Description
Principle of the Assay: HNF4alpha ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-HNF4alpha antibody and an HNF4alpha-HRP conjugate. The assay sample and buffer are incubated together with HNF4alpha-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 HNF4alpha concentration since HNF4alpha from samples and HNF4alpha-HRP conjugate compete for the anti-HNF4alpha antibody binding site. Since the number of sites is limited, as more sites are occupied by HNF4alpha From the sample fewer sites are left to bind HNF4alpha-HRP conjugate. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The comHNF4alpha concentration in each sample is interpolated from this standard curve