The assay is based on the competition between a 25-OH vitamin D conjugate coated on a microplate and the 25-OH Vitamin D content of a sample. Vitamin D is extracted (EX Reagent) from your samples and then mixed, in the coated microplate well breakaway plates, with reagent R1 which contains an antibody for 25-OH Vitamin D. After a first incubation, the microplate is washed and reagent R2 (HRP-labeled secondary antibody) is added. Following a second incubation and a washing step, reagent R3 (TMB substrate) is added. After a final incubation step, the reaction is stopped by adding the STOP solution and the colorimetric signal of the microplate is measured at 450 nm (primary wavelength) to which the background signal can be subtracted by using secondary wavelengths (620 to 650 nm). The 25-OH Vitamin D concentration of a patient sample is inversely proportional to the measured absorbance at 450 nm. The whole assay procedure takes about two hours.