Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture.

Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4. A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value. These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples. This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, "epitopes-ALL," and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.