Determination of affinity of monoclonal antibodies against human IgG

A method for determining the affinity and the concentration of mouse monoclonal antibodies against human IgG has been developed. The method comprises two steps. First, monoclonal antibodies are allowed to combine with radioiodinated human IgG. Secondly, mouse monoclonal IgG with and without complexed IgG is precipitated with rabbit IgG against mouse IgG. As the antigen is divalent complexes of varying composition are formed in this system leading to deviations from linearity in plots obtained in commonly employed analytical systems. The theoretical background of these systems has been studied by computer simulation, and a concentration effect on the formation of immune complexes was demonstrated. The affinities (in terms of the association constants) of 6 monoclonal antibodies were estimated and found to be in the range from 2 × 106 M−1 to 5 × 108 M−1.