Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy

Abstract

A procedure for constructing substrate-supported planar membrane using membrane fragments isolated from the macrophage-related cell line J774A.1 is described. Total internal reflection (TIR) fluorescence microscopy is employed to demonstrate that fluorescently labeled Fab fragments of a monoclonal antibody (2.4G2) with specificity for a murine macrophage cell-surface receptor for IgG (moFc.gamma.RII) bind to the planar model membranes. These measurements show that the planar membranes contain moFc.gamma.RII and yield a value for the association constant of 2.4G2 Fab fragments with moFc.gamma.RII equal to (9.6 .+-. 0.4) .times. 108 M-1 and indicate that the surface density of reconstituted moFc.gamma.RII is .apprx. 50 molecules/.mu.m2. In addition, TIR fluorescence microscopy is used to investigate the Fc-mediated competition of unlabeled, polyclonal murine IgG with labeled 2.4G2 Fab fragments for moFc.gamma.RII in the planar membranes. These measurements indicate that the reconstituted moFc.gamma.RII recognized by 2.4G2 Fab fragments also retains the ability to bind murine IgG Fc regions and yield a value for the association constant of polyclonal murine IgG with moFc.gamma.RII equal to (1-5) .times. 105 M-1. This work represents one of the first applications of TIR fluorescence microscopy to specific ligand-receptor interactions.