WO2006030866A1 - Procédé de détermination quantitative d’acide urique - Google Patents

Procédé de détermination quantitative d’acide urique Download PDF

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Publication number
WO2006030866A1
WO2006030866A1 PCT/JP2005/017055 JP2005017055W WO2006030866A1 WO 2006030866 A1 WO2006030866 A1 WO 2006030866A1 JP 2005017055 W JP2005017055 W JP 2005017055W WO 2006030866 A1 WO2006030866 A1 WO 2006030866A1
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WO
WIPO (PCT)
Prior art keywords
reagent
uric acid
peroxidase
uricase
test sample
Prior art date
Application number
PCT/JP2005/017055
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English (en)
Japanese (ja)
Inventor
Yuhko Hirao
Hiroshi Matsui
Original Assignee
Denka Seiken Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Denka Seiken Co., Ltd. filed Critical Denka Seiken Co., Ltd.
Priority to JP2006535202A priority Critical patent/JPWO2006030866A1/ja
Publication of WO2006030866A1 publication Critical patent/WO2006030866A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/62—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving uric acid

Definitions

  • the present invention relates to a reagent capable of accurately quantifying uric acid and a method for quantifying uric acid using the reagent. More specifically, the present invention relates to a reagent capable of accurately quantifying a low value in the determination of uric acid and an accurate quantification method of a low value of uric acid using the reagent.
  • Uric acid is a final metabolite of purines that are constituents of nucleic acids such as deoxyribonucleic acid and ribonucleic acid, ATP, which is an energy carrier, and other nucleotides.
  • Purines are synthesized mainly from amino acids and related compounds in the human body. Purine metabolism is involved in cell growth and plays a central role in maintaining life.
  • Uric acid is produced at a certain rate in the living body, temporarily stored in the body, and then is mainly excreted into the urine of the kidney, and the other part is excreted into the digestive tract and sweat as an extrarenal treatment. Production and excretion are almost the same, so production, storage and excretion are in equilibrium. In normal individuals, the daily production and excretion amount is approximately 700 mg, of which 500 mg is excreted in the kidney urine and approximately 20 Omg is excreted by extrarenal treatment. Extrarenal treatment increases and decreases in parallel with changes in serum uric acid levels.
  • Hyperuricemia is a state in which blood is saturated and urate is present, and is mainly caused by abnormal uric acid metabolism and decreased excretion of uric acid.
  • Gout is a typical disease that increases serum uric acid levels, but it also increases in cancer, leukemia, and myeloma.
  • causes of hypouricemia include insufficient intake of purines as raw materials for uric acid, decreased production of uric acid due to debilitating diseases throughout the body, decreased production of uric acid due to metabolic diseases, and increased uric acid excretion.
  • Metabolic diseases include xanthine oxidase deficiency, purine nucleoside phosphorylase deficiency, and 5-phosphoribosyl-1-pyrophosphate (PRPP) synthase deficiency.
  • PRPP 5-phosphoribosyl-1-pyrophosphate
  • the uric acid measurement method is roughly classified into a reduction method using the reducing property of uric acid under alkaline conditions and an enzymatic measurement method using uric acid-degrading enzyme uricase.
  • Reduction methods include a phosphotungstic acid deproteinization method and a phosphotungsten direct method.
  • Enzymatic methods using uricase include uricase 'catalase method, uricase' peroxidase method, and uricase UV method.
  • the uric acid content is obtained by colorimetric determination with a chromogenic reaction.
  • This method does not require deproteinization or sample blinding, and is a simple method in which color development is completed in a short time, and can be easily applied to various automatic analyzers.
  • Patent Document 1 Japanese Patent No. 2862817
  • Patent Document 2 Japanese Patent No. 3283348
  • Patent Document 3 Japanese Patent No. 3520874
  • Patent Document 4 Japanese Patent No. 3357667 Disclosure of the invention
  • An object of the present invention is to provide a quantification method and reagent composition excellent in accuracy at a low value over a uric acid quantification method using peroxidase, and in a wide measurement range. It is to provide a method for quantifying uric acid and a reagent composition with excellent accuracy.
  • the inventors of the present invention conducted extensive research on the problem of low accuracy in the uric acid quantification using the uricase 'peroxidase method. As a result, the peroxidase in the reagent reacted with uric acid to react with uric acid. It was found that uric acid in the low concentration range could not be accurately quantified.
  • Peroxidase is a reagent composition involved in the production of a quinone dye, and is usually used after a uricase enzyme reaction. Since the uricase enzyme is usually added to the second reagent, it is often added to the first reagent due to the stability and component balance of the whole reagent. Until the inventors of the present application found the above problems, the problems of the conventional reagent composition were overlooked.
  • the inventors of the present application transferred the first reagent force to the second reagent except for peroxidase so that uric acid and peroxidase do not contact before uric acid and uricase react.
  • the inventors have found that uric acid in a low concentration range can be accurately quantified, and have completed the present invention.
  • the present invention is as follows.
  • a method for quantifying uric acid characterized in that
  • a first reagent containing a hydrogen-donating compound is added to a test sample, then a second reagent containing 4-amaminoantipyrine is added, and then a third reagent containing uricase and peroxidase is added.
  • uric acid quantification method [1]
  • a uric acid determination reagent comprising a first reagent containing a hydrogen donor and a second reagent containing peroxidase and uricase, wherein the first reagent contains peroxidase, and
  • Second reaction 4AA + hydrogen donor + 2H 2 0 2 ⁇ quinone dye + 4H 2 0
  • the method of the present invention has a two-step reaction force.
  • uric acid in serum and urine is broken down into allantoin, carbon dioxide and hydrogen peroxide by uricase.
  • the peroxyhydrogen generated here produces a colored quinone (quinone dye) in the presence of peroxidase, 4-aminoantipyrine (4AA) and phenolic or alpha phosphorus hydrogen donor compounds in a second reaction. To do.
  • the method of the present invention can also be applied to a reaction system in which hydrogen peroxide generated in the first reaction oxidizes a leuco dye in the presence of a peroxidase and a leuco dye to produce a blue dye.
  • an automatic analyzer in a clinical test generally puts a predetermined amount of one or more reagents into a test sample in order, and measures the absorbance, scattered light, etc. at a certain wavelength after a certain amount of time for reagent loading, and within a predetermined time. By determining the amount of change and the rate of change, the amount of the test substance in the test sample can be quantified.
  • the reagent composition necessary for quantitative reaction is arbitrarily allocated to multiple reagents, but each reagent is kept for a long period of several months to several years. Therefore, the chemical reaction proceeds before being used for clinical testing, and the reagent composition necessary for the quantitative reaction is degraded, or a composition that inhibits the quantitative reaction is generated.
  • the reagent composition cannot be made by combining the compositions.
  • the method of the present invention is a method for quantifying uric acid using a reagent in which uric acid and peroxidase do not come into contact before uric acid and uricase react, and the reagent power first added to the test sample also excludes peroxidase. It is.
  • the negative effect of hemoglobin in the test sample is an adverse effect of removing the reagent force peroxidase first added to the test sample.
  • the bad effects of peroxidase can be avoided by first adding sodium azide as the reagent to be added to the test sample. Therefore, in the method of the present invention, sodium azide may be contained in the reagent initially added to the test sample.
  • the reagent composition used in the uric acid quantitative method of the present invention satisfies the following requirements (a) to (d): You may choose.
  • uricase and peroxidase are preferably included in the same reagent.
  • Azidosodium can also be included in the first reagent that is first added to the test sample.
  • the reagents added to the reaction system in which the reaction of the above formula occurs are referred to as the first reagent, the second reagent, and the third reagent in the order of addition.
  • the process of mixing and reacting the first reagent and the test sample is called the first process, and the process of adding the second reagent and reacting is called the second process.
  • the step of adding the third reagent to react is called the third step.
  • uricase and peroxidase are preferably included in the same reagent, but uricase and peroxidase are included in the same reagent. It is also possible to add uricase with the previous reagent and not add peroxidase with the later reagent. Further, any one of the first reagent, the second reagent, and the third reagent, and any two or three of them may contain a surfactant. Further, ascorbate oxidase may be contained in any one of the first reagent, the second reagent, and the third reagent, and any two or three of them.
  • Second reagent uricase, 4-aminoantipyrine, peroxidase (2)
  • Second reagent uricase, TOOS, peroxidase
  • TOOS contained in the first reagent is a hydrogen donor compound.
  • the hydrogen donor compounds N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyoxyline (HDAOS), N-ethyl-N-sulfopropyl- 3-methoxy-line (ADPS), N-ethyl-N-sulfopropyl-line (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxy-line (DAPS), N-sulfopropyl- 3,5-dimethoxy-line (HDAPS), N-ethyl-N-sulfopropyl-3,5-dimethyl-line (MAPS), N-ethyl-N-sulfopropyl-3-methyl-line (TOPS) , N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyline (ADOS), N-ethyl-N- (2-
  • Azidosodium that can be contained in the first reagent is added to avoid the influence of hemoglobin in the test sample. In order to avoid the effects of hemoglobin after adding the first reagent to the sample, it is desirable to react for a certain period of time.
  • Test samples used in the measurement method of the present invention are serum, plasma, and urine.
  • Automatic analyzer Hitachi, Model 7150
  • the amount of test sample and reagent used can be changed as appropriate depending on the analyzer used.
  • the concentration of sodium azide in the reaction solution in the first step is preferably 0.1 to 2 g / L (0.01% to 0.2% (w / v)).
  • the first step is preferably carried out in a pH 6.0 to pH 8.0 buffer solution, preferably a phosphate buffer solution or a Good buffer solution.
  • a pH 6.0 to pH 8.0 buffer solution preferably a phosphate buffer solution or a Good buffer solution.
  • 3-morpholinopropanesulfonic acid MOPS
  • 2-hydroxy-3-morpholinopropanesulfonic acid MOPSO
  • the first step preferably contains a surfactant.
  • the surfactant used in the first step include non-ionic surfactants such as polyoxyethylene alkyl ether and polyoxyethylene alkyl phenyl ether.
  • the concentration of the surfactant in the reaction solution in the first step is preferably about 0.1 to 10 g / L (0.01 to 1% (w / v)), more preferably 0.5 to 5 ⁇ / y (0.05 to 0.5). % (w / v)).
  • the buffer solution and surfactant used in the first step should be contained in the first reagent! However, after mixing the sample with the above buffer solution and / or surfactant-containing solution The first reagent may be added.
  • the reaction time (the time from the addition of the first reagent to the addition of the second reagent) is preferably about 10 minutes and 5 minutes.
  • the reagent configuration is three reagents
  • the second reagent and the third reagent are added in order, and the reactions in the second and third steps are performed. Just do it.
  • the second reagent and the third reagent may be added simultaneously, or the third reagent may be added 1 to 10 minutes after the addition of the second reagent.
  • uric acid in the test sample is decomposed into allantoin, carbon dioxide and hydrogen peroxide by the action of uricase.
  • the uric acid was quantified by converting the hydrogen peroxide produced in the first reaction into a colored quinone by peroxidase by an acid-acid condensation reaction of 4-aminoantipyrine with an ⁇ -phosphorus hydrogen donor compound. The measurement is performed at a wavelength of 400 to 700 nm.
  • the second step and the third step preferably include a surfactant.
  • the surfactant used in the second step and the third step include nonionic surfactant polyoxyethylene alkyl ether and polyoxyethylene alkyl ether.
  • the concentration of the surfactant in the reaction solution of the second step and the third step is preferably about 0.1 to 10 g / L (0.0 l to l% (w / v)), more preferably 0.5 to 5 8 / (0.05 to 0.5%).
  • the concentration of peroxidase when converting hydrogen peroxide to quinone dye in the reaction solution of the second step or the third step is preferably 0.5-10 IU / mL.
  • the concentration of 4-aminoantipyrine The concentration of hydrogen donating compounds such as aniline-based hydrogen donor compounds is preferably 0.1 to 5 mmol / L.
  • the concentration of uricase is preferably 50 to 500 IU / L.
  • Other preferable reaction conditions in the second step and the third step are the same as the preferable reaction conditions in the first step.
  • the amount of the produced quinone dye is measured by the absorbance at 550 to 650 nm in the sample. Measure by measuring. Absorbance may be measured using an absorptiometer.
  • reagent includes a reagent composition
  • reagent composition refers to an enzyme, a surfactant, or the like constituting the reagent. Say the substance.
  • Reagent compositions used in the first step and the second step (the first reagent composition and the first step, respectively)
  • Nonionic surfactant polyethylene glycol mono-p-isooctylphenyl ester manufactured by Nacalai Testa 0.1% (w / v) ascorbate oxidase 3000 IU / L
  • Nonionic surfactant 3 ⁇ 4 agent Polyethylene glycol mono-p-isococ / Refeninore Itel (Nacalai Tesque) 0. l3 ⁇ 4 (w / v) 4-aminoantipyrine 2.0 mmol / L
  • Nonionic surfactant Polyol mono-p isococtyl fe / Rhe Itel (Nacalai Tesque) 0.1% (w / v) ascorbate oxidase 3000 IU / L
  • Nonionic surfactant Poly-Mono-p-isooctylphenol Itel (manufactured by Nacalai Tesque) 0.1% (w / v) 4-aminoantipyrine 2.0 mmol / L
  • Nonionic surfactant Poly-mono-p-isooctylphenyl ester (Nacalai Testa) 0. l% (w / v) ascorbate oxidase 3000IU / L
  • Non-ionic surfactant Polyol Mono-p-isooctylphenol / Rye Itel (manufactured by Nacalai Tesque) 0. l% (w / v) T00S 4.5 mmol / L
  • the uric acid concentration in the sample was determined by the method using the first reagent containing peroxidase, which was conventionally used as a control.
  • the reagent compositions of the first reagent and the second reagent used were as follows.
  • Nonionic surfactant polyethylene glycol mono-P-isooctylphenyl ester manufactured by Nacalai Tesque 0. (w / v) ascorbate oxidase 3000 IU / L
  • Nonionic Surfactant Polyethylene Daricol Mono-P-Isooctylphenyl

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
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Abstract

L’invention concerne un procédé de détermination quantitative d’acide urique utilisant du peroxydase et d’une excellente précision à des valeurs basses ; et une composition réactive correspondante. L’invention porte sur un procédé de détermination quantitative d’acide urique dans un échantillon d’analyte, consistant à décomposer l’acide urique dans un échantillon d’analyte à l’aide d’uricase (enzyme) en peroxyde d’hydrogène et allantoïne et à mesurer la quantité de peroxyde d’hydrogène à l’aide de peroxydase, caractérisé en ce que tout contact de l’acide urique avec le peroxydase avant la décomposition de l’acide urique par l’uricase (enzyme) est évité pour ainsi obtenir une détermination quantitative précise de faibles valeurs d’acide urique.
PCT/JP2005/017055 2004-09-16 2005-09-15 Procédé de détermination quantitative d’acide urique WO2006030866A1 (fr)

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JP2006535202A JPWO2006030866A1 (ja) 2004-09-16 2005-09-15 尿酸の定量方法

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JP2004-270366 2004-09-16
JP2004270366 2004-09-16

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WO2006030866A1 true WO2006030866A1 (fr) 2006-03-23

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110082418A (zh) * 2019-05-27 2019-08-02 三诺生物传感股份有限公司 一种尿酸电化学测量方法
CN114441516A (zh) * 2021-12-20 2022-05-06 苏州百源基因技术有限公司 一种尿酸检测试剂盒及其制备方法
WO2022172343A1 (fr) 2021-02-10 2022-08-18 オリエンタル酵母工業株式会社 Activateur d'uricase et réactif de mesure d'acide urique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013081458A (ja) * 2011-09-30 2013-05-09 Shino Test Corp 測定試薬の安定化方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000028533A (ja) * 1998-04-23 2000-01-28 Shino Test:Kk 安定な測定試薬及び測定方法
JP2001255323A (ja) * 2000-01-05 2001-09-21 Hiromi Nanba 血液分離器具及び血液分離方法並びに生体試料調製方法、生体試料定量方法及び生体試料保存容器
JP2003043035A (ja) * 2001-08-01 2003-02-13 Toyobo Co Ltd 液状試薬原料の製造方法および組成物

Family Cites Families (4)

* Cited by examiner, † Cited by third party
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JP2794426B2 (ja) * 1988-11-18 1998-09-03 中外製薬株式会社 ペルオキシダーゼ含有試薬組成物及びペルオキシダーゼ酵素活性阻害防止方法
JP3711544B2 (ja) * 1994-08-02 2005-11-02 日立化成工業株式会社 化学発光測定試薬
JP3702476B2 (ja) * 1994-08-02 2005-10-05 日立化成工業株式会社 化学発光測定方法
JP2862817B2 (ja) * 1995-09-05 1999-03-03 株式会社エイアンドティー 液状濃縮酵素試薬

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000028533A (ja) * 1998-04-23 2000-01-28 Shino Test:Kk 安定な測定試薬及び測定方法
JP2001255323A (ja) * 2000-01-05 2001-09-21 Hiromi Nanba 血液分離器具及び血液分離方法並びに生体試料調製方法、生体試料定量方法及び生体試料保存容器
JP2003043035A (ja) * 2001-08-01 2003-02-13 Toyobo Co Ltd 液状試薬原料の製造方法および組成物

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110082418A (zh) * 2019-05-27 2019-08-02 三诺生物传感股份有限公司 一种尿酸电化学测量方法
CN110082418B (zh) * 2019-05-27 2021-10-15 三诺生物传感股份有限公司 一种尿酸电化学测量方法
WO2022172343A1 (fr) 2021-02-10 2022-08-18 オリエンタル酵母工業株式会社 Activateur d'uricase et réactif de mesure d'acide urique
CN114441516A (zh) * 2021-12-20 2022-05-06 苏州百源基因技术有限公司 一种尿酸检测试剂盒及其制备方法

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