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