Release reagent for vitamin d compounds

a technology of vitamin d and reagents, applied in the field of reagent composition, 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: 2016-01-28
ROCHE DIAGNOSTICS OPERATIONS INC
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0045]Without wanting to be bound to this theory, it may well be that the presence of one hydrogen carbonate salt and a substance capable of releasing hydrogen carbonate ions (HCO3−) upon hydrolysis in the reagent composition induces a pH shift. Lower concentrations of said hydrogen carbonate salt and / or said substance capable of releasing hydrogen carbonate ions (HCO3−) upon hydrolysis in a reagent composition cause a slower pH reduction of the reagent mixture during the pre-treatment reaction. Higher concentrations of said hydrogen carbonate salt and / or said substance capable of releasing hydrogen carbonate ions (HCO3−) upon hydrolysisin the reagent composition cause a faster pH reduction of the reagent mixture during the pre-treatment reaction. It also would appear that due to the concerted action of one hydrogen carbonate salt and a substance capable of releasing hydrogen carbonate ions (HCO3−) upon hydrolysis and reducing agent at alkaline buffer conditions an irreversible denaturation of vitamin D-binding protein is achieved and thereby later detection of a vitamin D compound is facilitated.
[0096]In the specific detection of a vitamin D compound further incubation steps follow after the pre-treatment step. The leftover of the reducing agent present in the reagent mixture can be blocked by addition of unspecific proteins, preferably e.g. human serum albumin (HSA). This unspecific proteins can be added separately or can be simply included in the solution also comprising the detecting reagent. By blocking the residual reducing capability of the reducing agent, a noncompromised detection of a vitamin D compound using a proteinaceous specific binding agent to a vitamin D compound is possible.

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.
The binding of 25-hydroxyvitamin D or other vitamin D compounds to the vitamin D-binding protein enormously complicates the determination of vitamin D compounds.
Despite immense efforts in recent years, all available methods for determining vitamin D have disadvantages such as laborious sample preparation, poor standardization, poor agreement between test procedures or bad recovery of spiked vitamin D (see for this in particular Zerwekh, J. E., supra).
It is particularly difficult to automate a test for a vitamin D compound.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Release reagent for vitamin d compounds
  • Release reagent for vitamin d compounds
  • Release reagent for vitamin d compounds

Examples

Experimental program
Comparison scheme
Effect test

example 1

Assays for the Detection of 25-hydroxyvitamin D

[0155]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.

[0156]The preparation of the ingredients and the general test procedure for a new test is described in the following:

1.1 Synthesis of hydroxyvitamin D2-3-2′-cyanoethyl ether

[0157]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 for 15 ...

example 2

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

[0169]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.

[0170]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.

Reagent Composition (A):

[0171]

 10 mMNaOH  4 mMEDTA6.7 mMDTT0.5Mof either EC, Na2CO3, NaCl or NaH2PO4

[0172]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 effect on the signal...

example 3

Alkaline Pretreatment with / without Carbonate Ester

[0173]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.

[0174]In aberrance to example 1.5 three different reagent compositions have been prepared containing either:

♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

▴: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT or

□: 10 mM NaOH, 4 mM EDTA.

[0175]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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular massaaaaaaaaaa
temperatureaaaaaaaaaa
temperatureaaaaaaaaaa
Login to view more

Abstract

A reagent composition for releasing vitamin D compounds bound to vitamin D-binding protein and 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 as well as the reagent mixture obtained in this manner. Also disclosed is the use of the reagent compositions to release vitamin D compounds as well as a kit for detecting a vitamin D compound.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 14 / 277,359 filed May 14, 2014, which is a continuation of International Application No. PCT / EP2012 / 072569 filed Nov. 14, 2012, which claims the benefit of European Patent Application No. 11189736.9 filed Nov. 18, 2011, the disclosures of which are hereby incorporated by reference in their entirety.BACKGROUND INFORMATION[0002]The present invention concerns 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. It also concerns 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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/82
CPCG01N33/82Y10T436/203332C07C401/00
Inventor ANTONI, SASCHAVOGL, CHRISTIAN
Owner ROCHE DIAGNOSTICS OPERATIONS INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap