Determination of Soluble Immune Complex Molar Composition and Antibody Association Constants by Ammonium Sulfate Precipitation

Abstract

The interaction between rabbit IgG anti-HSA and HSA was studied by ammonium sulfate precipitation of the complexes formed throughout a wide range of antigen excess. The Ag/Ab molar ratio varied between 0.85 and 1.20 in the range of five to 60 times antigen excess. In greater degrees of antigen excess (up to 500-fold) the molar ratio of the complexes remained at a plateau between 1.20 and 1.25. Identical results were obtained using antibodies isolated by immunoadsorbent techniques or using antibodies in antiserum. The molecular composition of the limiting complexes was primarily Ag₂Ab₂ and Ag₁Ab₁, but a small amount of Ag₂Ab₁ could have been present. The association constant between anti-HSA and HSA was determined with isolated antibodies and with anti-HSA in antiserum. The association constants were similar, ranging from 2.19 × 10⁷ to 3.20 × 10⁷ liters/mole. However, the values for the association constant were partially dependent upon the absolute amounts of antigen and antibodies present and upon the relative amounts of each.