Analysis of antibody-antigen interactions using size-exclusion high- performance (pressure) liquid chromatography

The development of methods to measure avidity of anti-sera reacting to antigen in solution using size-exclusion high-performance (pressure) liquid chromatography (SE-HPLC) was initiated using a model system composed of human serum albumin (HSA) and mouse anti-HSA IgG (mIgG). Mixtures containing varying concentrations of mIgG and HSA (mIgG, 0-2.4 μM, and HSA, 3.2 μM; mIgG, 0.80 μM, and HSA, 0-6.3 μM) were incubated for 1 h at 37°C. Mixture components were separated using a Bio-Sil TSK 250 size exclusion column (300 x 7.5 mm) equilibrated with 0.05 M sodium phosphate buffer (pH 6.8) containing 0.1 M sodium sulfate. Four separate peaks containing either HSA, mIgG, an intermediate molecular weight complex (C1) or a high-molecular- weight complex (C2) could be identified from the elution profiles at 280 nm. Plots of peak areas versus initial mIgG or HSA concentrations could be modeled mathematically using equations from the law of mass action. Apparent binding constants (1.1 x 10⁷ to 5.2 x 10⁷ liters/mol) and stoichiometry of binding are consistent with that reported in the literature.