Measurement of association constants in ELISA. Reactions between solid-phase antibody and fluid-phase biotinylated...

An ELISA procedure which utilized an avidin-peroxidase amplification reaction to detect and quantify the amount of fluid-phase biotinylated antigen bound to solid-phase antibody was developed to determine antibody association constants. The methodology required is uncomplicated, avoids the use of radioisotopes, and is theoretically amenable for use with any protein which can be biotinylated, and any receptor protein which can be immobilized on plastic wells. A plot of known immobilized biotinylated antigen concentrations, which ranged from approximately 100 ng/ml to 1000 ng/ml, versus avidin-peroxidase conjugate product formation, was used to establish a standard curve from which the amount of antibody-associated antigen in binding assays could be determined. Antibody association constants obtained with this method ranged from 10(5) to 10(9) M-1.