T he kit is a solid phase enzyme-linked immunoassay (ELISA),based on the principal of competitive binding. Anti-Vitamin D antibody coated wells are incubated with Vitamin D standards,controls, samples, and Vitamin D-Biotin conjugate at room temperature for 90 minutes. During the incubation, a fixed amount of biotin-labeled vitamin D competes with the endogenous Vitamin D in the sample, standard, or quality control serum for a fixed number of binding sites on the anti Vitamin D antibody. Following a wash step, bound Vitamin D-Biotin is detected with Streptavidin-HRP (SA-HRP). SA-HRP conjugate immunologically bound to the well progressively decreases as the concentration of Vitamin D in the specimen increases. Unbound SA-HRP conjugate is then removed and the wells are washed. Next, a solution of TMB Reagent is added and incubated at room temperature for 30 minutes,
resulting in the development of blue color. The color development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450 nm.A standard curve is obtained by plotting the concentration of the standard versus the absorbance. The color intensity will be inversely proportional to the amount of 25-OH Vitamin D in the sample. The assay measures both the 25-OH Vitamin D2 and
D3. The total assay procedure run time is 2.5 hours.