Affinity constants in immunoadsorptive chromatography: their simple determination and application to the selection of eluents in and optimization of the elution process

Abstract

A simple method is describe for the estimation of affinity constants of immobilised antibodies by measuring the ratio of bound antigen to immobilised antibody by the Mancini immunodiffusion technique, and representing the data in the form of a Langmuir adsorption isotherm plot of the reciprocal of the ratio of the bound antigen as abscissa. The slope of the resulting straight line is proportional to the equilibrium constant and the intercept on the ordinate is the reciprocal of the antibody valency. Sheep anti-human IgG immobilised on cyanogen bromide-activated Sepharose 4B® was employed in the measurement of the affinity constant fro the soluble affinity purified human IgG in several potential eluents. The qualitative and the quantitative effect of four potential eluents on human IgC antigen and sheep anti-human IgG antibody incubated in the four eluents for 24 h were also assessed by the Mancini technique. These results emphasise the importance of a knowledge of the affinity constant combined with quantitative data and effect of eluents and buffers on antibodies and antigens in immunoadsorption and their immunochemical procedures.