MEASURING 2-DIMENSIONAL RECEPTOR-LIGAND BINDING-KINETICS BY MICROPIPETTE

We report a novel method for measuring forward and reverse kinetic rat e constants, k(f)(o) and k(r)(o), for the binding of individual recept ors and ligands anchored to apposing surfaces in cell adhesion, Not on ly 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 ext ension of existing micropipette protocols. The analysis was based on a nalytical solutions to the probabilistic formulation of kinetics for s mall systems. This method was applied to examine the interaction betwe en Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary c ell transfectants and immunogobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a mono valent 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 cont act area, estimated to be a few percent of 3 mu m(2).