25-OH Vitamin D ELISA

vitamin d

Calbiotech's 25-OH Vitamin D ELISA features 100% Cross-reactivity to D2 & D3 and no outside extraction step! This is a sensitive, robust and automation-friendly assay, requiring no external sample preparation and no organic solvents with a 2.5 hour total assay time.

Clinical Significance

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 toxic.

Principle of the Test

The Calbiotech Vitamin D 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. Streptavidin-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 the amount of 25(OH)D in the sample. The assay measures both Vitamin D2 and D3). The total assay procedure run time is 2.5 hours.

Assay Procedure


Method Comparison

In an independent study, the Calbiotech 25(OH) Vitamin D ELISA showed excellent correlation to LC/MS/MS (R2=0.949; N=20), and performed better than a highly referenced, commercially available ELISA (R2=0.8314; N=20).

 methodcomptab1 methodtab2 

Standard Curve


Bench Marking



View a copy of "Clinical importance of 'Bioavailable' Vitamin D" here.