1,25-(0H)2 VITAMIN D ELISA KIT
INTENDED USE
The 1,25-(0H)2 Vitamin D ELISA kit is intended for the quantitative determination of 1,25 dihydroxy vitamin D in serum and plasma. It is for in vitro diagnostic use only.
SUMMARY AND EXPLANATION OF THE TEST
Vitamin D is either produced in the skin (under the influence of UV light) or taken up from nourishment. The storage type of vitamin D, namely 25-hydroxy vitamin D, is formed in the liver. The hormone 1,25-dihydroxy vitamin D (D hormone) is formed in a second hydroxylation step in the kidney. The responsible enzyme, the kidney 1α-hydroxylase, is subjected to a rigid control through hormones (especially parathyroid hormone) and its activity is influenced by the serum concentrations of calcium and phosphate.
The serum concentration of 1,25-dihydroxy vitamin D normally re-adjusts itself to the demands of metabolism. Deviations from the normal range of 1,25-dihydroxy vitamin D must therefore always be interpreted in the context of the remaining parameters of the calcium metabolism. The serum concentration of 1,25-dihydroxy vitamin D decreases only in seldom cases of vitamin D deficiency. For the diagnosis of vitamin D deficiency the precursor metabolite, 25-hydroxyvitamin D, should be measured.
The reason for a non-physiological deficiency of 1,25-dihydroxy vitamin D can be found in metabolic disturbances, caused either by genetic defects of the enzyme 1α-hydroxylase (rare) or kidney malfunctions (more common). Even a slightly impaired kidney function can lead to a decrease of the 1,25-dihydroxy vitamin D concentration.
Since 1,25-dihydroxy vitamin D has important functions in calcium metabolism as well as supplementing secretion of parathyroid hormone from the parathyroid glands, increasing kidney malfunctioning leads to development of renal osteopathy, which is characterized by osteomalacia and osteitis fibrosa.
Treatment of renal osteopathy consists of the administration of 1,25-dihydroxy vitamin D (calcitriol) or the prohormone 1α-hydroxy vitamin D. In renal tubules malfunctions decreased or relatively low levels of 1,25-dihydroxy vitamin D (e.g. diabetes insipidus, Fanconi-syndrom) are found. A non-physiological over-production of 1,25-dihydroxy vitamin D arises in granulomatosis (e.g. sarcoidosis), where extra-renal synthesis of 1,25-dihydroxy vitamin D occurs. This can lead to hypercalcaemia. Also in idiopathic hypercalciuria a relatively high level of 1,25-dihydroxy vitamin D is found. Increased concentrations of 1,25-dihydroxy vitamin D can be seen in case of non-functional vitamin D receptors (rare), duringcalcium deficient nutrition, as well as a result from overproduction of parathyroid hormone (primary hyperthyroidism).
PRINCIPLE OF THE TEST
1,25-(0H)2 Vitamin D ELISA Kit
The 1,25-(0H)2 Vitamin D ELISA kit is intended for the quantitative determination of 1,25 dihydroxy vitamin D in serum and plasma. The 1,25-(0H)2 Vitamin D ELISA Kit assay utilizes of a competitive Enzyme-Immuno-Assay (EIA) technique with a selected monoclonal antibody recognizing 1,25-dihydroxy vitamin D.
Standards, NSB (non-specific binding), controls and patient samples which are assayed for 1,25-dihydroxy vitamin D are incubated after the extraction step with the detection antibody. The pre-incubated solution is then transferred to the microplate coated with 1,25-dihydroxy vitamin D. During this incubation step, 1,25-dihydroxy vitamin D in the sample and a fixed amount of 1,25-dihydroxy vitamin D bound to the microtiter well compete for the binding of the detection antibodies. Then a peroxidase-conjugated anti-mouse antibody is added into each microplate well and a complex of 1,25-dihydroxy vitamin D - detection 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 1,25-dihydroxy 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. 1,25 dihydroxy vitamin D in the samples is determined from this curve.
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