Characterization of functional properties of C4-binding protein by monoclonal antibodies.
We prepared mouse monoclonal antibodies to human C4-binding protein (C4-bp) by fusing spleen cells from mice immunized with purified C4-bp to the mouse myeloma line P3U1. Of four monoclonal antibodies that reacted with intact C4-bp, two were specific for a 48K fragment, one of the chymotryptic cleavage products of C4-bp, and one was specific for another fragment (160K). The fourth monoclonal antibody did not react with either fragment. One of the monoclonals that reacted with the 48K fragment blocked the binding of C4-bp to cell-bound C4b. This monoclonal antibody (TK3) also inhibited two other functions of C4-bp, serving as an essential cofactor for C3b/C4b inactivator (I) in the cleavage of fluid-phase C4b and accelerating the decay of C2a from the C4b,2a complex. The other monoclonals had little or no effect on these activities of C4-bp. In addition, we found that the 48K fragment lost the binding affinity for C4b. However, it can function as a cofactor for I and as a decay-accelerator, although its activities were about 200 times weaker than intact C4-bp on a molar basis. The monoclonal antibody TK3 completely inhibited these activities of the 48K fragment. On the basis of these findings, we conclude that the functionally active site of C4-bp is located on the 48K fragment. Probably, the cofactor and decay-accelerating activities of C4-bp result from the binding of C4-bp to C4b.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D003181:Complement C4 / Q000378:metabolism; D015935:Complement C4b; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs; D006207:Half-Life; D006801:Humans; D016167:Integrin alphaXbeta2; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003181:Complement C4; D016167:Integrin alphaXbeta2; D010446:Peptide Fragments; D015935:Complement C4b