25(OH) Vitamin D ELISA
The Calbiotech, Inc. 25-hydroxy (25-OH) Vitamin D ELISA is intended for the quantitative determination of total 25-OH Vitamin D in human serum and plasma.
Vitamin D is a steroid hormone involved in the active intestinal absorption of calcium and in the regulation of its homeostasis. Vitamin D has two isomers: Vitamin D2 and Vitamin D3. Vitamin D2 is obtained from dairy products whereas Vitamin D3 is produced in the skin after exposure to ultraviolet light. In the liver, Vitamin D is hydroxylated at its carbon 25 to form 25-OH Vitamin D. This metabolite is the predominant circulating form of Vitamin D and is considered to be an accurate indicator of the general Vitamin D status of an individual. Vitamin D deficiency has been linked to many diseases including osteoporosis, rickets, osteomalacia, cancers, and cardiovascular diseases. Both dietary supplements of Vitamin D that are currently available in the market (Vitamin D2 and Vitamin D3) are converted to 25-OH Vitamin D in the liver. The sum of the concentrations of 25-OH Vitamin D2 and 25-OH Vitamin D3, in serum or plasma, is referred to as “Total 25-OH Vitamin D”. Accurate monitoring of total 25-OH Vitamin D level is critical in clinical settings. Vitamin D deficient patients who are prescribed a daily Vitamin D supplement should regularly monitor their serum or plasma Vitamin D levels in order to reach an optimal level and prevent their 25-OH Vitamin D concentrations from reaching excessive levels that are considered toxic1-5
The 25-OH Vitamin D is a solid phase ELISA based on the principal of competitive binding. Vitamin D calibrators, controls and samples are dispensed into pre-designated anti-Vitamin D coated microwells. After this, Biotin is dispensed into each well and Vitamin D competes with the endogenous Vitamin D in the sample, calibrator and control serum for a fixed number of binding sites on the anti-Vitamin D Antibody. The microwells are washed, and the bound Vitamin D Biotin is detected by the Streptavidin HRP. The Streptavidin HRP Conjugate bound to the wells decrease as the concentration of Vitamin D in the specimen increases; unbound Streptavidin HRP Conjugate is then removed and the microwells are once again washed. TMB is added and incubated, which results in the development of the blue color and stopped with the addition of stop solution. The absorbance is measured spectrophotometrically at 450nm. 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. This assay procedure run time is 100 minutes and measures both 25-OH Vitamin D2 and D3
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