Operational aspects of antibody affinity constants measured by liquid-phase and solid-phase assays

The association constant of monoclonal antibodies (Mabs) to tobacco mosaic virus has been determined in solution and solid-phase binding assays. The ELISA equilibrium titration method developed by Friguet et al. (1985) was found to be suitable for large antigens such as viruses. In the case of intact IgG antibody, it gave equilibrium constant (K) values ca 30% lower than those obtained by classical solution-phase assay while in the case of Fab', the same values were obtained in both assays. Solid-phase binding assays gave higher K values than solution-phase assays by a factor which varied with the Mab tested (1.5- to 5.4-fold higher). Furthermore, in solution-phase assay, K values were found to depend on the antibody concentration used in the assay. These results confirm the operational nature of antibody affinity constants and indicate that in order to compare the affinity of different Mabs in a meaningful way, it is necessary to use a single technique under standardized conditions.