SPECIES-SPECIFIC LONG-RANGE INTERACTIONS BETWEEN RECEPTOR LIGAND PAIRS/

Total internal reflection microscopy (TIRM) monitors Brownian fluctuat ions in elevation as small as 1 nm by measuring the scattering of a si ngle sphere illuminated by an evanescent wave when the sphere is levit ated by colloidal forces such as electrostatic double-layer repulsion. From the Boltzmann distribution of elevations sampled by the sphere o ver time, the potential energy profile can be determined with a resolu tion of similar to 0.1 of the thermal energy kT. Thus, the interaction between a receptor-coated (goat, horse, or rabbit immunoglobulin G (I gG)) latex sphere and a protein A (SpA)-coated glass microscope slide was studied. A typical TIRM potential energy profile measured between a bare sphere and a bare glass plate, where the sphere fluctuates arou nd the secondary potential energy minimum formed between double-layer repulsion and gravitational attraction, agrees well with DLVO theory. The interactions measured between IgG-coated spheres and SpA-coated sl ides, on the other hand, displayed a weaker repulsion compared with th at observed between bare surfaces under the same conditions. Analysis of the results obtained between the coated surfaces suggests an additi onal attractive force. The decay length of this attraction correlates with the known dissociation constants for the binding of IgG with SpA in free solution.