Measuring the Inhibition of Adhesion of Lectins to the Surface of Erythrocytes with Optically Controlled Collisions between Microspheres and Erythrocytes

This paper describes the use of the OPTCOL (optically controlled collision) assay, an assay that uses optical tweezers to cause two biologically relevant particles to collide, to measure the potency of inhibitors that block the adhesion of wheat germ agglutinin (WGA) to the surface of erythrocytes. WGA was attached covalently to polystyrene microspheres (3 μm in diameter). Optical tweezers were used to cause an erythrocyte and a WGA-coated microsphere to collide in buffer at a controlled velocity. In the absence of inhibitor, or at low concentrations of soluble inhibitor, the microsphere adhered to the cell through polyvalent, biospecific interactions between WGA and N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuAc) groups present on the surface of erythrocyte. At high concentrations of soluble inhibitors, adhesion was inhibited. The potency of inhibition was quantified by measuring the probability of adhesion as a function of the concentration of the inhibitor. The inhibition constants derived from measurements using OPTCOL agreed well with those obtained in hemagglutination inhibition assays, and they were also close to the dissociation constants measured by isothermal titration calorimetry. The experimental data suggest that the binding of WGA to erythrocyte is cooperative, but that the binding of WGA to soluble ligand is not. The ability to examine dynamic adhesion in a highly controlled fashion makes OPTCOL a useful bioassay with which to study inhibition of protein-cell adhesion under biologically relevant conditions.