Release reagent for vitamin d compounds

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.

What is claimed is: 
     
         1 . An in vitro method for releasing a vitamin D compound from a vitamin D-binding protein comprising the steps of:
 a) providing a sample to be investigated; and   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 (HCO 3   − ) 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.   
     
     
         2 . The method according to  claim 1 , wherein the reagent according to step (i) is soluble in an aqueous solution under the appropriate conditions for releasing a vitamin D compound from vitamin D-binding protein. 
     
     
         3 . The method according to  claim 1 , wherein the reagent according to step (i) contains a hydrogen carbonate salt in a concentration of 0.1 M to 2.0 M. 
     
     
         4 . The method according to  claim 1 , wherein the reagent according to step (i) contains a substance capable of releasing hydrogen carbonate ions (HCO 3   − ) upon hydrolysis in a concentration of 0.1 M to 2.0 M. 
     
     
         5 . The method according to  claim 1 , wherein the substance capable of releasing hydrogen carbonate ions (HCO 3   − ) upon hydrolysis is a cylic or non-cyclic carbonate ester or a hydroxylated or halogenized derivative thereof, respectively. 
     
     
         6 . The method according to  claim 1 , wherein the sample is a liquid sample. 
     
     
         7 . The method according to  claim 6 , wherein the sample is blood, serum or plasma. 
     
     
         8 . An in vitro method for measuring a vitamin D compound comprising the steps of:
 a) releasing a vitamin D compound from vitamin D-binding protein according to the method of  claim 1  and   b) measuring the vitamin D compound released in step (a).   
     
     
         9 . The method according to  claim 8 , wherein the vitamin D compound is selected from the group consisting of 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 , 24,25-dihydroxyvitamin D 2 , 24,25-dihydroxyvitamin D 3  and C3-epi 25-hydroxyvitamin D. 
     
     
         10 . The method according to  claim 9 , wherein at least one of the vitamin D compounds 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  are determined. 
     
     
         11 . A reagent composition for the release of a vitamin D compound from vitamin D-binding protein comprising a substance selected from the group consisting of a hydrogen carbonate salt or a substance capable of releasing hydrogen carbonate ions (HCO 3   − ) upon hydrolysis in a concentration of 0.1 M to 2.0 M and a reducing agent. 
     
     
         12 . The reagent composition according to  claim 11 , characterized in that the reducing agent is selected from the group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl, Dithiothreitol (DTT), N-Methylmaleimide, Ellman's Reagent and 1,2-dithiolane-3-carboxylic acid. 
     
     
         13 . The reagent composition according to  claim 11 , characterized in that the reducing agent is selected from the group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl and Dithiothreitol (DTT). 
     
     
         14 . The reagent composition according to  claim 11 , characterized in that the reducing agent has a concentration of 2 mM to 30 mM. 
     
     
         15 . A reagent mixture comprising:
 a sample to be investigated;   a reagent composition according to  claim 11 ; and   an alkalinising agent.   
     
     
         16 . The reagent mixture according to  claim 15 , characterized in that the alkalinising agent is selected from the group consisting of NaOH, KOH, Ca(OH) 2  and LiOH. 
     
     
         17 . A kit for detecting the release of a vitamin D compound from a vitamin D-binding protein, comprising:
 a reagent composition according to  claim 11 ; and   an alkalinising agent.