RELEASE REAGENT FOR VITAMIN D COMPOUNDS
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.
1 . 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 and
b) mixing the sample from step (a) with
i) a reagent containing one hydrogen carbonate salt and a substance capable of releasing hydrogen carbonate ions (HCO 3 − ) upon hydrolysis, wherein the total concentration of hydrogen carbonate ions (HCO 3 − ) from the hydrogen carbonate salt and released from the substance capable of releasing hydrogen carbonate ions (HCO 3 − ) is 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 substance capable of releasing hydrogen carbonate ions (HCO 3 − ) upon hydrolysis is a cyclic or non-cyclic carbonate ester or a hydroxylated or halogenized derivative thereof, respectively.
4 . The method according claim 1 , wherein the sample is a liquid sample.
5 . The method according to claim 1 , wherein the sample is blood, serum or plasma.
6 . 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).
7 . The method according to claim 6 , 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.
8 . The method according to claim 7 , wherein the vitamin D compounds 25-hydroxyvitamin D 2 and/or 25-hydroxyvitamin D 3 are determined.
9 . A reagent composition for the release of a vitamin D compound from vitamin D-binding protein comprising one hydrogen carbonate salt and a substance capable of releasing hydrogen carbonate ions (HCO 3 − ) upon hydrolysis, wherein the total concentration of hydrogen carbonate ions (HCO 3 − ) from the hydrogen carbonate salt and released from the substance capable of releasing hydrogen carbonate ions (HCO 3 − ) is 0.1 M to 2.0 M, and a reducing agent.
10 . The reagent composition according to claim 9 , 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.
11 . The reagent composition according to claim 9 , characterized in that the reducing agent is selected from the group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl and Dithiothreitol (DTT).
12 . The reagent composition according to claim 9 , characterized in that the reducing agent has a concentration of 2 mM to 30 mM.
13 . A reagent mixture comprising a sample to be investigated, a reagent composition according to claim 9 , and an alkalinising agent selected from the group consisting of NaOH, KOH, Ca(OH) 2 and LiOH, wherein the sample is blood, serum or plasma.