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 com posed of human serum albumin (HSA) and mouse anti-HSA IgG (mIgG). Mixt ures containing varying concentrations of mIgG; and HSA (mIgG, 0-2.4 m u M, and HSA, 3.2 mu M; mIgG, 0.80 mu M, and HSA, 0-6.3 mu M) were inc ubated for 1 h at 37 degrees C. Mixture components were separated usin g 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 s ulfate, Four separate peaks containing either HSA, mIgG, an intermedia te molecular weight complex (C1) or a high-molecular-weight complex (C 2) could be identified from the elution profiles at 280 nm. Plots of p eak areas versus initial mIgG; or HSA concentrations could be modeled mathematically using equations from the law of mass action. Apparent b inding constants (1.1 x 10(7) to 5.2 x 10(7) liters/mol) and stoichiom etry of binding are consistent with that reported in the literature. ( C) 1997 Academic Press.