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This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with VD3 protein. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to VD3. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ? 10nm. The concentration of VD3 in the samples is then determined by comparing the OD of the samples to the standard curve.