Description
Priciple of the assay: VB12 ELISA kit applies the competitive enzyme immunoassay technique utilizing a monoclonal anti-VB12 antibody and an VB12-HRP conjugate. The assay sample and buffer are incubated together with VB12-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 VB12 concentration since VB12 from samples and VB12-HRP conjugate compete for the anti-VB12 antibody binding site. Since the number of sites is limited, as more sites are occupied by VB12 from the sample, fewer sites are left to bind VB12-HRP conjugate. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The VB12 concentration in each sample is interpolated from this standard curve