Comparisons of pooled polyclonal rabbit anti-human C3d with four monoclonal mouse anti-human C3ds. II. Quantitation of RBC-bound C3d, and characterization of antiglobulin agglutination reactions against RBC from 27 patients with autoimmune hemolytic anemia

Abstract

Labelled polyclonal IgG anti-C3d and monoclonal IgM and IgG anti-C3d antibodies (MAs) were employed at increasing antibody excess to measure the number of C3d molecules on human red blood cells (RBC) coated by complement in vitro and in vivo. Values for the number of C3d sites per cell determined with polyclonal anti-C3d were at least 4-fold higher than when MAs were used. The results suggest that the molar combining ratio for polyclonal anti-C3d with a single RBC-bound C3d fragment is more likely greater than 4.0 than 1.0 as previously assumed. Antiglobulin agglutination studies compared polyclonal and monoclonal anti-C3d antibodies against C3d-coated RBC from 27 patients with autoimmune hemolytic anemia. All four MAs showed striking prozones, requiring their use over a 25-fold higher range of dilutions than polyclonal anti-C3d. Polyclonal anti-C3d produced stronger agglutination reactions than any of the IgG MAs. Only the IgM MA produced agglutination as strong as, or stronger than, polyclonal anti-C3d. While IgM MA always gave the strongest MA agglutination reactions, no consistent ranking of the three IgG MAs was observed. Agglutination was not enhanced when all IgG MAs were combined; addition of IgG MAs to IGM MA reduced the strength of agglutination seen with IgM alone, suggesting blocking of IgM binding by competing IgG anti-C3d.