25(OH)-Vitamin D (TR) ELISA Kit

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Enzyme Immunoassay for the Quantitative Determination of 25(OH)-Vitamin D Concentration in Human Serum

Introduction

25-hydroxy vitamin D (25-OH Vit D), also known as calciferol, is a steroid-like, fat-soluble pro-hormone with two primary forms: D2 (ergocalciferol) and D3 (cholecalciferol) (Holick, 2007). Vitamin D is essential for maintaining bone mineral density, facilitating calcium and phosphate absorption, and reducing renal excretion (Bouillon et al., 2019). In addition to bone health, sufficient vitamin D levels have been linked to reduced incidences of cancer, autoimmune diseases, and cardiovascular conditions (Pludowski et al., 2018).

Vitamin D is synthesized in the skin upon exposure to sunlight and can be obtained from dietary sources such as fortified grain products, fatty fish (e.g., sardines), egg yolks, and dairy products (Grant et al., 2022). Severe vitamin D deficiency can result in rickets in children and osteomalacia in adults, while insufficient levels are associated with secondary hyperparathyroidism, impaired immunity, abnormal cell metabolism, and osteoporosis (Forrest & Stuhldreher, 2011). However, excessive vitamin D supplementation can lead to toxicity and hypercalcemia, which may cause soft tissue calcification (Sahota, 2014).

Clinical Significance

Vitamin D plays a crucial role in calcium homeostasis and immune function. Research suggests that adequate 25(OH)-vitamin D levels contribute to the prevention of osteoporosis, fractures, and chronic diseases such as diabetes, multiple sclerosis, and certain cancers (Holick & Chen, 2008). Clinical studies have shown that 25(OH)-vitamin D measurements help assess deficiency, optimize supplementation, and monitor disease risk factors (Bouillon et al., 2019). Therefore, accurate quantification of serum 25(OH)-vitamin D is essential for clinical decision-making and public health strategies.

Test Principle

This assay is based on a competitive inhibition ELISA technique. The microtiter wells are coated with anti-25-OH vitamin D monoclonal antibodies. Patient serum and standards are combined with an extraction buffer to release vitamin D from its binding protein (DBP-complex). Following incubation and washing, biotinylated 25-OH vitamin D and HRP-conjugated streptavidin are added to the wells. The biotinylated 25-OH vitamin D competes with the endogenous serum vitamin D for binding sites on the coated antibodies. After incubation and washing, a chromogen-substrate solution is added, producing a blue color. The reaction is stopped with an acidic solution, changing the color to yellow, which is measured spectrophotometrically at 450 nm. The intensity of the color is inversely proportional to the concentration of 25-OH vitamin D in the sample. By referencing a standard curve, the 25-OH vitamin D concentration in the test sample is quantified.

References

  • Bouillon, R., Marcocci, C., Carmeliet, G., Bikle, D., White, J. H., Dawson-Hughes, B., … & Feldman, D. (2019). Skeletal and extraskeletal actions of vitamin D: Current evidence and outstanding questions. Endocrine Reviews, 40(4), 1109-1151. https://doi.org/10.1210/er.2018-00126
  • Forrest, K. Y., & Stuhldreher, W. L. (2011). Prevalence and correlates of vitamin D deficiency in US adults. Nutrition Research, 31(1), 48-54. https://doi.org/10.1016/j.nutres.2010.12.001
  • Grant, W. B., Boucher, B. J., Al Anouti, F., & Pilz, S. (2022). Comparing vitamin D status in different populations globally. Nutrients, 14(2), 333. https://doi.org/10.3390/nu14020333
  • Holick, M. F. (2007). Vitamin D deficiency. New England Journal of Medicine, 357(3), 266-281. https://doi.org/10.1056/NEJMra070553
  • Holick, M. F., & Chen, T. C. (2008). Vitamin D deficiency: A worldwide problem with health consequences. The American Journal of Clinical Nutrition, 87(4), 1080S-1086S. https://doi.org/10.1093/ajcn/87.4.1080S
  • Pludowski, P., Holick, M. F., Grant, W. B., Konstantynowicz, J., Mascarenhas, M. R., Haq, A., … & Vlassios, P. (2018). Vitamin D supplementation guidelines. Journal of Steroid Biochemistry and Molecular Biology, 175, 125-135. https://doi.org/10.1016/j.jsbmb.2017.01.021
  • Sahota, O. (2014). Understanding vitamin D deficiency. Aging Health, 10(1), 1-8. https://doi.org/10.2217/ahe.13.74
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