Assessment of the functional affinity constant of monoclonal antibodies using an improved enzyme-linked immunosorbent assay

The use of an enzyme-linked immunosorbent assay (ELISA) for the determination of affinity constants implies heterogeneous measurements. Therefore, despite their simplicity, direct solid-phase binding assays are not common. Many investigators have serious, and mostly justified, reservations about the application of solid-phase affinity methods. They refer to problems such as diffusion effects and difficulties in reaching equilibrium due to heterogeneous binding and co-operativity. Accordingly, functional affinity determinations are often described as meaningless. These objections apply to the measurement of the affinity of a monoclonal antibody using the enzyme-linked immunosorbent assay of Beatty et al. (J. Immunol. Methods (1987) 100, 173), which is based on the effect of antibody affinity on the sigmoidal dose response curve. The affinity constant is calculated by mathematical equations, based on the Law of Mass Action and the authors made a number of important assumptions - avoiding the above mentioned problems - in order to justify the use of the Law of Mass Action. By carefully examining these assumptions we have developed an improved ELISA procedure for functional affinity determinations on the basis of a primary coating with the antigen only. The coating conditions were validated by employing gold labelled colloidal particles and physical counting of the bound particles under the scanning electron microscope. Since monovalent binding between human chorionic gonadotropin and its monoclonal antibody could be achieved under equilibrium conditions, the application of the Law of Mass Action and hence of the Beatty formula became possible. We conclude that under these conditions functional affinity determinations are appropriate.