Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: The effect of hapten density

Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, KD_D1, was 2.46 × 10^-5 M, while the second was K_D2 = 1.37 × 10^-8 mol/m². This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 ± 2.7 J/(mol·K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.