Determination of intrinsic affinity constants of monoclonal antibodies against carcinoembryonic antigen

Abstract

A 2-phase method is described for the determination of the intrinsic affinity constants (K-values) for the interaction between monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) and CEA. The Mabs were coupled covalently to CNBr-activated paper discs. MAb coupled discs and a 2-fold dilution series of 125I-CEA were incubated at 20 degrees C until equilibrium was reached. Nonlinear curve-fitting was used for estimation of K-values and different calculation models were thereby tested. The K-values for 12 different anti-CEA MAbs were determined to be 0.3-52 X 10(6) M-1, which is 1-2 orders of magnitude lower than the values obtained in a previous study with some of these MAbs using the ammonium sulphate method to separate free from bound antigen. The K-values obtained by the paper disc method are probably better estimates of the intrinsic association constant than those obtained previously. There are two main reasons for this: (1) free and bound antigen are separated from each other under physiological conditions and (2) the opportunity for multipoint interaction between MAb and CEA is minimized when the MAb is coupled to a rigid carrier substance. Thus, even when the MAb reacts with greater than or equal to 2 epitopes in CEA, as seems to be the case with several of our anti-CEA MAbs, the intrinsic K-value should be obtained. The fundamental validity of 2-phase assays, sometimes questioned in the literature, is demonstrated.