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Accurate Vitamin D Results

25-Hydroxyvitamin D: A Difficult Analyte (Graham D. Carter, Clin. Chem. 2012, 58:3, 486-488)

The measurement of 25OH Vitamin D has tremendously evolved since the first competitive protein-binding assay that used tritiated 25OH Vitamin D3 and a Vitamin D binding protein (DBP) originating from rat [1]. The use of tritium based material, solvent extraction and chromatography certainly prevented the broad expansion of the assay at that time. Subsequent developments aimed at simplifying the assay procedure, while improving the overall quality of the measurement [2]. Simplifications have been tackled through the replacement of the tritium based label by 125I (RadioImmunoAssay, RIA) and enzymatic conjugates (Enzyme-Linked ImmunoSorbent Assay, ELISA) or by chemiluminescence based material (ChemiLuminescence ImmunoAssay, CLIA). Solvent extraction, used to solubilize the hydrophobic 25OH Vitamin D and to precipitate its binding proteins, gradually gave way to methods involving pH shifts, enzymatic digestion, displacement chemicals

Vitamin D 25-Hydroxyvitamin-D for accurate results by DIAsource

[1] Haddad JG, Chyu KJ. Competitive protein-binding radioassay for 25-hydroxycholecalciferol. J Clin Endocrinol Metab 1971; 33:554–7.
[2] Gomes FP, Shaw PN, Whitfield K, Koorts P, Hewavitharana AK. Recent trends in the determination of vitamin D. Bioanalysis 2013; 5(24):3063–3078.


Calibration of 25OH Vitamin D assays has long been an issue because no reference material was available until recently. A first step was taken by the NIST (National Institute of Standards and Technology) with the release of Standard Reference Material SRM972 in 2009. While this material proved to be useful for the calibration of liquid chromatography based methods, it was not useful in immunoassays due to added exogenous material in 3 out of the 4 vials provided. SRM972 was withdrawn in 2011 and replaced by SRM972a which is constituted of 3 unaltered samples and 1 spiked sample.

Reference Measurement Procedures (RMPs) were also developed at that time. The Belgian professor Linda Thienpont was one of the pioneers in this field. NIST and CDC (Centers for Disease Control and Prevention) further developed their own RMP, both based on the same technology as professor Thienpont’s method namely ID-LC-MS/MS (isotope dilution-liquid chromatography-tandem mass spectrometry). Although the acceptance of the Thienpont’s and NIST LC-MS/MS assays as reference methods [3], and the recent initiative towards standardization of the Vitamin D assays [4] have contributed to an improvement in the method-related variability in serum 25OH Vitamin D measurement, differences remain between assays. These variations can affect the 25OH Vitamin D clinical decision-making [5,6].

DIAsource 25OH Vitamin D Total ELISA, 25OH Vitamin D Total RIA and 25OH Vitamin D3 RIA are all three calibrated against the reference method ID-LC-MS/MS developed by professor Thienpont. This reference methodology is one of the RMP that is part of the Vitamin D Standardization Program (VDSP).

CAlibration for Vit D testing DIAsource

[5] Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, Hansen KE. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J. Clin. Endocrinol. Metab. 2004; 89:3152–7.
[6] Roth HJ, Schmidt-Gayk H, Weber H, Niederau C. Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography–tandem mass spectrometry as a reference. Ann. Clin. Biochem. 2008; 45:153–9.


Polyclonal antibodies, from sheep or rabbit, have been widely used from the 90’s, in RIA (RadioImmunoAssay), ELISA (Enzyme-Linked ImmunoSorbent Assay) and CLIA (ChemiLuminescence ImmunoAssay) assays.

While polyclonal antibodies are simple and inexpensive to produce, they usually lack specificity and suffer from batch to batch variability. Monoclonal antibody production requires high skills and is more expensive. However, a careful selection process ensures a superior specificity profile. Moreover, state of the art production techniques ensure a constant and renewable source of antibodies and all batches are identical.

In 2009, DIAsource Immunoassays patented Mouse Monoclonal Antibodies, based on a proprietary Vitamin D hapten, recognizing both 25OH Vitamin D3 and 25OH Vitamin D2. The patent was granted in Europe in 2013 (EP2316854) and is pending in the US (US20110097733). The patent covers any monoclonal antibody recognizing both 25OH Vitamin D3 and 25OH Vitamin D2. These monoclonal antibodies have been successfully used in commercial RIA (RadioImmunoAssay), ELISA (Enzyme-Linked ImmunoSorbent Assay), automated CLIA (ChemiLuminescence ImmunoAssay) and POCT (Point Of Care Test) assays by DIAsource Immunoassays (25OH Vitamin D Total ELISA and 25OH Vitamin D Total RIA) and many other companies. Amongst others, their main performance characteristics are a high sensitivity, a cross-reactivity towards 25OH Vitamin D2 close to 100% and an attractive specificity profile with i.e. virtually no cross-reactivity towards the C3-epimer molecule (See our Technical Reviews 2014-01 and 2014-02).

Antibody & Vitamin D testing

Vitamin D binding proteins

Because of its hydrophobic nature, 25OH Vitamin D is poorly soluble in aqueous media and is linked to binding proteins in human blood. Vitamin D Binding Protein (DBP) possesses a very high affinity for 25OH Vitamin D and links about 88% of its total amount [7]. Albumin exhibits a weaker affinity, about a 1000 times less than DBP, and binds about 12% of total 25OH Vitamin D thanks to its greater concentration. Only a tiny concentration, around 2-15pg/mL, circulates as the free form [8].

Displacing 25OH Vitamin D from its binding proteins has always been and remains a big challenge in 25OH Vitamin assay development. Early methods were based on extraction procedures involving organic solvents. While these methods were very efficient, the use of solvents and centrifugation steps prevented the automation of the assay. New methodologies were developed involving the denaturation of the binding proteins by pH shift (either acidic or alkaline), their enzymatic digestion, or the smooth release of 25OH Vitamin D by displacement reagents. The latter constitutes the state of the art method as it allows to work at neutral pH without any further neutralization or dilution step.

DIAsource 25OH Vitamin D Total ELISA and 25OH Vitamin D Total RIA are both based on a release solution that involves a displacement reagent. This solution works at pH 7.4 and takes place directly in the ELISA microtiter plate or in the RIA coated tube, eliminating the need for a tedious pre-treatment step that usually takes place in glass or plastic tubes. The DIAsource All-in-One (AIO) technology has been proven to be one of the most efficient release method of 25OH Vitamin D (See our Technical Review 2014-02).

[7] Haddad JG. The Vitamin D binding Protein and its clinical significance. In: Vitamin D: physiology, molecular biology and clinical applications. 1999; 101-107.
[8] Schwartz JB, Lai J, Lizaola B, Kane L, Markova S, Weyland P, Terrault NA, Stotland N, Bikle D. A comparison of direct and calculated free 25(OH) Vitamin D levels in clinical populations. J. Clin. Endocrinol. Metab. 2014; 99(5):1631-7.

Vitamin D binding proteins DIAsource


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