INTERACTION OF ANTIBODIES WITH FC-RECEPTORS IN SUBSTRATE-SUPPORTED PLANAR MEMBRANES MEASURED BY TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY

A procedure for constructing substrate-supported planar membranes 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 (moFcγRII) bind to the planar model membranes. These measurements show that the planar membranes contain moFcγRII and yield a value for the association constant of 2.4G2 Fab fragments with moFcγRII equal to (9.6 ± 0.4) ✕ 108 M‒1 and indicate that the surface density of reconstituted moFcγRII is ~50 molecules/μm2. 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 moFcγRII in the planar membranes. These measurements indicate that the reconstituted moFcγRII 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 moFcγRII equal to (1–5) ✕ 105 M‒1. This work represents one of the first applications of TIR fluorescence microscopy to specific ligand-receptor interactions. © 1990, American Chemical Society. All rights reserved.