25-Hydroxy Vitamin D3 ELISA Kit (A303874) employs the competitive enzyme immunoassay technique for the quantitative measurement of universal 25-Hydroxy Vitamin D3 in serum, plasma, tissue homogenates, and other biological fluids. The 96-well microtiter plate has been pre-coated with 25-Hydroxy Vitamin D3 antigen. During the incubation, 25-Hydroxy Vitamin D3 present in the samples or standards competes with the fixed amount of immobilized 25-Hydroxy Vitamin D3 for binding sites on the Biotinylated Anti-25-Hydroxy Vitamin D3 Antibody. The more 25-Hydroxy Vitamin D3 present in a sample or standard, the less Biotinylated Anti-25-Hydroxy Vitamin D3 Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-25-Hydroxy Vitamin D3 Antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is inversely proportional to the amount of 25-Hydroxy Vitamin D3 present in each sample or standard. The concentration of 25-Hydroxy Vitamin D3 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.