25-OH VITAMIN D EIA KIT
INTENDED USE
The 25-OH Vitamin D EIA Kit is intended for the quantitative determination of the 25-OH Vitamin D in plasma and serum. For in vitro diagnostic use only.
SUMMARY AND EXPLANATION OF 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 also by biosynthesis of 7-dehydrocholesterol and UV-light in skin because of 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.
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 lately 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 EIA Kit
25-OH Vitamin D EIA Kit is intended for the quantitative determination of the 25-OH Vitamin D in plasma and serum. This 25-OH Vitamin D EIA Kit test kit is a competitive protein binding assay for the measurement of 25-OH Vit D. It is based on the competition of 25-OH Vit D present in the sample with 25-OH Vit D tracer, for the binding pocket of vitamin D binding protein (VDBP, Gc-globulin). Since all circulating 25-OH Vit D is bound to VDBP in vivo, samples have to be precipitated with precipitation reagent to extract the analyte. The supernatant can be used without further treatment within the test.
In the first incubation step, sample, calibrator, control, the vitamin D binding protein and the VDBP-Antibody, an antibody specific for this protein is added to the solid phase. 25-OH Vitamin D present in the sample then competes with the tracer, coated on the well for the specific binding site of the binding protein and the VDBPAntibody is bound to the vitamin binding protein. Hence, with increasing concentrations of 25-OH Vit D in the sample, the amount of binding protein, immobilized to the well via the tracer, is reduced. After a washing step to remove unbound components, the quantitation of VDBP is achieved by incubation with a host specific peroxidase labeled antibody using TMB (tetramethylbenzidine) as enzyme substrate. An acidic stopping solution is then added to stop the reaction. The colour converts to yellow. The intensity of the yellow colour is indirectly proportional to the concentration of 25-OH Vitamin D in the sample. A dose response curve of the absorbance unit vs. concentration is generated using the results obtained from the calibrators. Concentrations of 25-OH Vitamin D, present in the patient samples, are determined directly from this curve.
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