Concurrent binding to multiple ligands: kinetic rates of CD16b for membrane-bound IgG1 and IgG2.
CD16b (FcgammaRIIIb) is the most common receptor for the Fc domain of IgG on leukocytes. The binding of Fc receptors to immunoglobulin triggers a wide 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 IgG2 was undetectable. Here we report the first measurement of kinetic rates of CD16b binding to membrane-bound IgG isotypes-a physically distinct and physiologically more relevant presentation-using a recently developed micropipette method. In contrast to the soluble data, we found clearly measurable IgG2 binding, with a forward kinetic rate six-fold lower than that of IgG1 but with an equilibrium affinity only threefold lower. This suggests a nonnegligible role for IgG2 in Fc-mediated immune responses, particularly in longer duration contacts. The binding constants were measured from two sets of experiments. Single-isotype experiments were analyzed by an existing model (, Biophys. J. 75:1553-1572). The resulting kinetic rates were used as input to an extended model (, Biophys. J. 79:1850-1857.) to predict the results of mixed-isotype experiments. This design enabled rigorous validation of the concurrent binding model through a test of its predictive ability.
Williams
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George W. Woodruff School of Mechanical Engineering and Georgia Tech/Emory Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0363, USA.
Selvaraj
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Zhu
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D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D006224:Cricetinae; D004912:Erythrocytes / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D017711:Nonlinear Dynamics; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014162:Transfection
D007074:Immunoglobulin G; D008024:Ligands; D017452:Receptors, IgG