Description
Principle of the Assay: HMW APN ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-HMW APN antibody and an HMW APN-HRP conjugate. The assay sample and buffer are incubated together with HMW APN-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 HMW APN concentration since HMW APN from samples and HMW APN-HRP conjugate compete for the anti-HMW APN antibody binding site. Since the number of sites is limited, as more sites are occupied by HMW APN from the sample, fewer sites are left to bind HMW APN-HRP conjugate. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The HMW APN concentration in each sample is interpolated from this standard curve