Use of monoclonal antibodies against factor H to investigate the role of a membrane-associated protein antigenically related to H in C3b-receptor function.
Three murine monoclonal IgG1 kappa-antibodies, MAH-1, MAH-2, and MAH-3, were raised against factor H purified from human plasma. In cross-inhibition studies MAH-3 did not compete with MAH-1 and MAH-2, and vice versa, for the binding to H, whereas MAH-1 and MAH-2 inhibited each other. MAH-1 and MAH-2 inhibited the binding of H to C3b attached to an ELISA plate as well as to C3b bound to sheep erythrocytes by means of the classical pathway convertase and of C3b to H attached to an ELISA plate. The determinant defined by MAH-1 and MAH-2 was no longer accessible on H bound to C3b. In contrast, MAH-3 interfere with the binding of H to C3b or vice versa only to a smaller extent but recognized a determinant still accessible on H bound to C3b and was able to agglutinate EAC14o23b-H in an indirect Coombs test. All three antibodies were shown to bind to tonsil cells and Raji cells in an indirect cell ELISA. The membrane-associated molecule detected by these antibodies had an apparent m.w. of 140,000 D in SDS-PAGE. All three antibodies partially inhibited the binding of EAC14o23b to tonsil lymphocytes and, in the presence of 0.1 mM DFP, to Raji cells; binding of EAC14o23bi and EAC14o23d to tonsil cells was not affected. We conclude that MAH-3 recognizes a determinant distinct from the ones recognized by MAH-1 and MAH-2, the latter possibly defining identical epitopes that are located close to the binding site for C3b. The fact that these two distinct epitopes could be detected on a 140,000-D membrane-associated protein from lymphoid cells strongly suggests that this molecule is at least antigenically related to serum H and shares with H a region carrying the binding site for C3b. The rosette inhibition studies imply that this structure is important for the binding of C3b-coated particles to lymphoid cells.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000276:immunology*; D001667:Binding, Competitive; D002460:Cell Line; D003180:Complement C3b Inactivator Proteins / Q000276:immunology* / Q000378:metabolism; D017242:Complement Factor H; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D014066:Palatine Tonsil / Q000166:cytology / Q000276:immunology; D011951:Receptors, Complement / Q000502:physiology*; D017463:Receptors, Complement 3b; D012397:Rosette Formation
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C493495:CFH protein, human; D003180:Complement C3b Inactivator Proteins; D000939:Epitopes; D008565:Membrane Proteins; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D017242:Complement Factor H