25-Hydroxyvitamin D Total ELISA | GDB7119

1. INSTRUCTION FOR USE
Immunoenzymetric assay for the in vitro quantitative measurement of 25-
hydroxyvitamin D2 and D3 (25OH-D2 and 25OH-D3) in serum and
plasma.
2. SUMMARY AND EXPLANATION

Vitamin D is the generic term used to designate Vitamin D2 or
ergocalciferol and Vitamin D3 or cholecalciferol.
Humans naturally produce Vitamin D3 when the skin is exposed to
ultraviolet sun rays.
In the liver mainly, Vitamin D3 is metabolised into 25-Hydroxyvitamin
D3 (25OH D3) which is the main form of Vitamin D circulating in the
body.
25OH D3 is a precursor for other Vitamin D metabolites and has also a
limited activity by itself.
The most active derivative is 1,25-hydroxyvitamin D3, produced in the
kidney (or placenta) by 1-hydroxylation of 25OH D3.
25OH Vitamin D stimulates the intestinal absorption of both calcium and
phosphorus and also bone resorption and mineralisation.
25OH Vitamin D might also be active in other tissues responsible for
calcium transport (placenta, kidney, mammary gland …) and endocrine
gland (parathyroid glands, beta cells…).
Vitamin D3 and Vitamin D2 are also available by ingestion through food
or dietary supplementation.
As Vitamin D2 is metabolised in a similar way to Vitamin D3, both
contribute to the overall Vitamin D status of an individual.
It is the reason why it is very important to measure both forms of 25OH
Vitamin D equally for a correct diagnosis of Vitamin D deficiency,
insufficiency or intoxication.
Vitamin D deficiency is an important risk factor for rickets, osteomalacia,
senile osteoporosis, cancer and pregnancy outcomes.
The measurement of both 25OH Vitamin D forms is also required to
determine the cause of abnormal serum calcium concentrations in
patients.
Vitamin D intoxication has been shown to cause kidney and tissue
damages.
3. PRINCIPLE OF THE TEST
The Global Diagnostics B 25OH Vitamin D Total ELISA is a solid phase
Enzyme Linked Immunosorbent Assay performed on microtiterplates.
During a first 1 hour incubation step, at room temperature, total 25OH
Vitamin D (D2 and D3) present in calibrators, controls and samples is
dissociated from binding serum proteins to fix on binding sites of a
specific monoclonal antibody. After 1 washing step, a fixed amount of
25OH Vitamin D-labelled with biotin in presence of horseradish
peroxidase (HRP), compete with unlabelled 25OH Vitamin D2 and 25OH
Vitamin D3 present on the binding sites of the specific monoclonal
antibody. After a 15 minutes incubation at room temperature, the
microtiterplate is washed to stop the competition reaction. TheChromogenic solution (TMB) is added and incubated for 15 minutes. The
reaction is stopped with the addition of Stop Solution and the
microtiterplate is then read at the appropriate wavelength. The amount of
substrate turnover is determined colourimetrically by measuring the
absorbance, which is inversely proportional to the total 25OH Vitamin D
(D2 and D3) concentration.
A calibration curve is plotted and the total 25OH Vitamin D (D2 and D3)
concentrations of the samples are determined by dose interpolation from
the calibration curve. 

4. REAGENTS

Note :Use Calibrator 0 for dilution of samples with values above the highest
calibrator.
No international reference material is available
5. MATERIALS NOT PROVIDED
The following material is required but not provided in the kit:
●Distilled water
●Pipettes for delivery of: 25 µl,75 μl, 100µl and 1 ml (the use of accurate
●pipettes with disposable plastic tips is recommended)
●Vortex mixer
●Magnetic stirrer
●Plate shaker (400 rpm)
●Washer for microtiterplates
●Microtiterplate reader capable of reading at 450 nm and 650
(bichromatic reading)