that the Len c 1 epitopes identified are exposed on the surface of the molecule. Conclusion: Several IgE-binding sequential epitopes of Len c 1 have been identified. Epitopes are located in the C-terminal region and are predicted to be exposed on the surface of the protein. Epitope diversity is positively correlated with IgE levels, pointing to a more polyclonal IgE response.