Measuring two-dimensional receptor-ligand binding kinetics by micropipette

We report a novel method for measuring forward and reverse kinetic rate constants, k(f)(o) and k(r)(o), for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion, Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunogobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are A(c)k(f)(o) = (2.6 +/- 0.32) x 10(-7) mu m(4) s(-1) and k(r)(o) = (0.37 +/- 0.055) s(-1) for the CD16A-hIgG interaction and A(c)k(f)(o) = (5.7 +/- 0.31) x 10-7 mu m(4) s(-1) and k(r)(o) = (0.20 +/- 0.042) s(-1) for the CD16A-rIgG interaction, respectively, where A(c) is the contact area, estimated to be a few percent of 3 mu m(2).