Principle of Assay
Vitamin D2 ELISA Kit (A3001) employs the competitive enzyme immunoassay technique for the quantitative measurement of Vitamin D2 in serum, plasma, tissue homogenates or other biological fluids. The 96-well microtiter plate has been pre-coated with Vitamin D2 antigen. During the incubation, Vitamin D2 present in the samples or standards competes with the fixed amount of immobilized Vitamin D2 for binding sites on the Biotinylated Anti-Vitamin D2 Antibody. The more Vitamin D2 present in a sample or standard, the less Biotinylated Anti-Vitamin D2 Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Vitamin D2 Antibody is removed by washing, and an HRP-Avidin 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 Vitamin D2 present in each sample or standard. The concentration of Vitamin D2 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
