Development of a novel peptide microarray for large-scale epitope mapping of food allergens

Background: The peptide microarray is a novel assay that facilitates high-throughput screening of peptides with a small quantity of sample. Objective: We sought to use overlapping peptides of milk allergenic proteins as a model system to establish a reliable and sensitive peptide microarray-based immunoassay for large-scale epitope mapping of food allergens. Methods: A milk peptide microarray was developed by using commercially synthesized peptides (20-mers, 3 offset) covering the primary sequences of α_s1-casein, α_s2-casein, β-casein, κ-casein, and β-lactoglobulin. Conditions for printing and immunolabeling were optimized using a serum pool of 5 patients with milk allergy. Reproducibility of the milk peptide microarray was evaluated using replicate arrays immunolabeled with the serum pool, whereas specificity and sensitivity were assessed by using serial dilution of the serum pool and a peptide inhibition assay. Results: Our results show that epitopes identified by the peptide microarray were mostly consistent with those identified previously by SPOT membrane technology, but with specific binding to a few newly identified epitopes of milk allergens. Data from replicate arrays were reproducible (r ≥ 0.92) regardless of printing lots, immunolabeling, and serum pool batches. Using the serially diluted serum pool, we confirmed that IgE antibody binding detected in the array was specific. Peptide inhibition of IgE binding to the same peptide and overlapping peptides further confirmed the specificity of the array. Conclusion: A reliable peptide microarray was established for large-scale IgE epitope mapping of milk allergens, and this robust technology could be applied for epitope mapping of other food allergens.