Anti-idiotypic antibodies in antisera against human C3 and factor H and their application in the enrichment of antibodies specific for H-binding domains of C3.
Antisera separately raised against C3 and factor H should contain antibodies against the binding domains by which these factors interact. Because these interacting domains are likely to be sterically complementary to each other, antibodies specific for these domains should also be sterically complementary or of anti-idiotypic specificity. To test this hypothesis, rabbit anti-human C3 IgG antibodies which bound to Sepharose-coupled rabbit anti-factor H IgG (anti-H-binding anti-C3) were separated by affinity chromatography. In a control experiment, the anti-human factor H column was found to bind 10 times more anti-C3 antibodies than a Sepharose column coupled with nonimmune rabbit IgG. The binding specificity of the control eluate was indistinguishable from the original anti-C3 preparation, whereas the anti-H-binding anti-C3 was shown to be enriched in regard to antibodies directed against the 42 kd fragment of the C3 alpha-chain of C3c, as demonstrated by immunoblotting of electrophoretically separated polypeptides of C3c and C3d in polyacrylamide gels. In competition binding studies, factor H was 25 times more potent as inhibitor of the anti-H-binding anti-C3 than of the original anti-C3 preparation. This similarity in binding specificity also paralleled a functional similarity in that anti-H-binding anti-C3 in the presence of factor I effected a 20% degradation of the alpha'-chain of C3b into fragments of 65, 45, and 42 kd which are the normal degradation products in the presence of factors I and H. These results suggest that the affinity procedure described resulted in the enrichment of anti-C3 antibodies with selective specificities for factor H-binding domains of C3, from a polyclonal antibody source.
D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D003176:Complement C3 / Q000276:immunology*; D003180:Complement C3b Inactivator Proteins / Q000276:immunology*; D017242:Complement Factor H; D017244:Complement Factor I; D010450:Endopeptidases / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000276:immunology*
C493495:CFH protein, human; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D000939:Epitopes; D007130:Immunoglobulin Idiotypes; D017242:Complement Factor H; D010450:Endopeptidases; D017244:Complement Factor I