Immunoglobulin Surface Binding Kinetics Studied with Total Internal Reflection/Fluorescence Correlation Spectroscopy.

Abstract

A new method for measuring the binding/unbinding rates and surface diffusion coefficient of fluorescent-labeled solute molecules at a surface is presented. In the technique, total internal reflection/fluorescence correlation spectroscopy (TIR/FCS), fluorescent-labeled molecules are in chemical equilibrium between solution and a surface to which they reversibly bind. A laser beam internally reflect at the solid/liquid interface, exciting primarily the fluorescence of surface-adsorbed molecules. Fluorescence collected by a microscope from a surface area (TURN)5 (mu)m('2) spontaneously fluctuates as single solute molecules bind to, unbind from, and surface diffuse along the surface. The fluorescence is detected by a photomultiplier and autocorrelated on-line by a minicomputer. The shape of the autocorrelation function depends in general on the bulk and surface diffusion coefficients, the binding rate constants, and the shape of the observed region. The size of the autocorrelation function is inversely proportional to the average number of fluorescent molecules within the observation area. Under appropriate experimental conditions, the binding constants and surface diffusion coefficient can be readily obtained from the autocorrelation function. The theory can be applied to nonspecific adsorption, lig and binding to immobilized receptor, and to a mixture of fluorescent and nonfluorescent species competing for the same surface binding sites. In contrast to other kinetics techniques, TIR/FCS requires no spectroscopic or thermodynamic change between dissociated and complexed states and no extrinsic perturbation from equilibrium. Rhodamine-labeled immunoglobulin and insulin reversibly adsorb to serum albumin - coated quartz with several rates; the most rapid observed is of characteristic time (TURN) 5 ms and is limited by the rate of bulk diffusion. Surface diffusion across the observation area during the short surface residency time is not observed. Rhodamine-labeled bivalent and univalent anti-dinitrophenyl antibodies bind to dinitrophenyl-coated quartz either too irreversibly or with too much nonspecific binding to obtain specific binding rates. TIR/FCS might prove useful on other systems containing fluorescent-labeled molecules in solution which interact with specific sites on a surface.