BINDING-KINETICS OF IMMOBILIZED ANTIBODIES IN A FLOW IMMUNOSENSOR

This study investigates the binding kinetics of immobilized antibody in a solid-phase displacement immunosensor conducted in flow under nonequilibrium conditions. The experimental system studied employs a monoclonal antibody, specific for cocaine and its metabolite, benzoylecgonine, immobilized onto agarose beads. After saturation of antibody binding sites with the fluorophore-labeled antigen, fluorescein-cadaverine-benzoylecgonine, the system is placed in a buffer Bow. Injection of unlabeled antigen causes displacement of proportional amounts of antibody-bound labeled antigen, which is detected downstream. Using a repetitive displacement technique, we have characterized the kinetics by determining the displacement efficiency and apparent dissociation constant at various flow rates and different antibody surface densities. The experiments were performed with several antigen concentrations to evaluate the reliability of the repetitive displacement technique. The amount of displaced labeled antigen was found to be a function of both the density of antibody-bound labeled antigen and the amount of injected unlabeled antigen. Upon repetitive sample injections, an exponential decrease of displaced labeled antigen is observed, corresponding to a similar decrease in the undissociated fraction. Further kinetics analysis reveals that the displacement efficiency is dependent on the Bow rate and the antibody surface density. The antibody immobilized at low density is much more sensitive to changes in the flow rate than the antibody immobilized at high density, as indicated by the decrease in the displacement efficiency and increase in the apparent dissociation constant (k(d)) with the increase in Bow rate.