25-OH Vitamin D ELISA Assay Kit


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. The 25-OH Vitamin D ELISA assay test kit is for research use only and not to be used in diagnostic procedures.

25-OH Vitamin D ELISA Assay Kit

25-OH Vitamin D ELISA Developed and Manufactured in Germany by DLD Diagnostika

Size: 1×96 wells
Sensitivity: 1.6 ng/mL
Dynamic Range: 4 – 120 ng/mL
Incubation Time: 2 hours, 45 minutes
Sample Type: Serum, Plasma
Sample Size: 20 µL
Alternative Names: 25-OH, Vitamin D, 25-hydroxy Vitamin D
For Research Use Only

Controls Included

Reference Range
8.2 to 37.4 ng/ml
It is suggested that each laboratory establish its own normal ranges

25-OH Vitamin D3 (ng/mL) x 2.5 = 25-OH Vitamin D3 (nmol/L)

Assay Background

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) [1, 2, 3, 4]. 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 circulating forms of the vitamin with little activity [1]. In contrast to other commercially available tests, the Eagle Biosciences 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 [5, 6, 7].

The new 25-OH Vitamin D ELISA assay test kit is designed for the determination of 25-OH Vitamin D in human serum or plasma samples. In the first analysis step, the calibrators and patient samples are diluted with biotin-labelled 25-OH vitamin D and added to microplate wells coated with monoclonal anti-25-OH Vitamin D antibodies. During the incubation an unknown amount of 25-OH Vitamin D in the patient sample and a known amount of biotin-labelled 25-OH vitamin D compete for the antibody binding sites in the microplate wells plate. Unbound 25-OH vitamin D is removed by washing. For the detection of bound biotin-labelled 25-OH vitamin D, a second incubation is performed using peroxidase-labelled streptavidin. In a third incubation using the peroxidase substrate tetramethylbenzidine (TMB) the bound peroxidase promotes a color reaction. The color intensity is inversely proportional to the 25-OH vitamin D concentration. D concentration.

Related Products

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Additional Information

Assay Procedure

  1. Pipette each 200 µl of prediluted Standards 1 – 6, prediluted Control1 and Control2 into the appropriate wells.
  2. Pipette each 200 µl of patient sample diluted in biotin/sample buffer into each well to be used in the assay.
  3. Incubate at room temperature (+18 °C to +25 °C) for 2 hours.
  4. After the 2 hour incubation, aspirate or discard the samples from the wells, add 300 µl of Wash Buffer and aspirate or discard again. Repeat washing with each 300 µl Wash Buffer two more times for a total of three washings. Tap the inverted wells gently on a clean dry absorbent surface to remove any droplets of Wash Buffer.
  5. Pipette 100 µl of Enzyme Conjugate into each well and incubate for 30 min at room temperature (+18 °C to +25 °C).
  6. After the 30 minute incubation, aspirate or discard the reagent from the wells, add 300 µl of Wash Buffer and aspirate or discard again. Repeat washing with each 300 µl Wash Buffer two more times for a total of three washings. Tap the inverted wells gently on a clean dry absorbent surface to remove any droplets of Wash Buffer.
  7. Pipette 100 µl of chromogen/ substrate solution into each well and incubate for 15 minutes at room temperature without shaking (protect from direct sunlight!).
  8. Stop the substrate reaction by addition of 100 µl of Stop Solution to each well (this will cause the blue colour to turn yellow).
  9. Photometric measurement of the colour intensity should be made at a wavelength of 450 nm and a reference wavelength between 620 nm and 650 nm within 30 minutes of adding the stop solution. Prior to measuring, slightly shake the microplate to ensure a homogeneous distribution of the solution.

Typical Standard Curve

25-OH Vitamin D ELISA


As there is no international standard, 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 with each 25-OH Vitamin D ELISA Assay Kit. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated.

Cross reactivity

This 25-OH Vitamin D ELISA assay kit detects 25-OH Vitamin D2 and D3 specifically. Cross reactions with other metabolites are given in the following table.

Cross reactivity (%)
25-OH Vitamin D3 100%
25-OH Vitamin D2 100%
Vitamin D3 (cholecalciferol) < 0.03%
Vitamin D2 (ergocalciferol) < 0.05%
24, 25-OH Vitamin D3 < 0.03%


Product Manual


Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.



Suarez, JC., Knape, K., Carey, JB. Effect of animal feed grade sodium bisulfate supplementation on performance, intestinal morphology and vitamin D status of broilers subjected to a coccidious vaccine challenge. Journal of Applied Poultry Research. (2021)


O’Brien, MB., McLoughlin, RM., Roche, C., et al. Effect of IL-8 haplotyple on temporal profile in circulating concentrations of interleukin 8 and 25(OH) vitamin D in Holstein-Friesian calves. Veterinary Immunology and Immunopathology. (2021)238:110287


McGillis, L., Bronte-Tinkew, DM., Dang, F., et al. Vitamin D deficiency enhances expression of autophagy-regulating miR-142-3p in mouse and ‘involved’ IBD patient intestinal tissues. American Journal of Physiology. (2021)


Daghestani, MH., Daghestani, MH., Warsy, A., et al. Adverse effects of Selected Markers on the Metabolicll and Endocrine Profiles of Obese Women With and Without PCOS. Frontiers in Endocrinology. (2021) 12:665446


Li, Y., Cook, KL., Yu, W., et al. Inhibition of Antiestrogen-Promoted Pro-Survival Autophagy and Tamoxifen Resistance in Breast Cancer through Vitamin D Receptor. Nutrients. (2021) 13(5):1715

Kreitman A, Schneider SH, Hao L, et al. Reduced Postprandial Bone Resorption and Greater Rise in GLP-1 in Overweight and Obese Individuals After an Alpha-Glucosidase Inhibitor: a Double-Blinded Randomized Crossover Trial. Osteoporos Int. 2021.

Pullinger S A, Varamenti E, Nikolovski Z, Elgingo M, Cardinale M. Seasonal Changes in Performance Related Characteristics and Biochemical Marker Variability of Adolescent Table Tennis Players, Asian J Sports Med. 2019 ; 10(1):e67278. doi: 10.5812/asjsm.67278.

Aboud, S F. “Correlation of Vitamin D Deficiency and Hyperparathyroidism with Anemic in Female Iraqi.” Iraqi National Journal of Chemistry, vol. 17, no. 1, 15 June 2017, doi:10.22317/iqnjc.06201703.

Zhang, Ning et al. (2018). Common Variants rs11191548 near the CYP17A1 Gene is associated with Hypertension and the Serum 25(OH) D levels in Han Chinese. Journal of Human Genetics.  63:731-737.

Lee, Rachel U., et al. (2018). 25-Hydroxyvitamin D Influenza Vaccine Response and Healthcare Encountes Among a Young Adult Population. PLoS One. 13. 

Pincikova, T., D. Paquin-Proulx, J. K. Sandberg, M. Flodström-Tullberg, and L. Hjelte. “Vitamin D treatment modulates immune activation in cystic fibrosis.” Clinical & Experimental Immunology(2017): n. pag. Web.

Willis, R., M. Smikle, K. Deceulaer, Z. Romay-Penabad, E. Papalardo, P. Jajoria, B. Harper, V. Murthy, M. Petri, and E. B. Gonzalez. “Clinical associations of proinflammatory cytokines, oxidative biomarkers and vitamin D levels in systemic lupus erythematosus.” Lupus(2017): 

Chatterton, J., A. Pas, S. Alexander, M. Leech, R. Jakob-Hoff, Bp Jensen, and A. Digby. “Concentrations of calcium and 25-hydroxycholecalciferol (vitamin D3) in plasma of wild kākāpō (Strigops habroptilus) living on two islands in New Zealand.” New Zealand Veterinary Journal65.4 (2017): 198-203. Web.

Mirhosseini, Naghmeh Z., Steven J. Knaus, Kaylee Bohaychuk, Jaswant Singh, Hassan A. Vatanparast, and Lynn P. Weber. “Both high and low plasma levels of 25-hydroxy vitamin D increase blood pressure in a normal rat model.” British Journal of Nutrition116.11 (2016): 1889-900. Web.

James, J;Weaver, V;Cantorna, MT;, (2016). Control of Circulating IgE by the Vitamin D Receptor In Vivo Involves B Cell Intrinsic and Extrinsic Mechanisms. J. Immunol. PubMed: 28003380

Crum-Cianflone, NF;Won, S;Lee, R;Lalani, T;Ganesan, A;Burgess, T;Agan, BK;, (2016). Vitamin D levels and influenza vaccine immunogenicity among HIV-infected and HIV-uninfected adults. Vaccine, 34(41), 5040-5046. doi:10.1016/j.vaccine.2016.06.019

Kenny, T;McCune, D;Kruskall, L;Navalta, J;Hickman, R;Young, J;, (2016). Vitamin D Status and Bone Mineral Density in Female Collegiate Dancers and Cheerleaders: 2670 Board #193 June 3, 9: 30 AM – 11: 00 AM (Dissertation, University of Las Vegas, 2016). Med Sci Sports Exerc.

VM Tsymbal; Determination of vitamin d metabolites in children with diabetic nephropathyActual problems of modern medicine: Bulletin; 4:52.

Jain, SK;Kanikarla-Marie, P;Warden, C;Micinski, D;L-cysteine supplementation upregulates glutathione (GSH) and vitamin D binding protein (VDBP) in hepatocytes cultured in high glucose and in vivo in liver, and increases blood levels of GSH, VDBP, and 25-hydroxy-vitamin D in Zucker diabetic fatty rats., Mol Nutr Food Res, Vol. 60, Iss. 5, pg. 1090-1098. doi: 10.1002/mnfr.201500667.

Marouj AL-Ajeeli, Jimenez Hector, CA Bailey;  Comparison of a commercial ELISA (25-OH-Vitamin D Kit, Eagle Biosciences®) and Liquid chromatography-mass spectrometry (LC/MS) assay to determine the serum 25-hydroxycholecalciferol in the broiler chicken.  Conference: Poultry Science Association Annual Meeting 2015, At Louisville, Kentucky, U.S.A.

Irani, M;Seifer, DB;Grazi, RV;Julka, N;Bhatt, D;Kalgi, B;Irani, S;Tal, O;Lambert-Messerlian, G;Tal, R. Vitamin D Supplementation Decreases TGF-β1 Bioavailability in PCOS: A Randomized Placebo-Controlled Trial. J. Clin. Endocrinol. Metab. 2015 Nov 1:4307-14. doi:10.1210/jc.2015-2580.

Matthews, DG;D’Angelo, J;Drelich, J;Welsh, J. Adipose-specific Vdr deletion alters body fat and enhances mammary epithelial density. J. Steroid Biochem. Mol. Biol. 2015 Sep 30. doi: 10.1016/j.jsbmb.2015.09.035

Assa, A;Vong, L;Pinnell, LJ;Rautava, J;Avitzur, N;Johnson-Henry, KC;Sherman, PM. Vitamin D deficiency predisposes to adherent-invasive Escherichia coli-induced barrier dysfunction and experimental colonic injury. Inflammatory Bowel Dis. 2015 Feb;21(2):297-306. doi: 10.1097/MIB.0000000000000282.

Jain Sushil K., Kahlon Gunjan, Bass Pat, Levine Steven N., and Warden Cassandra. Can l-Cysteine and Vitamin D Rescue Vitamin D and Vitamin D Binding Protein Levels in Blood Plasma of African American Type 2 Diabetic Patients? Antioxidants & Redox Signaling. 2015: doi:10.1089/ars.2015.6320.

Tamer Gheita, Safaa Sayed, Waleed Hammam, Gehan A. Hegazy.  Subclinical Hypovitaminosis D and Osteoporosis in Breast Cancer Patients. Middle East Journal of Internal Medicine. 2015: 8 (2): 12-17.

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