Release reagent for vitamin d compounds

Abstract

The present invention concerns a reagent composition for releasing vitamin D compounds bound to vitamin D-binding protein, a method for the detection of a 25-hydroxyvitamin D compound in which the 25-hydroxyvitamin D compound is released from vitamin D-binding protein using this reagent and the mixture obtained in this manner is analyzed, the use of the reagent to release vitamin D compounds as well as a kit for detecting 25-hydroxyvitamin D which contains the reagent for releasing vitamin D compounds in addition to the usual immunological reagents.

1 . A reagent composition for the release of vitamin D which has a pH value of 3.8 to 4.8 and contains 5 to 30 volume percent of one or more amphiphilic reagents selected from the group consisting of dimethyl sulfoxide (DMSO) and a liquid organic amide as well as optionally 0.7 to 8 volume percent of a short-chain (C1 to C3) alcohol. 
   
   
       2 . The reagent composition according to  claim 1 , characterized in that dimethylformamide (DMF), N,N-dimethylacetamide, tetramethylurea (TMU), N-methylpyrrolidone (N-MP), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone (DMPU) and hexamethylphosphoric acid triamide (HMPT) are used as the amphiphilic reagent. 
   
   
       3 . The reagent composition according to  claim 1 , characterized in that 7 to 20 volume percent amphiphilic reagent is present. 
   
   
       4 . The reagent composition according to  claim 1 , characterized in that ethanol is present as the short-chain alcohol. 
   
   
       5 . A reagent composition for releasing a vitamin D compound from a binding protein, the composition having a pH of 3.8 to 4.8 and comprising, in addition to the vitamin D compound and binding protein, one or more amphiphilic reagents selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N,N-dimethylacetamide, tetramethylurea (TMU), N-methylpyrrolidone (N-MP), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone (DMPU), and hexamethylphosphoric acid triamide (HMPT), wherein the one or more amphiphilic reagents are present in an amount of 5 to 30 volume percent. 
   
   
       6 . The reagent composition of  claim 5  further comprising 0.7 to 8 volume percent of an alcohol selected from the group consisting of methanol, ethanol, propanol, and isopropanol. 
   
   
       7 . The reagent composition of  claim 5  wherein the one or more amphiphilic reagents are present in an amount of 7 to 20 volume percent. 
   
   
       8 . A method for detection of a 25-hydroxyvitamin D compound in a sample comprising the steps of
 releasing the 25-hydroxyvitamin D compound from binding protein by combining the sample with a reagent composition having a pH of 3.8 to 4.8 and comprising one or more amphiphilic reagents selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N,N-dimethylacetamide, tetramethylurea (TMU), N-methylpyrrolidone (N-MP), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone (DMPU), and hexamethylphosphoric acid triamide (HMPT), wherein the one or more amphiphilic reagents are present in an amount of 5 to 30 volume percent,   adding to the released 25-hydroxyvitamin D compound a detection antibody and an antigen whereby the antigen is a defined amount of 25-hydroxyvitamin D that competes with the 25-hydroxyvitamin D in the sample to bind with the detection antibody, whereby the detection antibody comprises a moiety which produces a signal upon binding of the antibody to the antigen, and   determining the signal produced as a measure of the 25-hydroxyvitamin D compound in the sample.   
   
   
       9 . The method of  claim 8  wherein the 25-hydroxyvitamin D compound is selected from the group consisting of 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 , 1,25-dihydroxyvitamin D 2 , and 1,25-dihydroxyvitamin D 3 . 
   
   
       10 . A method for releasing a vitamin D compound from a binding protein in a sample, the method comprising
 combining the sample with a reagent composition having a pH of 3.8 to 4.8 and comprising one or more amphiphilic reagents selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N,N-dimethylacetamide, tetramethylurea (TMU), N-methylpyrrolidone (N-MP), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone (DMPU), and hexamethylphosphoric acid triamide (HMPT), wherein the one or more amphiphilic reagents are present in an amount of 5 to 30 volume percent, thereby releasing the vitamin D compound from the binding protein.   
   
   
       11 . The method of  claim 10  wherein the reagent composition further comprises 0.7 to 8 volume percent of an alcohol selected from the group consisting of methanol, ethanol, propanol, and isopropanol. 
   
   
       12 . A kit for the detection of 25-hydroxyvitamin D comprising components for immunological detection and a reagent composition having a pH value of 3.8 to 4.8 and comprising one or more amphiphilic reagents selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N,N-dimethylacetamide, tetramethylurea (TMU), N-methylpyrrolidone (N-MP), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone (DMPU), and hexamethylphosphoric acid triamide (HMPT), wherein the one or more amphiphilic reagents are present in an amount of 5 to 30 volume percent.