Epitope analysis of human monoclonal antibody specific for rice allergenic protein generated by in vitro immunization

We previously established an in vitro immunization protocol for generating antigen specific human monoclonal antibodies (mAbs). In vitro immunization was performed against the soluble protein of rice allergenic protein (RA), resulting in the generation of three B cell clones, AC7-1/F9, CB7-1/E2 and CB7-8/F5, all of which produce a RA-specific human monoclonal IgM antibody. We attempted to map the epitope regions recognized by these mAbs to characterize their specificities. We performed two rounds of epitope mapping, rough mapping using 10-mer peptides covering the full-length RA with 5 amino acids overlapping, and fine mapping using 8-mer peptides covering the putative epitope regions from the rough mapping with 1 amino acid overlapping. As a result of the fine mapping, we identified the epitope regions of these three mAbs as ⁴⁵QVWQDCCRQ⁵⁴L, ⁵⁶AVDDGWCRCGA⁶⁷L and ⁹¹FPGCRRG⁹⁸D on the RA molecule and found to be identical. Furthermore, we determined the putative core epitope regions, which are critical for mAb binding to each region, ⁴⁷WQDCC⁵²R and ⁶⁰GWC⁶³R. The information about the epitope region on the RA molecule, which might trigger the allergenic response, would be useful to establish a specific immunotherapy against rice allergy.