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 no nequilibrium conditions, the assay utilizes a monoclonal antibody to t he cocaine metabolite benzoylecgonine, which has been immobilized onto Sepharose beads and saturated with fluorophore-labeled antigen. Displ acement of antibody-bound labeled antigen by non-labeled antigen occur s when sample is introduced in the buffer flow. Comparison of matrices coated with two different antibody densities revealed that the displa cement efficiency is a function of the density of antibody-bound label ed antigen. A higher density of antibody provides a higher amount of d isplaced labeled antigen, but the displacement efficiency of the assay is decreased. The effect of antibody density on the immunoassay kinet ics was analyzed using a mathematical formulation developed to charact erize antibody-antigen interactions at solid-liquid interfaces. Higher antibody density proved to be associated with a lower apparent dissoc iation rate constant. The implications of these results on the design of immunoassays in flow are discussed.