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