ELISA-based determination of immunological binding constants.
An expression for the time-dependent concn of antibody in a hemispherical antigen-coated well is derived by taking the Laplace transformation of the diffusion equation. From this expression, and from antibody adsorption kinetics measured using ELISA, it is possible to evaluate the rate constant of bimolecular association, k1, the rate constant of first order antibody-antigen dissociation, k2, and their ratio, the binding equilibrium constant or affinity, Ka. For the interaction of an anti-arsanilate monoclonal antibody with arsanilate-coupled albumin, analysis yields k1 = 8.8 X 10(3) M-1 sec-1, k2 = 2.5 X 10(-4) sec-1 and Ka = 3.5 X 10(7) M-1, for mean values over 10 experiments. These results are discussed in reference to the conventionally-obtained values for binding constants, including the affinity of the anti-arsanilate monoclonal for the hapten (p-azobenzenearsonate)-N-acetyl-L-tyrosine, determined by equilibrium dialysis.
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001147:Arsanilic Acid / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008954:Models, Biological; D012710:Serum Albumin, Bovine / Q000276:immunology; D010132:p-Azobenzenearsonate / Q000276:immunology
D012710:Serum Albumin, Bovine; D010132:p-Azobenzenearsonate; D001147:Arsanilic Acid