Concurrent binding to multiple ligands: Kinetic rates of CD16b for membrane-bound IgG1 and lgG2

CD16b (Fc gamma RIIIb) is the most common receptor for the Fc domain of IgG on leukocytes. The binding of Fc receptors to immunoglobulin triggers a wi de array of immune responses. In published assays measuring the reaction of CD16b with isotypes of soluble IgG, the affinity for IgG1 was low and that for lgG2 was undetectable. Here we report the first measurement of kinetic rates of CD16b binding to membrane-bound IgG isotypes-a physically distinc t and physiologically more relevant presentation-using a recently developed micropipette method. in contrast to the soluble data, we found clearly mea surable lgG2 binding, with a forward kinetic rate six-fold lower than that of IgG1 but with an equilibrium affinity only threefold lower. This suggest s a nonnegligible role for lgG2 in Pc-mediated immune responses, particular ly in longer duration contacts. The binding constants were measured from tw o sets of experiments. Single-isotype experiments were analyzed by an exist ing model (Chesla et al., 1998, Biophys. J. 75:1553-1572), The resulting ki netic rates were used as input to an extended model (Zhu and Williams, 2000 , Biophys. J. 79:1850-1857.) to predict the results of mixed-isotype experi ments. This design enabled rigorous validation of the concurrent binding mo del through a test of its predictive ability.