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Release reagent for vitamin d compounds

a technology of vitamin d and reagents, which is applied in the field of vitamin d release reagents, can solve the problems of vitamin d having a relatively short biological half-life in the circulation, affecting the d status of patients, and the measurement of the vitamin d level itself is of little benefi

Inactive Publication Date: 2015-01-22
ROCHE DIAGNOSTICS OPERATIONS INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031]The present disclosure provides an in vitro method for releasing a vitamin D compound from vitamin D-binding protein comprising the step of a) providing a sample to be investigated, b) mixing the sample from step (a) with i) a reagent containing a hydrogen carbonate salt or a substance capable of releasing hydrogen carbonate ions (HCO3−) upon hydrolysis in a concentration of 0.1 M to 2.0 M, ii) a reducing agent, and iii) an alkalinising agent, thereby releasing the vitamin D compound from vitamin D-binding protein.

Problems solved by technology

Measuring the vitamin D level itself is of little benefit when determining the vitamin D status of a patient, because concentrations of vitamin D (vitamin D2 and vitamin D3) fluctuate greatly depending on food uptake or exposure to sunlight.
In addition vitamin D has a relatively short biological half-life in the circulation (24 hours) and it is therefore also for this reason not a suitable parameter for determining the vitamin D status of a patient.

Method used

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Examples

Experimental program
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Effect test

example 1

Assays for the Detection of 25-Hydroxyvitamin D

[0159]Commercial assays are used according to the manufacturer's instructions. The 25-hydroxyvitamin D determinations are carried out by means of HPLC (test for 25(OH)vitamin D3, from the “Immundiagnostik” Company, Bensheim, order No. KC 3400) or by means of LC-MS / MS (Vogeser, M. et al., Clin. Chem. 50 (2004) 1415-1417) as described in the literature.

[0160]The preparation of the ingredients and the general test procedure for a new test is described in the following:

[0161]1.1 Synthesis of hydroxyvitamin D2-3-2′-cyanoethyl ether

[0162]20.6 mg (50 μmol) 25-hydroxyvitamin D2 (Fluka No. 17937) is dissolved in a 25 ml three necked round bottom flask with an internal thermometer in 10 ml dry acetonitrile under an argon atmosphere. 1.5 ml tert.-butanol / acetonitrile (9:1) is added to the solution and cooled to 6° C. in an ice bath. Subsequently 820 μl of an acrylonitrile solution (86 μl acrylonitrile in 1.0 ml acetonitrile) is added and stirred f...

example 2

Comparison of Carbonate Ester to a Metal Salt, a Phosphate Buffer and a Carbonate

[0207]The sample to be investigated is measured using the Elecsys system from the Roche Diagnostics company. The total assay procedure is shown in example 1.5. In aberrance to example 1.5 the reagent composition (A) contains either 0.5 M ethylene carbonate (EC), 0.5 M Na2CO3, 0.5 M NaCl or 0.5 M NaH2PO4, respectively.

[0208]Reagent composition (A):

[0209]10 mM NaOH

[0210]4 mM EDTA

[0211]6.7 mM DTT

[0212]0.5 M of either EC, Na2CO3, NaCl or NaH2PO4

[0213]As control a reagent composition (A) containing 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT has been used. The results are shown in FIG. 5. The carbonate ester (0.5 M EC (♦) present in the alkaline pretreatment (reagent mixture) causes a signal enhancing effect in the competitive assay. Especially the signal dynamic is improved compared to a test without EC (□). A salt (0.5 M NaCl, (⋄)) shows no effect. The addition of 0.5 M Na2CO3 (◯) or 0.5 M NaH2PO4 (▴) shows a minor...

example 3

Alkaline Pretreatment with / without Carbonate Ester

[0214]The sample to be investigated is measured using the Elecsys system from the Roche Diagnostics company. The assay procedure is shown in example 1.5.

[0215]In aberrance to example 1.5 three different reagent compositions have been prepared containing either:[0216]♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or[0217]▴: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT or[0218]□: 10 mM NaOH, 4 mM EDTA.

[0219]After a 4 min pretreatment incubation of sample+either ♦ (reagent composition (A)+alkalinising agent (B) as described in example 1.5), ▴, or □, respectively, (=reagent mixture) and before addition of solution C the pH of the reagent mixture has been set to pH 9 by addition of bis-tris-propane pH 6.3 (FIG. 6). The carbonate ester EC present in the alkaline pretreatment (reagent mixture) causes a signal enhancing effect in the competitive assay. Especially the signal dynamic is improved compared to a test without EC.

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Abstract

A reagent composition for releasing vitamin D compounds bound to vitamin D-binding protein, an in vitro method for the detection of a vitamin D compound in which the vitamin D compound is released from vitamin D-binding protein by the use of this reagent composition and the reagent mixture obtained in this manner. The use of the disclosed reagent composition to release vitamin D compounds as well as a kit for detecting a vitamin D compound which contains the reagent composition for releasing vitamin D compounds in addition to common detecting reagents.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of International Application No. PCT / EP2011 / 058048, filed May 18, 2011, which claims the benefit of European Patent Application No. 10163453.3, filed May 20, 2010, and European Patent Application No. 11158296.1, filed Mar. 15, 2011, the disclosures of which are hereby incorporated by reference in their entirety.BACKGROUND OF THE DISCLOSURE[0002]An adequate supply of vitamin D is vital as the term “vitamin” already suggests. A deficiency of vitamin D leads to severe diseases such as rickets or osteoporosis. While vitamin D was still regarded as a single substance at the beginning of the last century, the vitamin D system has changed in the course of the last decades into a complex and manifold network of vitamin D metabolites. Nowadays more than 40 different vitamin D metabolic products are known (Zerwekh, J. E., Ann. Clin. Biochem. 41 (2004) 272-281).[0003]Humans can only produce D3 vitamins or calcifero...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/82
CPCG01N2440/00G01N33/82Y10T436/203332G01N33/48G01N33/50
Inventor ANTONI, SASCHAVOGL, CHRISTIAN
Owner ROCHE DIAGNOSTICS OPERATIONS INC
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