WO2014122974A1 - Procédé de mesure de la vitamine d faisant appel à un agent chaotropique - Google Patents

Procédé de mesure de la vitamine d faisant appel à un agent chaotropique Download PDF

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Publication number
WO2014122974A1
WO2014122974A1 PCT/JP2014/051006 JP2014051006W WO2014122974A1 WO 2014122974 A1 WO2014122974 A1 WO 2014122974A1 JP 2014051006 W JP2014051006 W JP 2014051006W WO 2014122974 A1 WO2014122974 A1 WO 2014122974A1
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vitamin
sample
reagent
present
chaotropic agent
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PCT/JP2014/051006
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English (en)
Japanese (ja)
Inventor
内田　好昭
和也 小見
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富士レビオ株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

Definitions

  • the present invention relates to a method for measuring vitamin D and the like.
  • Vitamin D (hereinafter simply referred to as vitamin D) is strongly bound to a binding protein (DBP: vitamin D binding protein, also referred to as Gc globulin) in blood. Therefore, in order to accurately measure vitamin D by the antigen-antibody method, a dissociation operation (pretreatment) between vitamin D and DBP is required.
  • an organic solvent eg, ethanol, methanol, DMSO
  • a denaturant eg, acid, protein denaturant, surfactant, hydrolase
  • the present invention provides a method for measuring vitamin D and the like.
  • the inventors of the present invention have intensively studied and found that vitamin D can be accurately measured by treating a sample with a chaotropic agent, thereby completing the present invention.
  • the present invention is as follows.
  • Method for measuring vitamin D including: 1) treating the sample with a chaotropic agent; and 2) detecting vitamin D in the treated sample.
  • Vitamin D measurement kit including: 1) Chaotropic agent; 2) Affinity substances for vitamin D and / or vitamin D preparations.
  • the method of the present invention is useful for the measurement of vitamin D. According to the present invention, vitamin D can be measured quickly and easily.
  • the kit of the present invention is useful for simple implementation of the method of the present invention, for example.
  • the present invention provides a method for measuring vitamin D in a sample.
  • the method of the present invention includes: 1) treating the sample with a chaotropic agent; and 2) detecting vitamin D in the treated sample.
  • the sample used in the method of the present invention is a sample containing or suspected of containing vitamin D or a metabolite thereof as shown below.
  • vitamin D metabolites include compounds in which a hydroxyl group is added to vitamin D, 25OH vitamin D2, 25OH vitamin D3, 1,25 (OH) 2 vitamin D2, 1,25 (OH) 2 vitamin D3. It is done.
  • vitamin D is intended to encompass vitamin D2 and vitamin D3, and drugs similar to vitamin D2 and vitamin D3, and their metabolites, unless otherwise specified.
  • the origin of the sample is not particularly limited, and may be a biological sample derived from a living organism or an environmental sample.
  • organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like. , Microorganisms, plants, fungi, and fish, preferably mammals, fungi, and fish, more preferably mammals, and even more preferably humans.
  • a biological sample can also be a blood-related sample (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extract, or a mixture thereof, which is the blood itself or a sample derived from blood.
  • a blood-related sample is preferred.
  • environmental samples include samples derived from soil, seawater, and fresh water.
  • the sample used in the method of the present invention is preferably a sample containing a complex of vitamin D and a molecule capable of binding thereto.
  • the method of the present invention has an advantage that vitamin D can be accurately measured even when a molecule (eg, protein) having a strong binding ability to vitamin D is present in the sample.
  • a molecule eg, protein
  • Kd 5 ⁇ 10 ⁇ 8
  • vitamin D can be measured accurately.
  • examples of other molecules having binding ability to vitamin D include albumin and lipid.
  • examples of the sample containing a complex of vitamin D and a molecule capable of binding thereto include blood-related samples (eg, whole blood, serum, plasma).
  • vitamin D in a sample can be measured regardless of the presence or absence of the molecule having binding ability to vitamin D, the type of the molecule, and the type of the sample. That is, the method of the present invention does not confirm information on these matters in advance, or even when an unknown molecule capable of binding to vitamin D is present in the sample, vitamin D in the sample. Can be measured. Therefore, the method of the present invention can be used as a standard method that can be widely used for the measurement of vitamin D.
  • the sample may be subjected to other treatments before being treated with the chaotropic agent.
  • treatment includes centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.
  • the volume of the sample to be treated with the chaotropic agent is not particularly limited as long as vitamin D can be measured, but is, for example, 0.1 to 1000 ⁇ l, preferably 0.5 to 100 ⁇ l, more preferably 1 to 50 ⁇ l.
  • chaotropic agents examples include guanidine, urea, thiosulfuric acid and iodide ions, and salts and mixtures thereof.
  • salt used in the present specification is an arbitrary salt, and examples thereof include inorganic salts, organic salts, and intramolecular salts.
  • inorganic salt include metal salts, halide salts, acid addition salts, and ammonium salts.
  • metal salt include a salt of an alkali metal (eg, lithium, sodium, potassium) and a salt of an alkaline earth metal (eg, magnesium, calcium).
  • halogen in the halide salt include fluorine, bromine, chlorine, and iodine.
  • Examples of acid addition salts that are inorganic salts include salts with inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, thiocyanic acid, and isothiocyanic acid.
  • Examples of the organic salt include salts with organic bases such as trimethylamine, triethylamine and pyridine, and salts with organic acids such as oxalic acid.
  • Preferable examples of the salt of the chaotropic agent include guanidine hydrochloride and guanidine isothiocyanate.
  • Sample processing may be performed using one or more (eg, two or three) chaotropic agents.
  • concentration of the chaotropic agent in the case where the sample is treated with the chaotropic agent is not particularly limited as long as it is an effective concentration for the measurement of vitamin D, and can be adjusted as appropriate, for example, 1 to 25M, preferably 1 to 20M, More preferably, it is 1 to 10M.
  • sample processing may be performed in combination with a reducing agent in addition to the chaotropic agent.
  • the method of the present invention may further comprise treating the sample with a reducing agent.
  • Treatment of the sample with the chaotropic agent and the reducing agent can be performed simultaneously or separately, but preferably is performed simultaneously.
  • Any reducing agent can be used as such a reducing agent, but a thiol group-containing reducing agent is preferred.
  • the thiol group-containing reducing agent include dithioerythritol, cysteine, 2-mercaptoethylamine, and dithiothreitol.
  • the concentration of the reducing agent in the case of treating the sample with a reducing agent is not particularly limited as long as it is an effective concentration for vitamin D measurement, and can be adjusted as appropriate, for example, 0.01 to 1000 mM, preferably 0. 05 to 100 mM, more preferably 0.1 to 10 mM.
  • the sample may be processed in combination with other components in addition to the chaotropic agent or the combination of the chaotropic agent and the reducing agent.
  • the method of the present invention may further comprise treating the sample with other components.
  • the treatment of the sample with the chaotropic agent, reducing agent and other components can be performed simultaneously or separately, but preferably is performed simultaneously.
  • examples of such other components include organic solvents (eg, alcohols such as ethanol, DMSO), surfactants (eg, anionic surfactants, cationic surfactants, amphoteric surfactants or nonionic surfactants). Surfactants).
  • the other component may be one kind, but may be plural (eg, two or three kinds). Such other components may be used at a concentration effective for the denaturing action, but may be used at a concentration effective for an action other than the denaturing action in anticipation of an action other than the denaturing action.
  • the affinity substance for vitamin D refers to a substance having an ability to bind to vitamin D, and examples thereof include antibodies to vitamin D and aptamers.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may also be a fragment of an antibody (eg, Fab, F (ab ′) 2 ), a recombinant antibody (eg, scFv).
  • the antibody may further be an antibody-like molecule (eg, affibody, anticalin, DARPins, monobody) produced by molecular biological techniques such as phage display and / or by protein engineering techniques using existing protein motifs. May be.
  • Sample processing includes, for example, (a) mixing the sample with an aqueous solution (eg, buffer) containing the components as described above to prepare a mixture, and (b) incubating the mixture. .
  • aqueous solution eg, buffer
  • Tris buffer eg, Tris-HCl buffer, TE buffer, TAE buffer, TBE buffer, Tris buffered saline
  • phosphate buffer eg, phosphate buffered saline
  • Water citrate-phosphate buffer
  • carbonate buffer eg, carbonate-sodium bicarbonate buffer
  • GOOD buffer eg, MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamide Glycine, tricine, glycinamide, bicine.
  • the sample can be treated under neutral conditions or under acidic or alkaline conditions, but preferably under neutral conditions.
  • the pH value employed in the treatment of the sample is, for example, 4.0 to 9.5, preferably 5.0 to 9.0, preferably 5.5 to 8.5, more preferably 6.0 to 8. .0.
  • the pH value in the sample processing can be adjusted using a buffer solution, an acidic substance, and an alkaline substance.
  • the temperature of the sample treatment is not particularly limited as long as the components such as the chaotropic agent are suitable for exerting the action, but for example, 15 to 60 ° C., preferably It is 20 to 50 ° C, more preferably 20 to 45 ° C.
  • the incubation time is, for example, 1 to 60 minutes, preferably 1 to 30 minutes or less, and more preferably 1 to 10 minutes.
  • the method of the present invention may comprise i) treating a sample with a reagent containing a chaotropic agent, and ii) treating the sample treated with a chaotropic agent with a diluent.
  • the reagent includes a chaotropic agent.
  • the reagent may also contain a reducing agent.
  • the reagent may further contain other components as described above.
  • the concentration of the chaotropic agent in the reagent is not particularly limited as long as the concentration is such that the action is exerted in the mixed solution of the reagent and the sample.
  • the concentration (as described above) in the mixed solution of the reagent and the sample The concentration is such that the concentration of the chaotropic agent when the sample is treated with the chaotropic agent). Therefore, the concentration of the chaotropic agent in the reagent can be appropriately set so as to achieve the above-described concentration based on the volume of the sample and the reagent.
  • the concentration of the reducing agent in the reagent is not particularly limited as long as the concentration is such that the action is exerted in the mixed solution of the reagent and the sample.
  • the concentration as described above in the mixed solution of the reagent and the sample The concentration is such that the concentration of the reducing agent when the sample is treated with the reducing agent can be achieved. Therefore, the concentration of the reducing agent in the reagent can be appropriately set so as to achieve the above-described concentration based on the volume of the sample and the reagent.
  • the reagent contains the above-described substances in an aqueous solution (eg, a buffer solution as described above).
  • the reagent can be a neutral solution or an acidic solution or an alkaline solution, but is preferably a neutral solution. Therefore, the pH value of the reagent is, for example, 4.0 to 9.5, preferably 5.0 to 9.0, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0.
  • the volume of the reagent can be appropriately determined depending on the volume and type of the sample, the purpose of the assay (eg, qualitative or quantitative measurement), etc. Is 1 to 10 times, more preferably 1 to 5 times.
  • the sample processing with the reagent is appropriately performed in an appropriate manner to exert the action of the components contained in the reagent.
  • the processing of the sample with the reagent can be performed in the same manner as the processing of the sample as described above.
  • (A1) The sample is mixed with the reagent to prepare the first mixed solution, and (b1) the first Incubating the mixture may include.
  • the temperature and time conditions in (a1) and (b1) are the same as those described above in (a) and (b), respectively.
  • the diluent may contain, for example, one or more (eg, two or more) of other components as described above.
  • the concentration of the component can be set as appropriate.
  • the diluent includes other components as described above in an aqueous solution (eg, a buffer as described above).
  • the diluent may be a neutral solution, an acidic solution or an alkaline solution, but is preferably a neutral solution.
  • the pH value of the diluted solution is, for example, 4.0 to 9.5, preferably 5.0 to 9.0, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. .
  • the volume of the diluent can be determined as appropriate depending on the volume and type of the sample and reagent, the purpose of the assay (eg, qualitative or quantitative measurement), etc. obtain. Specifically, the volume of the diluted solution is, for example, 1 to 20 times, preferably 1 to 10 times, more preferably 1 to 5 times the total volume of the sample and the reagent.
  • Processing of the sample with the diluent is appropriately performed in an appropriate manner.
  • the sample treatment with the diluent can be performed in the same manner as the sample treatment with the reagent, (a2) mixing the sample treated with the reagent with the diluent to prepare a second mixture, and (B2) Incubating the second mixed solution may be included.
  • the temperature and time conditions in (a2) and (b2) are the same as those described above in (a) and (b), respectively.
  • Vitamin D is detected in samples processed as described above. Vitamin D is detected qualitatively or quantitatively. In this step, when the above-described affinity substance is used, it may include adding the affinity substance to the treated sample.
  • Vitamin D can be detected by any method, for example, using an affinity substance for vitamin D. Detection of vitamin D may also be performed by immunological techniques. Examples of such immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples thereof include a magnetic particle method, an immunochromatography method, a luminescence immunoassay method, a spin immunoassay method, and a latex agglutination method. Examples of methods other than the above that enable detection of vitamin D include LC-MS.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FFA fluorescence immunoassay
  • a secondary antibody may be further used.
  • the secondary antibody may be an antibody against the primary antibody portion of an antibody against vitamin D (primary antibody), or an antibody against a complex of vitamin D and the primary antibody.
  • An antibody such as a secondary antibody may be linked to a detection substance.
  • the detection substance examples include enzymes (eg, horseradish peroxidase, alkaline phosphatase), affinity substances (eg, streptavidin, biotin), fluorescent substances (eg, fluorescein, fluorescein isothiocyanate, rhodamine), luminescent substances (eg, , Luciferin, aequorin), radioactive substances (eg, 3 H, 14 C, 32 P, 35 S, 125 I).
  • the antibody such as a secondary antibody may be immobilized on a support.
  • the support include particles (eg, magnetic particles), membranes (eg, nitrocellulose membrane), glass, plastic, metal, plates (eg, multiwell plates), and devices.
  • the antibody may also be provided in a form impregnated in a medium such as filter paper.
  • the present invention also provides a kit for measuring vitamin D.
  • the kit of the present invention comprises: 1) chaotropic agents; and 2) affinity substances for vitamin D and / or vitamin D preparations.
  • the kit of the present invention may also contain a reducing agent.
  • the chaotropic agent and the reducing agent may be dissolved in the same solution (eg, buffer).
  • the affinity substance for vitamin D is a primary antibody
  • the kit of the present invention may further contain a secondary antibody.
  • the kit of the invention comprises: 1) a reagent containing a chaotropic agent (which may further contain a reducing agent); 2) Diluent; and 3) Affinity substances and / or vitamin D preparations for vitamin D.
  • the kit of the present invention Details of the above-mentioned constituents contained in the kit of the present invention (eg, active ingredients and concentrations, preferred examples) are as described above in the method of the present invention.
  • the reagent and diluent may further contain other components described above in the method of the present invention.
  • the vitamin D preparation is an aqueous solution containing vitamin D at a predetermined concentration (single or plural) or vitamin D powder, and is useful as a control.
  • each component may be provided in a form accommodated in a different container (eg, tube, plate).
  • the kit of the present invention may be provided in the form of a device. Specifically, all of the components may be provided in a form housed in the device. Alternatively, some of the components may be provided in a form housed in the device, and the rest may be provided in a form that is not housed in the device (eg, a form housed in a different container). In this case, components that are not contained in the device may be used by being injected into the device during the measurement of vitamin D.
  • 25OH vitamin D2 and 25OH vitamin D3 are collectively abbreviated as 25OH vitamin D as needed as immunological methods for measuring vitamin D.
  • the primary antibody that recognizes was used.
  • the values measured in the following examples may correspond to the total amount of 25OH vitamin D2 and 25OH vitamin D3.
  • Example 1 Examination of 25OH Vitamin D Measurement Method Using Chaotropic Agent and Reducing Agent Human serum was treated with a combination of a reagent containing a chaotropic agent (guanidine hydrochloride) and a reducing agent (DTT) and a diluent over time. Then, 25OH vitamin D in human serum was measured by immunological method.
  • a reagent containing a chaotropic agent guanidine hydrochloride
  • DTT reducing agent
  • the method was performed as follows. 1) To the same serum (10 ⁇ l) collected from the same human, 4 volumes (40 ⁇ l) of various reagents [7.5 M, 5 M or 0 M guanidine hydrochloride, 4 mM in PBS buffer (pH 7.6), 2 mM or 0 mM reducing agent (dithiothreitol: DTT) + 10% (v / v) ethanol] was added to prepare a first mixed solution (50 ⁇ l) of serum and reagent. The concentration of guanidine hydrochloride in the first mixture is 6M, 4M, or 0M. The concentration of the reducing agent in the mixed solution is 3.2 mM, 1.6 mM, or 0 mM.
  • the first mixed solution was incubated at room temperature (25 ° C., the same applies hereinafter) for 10 minutes.
  • dilute solution (0.1% (w / v) BSA / PBS (pH 7.6) + 5% (v / v) ethanol + 5% (v / v) DMSO) (pH 7. 6) was added in 3 volumes (150 ⁇ l) to prepare a second mixture.
  • concentration of guanidine hydrochloride in the second mixture is 1.5M, 1M, or 0M.
  • the concentration of the reducing agent in the second mixed solution is 0.8 mM, 0.4 mM, or 0 mM.
  • the concentration of ethanol in the second mixture is 5.75% (v / v).
  • the concentration of DMSO in the second mixture is 3.75% (v / v).
  • 75 ⁇ l of the second mixed solution and 75 ⁇ l of anti-25OH vitamin D antibody-bound magnetic particle solution were mixed.
  • the solution obtained in 4) above was incubated at 37 ° C. for 10 minutes.
  • 6) After incubation, the magnetic particles in the sample were collected on the magnetic plate, and the magnetic particles were washed three times.
  • Alkaline phosphatase-labeled antibody an antibody against 25OH vitamin D-anti-25OH vitamin D antibody immune complex suspended in MES buffer was added to the magnetic particles excluding the washing solution.
  • the signal intensity (luminescence count) measured using a reagent containing a chaotropic agent is a signal measured using a reagent not containing a chaotropic agent (guanidine hydrochloride). It was significantly higher than the intensity (luminescence count). Further, the signal intensity measured using a reagent containing a chaotropic agent (guanidine hydrochloride) and a reducing agent (DTT) is more than the signal intensity measured using a reagent containing only a chaotropic agent (guanidine hydrochloride). Even higher values were shown.
  • the measurement sensitivity of 25OH vitamin D can be improved by using a chaotropic agent (guanidine hydrochloride), and a combination of chaotropic agent (guanidine hydrochloride) and reducing agent (DTT) is used. It was shown that the measurement sensitivity of 25OH vitamin D can be further improved.
  • a chaotropic agent guanidine hydrochloride
  • DTT reducing agent
  • Example 2 Comparison of correlation coefficient between measured value of vitamin D by the method of the present invention and measured value by an existing method using an organic solvent In order to verify the measurement accuracy of vitamin D by the method of the present invention, The correlation coefficient between the measured value of vitamin D by the above method and the measured value obtained by the existing method using an organic solvent was compared.
  • DiaSorin-RIA Measurement by an existing method (DiaSorin-RIA) As an existing method, DiaSorin-RIA was used as described later. DiaSorin-RIA was performed using a commercially available kit (25-Hydroxyvitamin D 125 I RIA KIT, manufactured by DiaSorin). The method was performed as follows. As a measurement sample, the same 13 human serum samples as in the above 3-1) were used.
  • Pretreatment operation a) A glass test tube is prepared. b) Dispense 500 ⁇ l of acetonitrile into each test tube. c) Add 50 ⁇ l of calibrator, control, or sample (such as serum). d) Stir the sample solution for 10 seconds. e) The sample solution is centrifuged at 1200 ⁇ g for 10 minutes at room temperature. f) The supernatant is used as a sample.
  • the method and kit of the present invention are useful for the measurement of vitamin D.

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Abstract

La présente invention concerne un procédé de mesure de la vitamine D. Plus particulièrement, la présente invention concerne un procédé de mesure de la vitamine D qui consiste à : (1) traiter un échantillon avec un agent chaotropique; et (2) détecter la vitamine D dans l'échantillon traité. La présente invention concerne également un kit de mesure de la vitamine D qui comprend : (1) un agent chaotropique; et (2) une substance qui a une affinité pour la vitamine D, et/ou une préparation de vitamine D. De préférence, l'agent chaotropique est la guanidine ou un sel associé. De préférence, l'échantillon est un échantillon prélevé chez l'homme ou un échantillon dérivé du sang.
PCT/JP2014/051006 2013-02-06 2014-01-20 Procédé de mesure de la vitamine d faisant appel à un agent chaotropique WO2014122974A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110494753A (zh) * 2017-03-28 2019-11-22 豪夫迈·罗氏有限公司 用于分析物测定的通用预处理试剂

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009510415A (ja) * 2005-09-29 2009-03-12 エフ．ホフマン−ラ ロシュ アーゲー ビタミンｄ化合物のための放出試薬
WO2011144661A1 (fr) * 2010-05-20 2011-11-24 Roche Diagnostics Gmbh Réactif de libération pour composés de vitamine d

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009510415A (ja) * 2005-09-29 2009-03-12 エフ．ホフマン−ラ ロシュ アーゲー ビタミンｄ化合物のための放出試薬
WO2011144661A1 (fr) * 2010-05-20 2011-11-24 Roche Diagnostics Gmbh Réactif de libération pour composés de vitamine d

Non-Patent Citations (1)

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MASANOBU KAWAKAMI ET AL.: "Effects of protein modification procedures on the interaction between 25-hybroxyvitamin D and the human plasma binding protein for vitamin D", BIOCHEMISTRY, vol. 20, 1981, pages 5881 - 5887 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110494753A (zh) * 2017-03-28 2019-11-22 豪夫迈·罗氏有限公司 用于分析物测定的通用预处理试剂
CN110494753B (zh) * 2017-03-28 2023-09-19 豪夫迈·罗氏有限公司 用于分析物测定的通用预处理试剂

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