Complement Fixation: The Effects of IgG and IgM Antibody Concentration on C1-Binding Affinity

Abstract

When red cells were reacted with increasing amounts of IgG antibody, the number of sites capable of binding C1 increased in a non-linear manner with constantly decreasing rate. At low antibody multiplicities, all of these sites bound C1 with approximately the same affinity, as shown by nearly complete C1 transfer at an ionic strength (μ) close to 0.06. As the amount of antibody increased, a heterogeneity in C1-binding affinities appeared, such that increasing amounts of C1 failed to transfer at μ = 0.059 but did transfer at μ = 0.140. It is suggested that this heterogeneity in C1 binding to IgG may reflect different C1-binding affinities of antibody doublets, triplets, etc. When red cells were reacted with increasing amounts of IgM antibody, the number of C1-binding sites increased linearly. The slope of the plotted curves of these data was close to 1. These sites bound C1 with a range of affinities that was constant, providing all sites were saturated with C1, over a wide range of IgM concentrations. If the antibody:C1 ratio exceeded approximately 1.5:1, a selection in favor of more firmly bound C1 appeared, so that the proportion of cell-bound C1 which transferred at any ionic strength below 0.14 became inversely proportional to the antibody concentration. It is suggested that this heterogeneity in C1 binding to IgM may reflect differences in the number of antibody combining sites which are occupied by antigen.