Effect of antibody density on the displacement kinetics of a flow immunoassay.

This study investigates the effect of antibody density on the kinetics of a solid-phase displacement immunoassay. Conducted in flow under nonequilibrium conditions, the assay utilizes a monoclonal antibody to the cocaine metabolite benzoylecgonine, which has been immobilized onto Sepharose beads and saturated with fluorophore labeled antigen. Displacement of antibody-bound labeled antigen by non-labeled antigen occurs when sample is introduced in the buffer flow. Comparison of matrices coated with two different antibody densities revealed that the displacement efficiency is a function of the density of antibody-bound labeled antigen. A higher density of antibody provides a higher amount of displaced labeled antigen, but the displacement efficiency of the assay is decreased. The effect of antibody density on the immunoassay kinetics was analyzed using a mathematical formulation developed to characterize antibody-antigen interactions at solid-liquid interfaces. Higher antibody density proved to be associated with a lower apparent dissociation rate constant. The implications of these results on the design of immunoassays in flow are discussed.