ELECSYS® VITAMIN D ASSAY
Electrochemiluminescence binding assay (ECLIA) for the
in-vitro determination of total 25-hydroxyvitamin D
Indication
Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to
sunlight. Vitamin D is biologically inert and must undergo hydroxylation steps to become active.1 Our
body can only synthesize vitamin D3. Vitamin D2 is taken up with fortified food or given by supplements.
Physiologically, vitamin D3 and D2 are bound to the vitamin D-binding protein (VDBP) in plasma and
transported to the liver to become 25-hydroxyvitamin D (vitamin D (25-OH)). As vitamin D (25-OH)
represents the major storage form, its blood concentration is used to assess the overall vitamin D status.
More than 95% of vitamin D (25-OH), measurable in serum, is vitamin D3 (25-OH) whereas vitamin D2
(25-OH) reaches measurable levels only in patients taking vitamin D2 supplements.1,2,3 Vitamin D is
essential for bone health. In children, severe deficiency leads to rickets. In elderly, the risk of falling has
been attributed to vitamin D deficiency due to muscle weakness. Moreover, low vitamin D
(25-OH) concentrations are associated with lower bone mineral density. Insufficiency has also been linked
to diabetes, cancer, cardiovascular disease, and autoimmune diseases.1 The Elecsys Vitamin D assay
employs VDBP to capture both 25-hydroxyvitamin D3 and D2. This assay is intended for the quantitative
determination of total vitamin D (25-OH) in human serum and plasma, as an aid in the assessment of
vitamin D sufficiency.
Test principle: Competitive protein binding assay
Natural VDBP
Denaturated VDBP
Ru
Ru
Pretreatment
+ Ruthenylated-VD-
Sample, PT1,
PT2, 9 min
BP R1, 9 min
VDBP = Vitamin D binding protein
PT1, PT2 = pre-treatment components
Ru
R2, Beads, 9min
Ru
Ru
Ruthenylated VDBP
Bi
+ 25(OH)D-Bi
Ru
Ru
Ru
25(OH)D-Bi =
biotinylated vitamin D (25-OH)
First, the sample is incubated with a pretreatment reagent for 9 minutes. Thereby, the natural VDBP in the
sample is denatured to release the bound vitamin D (25-OH). Second, the sample is further incubated
with a recombinant ruthenium-labeled VDBP to form a complex of vitamin D (25-OH) and the ruthenylatedVDBP. Third, with the addition of a biotinylated vitamin D (25-OH) a complex consisting of the rutheniumlabeled VDBP and the biotinylated vitamin D (25-OH) is formed. The entire complex becomes bound to the
solid phase (by the interaction of biotin and streptavidin-coated microparticles which are captured on
the surface of the elecrode). Unbound substances are removed. Applying voltage to the electrode induces
chemiluminescent emission which is measured by a photomultiplier. Results are determined via an
instrument-specific calibration curve which is generated by 2-point calibration and a calibration master
curve provided via the reagent barcode.
Bi
Elecsys® Vitamin D test characteristics
Testing time
27 minutes
Test principle
Competitive protein binding assay
Calibration
2 points
Sample material
Serum and plasma
Sample volume
1.5 μL
Limit of detection
3.00 ng/mL
Limit of quantitation (Functional sensitivity)
5.00 ng/mL (12.5 nmol/L) with CV ≤ 20%
Measuring range
5.00 - 60.0 ng/mL (12.5 - 150 nmol/L)
Dilution
1:2 (if concentration of diluted sample is > 25 ng/mL
or 62.5 nmol/L
Traceability
Standardized against LC-MS/MS which in turn is traceable to NIST
Repeatability
Within-run precision: LOQ - 15 ng/mL: SD ≤ 0.57 ng/mL
> 15 ng/mL: CV% ≤ 4.4%
Reproducibility
Intermediate precision: LOQ - 15 ng/mL: SD ≤ 0.85 ng/mL
> 15 ng/mL: CV% ≤ 5.7%
Expected values
Currently there is no standard definition of the optimal vitamin D status.
Many specialists consider the commonly used population based reference
values too low. Health based reference values are recommended to replace
population based reference values.1 Most experts agree that vitamin D
deficiency should be defined as vitamin D (25-OH) of ≤ 20 ng/mL (≤ 50
nmol/L).9 Vitamin D insufficiency is recognized as 21-29 ng/mL.9 Similarly,
the US National Kidney Foundation considers levels < 30 ng/mL to be
insufficient or deficient.10 The preferred level for vitamin D (25-OH) by many
experts is now recommended to be ≥ 30 ng/mL (≥ 75 nmol/L). 5,9,11,12
Order information
Elecsys® Vitamin D
100 tests
06506780 160
Vitamin D CalSet
4 x 1 mL
06506798 160
PreciControl Varia 3
3 x 3 mL each
06364829 160
Method comparison
40
Pearson r = 0.85
30
The sample concen-trations were
between approx. 6
ng/mL (15 nmol/L)
and 65 ng/mL (158
nmol/L).
20
10
0
0 10 20
30 40
50 60
Vitamin D (25-OH)
by LC-MS/MS [ng/mL]
70
2. A comparison of the Elecsys Vitamin D assay (y) using samples
measured with a commercially available 25-hydroxyvitamin D
immunoassay (x) gave the following correlation:
Number of samples
70
measured: 165
60
Deming
y = 0.992 x + 1.20
50
Elecsys® Vitamin D
[ng/mL]
Elecsys ® Vitamin D
[ng/mL]
1. A comparison of the Elecsys Vitamin D assay (y) using
samples measured with LC-MS/MS (x) gave the following
correlation:
Number of samples
70
measured: 290
60
Deming
y = 1.03 x + 3.07
50
Pearson r = 0.91
40
30
10
The sample concen-trations were
between approx. 13
ng/mL
0
(32.5 nmol/L) and 63
20
ng/mL (158 nmol/L).
01020
30 40
50 60 70
Commercially available vitamin D
(25-OH) immunoassay [ng/mL]
COBAS, COBAS E, ELECSYS, MODULAR and LIFE NEEDS ANSWERS are trademarks of Roche.
© 2016 Roche. PP-US-06958-0416
Roche Diagnostics
9115 Hague Road
Indianapolis, IN 46256
www.usdiagnostics.roche.com
References
1. Holick, M.F. (2007). Vitamin D deficiency. N Engl J Med, 357:266-281.
2. Houghton, L.A., Vieth, R. (2006). The case against ergocalciferol (vitamin
D2) as a vitamin supplement. Am J Clin Nutr, 84:694-697.
3. Hart, G.R., et al. (2006). Measurement of vitamin D Status: background,
clinical use and methodologies. Clin Lab,52(7-8):335-343
4. Bischoff-Ferrari, H.A., et al. (2006). Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin
Nutr, 84:18-28.
5. Dawson-Hughes, B., et al. (2005). Estimates of optimal vitamin D status.
Osteoporos Int, 16:713-716.
6. Vieth, R., et al. (2007). The urgent need to recommend an intake of
vitamin D that is effective. Am J Clin Nutr, 85:649-650.
7. Saintonge, S., et al. (2009). Implications of a New definition of Vitamin D
Deficiency in a Multiracial US Adolescent Population: The National Health
and Nutrition Examination Survey. Pediatrics, 123:197-803.
8. Adams, J.S. and Hewison M. (2010). Update in Vitamin D. J Clin. Endocrinol.Metab, 95:471-478.
9. Holick, M.F. (2009). Vitamin D status: measurement, interpretation, and
clinical application. Ann Epidemiol, 19:73-78.
10.KDOQI Clinical Practice Guidelines for Bone Metabolism and Disease in
Children with Chronic Kidney Disease. http://www.kidney.org/PROFESSIONALS/kdoqi/guidelines_pedbone/guide8.htm.
11.Souberbielle, J.C., et al. (2010). Vitamin D and musculoskeletal health,
cardiovascular disease, autoimmunity and cancer: Recommendations for
clinical practice. Autoimmun Rev, 9:709-715.
12.Vieth, R. (2011). Why the minimum desirable serum 25-hydroxyvitamin
D level should be 75 nmol/L (30 ng/mL). Best Pract Res Clin Endocrinol
Metabol, 25:681-691.