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

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

Contents

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 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:

3. Contents of the kit

• 12 x 8 well microtiter strips in stripholder, packed in zipped alubag with desiccant.
  Wells are coated with 25-OH-D-biotin bound to streptavidin
• Wash buffer; 10x concentrated
  The bottle contains 100 ml of wash buffer concentrate
• Assay buffer
  The bottle contains 15 ml of assay buffer, ready to use
• Vitamin D Binding Protein (blue cap)
  The vial contains Vitamin D Binding Protein sufficient for 96 tests, lyophilised
• 6 standards (white cap)
  The vials contain serum based 25-OH Vit D standards, ready to use. The concentrations are stated on the labels
• 2 controls (yellow cap)
  The vials contain serum based 25-OH Vit D controls, ready to use. The concentrations are stated on the labels
• Anti-Vitamin D Binding Protein Antibody (green cap)
  The vial contains 11 ml rabbit anti-VDBP antibody, ready to use, yellow solution
• Precipitating agent
  The vial contains 50 ml precipitating agent, ready to use, blue solution
• Conjugate
  The vial contains 22 ml goat anti-rabbit antibody conjugated to horseradish peroxidase (HRPO), ready to use (red solution)
• Substrate
  The vial contains 22 ml TMB solution, ready to use
• Stop solution
  The vial contains 7 ml of stop solution, ready to use.
• 2 self-adhesive plastic films
• Protocol sheet
• Instructions for use (package insert)

4. Additional material and equipment required

• Distilled water
• Deep freezer unit (-20 °C)
• Variable volume pipettes in the range of 20 µl to 200 µl
• Multichannel or multipette
• Manual or automatic microwell washer
• Temp-controlled centrifuge capable of 3000 rcf (rotational centrifugal force = g)
• Vortex mixer
• ELISA reader equipped with 450 nm filter
• Graph paper or software for calculation of results
• V - tubes, without safety lock lids

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:

• Dilute wash buffer concentrate to 1000 ml (add the 100 ml of concentrate to 900 ml of distilled water), mix well. Crystals in the buffer concentrate will dissolve at room temperature. This buffer is stable at 4 °C until expiry date stated on the label.
• Mark positions for blank, standards, controls and samples on the protocol sheet supplied.
• Take microtiter strips out of the bag and mark as appropriate. Store unused strips with the desiccant at 4 °C in the alubag. Strips are stable until expiry date stated on the label.
• All reagents should be kept at 4 °C until used in the assay. During the assay, all kit reagents can be left at room temperature.

Assay procedure:

• Dissolve vitamin D binding protein (VDBP, blue cap) in 13 ml assay buffer, mix well and leave at 4°C for at least 30 min., mix again before pipetting. Reconstituted VDBP is stable at -20 °C until expiry date stated on the label. Avoid repeated freeze-thaw cycles!
• Sample / Standard / Control preparation:
  V-tubes must be used for precipitation. Do not use round-bottom tubes or Eppendorf tubes with safety lock lids! Mix samples on Vortex mixer prior to pipetting.
  First put 50 µl of each sample, standard or control into V-tubes.
  Then add 400 µl precipitating agent to samples, standards and controls.
  Mix thoroughly on Vortex mixer.
  Incubate for 30 min. at -20°C.
  Centrifugation (rcf=rotational centrifugal force=g):
  • swing out rotor: 5 min. at ³3000 rcf (precool centrifuge to 4°C) or 2-3 min. at ³6000-8000 rcf (room temperature centrifuge, 18-26°C).
  • fixed angle rotor: 2-3 min. at maximum speed at room temperature or 4°C.

  Handle tubes carefully after centrifugation! The supernatant must be perfectly clear and well separated from the pellet at the bottom of the tubes. The supernatant contains the extracted 25-OH Vit D and should be assayed immediately. If not possible, keep at 4°C for max. 1 hour.

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 µl of supernatants of standards, controls and samples into the respective wells.
2. Mix vitamin D binding protein again and add 100 µl to all wells except blank.
3. Add 100 µl 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 26°C, incubate at 4°C. 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 µl 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 µl conjugate (red solution) to all wells.
7. Cover strips with plastic film and incubate 1 hour at room temperature (18-26°C). If room temperature exceeds 26°C, incubate at 4°C.
8. Discard contents of the wells and wash 4x with a minimum of 250 µl 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 µl of substrate to all wells.
10. Incubate for 20 min. at room temp. (18-26°C) in the dark.
11. Add 50 µl of stop solution to all wells, shake well.
12. Determine absorption with an ELISA reader at 450 nm against ³ 620 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 ³ 620 nm as reference.

6. Assay procedure scheme

Sample / Standard / Control 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:

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:

Sample volume / type:
50 µl 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.

Precision:

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

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

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

Incubation times:
3 h / 60 min / 20 min

Storage: 4 °C

Shelf life: 6 months from day of production

9. Technical hints

• Vitamin D assays use organic solvents for extraction. The adhesion of ethanolic solution (e.g. precipitating agent in the samples/standards/ controls) is different to that of aqueous solutions. Therefore:
• Make sure that the pipette tips are fixed well to the pipette to avoid dripping.
• Slowly flush the pipette tip once with the supernatant before pipetting it into the well of the microtiter plate.
• Do not leave the extracts at room temperature. Keep them at 4°C for max. 1 hour, if assaying isn't possible immediately.
• Never touch the pellet nor the side walls of the V-tube with the pipette.

General:

• To avoid cross-contamination, change pipette tips between addition of standards, controls, samples, tracer, conjugate, substrate and stop solution. Also use separate reservoirs for each reagent!
• Do not mix stoppers and caps of different reagents - contamination!
• Do not use reagents beyond expiry date.
• Protect reagents from direct sunlight.
• Do not mix or substitute reagents with those from other lots or sources.
• To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
• After incubation of the specific reagents, remove plate sealer slowly from microtiter plate to avoid cross-contamination.
• Substrate solution should remain colourless until added to the plate.
• Stop solution should be added to the plate in the same order as the substrate solution.

10. Precautions

All test components of human source were tested with 3^rd 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.

• All liquid reagents contain 0,01% thimerosal or 0,095% sodium azide as preservative.
  Thimerosal and sodium azide are toxic! Avoid contact with skin and mucous membrane.
• Do not pipette by mouth.
• Do not eat, drink, smoke or apply cosmetics where reagents are used.
• Avoid all contact with the reagents by using gloves.
• Stop solution contains diluted sulphuric acid. Irritation to eyes and skin is possible. Flush with water after contact!

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