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.
Rabbany
S Y
SY
Department of Engineering, Hofstra University, Hempstead, NY 11550.
Kusterbeck
A W
AW
Bredehorst
R
R
Ligler
F S
FS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D003042:Cocaine / Q000031:analogs & derivatives / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D051379:Mice; D012685:Sepharose
D000911:Antibodies, Monoclonal; D000941:Antigens; C005618:benzoylecgonine; D012685:Sepharose; D003042:Cocaine