Home Vitamin D

Today, the vitamin D levels in man are measured in clinical laboratories on a routine basis. "Routine" means, that in many cases the laboratory staff is not well-trained, and has to do a lot of work.
Unfortunately, the assay procedure for the measurement of vitamin D is rather complicated, and involves several crucial steps, as described below.

Assay procedure version: July 2000

Vitamin D

ELISA for the quantitative determination of
25-OH vitamin D in human plasma and serum
Cat.no. BI-20442 12 x 8 tests

This assay procedure is also available as single-page lab protocol, in the format MS WORD 97. Click here for download.


  1. Introduction
  2. Principle of the assay
  3. Contents of the kit
  4. Additional material and equipment required
  5. Reagents- and sample preparation - performance of the assay
  6. Assay procedure scheme
  7. Calculation of results
  8. Assay characteristics
  9. Technical hints
  10. Precautions
  11. Literature


1. Introduction

Vitamin D is a secosteroid hormone involved in the intestinal absorption of calcium and in the regulation of calcium homeostasis. There are two different forms of vitamin D, namely D3 and D2, which are structurally very similar. The latter one is a synthetic product, which is predominantly absorbed via fortified food. Physiological Vitamin D3 levels result from dietary uptake but also from biosynthesis in the skin from 7-dehydrocholesterol and UV-light due to sun exposure. The vitamin is then hydroxylated in the liver to 25-hydroxyvitamin D (25-OH Vit D) which is the major circulating metabolite of Vitamin D. Although the biological active form of vitamin D is 1,25(OH)2 Vit D, synthesised in the kidney, it is widely accepted that the measurement of circulating 25-OH Vit D provides better information with respect to the patients vitamin D status and is used for diagnosis of hypovitaminosis (1,2). The concentration of 25-OH Vit D decreases with age and deficiency is common among the elderly (3,4).

Clinical aspects:

Clinical applications of 25-OH Vit D measurements are the diagnosis and therapy control of postmenopausal osteoporosis (5,6), rickets, osteomalacia, renal osteodystrophy, pregnancy, neonatal hypocalcemia and hypoparathyroidism. In addition, a prevalence of subclinical vitamin D deficiency in different European countries has been discussed lately (7). Vitamin D intoxication mostly occurs from large intake of pharmaceutical preparations of vitamin D and may lead to hypercalcemia, hypercalcuria and nephrocalcinosis in susceptible infants (8).


2. Principle of the assay

This test kit is a competitive binding protein assay for the measurement of 25-OH Vit D. It is based on the competition of 25-OH Vit D present in the sample with biotinylated 25-OH Vit D (tracer) for the binding pocket of vitamin D binding protein (VDBP, Gc-globulin). Since all circulating 25-OH Vit D is bound to VDBP in vivo, samples have to be precipitated with organic solvents to extract the analyte. The supernatant is used without further treatment in the test.

In the first step, vitamin D binding protein, anti-vitamin D binding protein antibody and samples/standards/controls are added. 25-OH Vit D present in the sample then competes with the 25-OH-Vit D-Biotin, bound to the well, for the specific binding sites of the binding protein. Hence, with increasing concentrations of 25-OH Vit D in the sample the amount of binding protein immobilised to the well via the tracer is reduced. Simultaneous addition of an antibody specific for this protein yields a complex, which is finally quantitated by incubation with a host specific peroxidase labelled antibody using TMB as enzyme substrate. The amount of colour developed is inversely proportional to the amount of 25-OH Vit D present in the standards/samples/controls (i.e. the less vitamin D that is present in the sample, the higher the intensity of the colour developed).
A standard curve is plotted and the concentrations of 25-OH Vit D in the samples are calculated from this curve.

Below, the assay scheme is shown graphically:

Assay scheme

3. Contents of the kit


4. Additional material and equipment required


5. Reagent and sample preparation - performance of the assay

Serum- or plasma samples must be centrifuged within one hour. Store samples at -20 C if not assayed within 24 hours. Lipemic samples may give erroneous results and must be centrifuged for 10 min. at 13000 rcf (rotational centrifugal force = g) to seperate the lipids from the plasma. Samples should be mixed well before assaying. We recommend duplicates for all tests.

Reagent preparation:

Assay procedure:

  1. Pipette tips should be fixed well to the pipette to avoid dripping.
    Avoid touching the walls of the V-tube or the pellet with the pipette tip! Invisible thin pellet layer can stick to tube wall.
    Slowly flush the pipette tip once with the supernatant before pipetting the samples into wells.
    Place the supernatant at the bottom of the wells.
    Add 20 l of supernatants of standards, controls and samples into the respective wells.
  2. Mix vitamin D binding protein again and add 100 l to all wells except blank.
  3. Add 100 l antibody (green cap, yellow solution) into all wells, shake plate gently.
  4. Cover strips with plastic film and incubate 3 h at room temperature (18-26 C). If room temperature exceeds 26C, incubate at 4C. Make sure all wells are sealed well with the film to avoid evaporation.
  5. Remove plate sealer slowly from microtiter plate. Discard contents of the wells completely and wash 4x with a minimum of 250 l diluted wash buffer. Make sure that residual buffer is removed completely after the last wash, e.g. by inverting the plate and tapping firmly on absorbent paper.
  6. Add 200 l conjugate (red solution) to all wells.
  7. Cover strips with plastic film and incubate 1 hour at room temperature (18-26C). If room temperature exceeds 26C, incubate at 4C.
  8. Discard contents of the wells and wash 4x with a minimum of 250 l diluted wash buffer. Make sure that residual buffer is removed completely after the last wash, e.g. by inverting the plate and tapping firmly on absorbent paper.
  9. Add 200 l of substrate to all wells.
  10. Incubate for 20 min. at room temp. (18-26C) in the dark.
  11. Add 50 l of stop solution to all wells, shake well.
  12. Determine absorption with an ELISA reader at 450 nm against 620 nm as reference.

If no reference wavelength is available, read only at 450 nm. If the extinction of the zero standard exceeds the measurement range of the photometer, absorption must be measured immediately at 405 nm against 620 nm as reference.


6. Assay procedure scheme

Sample / Standard / Control preparation:
Sample preparation

Assay procedure:


7. Calculation of results

A calibration curve is constructed from the standards. Commercially available software can be used as well as graph paper. Results of the samples are read from this calibration curve.

THE CALIBRATION CURVE IS NOT LINEAR, therefore a spline- or 4PL algorithm is recommended.


8. Assay characteristics

Standard range:
2.5 - 100 ng/ml
1 ng/ml = 2.5 nmol/l
1 nmol/l = 0.4 ng/ml

A typical standard curve looks like this:

Calibration curve

Detection limit:
The detection limit is the concentration of 25-OH Vit D at 95% B/B0.
For this assay the detection limit was determined as 0.6 ng/ml.

Cross reactivity:

24,25-(OH)2 vitamin D3
25-OH vitamin D2
1,25-(OH)2 vitamin D3
vitamin D2 & D3

Sample volume / type:
50 l of human plasma or serum. EDTA or Heparin plasma are the preferred matrices for this assay to compare results to other 25-OH-D analysis systems.


4 samples were tested in 16 replicates. Two repeats of the experiments are shown.


Run 1 (n=16)

Run 2 (n=16)


Mean (ng/ml)


Mean (ng/ml)




8 %


10 %



9 %


11 %



17 %


14 %



9 %


9 %


6 patient samples were tested in duplicates (3 runs).

Sample Nr:

Run 1

Run 2

Run 3

Mean (ng/ml)







6.6 %






13.3 %






11.3 %






7.8 %






9.2 %






15.0 %

10 plasma samples were spiked with 62.4 ng/ml 25-OH Vit D - plasma standard and measured in the assay.




62.4 ng/ml

58.7 ng/ml

94 %

Incubation times:
3 h / 60 min / 20 min

Storage: 4 C

Shelf life: 6 months from day of production


9. Technical hints



10. Precautions

All test components of human source were tested with 3rd generation tests against HIV-Ab and HBsAG; all components were found to be negative. However, they should be handled and disposed as if they were infectious, since no test method can offer complete assurance.


11. Literature

  1. Hollis B.W. et al., Calcified Tissue International (1996) 58: 4-5
  2. Thomas M.K. et al., New England Journal of Medicine (1998) 338: 777-783
  3. Waern E. et al., Osteoporos. Int. (1996) 6: 127
  4. Parfitt A.M. et al., Am. Clin. Nutr. (1982) 36: 1014-1031
  5. Khaw K.T. et. al., Brit. Med. J. (1992) 305: 273-277
  6. Villareal D.T. et. al., J. Clin. Endocrinol. Metab. (1991) 72: 628-634
  7. Scharla et. al., Osetoporosis Int. (1998) 8: 7-12
  8. Misselwitz J et al., Acta. Paediatr. Scand. (1990) 79: 637-643

Last modified: 19. Sep 04