This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with VD. Standards or samples are added to the appropriate micro ELISA plate wells then with a biotin-conjugated antibody specific to VD. 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 VD in the samples is then determined by comparing the OD of the samples to the standard curve.