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Method and reagent for determining vitamin d metabolites

A technology of vitamins and hydroxyvitamins, applied in biological testing, material inspection products, etc.

Inactive Publication Date: 2016-01-13
ORGENTEC DIAGNOSTIKA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] It was therefore an object of the present invention to provide reagents for the release of conjugated vitamin D which solve the current problems of releasing conjugated vitamin D from protein complexes and which are also suitable for use in automated direct quantitative determinations of vitamin D. application in the method

Method used

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  • Method and reagent for determining vitamin d metabolites
  • Method and reagent for determining vitamin d metabolites
  • Method and reagent for determining vitamin d metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1: Preparation of vitamin D releasing agent

[0075] Sodium toluenesulfonate was dissolved in water and adjusted to a final concentration of 1M sodium toluenesulfonate, 100 mM sodium citrate, and 50 mM iron(III) chloride with a stock solution of 1 M sodium citrate and 1 M iron(III) chloride.

Embodiment 2

[0076] Example 2: Binding of 25-OHD antibody on solid phase

[0077] Antibodies to 25-OH-vitamin D3 or 25-OH-vitamin D2 were diluted to a concentration of 1 μg / ml in Tris salt buffer at pH 8.0. The cavity of a microtiter plate (Maxisorb, Nunc) was coated with 100 μl of antibody dilution, dried at 37°C and left at 4°C until assayed.

Embodiment 3

[0078] Example 3: Competitive binding assay

[0079] Serum matrix without vitamin D (Seracon, Seracare Inc.) had been mixed with the established 25-OHD3 concentrations. 80 μl of each concentration series were filled into microtiter plate cavities pre-coated with 25-OHD-biotin tracer and incubated for 5 minutes at RT (20-27° C.) to allow tracer resolubility. Next, 80 [mu]l of the vitamin D releasing agent of Example 1 was added to the sample and mixed. 100 μl of the sample diluted with vitamin D releaser was transferred into the 25OHD-antibody coated cavity and incubated at room temperature for 30 minutes. Next, the sample mixture was removed and the cavities were washed 3 times with 200 μl each of wash buffer. The biotin-25OHD tracer bound to the antibody was detected by means of a peroxidase-labeled streptavidin and tetramethylbenzidine (TMB)-chromogenic reaction. Optical density was measured at 450 nm after the substrate reaction was stopped by adding 100 μl of 50 mM phos...

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Abstract

The present invention relates to a method for releasing bound vitamin D by bringing a sample containing vitamin D into contact with a releasing reagent containing at least one hydrotropic substance and at least one transition metal salt. The released vitamin D can then be quantitatively determined. The present invention also relates to a reagent for releasing and possibly determining vitamin D, containing at least one hydrotropic substance and at least one transition metal salt, and to the use of such a releasing reagent for releasing and possibly determining vitamin D.

Description

technical field [0001] The present invention relates to a method for releasing bound vitamin D by bringing a vitamin D-containing sample into contact with a releasing agent comprising at least one hydrotropic substance and at least one transition metal Salt. The released vitamin D can then be quantified. In addition, the invention also relates to a reagent for the release and optional determination of vitamin D comprising at least one hydrotropic substance and at least one transition metal salt, and to such a reagent for the release and optional determination of vitamin D Application of release agent. Background technique [0002] It is very important for humans to provide adequate vitamin D. The most common physiological form of vitamin D is vitamin D3 or vitamin D2. The most important derivatives of vitamin D3 (Cholecalciferol) with physiological effects are 25OHD3 (25-hydroxyvitamin D3 or calcifediol) and 1,25(OH) 2 D3 (1-alpha, 25-dihydroxyvitamin D3 or calcitriol)....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82
CPCG01N33/82
Inventor R·波佩V·索斯凯克
Owner ORGENTEC DIAGNOSTIKA
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