Description
Principle of the Assay: Big ET ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-Big ET antibody and an Big ET-HRP conjugate. The assay sample and buffer are incubated together with Big ET-HRP conjugate in pre-coated plate for one hour. After the incubation period, the wells are decanted and washed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stop solution is added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the Big ET concentration since Big ET from samples and Big ET-HRP conjugate compete for the anti-Big ET antibody binding site. Since the number of sites is limited, as more sites are occupied by Big ET from the sample, fewer sites are left to bind Big ET-HRP conjugate. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The Big ET concentration in each sample is interpolated from this standard curve