The 25-OH Vitamin D ELISA assay test kit is designed for the determination of 25-OH Vitamin D in human serum or plasma samples.
Contents of Kit
1. MT-Strips, 12 strips 8 wells per strip, single break apart, coated with monoclonal antibody (vitamin D2 and D3) 2. Standards 1-6, 6 vials 1 ml each, colored red-brown; To be diluted 1:26 in working strength biotin. 3. Control 1+2, 2 vials 1 ml each, colored red-brown, To be diluted 1:26 in working strength biotin. values for the Control are given on the vial label 4. Biotin Biotin, 1.2 ml, 100 × concentrated; colored blue 1 vial 5. Sample Buffer, 1 vial 100 ml, colored yellow 6. Enzyme Conjugate, 1 vial 12 ml, colored blue, ready for use 7. Substrate, 1 vial 12 ml tetramethyl benzidine (TMB)/ H2O2, ready for use 8. Wash Buffer, 1 vial 100 ml, 10 × concentrated 9. Stop Solution, 1 vial 12 ml, ready for use, 0.5M sulphuric acid
Storage
On arrival, store the 25-OH Vitamin D ELISA assay kit at 2-8 °C. Once opened the kit is stable until its expiry date. Do not use components beyond the expiration date shown on the kit labels.
Performance Characteristics
Calibration As there is no international standard for 25-OH Vitamin D, the standards and controls are calibrated gravimetrically using UV-Vis (264nm) verified stock standards and compared with NIST standards (National Institute of Standards and Technology, USA), DEQAS (Vitamin D External Quality Assessment Scheme, UK) quality assessment data and in-house quality control sera. For every group of tests performed, the values of the concentrations must lie within the limits stated for the relevant test kit lot. A quality control certificate containing these reference values is included. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated. Antibodies The reagent wells of the 25-OH Vitamin D ELISA assay kit are coated with monoclonal antibodies which identify specifically 25-OH vitamin D3 and 25-OH vitamin D2.
Precision
The reproducibility of the 25-OH Vitamin D ELISA assay kit was investigated by determining the intra- and interassay coefficients of variation using 3 sera from different areas of the calibration curve. The intra-assay CVs are based on 40 measurements for each serum and the inter-assay CVs on four measurements performed in six different test runs.
Detection Limit
The lower detection limit of the 25-OH Vitamin D ELISA assay kit is defined as the mean value of an analyte-free sample minus three times the standard deviation and is the smallest detectable 25-OH Vitamin D concentration. The detection limit of 25-OH Vitamin D ELISA is 1.6 ng/ml.
General Description
The 25-OH Vitamin D ELISA assay kit is designed for the serological determination of the vitamin D concentration in the human organism types of vitamin D that are differentiated are vitamin D2 (ergocalciferol) that is contained in plant food (mushrooms, avocado) and vitamin D3 (cholecalciferol) that is produced from 7-dehydrocholesterol in the skin under ultra-violet irradiation or found in animal food or products (sea fish, egg yolk, butter). These two forms of vitamin D, which are not yet biologically active, are bound by a protein called VDBP (vitamin D binding protein) in the bloodstream, then metabolised in the liver and converted into 25-OH vitamin D2 (calcidiol) and 25-OH vitamin D3 (calcitriol), respectively, which are storage forms of the vitamin with little activity. In contrast to other commercially available tests, the 25-OH Vitamin D ELISA assay kit uses a newly designed monoclonal antibody which is specific for both vitamin D2 and vitamin D3 at 100% specificity. This is necessary because sometimes vitamin D2 instead of D3 is used in therapy.
Standard Curve
If the extinction of a sample lies below the value of calibrator 6 (120 ng/ml), the result should be given as ">120 ng/ml". It is recommended that the sample be re-tested at a dilution of 1:2 with calibrator 1 before following the test instructions. The result in ng/ml read from the calibration curve for this sample must then be multiplied by a factor of 2. For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another the sample should be retested.
Citations
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Background
As a fat-soluble steroid, vitamin D plays a role in calcium, phosphate, and bone metabolism, influencing intestinal calcium absorption and the development of rickets or osteomalacia, in addition to a variety of extra-skeletal roles that researchers have found it may have. Vitamin D is derived from ultraviolet light exposure at the skin site, which converts 7-dehydrocholesterol to vitamin D3, a process in which the effect of vitamin D synthesis is influenced by light intensity and air pollution. Dietary intake of vitamin D does not exceed 20% of the daily requirement, generally from dairy products, grain products, fish oil and other foods to obtain.
Food-source vitamin D is absorbed in the small intestine, where it binds to celiac particles and is transported to the lymphatics, where it subsequently enters the circulation bound to vitamin D-binding protein (VDBP). The bioactive form of vitamin D is obtained through successive hydroxylation steps in the liver and kidneys. 25(OH)D in the circulatory system has a long half-life, is easy to analyze, and is widely considered to be the best indicator of vitamin D status. Many studies have evaluated serum 25(OH)D concentrations after vitamin D supplementation at different ages and concluded that changes in serum 25(OH)D depend on baseline vitamin D status. Also, the amount of vitamin D supplemented needs to be determined based on the patient's age, weight, genetic polymorphisms, and assay method.
Figure 1. Vitamin D metabolism and its alterations in different clinical conditions (Source: Bilezikian JP, et al. 2021)
Several studies have found that vitamin D may have an extra-skeletal role, and that vitamin D signaling is active in the immune system and has an important physiological role in protecting the human host from bacterial and viral invasion. Vitamin D innate immune signaling mechanisms include the production of cytokines, antimicrobial proteins, and pattern recognition receptors. Some studies have shown that vitamin D supplementation can help fight bacterial infections, and in studies on COVID-19 researchers have also found that vitamin D may alleviate viral respiratory infections, and that vitamin D supplementation in the elderly and in patients with low levels of vitamin D may help prevent COVID-19 infections and the development of severe symptoms after infection. In conclusion, more research on the extra-skeletal effects of vitamin D is needed to demonstrate its importance.
Alternative Names
25(OH)D 25(OH) Vitamin D
References
1. Bilezikian JP, et al. Vitamin D: Dosing, levels, form, and route of administration: Does one approach fit all? Rev Endocr Metab Disord. 2021 Dec;22(4):1201-1218.
2. Ismailova A, et al. Vitamin D, infections and immunity. Rev Endocr Metab Disord. 2022 Apr;23(2):265-277
Q: We were curious if the vitamin D kit comes with any supplied quality control materials.
A: This kit does come with controls. While we don’t have a recommendation for another vendor’s products, controls designed for this type of assay should work.
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References
Make hey while sun shines! Hormone 25 OH D (vitamin D3) in follicular fluid: a determinant factor for top grade blastocyst formation?
The vitamin D receptor (VDR) and its ligand 1,25(OH)(2)D-3 (1,25D) exert anti-tumor effects, but considerable heterogeneity has been reported in different model systems. In general, cell lines derived from aggressive tumor subtypes such as Triple Negative Breast Cancer (TNBC) express low levels of VDR and are less sensitive to 1,25D than those derived from more differentiated tumor types. We have previously reported that 1,25D inhibits hyaluronic acid synthase 2 (HAS2) expression and hyaluronic acid (HA) synthesis in murine TNBC cells. Here we confirmed the inhibitory effect of 1,25D on HA synthesis in human Hs578T cells representative of the mesenchymal/stem-like (MSL) subtype of TNBC. Because HA synthesis requires the production of hexoses for incorporation into HA, we predicted that the high HA production characteristic of Hs578T cells would require sustained metabolic changes through the hexosamine biosynthetic pathway (HBP). We thus examined metabolic gene expression in Hs578T cell variants sorted for High (HA(High)) and Low (HA(Low)) HA production, and the ability of 1,25D to reverse these adaptive changes. HA(High) populations exhibited elevated HA production, smaller size, increased proliferation and higher motility than HA(Low) populations. Despite their more aggressive phenotype, HA(High) populations retained expression of VDR protein at levels comparable to that of parental Hs578T cells and HA(Low) subclones. Treatment with 1,25D decreased production of HA in both HA(High) and HA(Low) populations. We also found that multiple metabolic enzymes were aberrantly expressed in HA(High) cells, especially those involved in glutamine and glucose metabolism. Notably, Glutaminase (GLS), a known oncogene for breast cancer, was strongly upregulated in HA(High) vs. HA(Low) cells and its expression was significantly reduced by 1,25D (100 nM, 24 h). Consistent with this finding, Seahorse extracellular flux analysis indicated that respiration in HA(High) cells was significantly more dependent on exogenous glutamine than HA(Low) cells, however, acute 1,25D exposure did not alter metabolic flux. In contrast to GLS, the glutamate transporter SLC1A7 was significantly reduced in HA(High) cells compared to HA(Low) cells and its expression was enhanced by 1,25D. These findings support the concept that 1,25D can reverse the metabolic gene expression changes associated with HA production in cancer cells with aggressive phenotypes.
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Figure 1. Vitamin D metabolism and its alterations in different clinical conditions
