Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in...

Identification of allergenic IgE epitopes is instrumental for the development of novel diagnostic and prognostic methods in food allergy. In this work, we present the quantification and validation of a Bead-Based Epitope Assay (BBEA) that through multiplexing of epitopes and multiple sample processing enables completion of large experiments in a short period of time, using minimal quantities of patients' blood. Peptides that are uniquely coupled to beads are incubated with serum or plasma samples, and after a secondary fluorophore-labeled antibody is added, the level of fluorescence is quantified with a Luminex reader. The signal is then normalized and converted to epitope-specific antibody binding values. We show that the effect of technical artifacts, i.e. well position or reading order, is minimal; and batch effects - different individual microplate runs - can be easily estimated and eliminated from the data. Epitope-specific antibody binding quantified with BBEA is highly reliable, reproducible and has greater sensitivity of epitope detection compared to peptide microarrays. IgE directed at allergenic epitopes is a sensitive biomarker of food allergy and can be used to predict allergy severity and phenotypes; and quantification of the relationship between epitope-specific IgE and IgG4 can further improve our understanding of the immune mechanisms behind allergic sensitization.