1,25-dihydroxy Vitamin D ELISA
The DRG 1,25-Dihydroxy Vitamin D kit is a complete assay system intended for the purification of 1,25-dihydroxy Vitamin D (1,25D) in human serum or plasma by immunoextraction followed by quantitation by enzyme immunoassay. Results are to be used in conjunction with other clinical and laboratory data to assist the clinician in the assessment of 1,25D deficiency associated with renal disease in adult populations.
Vitamin D is a commonly used collective term for a family of closely related molecules derived from naturally occurring 7-dehydrocholesterol (pro-vitamin D3). Pro-vitamin D3 undergoes photolytic conversion in the skin to ‘parent’ vitamin D3 (cholecalciferol) upon exposure to sunlight. This compound is biologically inactive, but enters the circulation and is hydroxylated in the liver to active 25-hydroxyvitamin D (25D). A small proportion of this becomes further hydroxylated in the kidney to the highly potent calciotropic hormone 1,25D.
1,25D is largely bound to Vitamin D Binding Protein and albumin in the circulation.
1,25D is one of the major regulators of calcium (and phosphate) metabolism, stimulating intestinal calcium absorption and increasing bone resorption. It also inhibits parathyroid hormone (PTH) production both by direct action on the parathyroid glands and indirectly by raising serum calcium levels. 1,25D production is itself stimulated by parathyroid hormone (PTH), thus providing an effective control loop.
Hypovitaminosis D is commonly associated with dietary insufficiency, most frequently with vegetarianism, and is also associated with low exposure to sunlight (e.g. the elderly and institutionalized) and skin pigmentation.
1,25D production appears to be impaired in early renal failure though this may not be a renal effect. In late-stage renal failure, 1α-hydroxylation may be impaired, with low 1,25D levels as a result.
The DRG 1,25-Dihydroxy Vitamin D kit is a complete assay system for the purification of 1,25D in patient samples by immunoextraction followed by quantitation by EIA. Patient samples are delipidated and 1,25D extracted from potential cross-reactants by incubation for 90 minutes with a highly specific solid phase monoclonal anti-1,25D. The immunoextraction gel is then washed and purified 1,25D eluted directly into glass assay tubes. Reconstituted eluates and Standards are incubated overnight with a highly specific sheep anti-1,25D. Then a portion of this is incubated for 90 minutes with shaking in microplate wells which are coated with a specific anti-sheep antibody. 1,25D linked to biotin is then added and the plate shaken for a further 60 minutes before aspiration and washing. Enzyme(horseradish peroxidase) labelled avidin is added and binds selectively to complexed biotin and, following a further wash step, colour is developed using a chromogenic substrate (TMB). The absorbance of the stopped reaction mixtures are read in a microtiter plate reader, colour intensity developed being inversely proportional to the concentration of 1,25D.