A simple method for determining K(A)s of both low and high affinity IgG antibodies

A rapid and convenient method for measuring affinity constants (K(A)) of IgG antibody-hapten complexes is described. A key advantage of this method is it suitability for quantification of both low and high affinity interactions. A comparison is made of the K(A)s of the low affinity anti-phosphocholine (PC) antibody T15 (K(A) = 2.9 x 10⁵ M^-1) and five heavy chain complementarity determining region 2 (HCDR2) mutant antibodies expressed as IgG2b transfectants. As a demonstration of the general applicability of the technique, colchicine binding to the high affinity monoclonal IgG2a antibody C44 (K(A) = 1.5 x 10⁹ M^-1) is measured also; thus the assay is applicable over a four-log range of affinities. The assay, based upon use of fixed Staphylococcus aureus Cowan I strain cells as an adsorbent for antibody- radiolabeled antigen complexes, is conducted over a range of hapten concentrations at constant antibody concentration. The K(A) is obtained by Scatchard analysis.