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. A reagent composition for releasing a vitamin D compound from a vitamin D-binding protein comprising: one or more hydrogen carbonate salt(s), one or more carbonate ester(s), and; 2 mM to 30 mM of a reducing agent selected from the group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, tris(2-carboxyethyl)phosphine (TCEP), Cystein-HCl, Dithiothreitol (DTT), N-Methylmaleimide, Ellman's Reagent and 1,2-dithiolane-3-carboxylic acid.

2. The reagent composition according to claim 1, wherein the reducing agent is selected from the group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl and Dithiothreitol (DTT).

3. A reagent mixture comprising a sample to be investigated, a reagent composition according to claim 1, 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.

4. The reagent composition of claim 1, wherein the one or more hydrogen carbonate salt(s) is selected from the group consisting of sodium hydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogen carbonate, calcium hydrogen carbonate, magnesium hydrogen carbonate, and combinations thereof.

5. The reagent composition of claim 1, wherein the one or more carbonate ester(s) selected from the group consisting of ethylene carbonate, dim ethyl carbonate, propylene carbonate, vinylene carbonate, trimethylene carbonate, erythritol bis-carbonate, glycerol 1,2-carbonate, 4-chloro-1,3-dioxolan-2-one, 4,5-dichloro-1,3-dioxolan-2-one, 2,5-dioxahexanedioic acid dimethyl ester, 1,2 butylene carbonate, cis 2,3 butylene carbonate, trans 2,3 butylene carbonate, hydroxylated or halogenized derivatives thereof, and combinations thereof.

6. The reagent composition of claim 1, wherein the one or more hydrogen carbonate salt(s) has a concentration of 0.1 to 1.5 M. 1.

7. The reagent composition of claim 1, wherein the one or more carbonate ester(s) has a concentration of 0.5 M.

Description

DESCRIPTION OF THE FIGURES

(1) 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 (?), 0.30 M (?), 0.50 M (?), 0.75 M (?), 1.00 M (?), 1.50 M (?) EC. The X axis shows the time in minutes, the Y axis the pH.

(2) 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 (?), 0.50 M (?), 0.30 M (?) 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.

(3) 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 (?), 6.7 mM (?), 10.0 mM/12.0 mM (?), 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.

(4) 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.

(5) () y=x

(6) () Linear regression Vitamin D assay=2.0116+0.9036*x, Pearsons r=0.9509

(7) 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.

(8) () y=x

(9) () Linear regression Vitamin D assay=0.7496+0.7338*x, Pearsons r=0.7914

(10) FIG. 5: Calibration curves of a Vitamin D assay as described in example 2 with reagent composition (A) containing 0.5 M ethylene carbonate (?), 0.5 M Na.sub.2CO.sub.3 (?), 0.5 M NaH.sub.2PO.sub.4 (?), 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.

(11) FIG. 6: Calibration curves of a Vitamin D assay as described in example 3 with reagent composition (A) containing: ?: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 MEC, or ?: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, or ?: 10 mM NaOH, 4 mM EDTA. 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.

(12) FIG. 7: Calibration curves of a Vitamin D assay as described in example 4 with reagent composition (A) containing: ?: 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 dimethyl carbonate. 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.

(13) FIG. 8: Calibration curves of a Vitamin D assay as described in example 5 with reagent composition (A) containing: ?: 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 ?: 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. 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.

(14) FIG. 9: Calibration curves of a Vitamin D assay as described in example 4 with reagent composition (A) containing: ?: 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. 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

Assays for the Detection of 25-Hydroxyvitamin D

(15) 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.

(16) The preparation of the ingredients and the general test procedure for a new test is described in the following:

1.1 Synthesis of Hydroxyvitamin D.SUB.2.-3-2-Cyanoethyl Ether

(17) 20.6 mg (50 ?mop 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 ?l of an acrylonitrile solution (86 ?l acrylonitrile in 1.0 ml acetonitrile) is added and stirred for 15 minutes at 6? C. Then 205 ?l 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.

(18) 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

(19) 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.

(20) 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. eluent A=Millipore-H.sub.2O+0.1% trifluoroacetic acid; eluent B=95% acetonitrile+5% Millipore-H.sub.2O+0.1% TFA; gradient: from 50% B to 100% B in 100 min flow rate: 30 ml/min temperature: room temperature column dimension: ?=5.0 cm; L=25 cm column material: Vydac C18/300 ?/15-20 ?m det. wavelength: 226 nm

(21) Fractions whose product content is larger than 85% according to analytical HPLC (Vydac C18/300 ?/5 ?m; 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 D.SUB.2.-3-3-N-(hemisuberyl)aminopropyl-ether-biotin-(beta-Ala)-Glu-Glu-Lys(epsilon) conjugate (?AgBi)

(22) 13.7 mg (25 ?mol) hydroxyvitamin D.sub.2-3-3-aminopropyl ether is dissolved in 3.5 ml DMSO, 28.7 mg (30 ?mol) biotin-(beta-Ala)-Glu-Glu-Lys(epison)-hemi-suberate-N-hydroxysuccinimide ester (Roche Applied Science, No. 11866656) and 12.5 ?l 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 ?m 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

(23) 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 l-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

(24) The sample to be investigated is measured using the Elecsys? system from the Roche Diagnostics company.

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

(26) In this example the reagent mixture is formed of 15 ?l sample mixed with 15 ?l of the reagent composition (A) and 10 ?l of the alkalinising agent (B). The reagent mixture is incubated for 9 minutes. In the next step 70 ?l 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 ?l) as well as 30 ?l of magnetizable polystyrene particles coated with streptavidin (SA) (30 ?l) (Suspension E) are added. After a further 9 minutes incubation the amount of bound ruthenylated vitamin D-binding protein is determined as usual (see FIG. 1, 2, 3, 4a, 4b).

(27) Reagent Composition (A) Contains:

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

(29) Alkalinising agent (B) contains:

(30) TABLE-US-00002 1.375 M NaOH

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

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

(33) Solution D with the biotinylated wall antigen contains:

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

(35) Suspension E with SA-coated latex particles contains:

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

EXAMPLE 2

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

(37) 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.

(38) 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.

(39) Reagent Composition (A):

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

(41) 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 (?) 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 (?) shows a minor effect on the signal.

EXAMPLE 3

Alkaline Pretreatment with/without Carbonate Ester

(42) 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.

(43) In aberrance to example 1.5 three different reagent compositions have been prepared containing either: ?: 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 or ?: 10 mM NaOH, 4 mM EDTA.

(44) After a 4 min pretreatment incubation of sample+ either ? (reagent composition (A)+alkalinising agent (B) as described in example 1.5), ?, 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

Ethylene Carbonate vs Dimethyl Carbonate

(45) 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.

(46) In aberrance to example 1.5 two different reagent compositions (A) have been prepared containing either: ?: 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 dimethyl carbonate.

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

EXAMPLE 5

Effect of the Hydrolysis Products of Ethylene Carbonate

(48) 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.

(49) In aberrance to example 1.5 five different reagent compositions (A) have been prepared containing either: ?: 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 ?: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO.sub.3+0.5 M ethylene glycol ?: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT.

(50) The alkaline hydrolysis product of EC is ethylene glycol, which has no influence on the assay (?). 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

Ethylene Carbonate vs Glycerol 1,2 Carbonate

(51) 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.

(52) In aberrance to example 1.5 two different reagent compositions (A) have been prepared containing either: ?: 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.

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