Abstract
We developed a noncompetitive two-site sandwich ELISA to quantitate monoclonal antibodies in culture supernatant. This assay measures the initial enzyme activity rate during the first minute of the reaction, which ensures linear velocity relative to time and a progress curve slope proportional to analyte concentration. During this period, the enzyme substrate is in large excess relative to the analyte/antibody-enzyme complex, and enzyme catalysis proceeds in steady-state conditions. Analyses of repeatability gave coefficients of variation between 4.4 and 9.7 (interassay) and 4.4 and 6.4 (intra-assay), and analyte detectability ranged from 5.8 to 12 ng/ml. The Z-factor calculated for analyte samples at their end dilution yielded mean values from 0.57 to 0.87, which confirmed assay robustness. This initial velocity-based sandwich ELISA is a simple, sensitive, reproducible method to quantitate bi-epitopic antigens.