RELEASE REAGENT FOR VITAMIN D COMPOUNDS

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.

Claims

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 that releases hydrogen carbonate ions (HCO.sub.3.sup.−) upon hydrolysis, wherein the total concentration of hydrogen carbonate ions (HCO.sub.3.sup.−) from the hydrogen carbonate salt and released from the substance capable of releasing hydrogen carbonate ions (HCO.sub.3.sup.−) 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.sub.3.sup.−) upon hydrolysis is a cylic 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 claims 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.sub.2, 25-hydroxyvitamin D.sub.3, 24,25-dihydroxyvitamin D.sub.2, 24,25-dihydroxyvitamin D.sub.3 and C3-epi 25-hydroxyvitamin D.

8. The method according to claim 7, wherein the vitamin D compounds 25-hydroxyvitamin D.sub.2 and/or 25-hydroxyvitamin D.sub.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 that releases hydrogen carbonate ions (HCO.sub.3.sup.−) upon hydrolysis, wherein the total concentration of hydrogen carbonate ions (HCO.sub.3.sup.−) from the hydrogen carbonate salt and released from the substance capable of releasing hydrogen carbonate ions (HCO.sub.3.sup.−) 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).sub.2 and LiOH, wherein the sample is blood, serum or plasma.

Description

DESCRIPTION OF THE FIGURES

[0128] FIG. 1: pH change of the reagent mixture during the pre-treatment step. The assay was performed as outlined in example 1.5. Reagent composition (A) contains various concentrations of ethylene carbonate (EC): 0.00 M (), 0.10 M (.diamond-solid.), 0.30 M (□), 0.50 M (.box-tangle-solidup.), 0.75 M (◯), 1.00 M (.square-solid.), 1.50 M (⋄) EC. The X axis shows the time in minutes, the Y axis the pH.

[0129] FIG. 2: Calibration curves of a Vitamin D assay as described in example 1.5 with reagent composition (A) containing various concentrations of ethylene carbonate (EC): 1.50 M (), 1.00 M (□), 0.75 M (.diamond-solid.), 0.50 M (◯), 0.30 M (.box-tangle-solidup.) and 0.10 M (⋄) EC. The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

[0130] FIG. 3: Calibration curves of a Vitamin D assay as described in example 1.5 with reagent composition (A) containing various concentrations of the reducing agent dithiothreitol (DTT): 1.0 mM (□), 2.0 mM (), 4.0 mM (.diamond-solid.), 6.7 mM (◯), 10.0 mM/12.0 mM (.box-tangle-solidup.), 15.0 mM (⋄). The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

[0131] FIG. 4a: Method comparison: Vitamin D assay (example 1) and liquid chromatography-tandem mass spectrometry (LC-TMS) 25-hydroxyvitamin D was determined by means of LC-TMS as well as by means of the vitamin D assay of example 1.5, where reagent composition (A) with 0.5 M ethylene carbonate (EC) was used for the incubation. The results in ng/ml for multiple serum samples are plotted on the X axis for the LC-TMS and on the Y axis for the vitamin D assay of example 1.5. [0132] (---) y=x [0133] ()Linear regression [0134] Vitamin D assay=2.0116+0.9036*x, Pearsons r=0.9509

[0135] FIG. 4b: Method comparison: Vitamin D assay (example 1) and LC-TMS 25-hydroxyvitamin D was determined by means of LC-TMS as well as by means of the vitamin D assay of example 1.5, where reagent composition (A) without ethylene carbonate (EC) was used for the incubation. The results in ng/ml for multiple serum samples are plotted on the X axis for the LC-TMS and on the Y axis for the vitamin D assay of example 1.5. [0136] (---) y=x [0137] ()Linear regression

[0138] Vitamin D assay=0.7496+0.7338*x, Pearsons r=0.7914

[0139] FIG. 5: Calibration curves of a Vitamin D assay as described in example 2 with reagent composition (A) containing 0.5 M ethylene carbonate (.diamond-solid.), 0.5 M Na.sub.2CO.sub.3 (◯), 0.5 M NaH.sub.2PO.sub.4 (.box-tangle-solidup.), 0.5 M NaCl (⋄), and control (□). The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

[0140] FIG. 6: Calibration curves of a Vitamin D assay as described in example 3 with reagent composition (A) containing: [0141] .diamond-solid.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC, or [0142] .box-tangle-solidup.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, or [0143] □: 10 mM NaOH, 4 mM EDTA. [0144] The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

[0145] FIG. 7: Calibration curves of a Vitamin D assay as described in example 4 with reagent composition (A) containing: [0146] ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5), or [0147] .diamond-solid.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M dimethyl carbonate. [0148] The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

[0149] FIG. 8: Calibration curves of a Vitamin D assay as described in example 5 with reagent composition (A) containing: .diamond-solid.: 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, 0.5 M NaHCO.sub.3, or .box-tangle-solidup.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO.sub.3 +0.5 M ethylene glycol, or □: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT. [0150] The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

[0151] FIG. 9: Calibration curves of a Vitamin D assay as described in example 4 with reagent composition (A) containing: .diamond-solid.: 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, 0.5 M glycerol 1,2 carbonate. [0152] The X axis shows the concentration in ng/ml, the Y axis shows the counts determined as usual using the Elecsys® system from the Roche Diagnostics company.

EXAMPLE 1

[0153] Assays for the Detection of 25-Hydroxyvitamin D

[0154] 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 D.sub.3, 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.

[0155] 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

[0156] 20.6 mg (50 82 mol) 25-hydroxyvitamin D.sub.2 (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 minutes at 6° C. Then 205 μl of a potassium hydride solution (25 mg KH in 0.5 ml tert.-butanol/acetonitrile 9:1) is added. A brief flocculation occurs after which a clear solution is obtained. The reaction solution is stirred for a further 45 minutes at 6° C. and subsequently for 60 minutes at 4° C.

[0157] Subsequently the reaction solution is diluted with 10 ml methyl-tert.-butyl ether and washed twice with 10 ml H.sub.2O each time. The organic phase is dried with about 1 g anhydrous sodium sulfate, filtered over a G3 glass frit and evaporated on a rotary evaporator. It is dried in a high vacuum to form a viscous clear residue with a mass of about 55 mg.

1.2 Synthesis of hydroxyvitamin D.SUB.2.-3-3-aminopropyl ether

[0158] The entire nitrile obtained above is dissolved in 15 ml diethyl ether and admixed with a suspension of 7.5 mg lithium hydride in 7.5 ml diethyl ether while stirring. The reaction mixture is stirred for 1 hour at room temperature. Afterwards a suspension of 38.4 lithium aluminium hydride in 6.6 ml diethyl ether is added. This results in a strong turbidity of the mixture. The reaction mixture is stirred for a further hour at room temperature, then the reaction mixture is cooled to 0-5° C. in an ice bath and 35 ml water is carefully added. The pH is made strongly basic by addition of 6.6 ml 10 M potassium hydroxide solution.

[0159] It is extracted three times with 65 ml methyl-tert.-butyl ether each time. The combined organic phases are dried using about 5 g anhydrous sodium sulfate, filtered and evaporated at room temperature on a rotary evaporator. The residue is dried to mass constancy using an oil pump. The crude product is dissolved in 5 ml DMSO and 3.0 ml acetonitrile and purified by means of preparative HPLC.

[0160] eluent A=Millipore-H.sub.2O+0.1% trifluoroacetic acid;

[0161] eluent B=95% acetonitrile+5% Millipore-H.sub.2O+0.1% TFA;

[0162] gradient: from 50% B to 100% B in 100 min

[0163] flow rate: 30 ml/min

[0164] temperature: room temperature

[0165] column dimension: Ø=5.0 cm; L=25 cm

[0166] column material: Vydac C18/300 Å/15-20 μm

[0167] det. wavelength: 226 nm

[0168] Fractions whose product content is larger than 85% according to analytical HPLC (Vydac C18/300 Å/5 μm; 4.6×250 mm) are pooled in a round bottom flask and lyophilized. 13.7 mg (yield: 58%) is obtained as a colourless lyophilisate.

1.3 Synthesis of hydroxyvitamin D2-3-3′-N-(hemisuberyl)aminopropyl-ether-biotin-(beta-Ala)-Glu-Glu-Lys(epsilon) conjugate (=Ag—Bi)

[0169] 13.7 mg (25 μmol) hydroxyvitamin D.sub.2-3-3′-aminopropyl ether is dissolved in 3.5 ml DMSO, 28.7 mg (30 μmol) biotin-(beta-Ala)-Glu-Glu-Lys(epison)-hemi-suberate-N-hydroxysuccinimide ester (Roche Applied Science, No. 11866656) and 12.5 μl triethylamine are added and it is stirred overnight at room temperature. The reaction solution is diluted with 4.5 ml DMSO, filtered through a 0.45 μm microfilter and subsequently purified by means of preparative HPLC (conditions see example 2.3 b)). Fractions that contain more than 85% product according to analytical HPLC are pooled and lyophilized. 9.8 (yield: 30%) purified biotin conjugate is obtained.

1.4 Ruthenylation of Vitamin D-Binding Protein and Purification by Gel Filtration Chromatography

[0170] The vitamin D-binding protein is transferred to 100 mM potassium phosphate/150 mM sodium chloride buffer, pH 8.5 and the protein concentration is adjusted to 5-10 mg/ml. The ruthenylation reagent (ruthenium (II) tris (bipyridyl)-N-hydroxysuccinimide ester) is dissolved in DMSO and added to the antibody solution at a molar ratio of 3 to 1. After a reaction time of 45 min the reaction is stopped by addition of 1-lysine and the ruthenylated vitamin D-binding protein (=DBP-Ru) is purified by gel filtration on a Superdex 200 column.

1.5 Test Procedure in the Assay

[0171] The sample to be investigated is measured using the Elecsys® system from the Roche Diagnostics company.

[0172] The reagent mixture is formed by mixing a sample to be investigated with the reagent composition (A) and an alkalinising agent (B).

[0173] In this example the reagent mixture is formed of 15 μl sample mixed with 15 μl of the reagent composition (A) and 10 μl of the alkalinising agent (B). The reagent mixture is incubated for 9 minutes. In the next step 70 μl of detecting reagent (Solution C) is added to the reagent mixture and incubated for further 9 minutes. In the last step biotinylated wall antigen (Solution D) (60 μl) as well as 30 μl of magnetizable polystyrene particles coated with streptavidin (SA) (30 μl) (Suspension E) are added. After a further 9 minutes incubation the amount of bound ruthenylated vitamin D-binding protein is determined as usual (see FIGS. 1, 2, 3, 4a, 4b).

[0174] Reagent composition (A) contains:

TABLE-US-00001  10 mM NaOH   4 mM EDTA 6.7 mM dithiothreitol (DTT) 0.5M ethylene carbonate (EC) pH 5.5

[0175] Alkalinising agent (B) contains:

TABLE-US-00002 1.375M NaOH

[0176] Solution C with the ruthenylated vitamin D-binding protein (DBP-Ru) contains:

TABLE-US-00003 0.2M bis-tris-propane (pH 7.5) 2.5% human serum albumin (HSA) 50 mM NaCl   1% mannit 0.1% oxypyrion 0.12 μg/mL DBP-Ru

[0177] Solution D with the biotinylated wall antigen contains:

TABLE-US-00004 0.2M bis-tris-propane (pH 8.6) 0.5% tween-20 solution 0.1% oxypyrion 30 ng/ml biotin 0.0108 μg/mL Ag-Bi (from example 1.1)

[0178] Suspension E with SA-coated latex particles contains:

TABLE-US-00005 0.72 mg/ml SA-coated magnetizable polystyrene particles having a binding capacity of 470 ng/ml.

EXAMPLE 2

[0179] Comparison of Carbonate Ester to a Metal Salt, a Phosphate Buffer and a Carbonate

[0180] 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.

[0181] In aberrance to example 1.5 the reagent composition (A) contains either 0.5 M ethylene carbonate (EC), 0.5 M Na.sub.2CO.sub.3, 0.5 M NaCl or 0.5 M NaH.sub.2PO.sub.4, respectively.

[0182] Reagent composition (A):

TABLE-US-00006  10 mM NaOH   4 mM EDTA 6.7 mM DTT 0.5M of either EC, Na.sub.2CO.sub.3, NaCl or NaH.sub.2PO.sub.4

[0183] 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 (.diamond-solid.) 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 Na.sub.2CO.sub.3 (◯) or 0.5 M NaH.sub.2PO.sub.4 (.box-tangle-solidup.) shows a minor effect on the signal.

EXAMPLE 3

[0184] Alkaline Pretreatment with/without Carbonate Ester

[0185] 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.

[0186] In aberrance to example 1.5 three different reagent compositions have been prepared containing either:

[0187] .diamond-solid.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

[0188] .box-tangle-solidup.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT or

[0189] □: 10 mM NaOH, 4 mM EDTA.

[0190] After a 4 min pretreatment incubation of sample+either .diamond-solid. (reagent composition (A)+alkalinising agent (B) as described in example 1.5), .box-tangle-solidup., 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.

EXAMPLE 4

[0191] Ethylene Carbonate Vs Dimethyl Carbonate

[0192] 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.

[0193] In aberrance to example 1.5 two different reagent compositions (A) have been prepared containing either:

[0194] ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

[0195] .diamond-solid.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M dimethyl carbonate.

[0196] Both carbonate ester, ethylene carbonate or dimethyl carbonate, respectively, show the same assay performance (FIG. 7).

EXAMPLE 5

[0197] Effect of the Hydrolysis Products of Ethylene Carbonate

[0198] 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.

[0199] In aberrance to example 1.5 five different reagent compositions (A) have been prepared containing either:

[0200] .diamond-solid.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

[0201] ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO.sub.3 or

[0202] .box-tangle-solidup.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO.sub.3 +0.5 M ethylene glycol

[0203] □: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT.

[0204] The alkaline hydrolysis product of EC is ethylene glycol, which has no influence on the assay (.box-tangle-solidup.). A hydrogene carbonate salt (NaHCO.sub.3) shows also a signal enhancing effect, but not as much as a carbonate ester (FIG. 8).

EXAMPLE 6

[0205] Ethylene Carbonate Vs Glycerol 1,2 Carbonate

[0206] 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.

[0207] In aberrance to example 1.5 two different reagent compositions (A) have been prepared containing either:

[0208] .diamond-solid.: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

[0209] ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M glycerol 1,2 carbonate.

[0210] Both carbonate ester, ethylene carbonate or glycerol 1,2 carbonate, respectively, show the same assay performance (FIG. 9).