Product name25(OH) Vitamin D ELISA kit
Intra-assay Sample n Mean SD CV% Sample 1 20 225.8ng/ml 1.6% Sample 2 20 37.3ng/ml 2.1% Sample 3 20 5.3ng/ml 3.4% Inter-assay Sample n Mean SD CV% Sample 1 30 297.2ng/ml 11.5% Sample 2 30 38.4ng/ml 15.8%
Sample typeSerum, Plasma
Range0.5 ng/ml - 1010 ng/ml
Sample specific recovery Sample type Average % Range Serum 91.9 84% - 97.9%
Assay time1h 30m
Assay durationMultiple steps standard assay
25(OH) Vitamin D ELISA kit is a complete kit for the quantitative determination of 25(OH) Vitamin D3 and 25(OH) Vitamin D2 and was tested in human plasma and serum samples. Nevertheless, ab213966 is not species specific.
It is recommended that you read the entire kit insert before proceeding with the assay.
Recent research efforts have shown that Vitamin D levels affect various disease states and are being linked with numerous indicators of well-being in humans. These include bone diseases such as osteoporosis and arthritis, but also additional disease including hypertension, diabetes, cancer and heart disease to name a few. Our Vitamin D ELISA kit offers an alternative to labor intensive and/or costly methods of testing for Vitamin D levels in human plasma and serum.
The transformation to the active form of Vitamin D begins with 7-dehydrocholesterol being acted upon by UV rays from the sun to form parent Vitamin D3. Alternatively, Vitamin D can be ingested as parent Vitamin D2 from various food sources, native or fortified. These parent compounds are transported to the liver and undergo hydroxylation to 25(OH) Vitamin D. This metabolite is then transported to the kidney where it undergoes a second hydroxylation to 1,25(OH)2 Vitamin D, the biologically active form of Vitamin D. It is important to note that levels of Vitamin D metabolites increase proportionately with increased uptake of parent Vitamin D. This combined with the greater half-life and stability of 25(OH) Vitamin D in circulation versus the active form (25 days versus 8 hours) are the reasons that the detection of the 25(OH) Vitamin D metabolite is used as the indicator for total Vitamin D concentration.
Dissociation Buffer is added to wells coated with donkey anti-sheep IgG antibody. Standards and Samples are then added to these wells and dissociation of 25(OH) Vitamin D from Vitamin D Binding protein allowed to occur.
A solution of alkaline phosphatase conjugated 25(OH) Vitamin D3 is then added, followed by a solution of sheep monoclonal antibody to 25(OH) Vitamin D.
During incubation at room temperature the antibody binds the 25(OH) Vitamin D from the sample/standard or conjugate in a competitive manner and is itself captured by the anti-sheep IgG antibody. The plate is then washed, leaving a complex with bound 25(OH) Vitamin D from samples or the Alkaline phosphatase conjugate.
A pNpp substrate solution is added initiating an alkaline phosphatase catalyzed reaction that generates a yellow color in the solution.
Stop solution is added to stop the substrate reaction and the resulting yellow color is read at 405nm. The amount of signal is inversely proportional to the level of 25(OH) Vitamin D in the sample.
PlatformPre-coated microplate (12 x 8 well strips)
Storage instructionsStore at -20°C. Please refer to protocols.
Components 1 x 96 tests 25(OH) Vitamin D Antibody 1 x 5ml 25(OH) Vitamin D Conjugate (100X) 1 x 50µl 25(OH) Vitamin D Standard 1 (1010 ng/mL) 1 x 40µl 25(OH) Vitamin D Standard 2 (279 ng/mL) 1 x 40µl 25(OH) Vitamin D Standard 3 (71.6 ng/mL) 1 x 40µl 25(OH) Vitamin D Standard 4 (24.4 ng/mL) 1 x 40µl 25(OH) Vitamin D Standard 5 (4.8 ng/mL) 1 x 40µl 25(OH) Vitamin D Standard 6 (0.5 ng/mL) 1 x 40µl 25(OH) Vitamin D3 Conjugate Diluent 1 x 6ml Dissociation Buffer 1 x 10ml Donkey anti-sheep IgG Microplate (12 x 8 wells) 1 x 96 tests Plate Sealer 3 units pNpp Substrate 1 x 20ml Sample Diluent 1 x 1.8ml Stop Solution 1 x 5ml Wash Buffer 4 Concentrate (20X) 1 x 20ml
- Pathways and Processes
- Metabolic signaling pathways
- Lipid and lipoprotein metabolism
FunctionHas a D-25-hydroxylase activity on both forms of vitamin D, vitamin D(2) and D(3).
Involvement in diseaseDefects in CYP2R1 are the cause of rickets vitamin D-dependent type 1B (VDDR1B) [MIM:600081]; also known as pseudovitamin D(3) deficiency rickets due to 25-hydroxylase deficiency. A disorder caused by a selective deficiency of the active form of vitamin D (1,25-dihydroxyvitamin D3) and resulting in defective bone mineralization and clinical features of rickets. The patients sera have low calcium concentrations, low phosphate concentrations, elevated alkaline phosphatase activityand low levels of 25-hydroxyvitamin D.
Sequence similaritiesBelongs to the cytochrome P450 family.
Cellular localizationEndoplasmic reticulum membrane. Microsome membrane.
- Information by UniProt
- Cytochrome P450 2R1
ab213966 has been referenced in 1 publication.
- Benetti E et al. Effects of vitamin D on insulin resistance and myosteatosis in diet-induced obese mice. PLoS One 13:e0189707 (2018). PubMed: 29342166