Mapping of the IgE and IgG4 sequential epitopes of milk allergens with a peptide microarray–based immunoassay

BackgroundPeptide microarray analysis is a novel method that can provide useful information on the nature of specific allergies.ObjectiveWe sought to determine the specificity and diversity of IgE and IgG4 antibodies binding to sequential epitopes of αs1-, αs2-, β-, and κ-caseins and β-lactoglobulin by using a peptide microarray–based immunoassay.MethodsA microarray immunoassay was performed with sera from 31 children with IgE-mediated milk allergy (16 with positive oral milk challenge results [ie, the reactive group] and 15 with negative oral milk challenge results [ie, the tolerant group]). A library of peptides, consisting of 20 amino acids (AAs) overlapping by 17 (3-offset), corresponding to the primary sequences of αs1-, αs2-, β-, and κ-caseins and β-lactoglobulin was printed on epoxy-coated slides. A region was defined as an epitope if it was statistically associated with reactive groups and recognized by at least 75% of reactive patients.ResultsBy using this method, a total of 10 epitopes were identified: αs1, AAs 28 to 50, 75% reactive and 26.7% tolerant; αs2, AAs 1 to 20, 75% reactive and 13.3% tolerant; AAs 13 to 32, 75% reactive and 26.7% tolerant; AAs 67 to 86, 75% reactive and 33.3% tolerant; and AAs 181 to 207, 75% reactive and 20% tolerant; β-casein, AAs 25 to 50, 75% reactive and 33.3% tolerant, AAs 52 to 74, 81.3% reactive and 26.7% tolerant; and AAs 154 to 173, 75% reactive and 33.3% tolerant; β-lactoglobulin, AAs 58 to 77, 81.3% reactive and 40% tolerant; and κ-casein, AAs 34 to 53, 87.5% reactive and 40% tolerant.ConclusionSeveral regions have been defined as epitopes, which showed differential recognition patterns between reactive and tolerant patients. Further studies are needed to validate the utility of this assay in clinical practice.