WO2004029613A1 - Procede pour mesurer la teneur en glycoproteine - Google Patents

Procede pour mesurer la teneur en glycoproteine Download PDF

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Publication number
WO2004029613A1
WO2004029613A1 PCT/JP2003/012232 JP0312232W WO2004029613A1 WO 2004029613 A1 WO2004029613 A1 WO 2004029613A1 JP 0312232 W JP0312232 W JP 0312232W WO 2004029613 A1 WO2004029613 A1 WO 2004029613A1
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WIPO (PCT)
Prior art keywords
fructosylamine
measuring
glycated protein
amount
glycated
Prior art date
Application number
PCT/JP2003/012232
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English (en)
Japanese (ja)
Inventor
Koji Sode
Original Assignee
Koji Sode
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.)
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Publication date
Application filed by Koji Sode filed Critical Koji Sode
Priority to JP2004539527A priority Critical patent/JP4330534B2/ja
Priority to AU2003266612A priority patent/AU2003266612A1/en
Publication of WO2004029613A1 publication Critical patent/WO2004029613A1/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
    • 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
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/723—Glycosylated haemoglobin

Definitions

  • the present invention relates to a method for measuring glycated protein and hemoglobin A 1c used as a marker for diagnosing diabetes in the field of clinical examination. More specifically, the present invention provides a method for quantifying glycated protein and hemoglobin A1c based on the fact that glycated protein and hemoglobin A1c antagonistically inhibit the fructosylamine oxidation reaction catalyzed by fructosylamine oxidation. On how to do. Furthermore, the present invention relates to a glycated protein and a kit for measuring hemoglobin A1c and a sensor constructed based on the method of the present invention. Background art
  • the amino groups of the protein main chain and side chains are non-enzymatically linked to the reducing end of a reducing sugar such as glucose to produce an Amadori compound, ie, a glycated protein.
  • a reducing sugar such as glucose
  • an Amadori compound ie, a glycated protein.
  • hemoglobin is glycated in blood to produce glycated hemoglobin (glycohemoglobin; HbAlc). Since the presence of HbAlc in hemoglobin is higher in diabetic patients than in healthy subjects, and the blood concentration of HbAlc reflects blood glucose levels in the past few weeks, the HbAlc blood concentration can be used for diagnosis of diabetes and diabetes. It is extremely important in clinical trials as an indicator of glycemic control in patients.
  • fructosylamine oxidation catalysts reported so far cannot oxidize unhydrolyzed sugarcane protein as a substrate. For this reason, glycated protein is first hydrolyzed, and the resulting low-molecular-weight glycated peptide or fructosylamine is oxidized using a fructosylamic acid catalyst to obtain a glycated protein. The protein was being measured. Therefore, there is a need in the art for a method for measuring a glycated protein without decomposing it.
  • An object of the present invention is to provide a novel method for measuring a saccharified protein and a saccharified protein in which the concentrations of glycated proteins and hemoglobin A 1c are measured without hydrolysis. More specifically, the present invention provides a method for measuring a glycated protein, particularly hemoglobin A1c (HbAlc) or glycated albumin, which is used in the field of clinical testing and the like, and a measuring reagent and a sensor constructed based on the method. The purpose is to: Disclosure of the invention
  • the present inventors have found that glycated proteins can competitively inhibit the fructosylamine oxidation reaction catalyzed by a fructosylamine oxidation catalyst, and have completed the present invention. That is, the present invention relates to a method for measuring a glycated protein in a sample, comprising contacting the sample with a fructosylamic acid catalyst in the presence of a fixed amount of fructosylamine. A method is provided wherein the amount of the glycated protein is measured by measuring the amount of fructosylamine oxidized by the catalyst. According to the present invention, glycated protein can be measured without hydrolyzing the glycated protein, using the competitive inhibition of the fructosylamine oxidation catalyst by the glycated protein as an index.
  • fructosylamine oxidation catalyst refers to a catalyst that catalyzes the oxidation reaction of fructosylamine, which inhibits the reaction in a concentration-dependent manner in the presence of glycated protein. Represents the catalyst to be used.
  • examples of fructosylamine oxidation catalysts include fructosylamine oxidase and polymers that catalyze the oxidation of fructosylamine in the presence of an electron acceptor, for example, polymers containing imidazole groups.
  • the present invention provides a method for measuring a glycated protein, wherein the fructosylamine oxidation catalyst described above is a molecule having an imidazole group. In a preferred embodiment, the present invention provides a method for measuring a glycated protein, wherein the fructosylamine oxidation catalyst is fructosylamine oxidase. More preferably, the fructosylamine oxidation catalyst is selective for fructosyl valine.
  • the step of measuring the amount of fructosylamine is performed by spectrophotometrically measuring the amount of an electron acceptor reduced as the fructosylamine is oxidized.
  • the step of measuring the amount of fructosylamine is performed by electrochemically measuring the amount of an electron acceptor reduced with the oxidation of fructosylamine using an electrode. Done.
  • the present invention provides a method for measuring a glycated protein, wherein the glycated protein described above is hemoglobin A1c.
  • the present invention also provides a glycated protein measurement kit based on the above principle.
  • the kit comprises a fructosylamine oxidation catalyst and fructosylamine.
  • the present invention also provides a hemoglobin A1c measurement kit based on the above-described principle.
  • the present invention provides a glycated protein measuring sensor based on the above principle.
  • the present invention also provides a sensor for measuring hemoglobin A1c based on the principle described above.
  • FIG. 1 shows the sensor system constructed in the second embodiment.
  • FIG. 2 shows a decrease in the steady-state current value when the HbAlc sample was added to the sensor system constructed in Example 2.
  • Fig. 3 shows that various proteins were added to the sensor shown in The graph shows the dependence of the decrease in the steady-state current value upon the addition of white matter and HbAlc on the sample concentration.
  • FIG. 4 shows the results of measuring HbA1c using the sensor system created in Example 2.
  • FIG. 5 shows the results of measuring HbA1c using the sensor system created in Example 5.
  • the method for measuring a glycated protein of the present invention is based on the discovery that glycated proteins can competitively inhibit the fructosylamine oxidation reaction catalyzed by a fructosylamine oxidation catalyst. That is, when a sample containing a glycated protein is brought into contact with a fructosylamine oxidation catalyst in the presence of a fixed amount of fructosylamine, the amount of fructosylamine oxidized by the fructosylamine oxidation catalyst becomes saccharified. It varies depending on the protein concentration. Therefore, by measuring the amount or oxidation rate of oxidized fructosylamine, the concentration of glycated protein in a sample can be determined.
  • a fructosylamine oxidation catalyst for example, a synthetic polymer having an imidazole group or a fructosylamine oxidase can be used.
  • a catalyst having an activity of oxidizing fructosylamine and being antagonized by glycated protein is used. If there is, it is not limited.
  • imidazole it is possible to use a polymer obtained by polymerizing a functional group that functions as a general base catalyst, for example, a monomer containing bipyridyl containing pyridine.
  • HbAlc has a fructosyl rubulin moiety in its molecule and acts antagonistically to a fructosylamine oxidation catalyst selective for fructosyl valine.
  • a fructosylamine oxidation catalyst selective for fructosyl valine.
  • the enzyme can be used, but it is limited as long as it has the oxidizing activity of fructosyl valine and is subject to competitive inhibition by HbAlc. It is not something to be done.
  • fructosyl valine but also a catalyst polymer having an imidazole group, which is synthesized using an analog, methyl valine, as ⁇ -type can be used as the catalyst.
  • the measurement of the glycated protein in the present invention can be easily performed by measuring the amount or reduction rate of the electron acceptor reduced by the fructosylamine oxidation by the fructosylamine oxidation catalyst.
  • a sample is added to a solution containing a fixed amount of a fructosylamine oxidation catalyst, fructosylamine, and an electron acceptor, and the amount or reduction rate of the electron acceptor reduced by oxidation of fructosylamine is measured.
  • the amount of the electron acceptor or the reduction rate in the absence of the sample the glycated protein in the sample can be measured.
  • Electron acceptors can be measured by spectroscopic methods based on the characteristic spectrum of the reduced state of the electron acceptor, or by reoxidizing the electron acceptor in the reduced state on the electrode. An electrochemical method by measuring the obtained current can be used.
  • an unknown concentration of HbA1c can be quantified with a sensitivity of 10 nM or less in the presence of PMS and DCIP as electron acceptors.
  • various artificial electron acceptors may be used.
  • the electron acceptor potassium phenocyanide, phlegmene, an osmium derivative, or the like can be used.
  • oxygen can be used as an electron acceptor in addition to the above-mentioned electron acceptor.
  • the amount of oxygen decrease and the rate of decrease or the amount of hydrogen peroxide produced
  • the inhibition of the enzyme by glycated protein can be measured, and the concentration of glycated protein can be measured.
  • the present invention features a kit for measuring a glycated protein such as HbA1c or glycated albumin, which includes a measuring method using the principle of the present invention.
  • the glycated protein measurement kit of the present invention contains the fructosylamine oxidizing catalyst according to the present invention, fructosylamine, and an electron acceptor in an amount sufficient for at least one time of the assay.
  • the kit contains a fructosylamic acid catalyst; a buffer adjusted to pH 6.0 to 10; a suitable mediator; and a glycated protein or a derivative thereof for preparing a calibration curve. Includes standard solutions, as well as guidelines for use.
  • the glycated protein analysis kit according to the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Sensor
  • the present invention features a sensor for measuring a glycated protein such as glycated albumin, HbAle, or the like.
  • the glycated protein sensor of the present invention electrochemically oxidizes a mediae, which is reduced as fructosylamine is oxidized by a fructosylamine oxidation catalyst, on an electrode, and determines a current value at this time. Measure.
  • the oxidation reaction is competitively inhibited depending on the concentration of glycated protein.Therefore, a current curve was measured in the presence of various concentrations of glycated protein, and a standard curve was prepared.
  • the glycated protein concentration in the sample can be determined.
  • the electrode a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and a fructosylamine oxidation catalyst is fixed on the electrode.
  • the immobilization method include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, and a redox polymer. May be used.
  • a sensor for measuring HbAlc can be constructed as follows. Measured in an ambrometric system using a carbon electrode, gold electrode, platinum electrode, etc. In this case, use an electrode on which a fructosylamine oxidation catalyst is immobilized as a working electrode, and use a buffer solution containing a mediator together with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an AgZAgC1 electrode). And keep it at a constant temperature. Apply a constant voltage to the working electrode, add a fixed amount of fructosylamine and a sample, and measure the increase in current. As a medium, potassium ferricyanide, phlegmene, osmium derivatives, phenazine methosulfate and the like can be used.
  • an electrode in which a fructosylamine oxidation catalyst and an electron mediator such as potassium ferricyanide, phenocene, osmium derivatives, and phenazine methosulfate are immobilized on a polymer matrix by adsorption or covalent bonding as a working electrode.
  • a buffer insert it into a buffer with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCI electrode) and keep it at a constant temperature. Apply a constant potential to the working electrode, add a fixed amount of fructosylamine and sample, and measure the increase in current.
  • the concentration of glycated protein in the sample can be determined according to calibration curves prepared using glycated protein solutions of various concentrations.
  • HbAlc was measured using the inhibition of fructosyl valine response observed in the present sensor system as an index. That is, in this sensor system, the response current value to fructosyl valine 0. ImM in the absence of HbAlc decreases as the added HbAlc concentration increases. From the response current value for fructosyl valine 0.1 mM, the decrease in the non-specific response value observed in the presence of various proteins was subtracted to obtain the response current value for fructosyl palin 0.1 ImM. Fructosylvaline, which is observed in the presence of various concentrations of HbA1c, is subtracted from this value.
  • the response current to ImM is subtracted, and the difference in fructosylvaline in the absence of HbA1c is calculated.
  • the ratio of the response current to sylvaline 0.1 mM was defined as the inhibition rate.
  • the inhibition rate is plotted on the vertical axis, and the results of measurement of HbAlc are shown in FIG.
  • HbAlc could be detected at a sensitivity of 10 nM without denaturing or hydrolyzing using this sensor.
  • HbAlc hemoglobin Alc
  • HbAo hemoglobin Ao
  • FIG. 5 shows the result of plotting the increased current value against the Hb A 1c concentration. This measurement was carried out by varying the mixing ratio of 1181 cZHbAo in the HbA1.711180 mixture, which is the sample to be added.

Abstract

La présente invention concerne un procédé pour mesurer la teneur en une glycoprotéine telle que l'hémoglobine Alc dans un échantillon. Ce procédé se caractérise en ce qu'il comprend la mise en contact de l'échantillon avec un catalyseur d'oxydation de fructosylamine en la présence d'une quantité définie de fructosylamine, puis la quantification de la fructosylamine ainsi oxydée par le catalyseur pour déterminer le teneur en glycoprotéine. Comme catalyseur d'oxydation de fructosylamine peut être utilisée une molécule qui présente imidazole, une fructosylamine oxydase, etc.
PCT/JP2003/012232 2002-09-26 2003-09-25 Procede pour mesurer la teneur en glycoproteine WO2004029613A1 (fr)

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JP2004539527A JP4330534B2 (ja) 2002-09-26 2003-09-25 糖化蛋白質測定方法
AU2003266612A AU2003266612A1 (en) 2002-09-26 2003-09-25 Method of measuring glycoprotein

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JP2002-319040 2002-09-26
JP2002319040 2002-09-26

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014102144A (ja) * 2012-11-20 2014-06-05 Nipro Corp 糖化ヘモグロビン測定キットおよび糖化ヘモグロビン測定方法
WO2015005257A1 (fr) * 2013-07-09 2015-01-15 協和メデックス株式会社 Procédé de mesure de l'hémoglobine glyquée

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001054398A (ja) * 1999-08-18 2001-02-27 Asahi Chem Ind Co Ltd タンパク質の選択的断片化方法
JP2001204494A (ja) * 2000-01-25 2001-07-31 Koji Hayade 糖化ヘモグロビンの測定キット
JP2001215229A (ja) * 2000-02-02 2001-08-10 Wako Pure Chem Ind Ltd 糖化蛋白質測定試薬
WO2001090735A1 (fr) * 2000-05-23 2001-11-29 Koji Sode Trousse permettant d'analyser des proteines glyquees
WO2002022698A1 (fr) * 2000-09-12 2002-03-21 Koji Sode Polymères agissant en tant qu'enzymes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001054398A (ja) * 1999-08-18 2001-02-27 Asahi Chem Ind Co Ltd タンパク質の選択的断片化方法
JP2001204494A (ja) * 2000-01-25 2001-07-31 Koji Hayade 糖化ヘモグロビンの測定キット
JP2001215229A (ja) * 2000-02-02 2001-08-10 Wako Pure Chem Ind Ltd 糖化蛋白質測定試薬
WO2001090735A1 (fr) * 2000-05-23 2001-11-29 Koji Sode Trousse permettant d'analyser des proteines glyquees
WO2002022698A1 (fr) * 2000-09-12 2002-03-21 Koji Sode Polymères agissant en tant qu'enzymes

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* Cited by examiner, † Cited by third party
Title
CHIE NAKAJIMA: "Kecchu fructosamine no kosoteki sokutei shiyaku (FOD-POD-TOOS-Kei), ni kansuru kisoteki kento", THE JOURNAL OF MEDICINE, vol. 40, no. 05, 25 November 1998 (1998-11-25), pages 897 - 902, XP002977410 *
FUMIMASA ISHIMURA: "Kaiyo kobo yurai fructosylamine sanka koso o mochiiru toka hemoglobin keisoku", THE ELECTROCHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU, vol. 79, no. 02, 15 March 2001 (2001-03-15), pages 877, XP002977408 *
KISHIRI KOMIYAMA: "Kosoteki hoho ni yoru glycated protein no shinki sokuteiho no kaihatsu", RINSHO KAGAKU, vol. 27, no. 02, 30 June 1998 (1998-06-30), pages 99 - 106, XP002977409 *
SHIGENORI OTA: "Molecular inprinting catalysis o mochiita toka tanpaku-shitsu no sokutei", THE ELECTROCHEMICAL SOCIETY OF JAPAN TAIKAI KOEN YOSHISHU, vol. 96, 25 March 2002 (2002-03-25), pages 266, XP002977407 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014102144A (ja) * 2012-11-20 2014-06-05 Nipro Corp 糖化ヘモグロビン測定キットおよび糖化ヘモグロビン測定方法
WO2015005257A1 (fr) * 2013-07-09 2015-01-15 協和メデックス株式会社 Procédé de mesure de l'hémoglobine glyquée
US10006923B2 (en) 2013-07-09 2018-06-26 Kyowa Medex Co., Ltd. Method for measuring glycated hemoglobin

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JPWO2004029613A1 (ja) 2006-01-26
AU2003266612A1 (en) 2004-04-19
JP4330534B2 (ja) 2009-09-16

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