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 gp91Phox and p22phox 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 gp91Phox. Phage selected by mAb 449 contributed the consensus WRWPGPQVL, resembling in part 182GPQV185 of p22phox. 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.
Burritt
J B
JB
Department of Microbiology, Montana State University, Bozeman 59717, USA. jburritt@gemini.oscs.montana.edu
Fritel
G N
GN
Dahan
I
I
Pick
E
E
Roos
D
D
Jesaitis
A J
AJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D016384:Consensus Sequence; D003573:Cytochrome b Group / Q000737:chemistry / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000097:blood / Q000737:chemistry*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000276:immunology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009504:Neutrophils / Q000737:chemistry*; D019151:Peptide Library; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D021122:Protein Subunits
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D000939:Epitopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D019151:Peptide Library; D010750:Phosphoproteins; D021122:Protein Subunits; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase