title: The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.
abstract: The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000502:physiology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D003166:Complement Activating Enzymes / Q000276:immunology / Q000378:metabolism; D011415:Complement Factor B / Q000378:metabolism; D011416:Complement Factor D / Q000276:immunology / Q000378:metabolism; D003170:Complement Pathway, Alternative; D006461:Hemolysis; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D009842:Oligopeptides / Q000378:metabolism; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D003166:Complement Activating Enzymes; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B