An enzyme immunoassay for the quantitative measurement of total 25-OH Vitamin D (Vitamin D2 and Vitamin D3) in serum. This product is for Research Use Only; International customers contact your representative to receive information about the CE marked version. The DRG 25-OH Vitamin D total ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle of competitive binding. In the first step, samples have to be pretreated in separate vials with denaturation buffer to extract the analyte, since most circulating 25-OH Vit D is bound to VDBP in vivo. After neutralization, biotinylated 25-OH Vitamin D (enzyme conjugate) and peroxidase-labeled streptavidin- (enzyme complex) are added. After careful mixing, the solution is transferred to the wells of the microtiter plate. Endogenous 25-OH Vitamin D of a patient sample competes with a 25-OH Vitamin-D3-biotin conjugate for binding to the VDBG that is immobilized on the plate. Binding of 25-OH Vitamin D-biotin is detected by peroxidase-labeled streptavidin. Incubation is followed by a washing step to remove unbound components. The color reaction is started by addition enzyme substrate and stopped after a defined time. The colour intensity is inversely proportional to the concentration of 25-OH Vitamin D in the sample.