25-0H VITAMIN D ELISA KIT
INTENDED USE
The 25-0H Vitamin D ELISA kit is intended for the quantitative determination of the 25-OH Vitamin D in serum and fresh plasma. For in vitro diagnostic use only.
SUMMARY AND EXPLANATION OF THE TEST
Vitamin D is a steroid hormone involved in the intestinal absorption of calcium and the regulation of calcium homeostasis. There are two different forms of vitamin D, named D3 and D2, which are very similar in structure. The D2 is a synthetic product, which is predominantly absorbed by fortified food.
Physiological vitamin D3 levels result not only from dietary uptake but can also be produced from a cholesterol precursor, 7-dehydrocholesterol, in the skin during sun exposure. In the liver, the vitamin is hydroxylated to 25-hydroxyvitamin D (25(OH)-vitamin D), the major circulating metabolite of vitamin D. Although 1,25-(OH)2 vitamin D portrays the biological active form of vitamin D, which is synthesized in the kidney, it is widely accepted that the measurement of circulating 25(OH)-vitamin D provides better information with respect to patients vitamin D status and allows its use in diagnose hypovitaminosis.
Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: solid as a rock (Wielders and Wijnberg, 2009). The concentration of 25(OH)-vitamin D decreases with age and a deficiency is common among elderly persons.
Clinical applications of 25(OH)-vitamin D measurements are the diagnosis and therapy control of postmenopausal osteoporosis, rickets, osteomalacia, renal osteodystrophy, pregnancy, neonatal hypocalcemia and hyperparathyroidism. In addition, a prevalence of subclinical vitamin D deficiency has been discussed in different European countries.
Vitamin D intoxication mostly occurs during a large intake of pharmaceutical preparations of Vitamin D and may lead to hypercalcemia, hypercalcuria and nephrocalcinosis in susceptible infants.
PRINCIPLE OF THE TEST
25-OH Vitamin D ELISA Kit
The 25-0H Vitamin D ELISA kit is intended for the quantitative determination of the 25-OH Vitamin D in serum and fresh plasma. The 25-0H Vitamin D ELISA kit assay utilizes of a competitive ELISA technique with a selected monoclonal antibody recognizing 25(OH)-vitamin D. For a reliable determination of 25(OH)-vitamin D, it is necessary to release it from the 25(OH)-vitamin D-DBP-complex.
Standards, controls and patient samples which are assayed for 25(OH)-vitamin D are incubated with the releasing reagent. The pre-incubated solutions are then transferred to the microplate coated with 25(OH)-vitamin D, and an anti-25(OH)-vitamin D antibody is added. During an over night incubation step, 25(OH)-vitamin D in the sample and a fixed amount of 25(OH)-vitamin D bound to the microtiter well compete for the binding of the antibody. Then a peroxidase-conjugated antibody is added into each microplate well. A complex of 25(OH)-vitamin D - anti-25(OH)-vitamin D antibody – peroxidase conjugate is formed.
Tetramethylbenzidine (TMB) is used as a peroxidase substrate. Finally, an acidic stop solution is added to terminate the reaction, whereby the color changes from blue to yellow. The intensity of the yellow color is inversely proportional to the concentration of 25(OH)-vitamin D. A dose response curve of the absorbance unit (optical density, OD at 450 nm) vs. concentration is generated using the values obtained from the standard. 25(OH) -vitamin D in the samples is determined from this curve.
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