Epitope identification for human neutrophil flavocytochrome b monoclonals 48 and 449

Flavocytochrome b(558) (CYt b) is important in generating superoxide and ot her toxic oxygen species involved in inflammation and host defense. Monoclo nal 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 com plex. Until now, data were unavailable to predict which regions of the prot ein were bound by each antibody. Random sequence phage-display peptide libr ary analysis of each antibody was used to select peptides that mimic the se quence of each protein epitope. Phage sequences selected by mAb 48 presente d the consensus peptide sequence DRDVXTGL, which closely resembles (498)EKD VITGL(505) Of gp91(phox). Phage selected by mAb 449 contributed the consens us WRWPGPQVL, resembling in part (182)GpQV(185) Of p22(phox). Confirmation for this second epitope was provided by peptide walking analysis. Identifyi ng the protein residues bound by these antibodies makes each a more informa tive probe for Cyt b analysis.