Characterization of monoclonal antibodies to cytochrome c: analysis of the antigenic structure of holo- and apo-cytochromes, and CNBr-peptide fragments of horse cytochrome c.
Five mouse hybridoma cell lines secreting SA, SB, SC, SD, and SE monoclonal antibodies (McAb) to cytochrome c have been produced. From the cross-reactivities of these McAb with various vertebrate cytochromes c, the antigenic sites for SA and SB McAb were proposed to be at Thr(89)-Glu(92)-Ala(96) and Asn(103), respectively. The binding site for other McAb have not been determined. Cross-reactivity studies based on enzyme-linked immunosorbent assays and dot immunobinding assays indicated that SA, SB, and SC McAb did not bind to apo-cytochrome c nor to any of the three CNBr-peptide fragments. This observation suggests that (i) the antigenic specificity of these McAb is dependent on the conformatiuon of the antigenic site which is inherent to the native holoprotein molecule and (ii) the ordered conformation in the C-terminal regions of holo-cytochrome c is destroyed during CNBr-peptide fragmentation. On the other hand, the lack of binding of SD and SE McAb to apo-cytochrome c indicates that these McAb are also specific for conformational sites. The binding of SD and SE McAb to the heme-containing A-peptide fragment (residues 1-65) suggests that the conformation around the heme, as possible antigenic sites, are stable because of the thioether linkages by the Cys residues.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D000941:Antigens / Q000032:analysis*; D001059:Apoproteins / Q000276:immunology; D002460:Cell Line; D003429:Cross Reactions; D003574:Cytochrome c Group / Q000032:analysis / Q000276:immunology*; D045304:Cytochromes c; D000939:Epitopes; D006736:Horses; D006825:Hybridomas; D051379:Mice
D000911:Antibodies, Monoclonal; D000941:Antigens; D001059:Apoproteins; D003574:Cytochrome c Group; D000939:Epitopes; D045304:Cytochromes c