This assay employs the competitive inhibition enzyme immunoassay technique. An antibody specific for Vitamin D2 (VD2) has  been  pre-coated  onto  a  microplate.  A  competitive  inhibition  reaction  is  launched  between Horseradish Peroxidase (HRP) labeled Vitamin D2 (VD2) and unlabeled Vitamin D2 (VD2) (Standards or samples) with the pre-coated antibody specific for Vitamin D2 (VD2). After incubation the unbound conjugate is washed off. The amount of bound HRP conjugate is reverse proportional to the concentration of  Vitamin D2 (VD2) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of  Vitamin D2 (VD2) in the sample.