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Technical Literature

25-Hydroxy Vitamin D EIA
Enzyme immunoassay for the quantitative determination of 25-hydroxyvitamin D and other hydroxylated metabolites in serum or plasma

Intended Use
For In Vitro Diagnostic Use
The IDS 25-Hydroxy Vitamin D EIA kit is an enzyme immunoassay intended for the quantitative determination of 25-hydroxyvitamin D (25-OH D) and
other hydroxylated metabolites in human serum or plasma.

Summary and Explanation
Vitamin D is a commonly used collective term for a family of closely related seco-steroids. Upon exposure to sunlight, 7-dehydro-cholesterol, located deep in the actively growing layers of the epidermis, undergoes photolytic cleavage of the “B” ring to yield pre-vitamin D3 which is isomerised to vitamin D3 (cholecalciferol). Vitamin D3 and vitamin D2 (ergocalciferol) may also be obtained by dietary supplementation or from a limited number of foods. Vitamin D2 is metabolised in a similar way to vitamin D3.Vitamin D is stored in adipose tissue and enters the circulation bound to vitamin D binding protein (VDBP) and albumin. In the liver, vitamin D is hydroxylated to give 25-hydroxyvitamin D which also circulates as a complex with VDBP. A small proportion of the 25-OH D is further hydroxylated in the kidney, under direct regulation by parathyroid hormone and ionised calcium levels, to form the biologically-active calcitropic hormone 1,25-dihydroxyvitamin D. Further hydroxylation and metabolism of vitamin D produces compounds that are water soluble and readily excreted. Hepatic vitamin D 25-hydroxylase activity is not tightly regulated, and changes in cutaneous production of vitamin D3, or ingestion of vitamin D (D3 or D2), will result in changes in circulating levels of 25-OH D (1). Serum concentration of 25-OH D is considered to be the most reliable measure of overall vitamin D status and thus can be used to determine whether a patient is vitamin D sufficient(2). Assessment of vitamin D status may be required to determine the cause of abnormal serum calcium concentrations in patients.

Method Description
The IDS 25-Hydroxy Vitamin D EIA kit is an enzyme immunoassay for the quantitation of 25-OH D and other hydroxylated metabolites in serum or plasma. Calibrators, controls and samples are diluted with biotin labelled 25-OH D. The diluted samples are incubated in microtitre wells which are coated with a highly specific sheep 25-OH D antibody for 2 hours at room temperature before aspiration and washing. Enzyme (horseradish peroxidase) labelled avidin, is added and binds selectively to complexed biotin and, following a further wash step, colour is developed using a chromogenic substrate (TMB). The absorbance of the stopped reaction mixtures are read in a microtitre plate reader, colour intensity developed being inversely proportional to the concentration of25-OH D.


Limitations of Use

  • Samples suspected of containing analyte concentrations in excess of the highest calibrator should be assayed in dilution.
  • As in the case of any diagnostic procedure results must be interpreted in
    conjunction with the patient's clinical presentation and other information available.
  • The performance characteristics of this assay have not been established in a
    paediatric population.
  • In rare cases, interference due to extremely high titres of antibodies to avidin
    can occur.
  • The following substances have been tested and found not to interfere in the
    IDS 25-Hydroxy Vitamin D assay:
    • Haemoglobin tested up to 1470 mg/dL
    • Bilirubin tested up to 513 μmol/L
    • Lipid tested up to 5.6 mmol/L triglyceride

Expected Values
Each laboratory determines its own ranges for their local population.
There is no universal agreement on the optimal concentration of 25-OH D. Ranges should be based on clinical decision values that apply to both sexes of all ages rather than population based reference ranges for 25-OH D. To that end, a large study examined the relationship of intact PTH with vitamin D levels in serum. A plateau for iPTH was seen at ~30 ng/mL. Similarly, Calcium (Ca) absorption increased with increasing 25-OH D level until ~30 ng/mL 25-OH D was reached. Optimal Ca absorption requires levels of 25-OH D exceeding 30 ng/mL.

In the case of 25-OH D, there are also many other factors that may influence values: diet, time of day, sun exposure, season of year, geographic location, age, use of sunscreen and/or protective clothing and skin pigmentation. Thus, sampling a group of apparently healthy individuals is not the ideal way to establish the reference range. The US National Osteoporosis Foundation recommends a level >30 ng/mL to protect bone health. Similarly, the US National Kidney Foundation considers levels <30 ng/mL to be insufficient or deficient12.

From a review of the available literature, the recommendations for 25-OH D levels are:

Level   nmol/L ng/mL
Deficient   <25  <10
Insufficient  25-74 10-29
Sufficient  75-250  30-100
Potential Intoxication >250  >100

The following range has been determined using the IDS 25-Hydroxy Vitamin D EIA kit and is provided for guidance only. Each laboratory should determine ranges for their local population.

Normal adults 47.7 - 144 nmol/L (n = 36)

Performance Data

The IDS 25-Hydroxy Vitamin D EIA kit was compared against a recognised radioimmunoassay for the quantitative determination of 25-hydroxyvitamin D and
other hydroxylated metabolites. A population of 180 samples, selected to represent a wide range of 25-hydroxyvitamin D [9.3 - 151.2 nmol/L], were assayed by each method. Least squares regression analysis was performed on the comparative data:
IDS = 1.01(x) + 0.7; correlation coefficient (r) = 0.9

The sensitivity, defined as the concentration corresponding to the mean minus 2 standard deviations of 10 replicates of the zero calibrator, is 5 nmol/L.


Intra assay Inter assay
Mean (nmol/L) N=10% CV Mean (nmol/L) N=11% CV
39.0 5.3 40.3 4.6
67.1 5.6 72.0 6.4
165 6.7 132 8.7

25 Recovery was assessed by adding -OH D to samples prior to assay.

Sample Measured (nmol/L) Expected (nmol/L) Recovery %
A 122 126 97
A 95.6 98.4 97
B 147 141 104
B 123 118 105
Mean 101