25-OH Vitamin D ELISA Assay Kit
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
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)
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 . 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.
Mouse Rat 25-OH Vitamin D ELISA Assay
25-OH Vitamin D HPLC Assay Kit
25-OH Vitamin D HPLC Column
- Pipette each 200 µl of prediluted Standards 1 – 6, prediluted Control1 and Control2 into the appropriate wells.
- Pipette each 200 µl of patient sample diluted in biotin/sample buffer into each well to be used in the assay.
- Incubate at room temperature (+18 °C to +25 °C) for 2 hours.
- 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.
- Pipette 100 µl of Enzyme Conjugate into each well and incubate for 30 min at room temperature (+18 °C to +25 °C).
- 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.
- Pipette 100 µl of chromogen/ substrate solution into each well and incubate for 15 minutes at room temperature without shaking (protect from direct sunlight!).
- Stop the substrate reaction by addition of 100 µl of Stop Solution to each well (this will cause the blue colour to turn yellow).
- 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
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.
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
|25-OH Vitamin D2
|Vitamin D3 (cholecalciferol)
|Vitamin D2 (ergocalciferol)
|24, 25-OH Vitamin D3
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.