This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with VD3. During the reaction, VD3 in the sample or standard competes with a fixed amount of VD3 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VD3. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The concentration of VD3 in tested samples can be calculated by comparing the OD of the samples to the standard curve.