Epitope identification for human neutrophil flavocytochrome b monoclonals 48 and 449

Flavocytochrome b₅₅₈ (Cyt b) is important in generating superoxide and other toxic oxygen species involved in inflammation and host defense. Monoclonal antibodies (mAbs) 48 and 449 bind the gp91(phox) and p22(phox) subunits of Cyt b, respectively, and have been used to characterize this enzyme complex. Until now, data were unavailable to predict which regions of the protein were bound by each antibody. Random sequence phage-display peptide library analysis of each antibody was used to select peptides that mimic the sequence of each protein epitope. Phage sequences selected by mAb 48 presented the consensus peptide sequence, DRDVXTGL, which closely resembles ⁴⁹⁸EKDVITGL⁵⁰⁵ of gp91(phox). Phage selected by mAb 449 contributed the consensus WRWPGPQVL, resembling in part ¹⁸²GPQV¹⁸⁵ of p22(phox). Confirmation for this second epitope was provided by peptide walking analysis. Identifying the protein residues bound by these antibodies makes each a more informative probe for Cyt b analysis.