WO2022054890A1 - 測定誤差低減方法 - Google Patents
測定誤差低減方法 Download PDFInfo
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- WO2022054890A1 WO2022054890A1 PCT/JP2021/033235 JP2021033235W WO2022054890A1 WO 2022054890 A1 WO2022054890 A1 WO 2022054890A1 JP 2021033235 W JP2021033235 W JP 2021033235W WO 2022054890 A1 WO2022054890 A1 WO 2022054890A1
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- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
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- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
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- WYACBZDAHNBPPB-UHFFFAOYSA-N diethyl oxalate Chemical compound CCOC(=O)C(=O)OCC WYACBZDAHNBPPB-UHFFFAOYSA-N 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
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- 108010054790 glycerol-3-phosphate oxidase Proteins 0.000 description 1
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- 108010036302 hemoglobin AS Proteins 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000003707 hexyloxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- HBSYJULDYOVADU-UHFFFAOYSA-N hydrazine;3-methyl-1,3-benzothiazol-2-one Chemical compound NN.C1=CC=C2SC(=O)N(C)C2=C1 HBSYJULDYOVADU-UHFFFAOYSA-N 0.000 description 1
- LEUJVEZIEALICS-UHFFFAOYSA-N hydrogen sulfate;1h-imidazol-2-ylazanium Chemical compound OS(O)(=O)=O.NC1=NC=CN1 LEUJVEZIEALICS-UHFFFAOYSA-N 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- 229940040102 levulinic acid Drugs 0.000 description 1
- 125000000040 m-tolyl group Chemical group [H]C1=C([H])C(*)=C([H])C(=C1[H])C([H])([H])[H] 0.000 description 1
- 108010077372 mast cell degranulating peptide Proteins 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- XPIWVCAMONZQCP-UHFFFAOYSA-N methyl 2-oxobutanoate Chemical compound CCC(=O)C(=O)OC XPIWVCAMONZQCP-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- CWOMTHDOJCARBY-UHFFFAOYSA-N n,n,3-trimethylaniline Chemical compound CN(C)C1=CC=CC(C)=C1 CWOMTHDOJCARBY-UHFFFAOYSA-N 0.000 description 1
- ZPEDSBOBSNNATM-UHFFFAOYSA-N n-[2-(n-ethyl-3-methylanilino)ethyl]acetamide Chemical compound CC(=O)NCCN(CC)C1=CC=CC(C)=C1 ZPEDSBOBSNNATM-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- JSABTPDNEXHNOQ-UHFFFAOYSA-N n-phenylaniline;sodium Chemical compound [Na].C=1C=CC=CC=1NC1=CC=CC=C1 JSABTPDNEXHNOQ-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000005691 oxidative coupling reaction Methods 0.000 description 1
- 125000003854 p-chlorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1Cl 0.000 description 1
- 125000004115 pentoxy group Chemical group [*]OC([H])([H])C([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 150000002990 phenothiazines Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108010089000 polyamine oxidase Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 108010001816 pyranose oxidase Proteins 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- SVLRFMQGKVFRTB-UHFFFAOYSA-M sodium;3-(3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].COC1=CC(NCC(O)CS([O-])(=O)=O)=CC(OC)=C1 SVLRFMQGKVFRTB-UHFFFAOYSA-M 0.000 description 1
- HDARHUHTZKLJET-UHFFFAOYSA-M sodium;3-(n-ethyl-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC(OC)=CC(OC)=C1 HDARHUHTZKLJET-UHFFFAOYSA-M 0.000 description 1
- HLXGRHNZZSMNRX-UHFFFAOYSA-M sodium;3-(n-ethyl-3,5-dimethylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC(C)=CC(C)=C1 HLXGRHNZZSMNRX-UHFFFAOYSA-M 0.000 description 1
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- ZPCAZHPYLUKSMY-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 ZPCAZHPYLUKSMY-UHFFFAOYSA-M 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 229940045136 urea Drugs 0.000 description 1
Classifications
-
- 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
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
- C07C49/86—Ketones containing a keto group bound to a six-membered aromatic ring containing —CHO groups
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
-
- 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
-
- 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
- G01N33/725—Haemoglobin using peroxidative activity
-
- 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
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
Definitions
- the present invention relates to a method of measuring a measurement target in a sample based on an enzymatic method. More specifically, the present invention relates to a measurement method for quantifying a measurement target by quantifying hydrogen peroxide generated by reacting the measurement target in a sample with an enzyme.
- a measuring method in which the amount of a component in a sample derived from a biological sample such as whole blood, serum, plasma, or urine is detected by using an enzymatic reaction.
- This measurement method is a so-called enzyme method, in which a component to be measured in a sample is reacted with an enzyme using it as a substrate to generate hydrogen peroxide, and the generated hydrogen peroxide is peroxidized.
- This is a measurement method for quantifying a component to be measured by allowing a color former to act in the presence of (POD) and detecting a change in color development.
- Measurement of the component to be measured in a sample derived from a biological sample based on such an enzyme method is often used for continuous measurement using an automatic analyzer for reasons such as simplicity of operation.
- the enzyme method is susceptible to the influence of components other than the measurement target derived from biological samples and reagents, and the problem is that the measured value is positively affected to be higher than the theoretical value or negatively affected to be lower than the theoretical value. ing.
- the component other than the component to be measured is faster than the "main reaction" in which the enzyme reacts with the component to be measured.
- a method of eliminating hydrogen peroxide derived from hydrogen peroxide by adding catalase is known (Patent Document 1).
- catalase may be destabilized by these.
- a surfactant is often used as the measurement reagent of the enzyme method for controlling the reactivity of the enzyme, pretreatment of the sample, etc.
- the surfactant may be used during storage of the surfactant.
- Peroxide is easily generated from the surfactant, and in such a case, the peroxide affects the main reaction, and there is also a problem that the measured value becomes high.
- the enzymatic reaction is carried out in an aqueous solution containing ⁇ -keto acid to eliminate the peroxide contained in the components in the reagent, thereby quantifying hydrogen peroxide generated from the measurement target.
- Patent Document 2 A method for preventing the influence on the color change due to hydrogen peroxide is known (Patent Document 2).
- acatalasia, Acatalasemia, or Takahara's disease is a constitutional abnormality of an autosomal recessive gene that is almost deficient in catalase, discovered by the plateau in 1946 (Non-Patent Document 1).
- Catalase is not contained in the biological samples of patients with acatalasia hematosis (Non-Patent Documents 1 and 2).
- the biological sample of a patient who does not have acatalasia hematosis contains endogenous catalase, if an endogenous peroxide is present in the biological sample of the patient, the patient's own endogenous catalase.
- the peroxide is eliminated in advance and does not affect the color change due to the quantification of hydrogen peroxide generated from the measurement target.
- catalase is not contained in the biological sample of the patient with acatalasia hematology
- the endogenous peroxide contained in the biological sample cannot be eliminated in advance. Therefore, if an endogenous peroxide derived from the patient's biological sample is present, it will have a positive effect on the color change due to hydrogen peroxide other than hydrogen peroxide derived from the measurement target in the enzymatic method, and the measurement will be performed. There was a problem that the value became high.
- the inventors of the present application have investigated the positive effects of endogenous peroxides derived from such patient biological samples using pyruvic acid, which is an example of ⁇ -keto acid disclosed in Patent Document 2.
- pyruvic acid which is an example of ⁇ -keto acid disclosed in Patent Document 2.
- a sample derived from a biological sample of a patient with acatalasia hematology hereinafter, may be simply referred to as a non-catalase sample
- a sample derived from a biological sample of a healthy person or a calibrator we have also found a problem that the measured absorbance value decreases even in the above-mentioned (comparative example described in Examples described later in the present specification). The reason for this is not clear, but it is highly probable that ⁇ -keto acid interfered with the main reaction itself, which is the reaction between hydrogen peroxide and peroxidase and the color former, which is caused by the measurement target.
- An object of the present invention is to provide a measuring method capable of suppressing the positive influence of a peroxide derived from a sample in a method of measuring a measurement target in a sample by an enzymatic method. More specifically, it is an object to provide a measuring method and a measuring reagent that can suppress the increase in the value in a catalase-free sample without affecting the color development of a healthy person's sample or a calibrator.
- the present invention is for solving the above-mentioned problems, and is a method for measuring a measurement target component by quantifying hydrogen peroxide produced by reacting a measurement target component in a sample with an enzyme, in a healthy person.
- the compound represented by the following general formula (I) has an electron donating property at the second position. Hydrogen peroxide derived from the measurement target without being affected by the sample by contacting the sample with the enzyme in the presence of one or more compounds selected from the group consisting of a benzoimidazole derivative having a substituent and histidine.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown. That is, the present invention has the following configuration.
- a method for measuring a measurement target component by quantifying hydrogen peroxide produced by reacting the measurement target component in a sample with an enzyme including the following steps; (A) In the presence of one or more compounds selected from the group consisting of the compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown.
- R1 and R2 of the formula (I) are hydrogen, a linear alkyl group having 1 to 4 carbon atoms, a phenyl group, or an alkyloxy group having 1 to 4 carbon atoms.
- R1 or R2 of the formula (I) is a hydrogen or a methyl group, and the other is selected from the group consisting of a hydrogen, a methyl group, an ethyl group, a phenyl group, a methoxy group and an ethoxy group.
- ⁇ 4> The measuring method according to ⁇ 1>, wherein the compound represented by the formula (I) is phenylglyoxal, glyoxal, diacetyl, 2,3-pentanedione, methyl pyruvate or ethyl pyruvate.
- ⁇ 5> The measuring method according to ⁇ 1>, wherein the benzimidazole derivative having an electron-donating substituent at the 2-position is 2-aminobenzimidazole.
- ⁇ 6> The measuring method according to any one of ⁇ 1> to ⁇ 5>, wherein the component to be measured is HbA1c.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown.
- R1 and R2 of the formula (I) are hydrogen, a linear alkyl group having 1 to 4 carbon atoms, a phenyl group, or an alkyloxy group having 1 to 4 carbon atoms.
- R1 or R2 of the formula (I) is a hydrogen or a methyl group, and the other is selected from the group consisting of a hydrogen, a methyl group, an ethyl group, a phenyl group, a methoxy group and an ethoxy group.
- the enzyme that acts on the measurement target component to generate hydrogen peroxide is a protease or a fructosyl peptide oxidase.
- It consists of an enzyme that acts on the component to be measured to generate hydrogen peroxide, a color former, a peroxidase, a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine.
- the first reagent contains a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and one or more compounds selected from the group consisting of histidine.
- the second reagent contains an enzyme, The measurement reagent kit.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown.
- It is a measurement error reduction method in It comprises contacting a sample with a compound represented by the following general formula (I), a benzimidazole derivative having an electron donating substituent at the 2-position, and one or more compounds selected from the group consisting of histidine.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown.
- the present invention is a measurement method based on an enzymatic method in which a component to be measured in a sample is reacted with an enzyme to react with a hydrogen peroxide produced by reacting a color former with a color former to measure the component to be measured, in the presence of a specific compound.
- the present invention provides a measuring method and a measuring reagent capable of suppressing an increase in price derived from a component other than the measurement target component in the acatalase-free sample without affecting the color development of the measurement target component such as a sample other than the acatalase-free sample or a calibrator. be able to. That is, according to the present invention, regardless of whether the sample is a catalase-free sample or not, accurate measurement can be performed by an enzymatic method without being affected by sample-derived components other than the measurement target component.
- the measuring method of the present invention is a method for measuring a measurement target component by quantifying hydrogen hydrogen produced by reacting the measurement target component in a sample with an enzyme, and is a method including the following steps.
- a sample and an enzyme in the presence of one or more specific compounds selected from the group consisting of the compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine.
- a step of generating hydrogen peroxide by contacting a reagent containing the above (B) a step of reacting the generated hydrogen peroxide with a color former in the presence of peroxidase (C) a step of detecting a change in color tone.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown.
- the reaction between the measurement target component and the enzyme performed by contacting the sample with the enzyme that acts on the measurement target component and produces hydrogen peroxide in the presence of the specific compound produces hydrogen peroxide.
- Any reaction condition may be used, for example, 10 to 50 ° C., preferably 20 to 40 ° C., and the reaction time is 5 seconds to 60 minutes, preferably 30 seconds to 15 minutes, more preferably 1 minute to 10 minutes. Minutes, most preferably 3-5 minutes.
- the concentration of the specific compound in this reaction may be any concentration that can avoid the influence of hydrogen peroxide derived from components other than the measurement target component contained in the sample and reduce the measurement error.
- the concentration of the specific compound when prescribed in the measurement reagent may be formulated so as to be the above concentration in the solution in which the reaction is carried out.
- the color former in the step (B) may be any as long as it reacts with hydrogen peroxide in the presence of peroxidase to produce a dye.
- Examples of the color former include an oxidation coupling type chromogen, a leuco type chromogen and the like.
- Leuco-type chromogen is a substance that is independently converted into a dye in the presence of hydrogen peroxide and peroxidase.
- leuco-type chromogens examples include phenothiazine-based chromogens, triphenylmethane-based chromogens, diphenylamine-based chromogens, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, tetramethylbenzidine, and the like. Phenothiazines are preferred.
- phenothiazine-based chromogen examples include 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP) and 10-N-methylcarbamoyl-3,7-bis (dimethylamino).
- CCAP 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine
- CCAP 10-N-methylcarbamoyl-3,7-bis (dimethylamino).
- -10H-phenothiazine (MCDP) 10-N- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67) and the like can be mentioned.
- 10-N- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67) is particularly preferable.
- triphenylmethane-based chromogens examples include N, N, N', N', N'', N''-hexa (3-sulfopropyl) -4,4', 4''-triaminotriphenyl. Examples thereof include methane (TPM-PS).
- diphenylamine-based chromogen examples include N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 4,4'-bis (dimethylamino) diphenylamine, and bis.
- DA-64 N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt
- DA-64 4,4'-bis (dimethylamino) diphenylamine
- BCMA [3-Bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine
- the oxidative coupling type chromogen is a substance in which two compounds oxidatively couple to form a dye in the presence of hydrogen peroxide and an active peroxide substance.
- Examples of the combination of the two compounds include a combination of a coupler and anilines (Trinder's reagent), a combination of a coupler and phenols, and the like.
- coupler examples include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazine and the like.
- anilins examples include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), and N-ethyl-N- (2-hydroxy).
- phenols examples include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.
- the change in the color tone of the produced dye in the step (C) can be detected by measuring the absorbance.
- Examples of the measurement of the absorbance include a method of measuring using a spectrophotometer.
- the color tone change can be calculated by applying the absorbance measured in step (C) to a calibration curve showing the relationship between the concentration of the component to be measured and the absorbance, which was created using the component to be measured with a known concentration. can.
- hydrogen peroxide derived from the measurement target can be correctly quantified while avoiding the positive influence derived from the components other than the measurement target component in the sample.
- the component other than the component to be measured in the sample include peroxides other than the component to be measured in the biological sample, peroxide contained in the measurement reagent, and peroxide generated by pretreatment of the biological sample. Since catalase is not contained in the biological sample of patients with acatalasia hematosis, if an endogenous peroxide contained in the biological sample is present, it cannot be eliminated in advance and is positive.
- the present invention is particularly preferred because it causes an effect and can avoid the effect of such an endogenous peroxide.
- the sample used in the method of the present invention may be any sample derived from a living body, such as whole blood, serum, plasma, urine, erythrocytes separated from whole blood, and erythrocytes separated from whole blood and further washed. And so on.
- the biological sample is directly or subjected to certain pretreatment, dilution, etc., and is used as the sample of this measurement method.
- the pretreatment includes any of the pretreatments necessary for measuring the object to be measured in the biological sample, such as filtration with a filter, centrifugation, treatment with an anticoagulant, treatment with a preservative, heating or cooling treatment.
- Specific compound Specific compounds of the present invention include the following 1. ⁇ 3. Compounds include. 1. 1. The compound represented by the following general formula (I)
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent.
- An aryl group which may have a substituent or an alkyloxy group having 1 to 6 carbon atoms is shown.
- Examples of the linear or branched alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a pentyl group, and a hexyl group.
- methyl glyoxal hydrogen
- R2 methyl group, CAS number [78-98-8]
- R2 methyl group, CAS number [431-03-8]).
- R1 and R2 may be the same or different, and a compound having a hydrogen, a methyl group or an ethyl group is preferable.
- substituents include a halogen atom, a methyl group, an amino group, a sulfo group, a carboxyl group and the like.
- aryl group examples include a phenyl group and a naphthyl group
- substituent examples include a halogen atom, a methyl group, an amino group, a sulfo group, a carboxyl group and the like.
- R2 phenyl group, CAS number [1075-06-5]
- R2 phenyl group, CAS.
- alkoxy group having 1 to 6 carbon atoms examples include a methoxy group, an ethoxy group, a propioxy group, a butoxy group, a pentoxy group, and a hexoxy group.
- R1 and R2 are hydrogen, a linear or branched alkyl group having 1 to 4 carbon atoms, a phenyl group which may have a substituent, and 1 carbon atom. It is more preferable that it is any of 4 to 4 alkoxy groups. Even more preferably, R1 and R2 are hydrogen, a linear or branched alkyl group having 1 to 4 carbon atoms, a phenyl group, or an alkoxy group having 1 to 4 carbon atoms.
- the benzimidazole derivative having an electron-donating substituent at the 2-position of the present invention has a benzimidazole skeleton and the 2-position is substituted with a substituent. Any compound may be used as long as it has an action of suppressing the positive influence of the peroxide derived from the sample in the method of measuring the measurement target in the sample by the enzymatic method.
- the electron-donating substituent bonded to the 2-position of the derivative having the above action include an amino group (-NH 2 , -NR 2 : R is an alkyl group), an alkoxy group, an alkyl group, an aryl group and the like. ..
- Such compounds include 2-aminobenzimidazole (CAS number [934-32-7]) whose 2-position is substituted with an amino group and 2-ethyl-1H-benzimidazole (CAS number) substituted with an ethyl group. [1848-84-6]). Of these, 2-aminobenzimidazole is preferable.
- Histidine used in the present invention is a compound defined by CAS No. [71-00-1].
- a component to be measured in a sample is reacted with an enzyme using it as a substrate to generate hydrogen peroxide, and the generated hydrogen peroxide acts on a color former in the presence of peroxidase (POD).
- POD peroxidase
- Enzymes that react with the component to be measured to generate hydrogen peroxide include, for example, an enzyme that directly converts the component to be measured into hydrogen peroxide, an enzyme that indirectly converts the component to be measured into hydrogen peroxide, and an enzyme to be measured. Examples thereof include an enzyme that directly produces hydrogen peroxide, an enzyme that indirectly produces hydrogen peroxide from a component to be measured, and the like. Therefore, all the components measurable by such an enzymatic reaction can be the measurement target of the present invention.
- hemoglobin A1c HbA1c
- glycoalbumin G
- uric acid creatinine, cholesterol, triglyceride, polyamine, bile acid, 1,5-anhydroglucitol, pyruvate, lactic acid, phospholipids, urea, Glucose, choline, creatine, free fatty acid and the like
- oxidase specific to the above-mentioned component to be measured or a derivative thereof, the following is listed in the form of "component to be measured (enzyme)".
- Hemoglobin A1c proteosyl amino acid oxidase or fructosyl peptide oxidase
- GA protease, ketoamine oxidase
- uric acid uricase
- creatinine creatininase, creatinase, sarcosine oxidase
- cholesterol cholesterol
- Triglyceride lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase
- polyamine polyamineamide hydrolase, polyamineoxidase, ptolessinoxidase
- bile acid (3- ⁇ -hydroxysteroid dehydrogenase, diahorase), 1, 5-Anhydroglucitol (1,5-anhydroglucitol oxidase, pyranose oxidase), pyruvate (pyruvate oxidase), lactic acid (lactic acid (lactic acid
- the reaction between the measurement target component and the enzyme that acts on the measurement target component to generate hydrogen peroxide is preferably carried out in an aqueous medium.
- the aqueous medium include deionized water, distilled water, a buffer solution and the like.
- the pH of the buffer solution is pH 4.0 to 10.0, preferably pH 6.0 to 8.0.
- the buffer used for the buffer include a phosphate buffer, a boric acid buffer, and Good's buffer.
- the reaction between the component to be measured and the enzyme can be carried out in the coexistence of a stabilizer, a preservative, an interfering substance scavenger, a reaction accelerator and the like.
- Examples of the measurement reagent configuration for carrying out the measurement method of the present invention include a two-reagent system and a three-reagent system kit.
- Examples of the three reagents include a configuration consisting of a pretreatment liquid for pretreating a sample, a first reagent, and a second reagent
- examples of the two reagents include a configuration consisting of a first reagent and a second reagent.
- the color former and POD are contained in different reagents, and the specific compound component of the present invention is contained in the pretreatment liquid or the first reagent.
- a specific mode of reagent formulation is shown below.
- the measuring reagent of the present invention can be applied to various automatic analyzers as before. .. By applying it to various automatic analyzers, it is possible to provide a method for measuring each component to be measured excluding the positive influence derived from the component other than the component to be measured in the sample.
- the component to be measured in the present invention is not particularly limited, and all components that can be measured by quantifying hydrogen peroxide generated by an enzymatic reaction can be measured, and among them, hemoglobin A1c is particularly measured. The case of doing so will be described.
- Examples of the HbA1c measuring method according to the present invention include a method using the following hemoglobin A1c measuring reagent kit.
- First reagent protease, color former, specific compound of the present invention Second reagent fructosyl peptide oxidase, POD First, the first reagent is added to the sample, and the saccharified dipeptide at the N-terminal of the HbA1c ⁇ chain is digested and cleaved by the protease. Next, a second reagent is added, an oxidase specific for the saccharified dipeptide (fructosylpeptide oxidase) is allowed to act, and a coloring agent is allowed to act on the produced hydrogen peroxide in the presence of peroxidase, and the ratio is changed. A method of color quantification can be mentioned. The reaction of the enzyme that acts on the component to be measured and produces hydrogen peroxide is carried out in the presence of the specific compound of the present invention contained in the first reagent to suppress the positive influence derived from the component to be measured. Can be done.
- Hemoglobin A1c is generally determined as HbA1c%.
- HbA1c% means the ratio (%) of the A1c concentration to the hemoglobin concentration in the sample, which is usually used clinically.
- the method for measuring HbA1c% is basically a first step of optically measuring the hemoglobin concentration in the sample, a second step of optically measuring the HbA1c concentration in the sample, and a hemoglobin in the sample. The step of calculating the ratio of the HbA1c concentration to the concentration (HbA1c%) is included.
- the above-mentioned measurement of hemoglobin A1c corresponds to the second step of the method for measuring HbA1c%.
- the method for measuring A1c% is that in the first step, the optical measurement of the sample is performed by measuring the absorbance using light of the first wavelength and light of the second wavelength, respectively.
- the hemoglobin concentration measured at the first wavelength is corrected using the measured value at the second wavelength.
- the corrected hemoglobin concentration is used in the HbA1c% calculation step.
- a known method such as methization may be used to stabilize the absorbance of hemoglobin as a constant structure.
- a treatment may be performed to facilitate digestion and cleavage of the saccharified dipeptide from the HbA1c ⁇ chain by coexisting a surfactant or the like at the time of digestion or cleavage with a protease.
- the term "first wavelength” means a wavelength for measuring hemoglobin, preferably the first light used for optical measurement of hemoglobin concentration in the first step of the method of the present invention.
- the first wavelength can be appropriately selected from the range of 450 nm to 610 nm.
- the term "second wavelength" means a wavelength for correcting the apparent hemoglobin concentration measured at the first wavelength to the true hemoglobin concentration, and is preferably the first step of the method of the present invention. It is the wavelength of the second light used for the optical measurement in.
- the second wavelength can be appropriately selected from the range of 690 nm to 900 nm.
- the measuring method of the present invention avoids the influence of hydrogen peroxide derived from components other than the measurement target component contained in the sample, and quantifies hydrogen peroxide derived from the measurement target component by an enzymatic method. Therefore, it can be said that it is a method for reducing measurement error in the method for measuring the component to be measured.
- the method for reducing measurement error of the present invention is one or more selected from the group consisting of a sample, a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine.
- the method comprises a step of bringing the compounds into contact with each other, and each configuration is as described above.
- R1 and R2 may be the same or different, hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and a substituent.
- An aryl group which may have a group or an alkyloxy group having 1 to 6 carbon atoms is shown.
- Example 1 Examination using a catalase-free model sample 1. Measuring reagents and measuring samples: ⁇ Protease-containing substrate reagent (R1)> 50 mM MES pH 6.0 1.0% Emar 20C (Kao) Protin PC10F (Daiwa Kasei Kogyo Co., Ltd.) DA-67 (10- (Carboxamide Aminocarbonyl) -3,7-Bis (Dimethylamino) Phenothiazine Sodium, Wako Pure Chemical Industries, Ltd.) Additives shown in Tables 1 to 4 The manufacturers and distributors of the additives shown in Tables 1 to 4 are as follows. TCI: Tokyo Chemical Industry Co., Ltd. Kishida: Kishida Chemical Co., Ltd. Fuji: Fujifilm Wako Pure Chemical Industries, Ltd.
- the relative value (%) of the absorbance of HbA1c (%) measured under each condition to the absorbance obtained under condition 1 (without additives) was calculated and shown in Tables 1 to 4.
- the relative value of the measured absorbance of the calibrator 1 and the calibrator 2 is preferably a value close to 100% with respect to the condition 1. Further, it is preferable that the relative values of the measured absorbances of the acatalase-free model samples A and B are lower than those of the condition 1.
- Conditions 13-21 and 38-40 will be verified for benzimidazole derivatives having an electron-donating substituent at the 3-2.2 position.
- imidazole was added, 0.01% (condition 13) did not cause a change in absorbance for either the calibrator or the acatalase-free model sample.
- 0.1 to 0.2% is added (conditions 14 and 15)
- the decrease in the relative absorbance value of the calibrator is larger than the decrease in the relative absorbance value of the acatalase-free model sample, which is not suitable for measurement. It was considered.
- benzimidazole was added in an amount of 0.01% to 0.2% (conditions 16 to 18), the relative absorbance of the acatalasia model sample decreased at any concentration, but the relative absorbance of the calibrator further decreased.
- the relative absorbance of the sample of 2-aminoimidazole sulfate did not decrease, it was considered that a benzimidazole skeleton was required as a compound having the function of the present invention.
- the benzimidazole derivative having an electron-donating substituent at the 2-position specifically eliminates the endogenous peroxide in the sample, while affecting the main reaction caused by hydrogen peroxide generated from the substance to be measured. It was found that the compound had the desired function of the present invention.
- histidine was found to be a compound having the desired function of the present invention.
- Table 5 shows the judgment results of the relative values shown in Tables 1 to 4.
- the evaluation criteria are as follows. 4-1. Evaluation Criteria for Calibrator A: The relative values of the measured absorbances of the calibrators 1 and 2 are both 90 to 110% B: The relative values of the measured absorbances of the calibrators 1 and 2 are both 80 to 120% C: Calibrator 1 Since one of the relative values of the measured absorbances of 2 is less than 80% or greater than 120%, the relative values of the measured absorbances of the calibrator 1 and the calibrator 2 are preferably close to 100% with respect to no additive. It was determined that A was the most preferable, then B was preferable, and C was not preferable.
- A The relative value of both samples A and B is 60% or less
- B The relative value of both samples A and B is 90% or less
- C The relative value of both samples A and B is 95% or less
- D The relative value of both samples A and B is 95% or more.
- the relative value of the measured absorbance of the acatalase-free model samples A and B is preferably lower than that of condition 1, so A is the most. It was determined that B and C were preferable in that order, and D did not have the required performance. If the calibrator's judgment was "C", it was excluded from the sample evaluation (indicated as "-" in the table).
- phenylglyoxal, methyl pyruvate, and ethyl pyruvate which contain the comprehensive evaluation A, are the most preferable compounds.
- the compound represented by the general formula (I) found in the present invention a benzimidazole derivative having an electron-donating substituent at the 2-position, or histidine is a method for measuring a component to be measured in a sample by an enzymatic method. In the above, by allowing it to exist in the measurement system, all of them specifically eliminate the endogenous peroxide in the sample, but do not affect the main reaction caused by hydrogen peroxide generated from the component to be measured. all right.
- Example 2 Examination using an acatalase-free sample 1.
- DA-67 (10- (Carboxamide Aminocarbonyl) -3,7-Bis (Dimethylamino) Phenothiazine Sodium, Wako Pure Chemical Industries, Ltd.)
- Test sample ⁇ calibrator> A Nordia NHbA1c calibrator was used. ⁇ Diluted solution> Purified water was used. ⁇ Measurement sample> The normal specimens C and D and the acatalase-free specimen E prepared in Reference Example 2 were diluted 26-fold with purified water and measured.
- the healthy sample C had a slightly higher value of 5.29% with respect to the HPLC value of 4.95%
- the healthy sample D had a slightly higher value of 6.65% with respect to the HPLC value of 6.50%. did. It is presumed that this is a measurement error caused by using a calibrator optimized for the R1-A reagent when calibrating with the R1-B reagent. Therefore, if the calibrator is optimized separately for the R1-B reagent, it is considered that more accurate measurement is possible.
- a measuring method and a measuring reagent capable of accurately quantifying hydrogen peroxide derived from a measurement target without being affected by a sample-derived component other than the measurement target in the measurement method based on the enzyme method are provided. be able to. In particular, regardless of whether the sample is acatalase-free or not, it is possible to accurately measure the measurement target by the enzymatic method without being affected by the components derived from the sample other than the measurement target.
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Abstract
Description
すなわち、本発明は以下の構成を有する。
<1>
検体中の測定対象成分を酵素と反応させて生成される過酸化水素を定量することにより測定対象成分を測定する方法であって、以下の工程を含む前記測定方法;
(A)以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物の存在下、
検体と酵素を接触させることにより過酸化水素を生成する工程
(B)生成した過酸化水素をパーオキシダーゼ存在下、発色剤と反応させる工程
(C)色調変化を検出する工程
<2>
式(I)のR1、R2が、水素、炭素数1~4の直鎖アルキル基、フェニル基又は炭素数1~4のアルキルオキシ基のいずれかである、<1>に記載の測定方法。
<3>
式(I)のR1またはR2のいずれか片方が水素又はメチル基であって、もう片方が水素、メチル基、エチル基、フェニル基、メトキシ基及びエトキシ基からなる群から選択されるいずれかである、<1>に記載の測定方法。
<4>
式(I)で示される化合物が、フェニルグリオキサール、グリオキサール、ジアセチル、2,3-ペンタンジオン、ピルビン酸メチル又はピルビン酸エチルである<1>に記載の測定方法。
<5>
2位に電子供与性置換基を有するベンゾイミダゾール誘導体が、2-アミノベンゾイミダゾールである<1>に記載の測定方法。
<6>
測定対象成分がHbA1cである<1>~<5>のいずれかに記載の測定方法。
<7>
測定対象成分に作用し過酸化水素を生成する酵素、発色剤、パーオキシダーゼ並びに以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を含む測定試薬。
<8>
式(I)のR1、R2が、水素、炭素数1~4の直鎖アルキル基、フェニル基又は炭素数1~4のアルキルオキシ基のいずれかである<7>に記載の測定試薬。
<9>
式(I)のR1又はR2のいずれか片方が水素又はメチル基であって、もう片方が水素、メチル基、エチル基、フェニル基、メトキシ基及びエトキシ基からなる群から選択されるいずれかである、<7>に記載の測定試薬。
<10>
式(I)で示される化合物が、フェニルグリオキサール、グリオキサール、ジアセチル、2,3-ペンタンジオン、ピルビン酸メチル又はピルビン酸エチルである<5>に記載の測定試薬。
<11>
2位に電子供与性置換基を有するベンゾイミダゾール誘導体が、2-アミノベンゾイミダゾールである<7>に記載の測定試薬。
<12>
測定対象成分がHbA1cであり、測定対象成分に作用し過酸化水素を生成する酵素が、プロテアーゼ及びフルクトシルペプチドオキシダーゼである<7>~<11>のいずれかに記載の測定試薬。
<13>
測定対象成分に作用し過酸化水素を生成する酵素、発色剤、パーオキシダーゼ並びに以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を含む測定試薬キットであって、
第1試薬に以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を含み、
第2試薬に酵素を含む、
前記測定試薬キット。
<14>
検体中に含まれる測定対象成分以外の成分に由来する過酸化水素の影響を回避して、測定対象成分に由来する過酸化水素を酵素法により定量することにより、前記測定対象成分を測定する方法における測定誤差低減方法であって、
検体と、以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を接触させる工程を含むことを特徴とする測定誤差低減方法。
本発明の測定方法は、検体中の測定対象成分を酵素と反応させて生成される過酸化水素を定量することにより測定対象成分を測定する方法であって、以下の工程を含む方法である。
(A)以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の特定化合物の存在下、検体と酵素を含む試薬を接触させることにより過酸化水素を生成する工程
(B)生成した過酸化水素をパーオキシダーゼ存在下、発色剤と反応させる工程
(C)色調変化を検出する工程
この反応における特定化合物の濃度は、検体中に含まれる測定対象成分以外の成分に由来する過酸化水素の影響を回避して、測定誤差を低減できるような濃度であればよい。具体的には、反応が行われる溶液中で0.001~1v/v%であればよく、0.01~0.5v/v%が好ましく、0.05~0.3%がよりいっそう好ましい。
また、測定試薬に処方した際の特定化合物の濃度は、反応が行われる溶液中で上記濃度となるように処方すればよい。
色調変化は、既知濃度の測定対象成分を用いて作成された、測定対象成分の濃度と吸光度との関係を表す検量線に、工程(C)で測定した吸光度をあてはめて濃度を算出することができる。
本発明の方法で使用する試料としては、生体由来の試料であればいずれでもよく、全血、血清、血漿、尿、全血より分離された赤血球、全血より分離され、さらに洗浄された赤血球等が挙げられる。
生体試料は、直接、あるいは一定の前処理、希釈などが行われ、本測定方法の検体として用いられる。前処理としては、フィルターによるろ過、遠心分離、抗凝固剤による処理、防腐剤による処理、加熱あるいは冷却処理など、生体試料中の測定対象を測定するために必要な前処理のいずれをも含む。
本発明の2位に電子供与性置換基を有するベンゾイミダゾール誘導体としては、ベンゾイミダゾール骨格を有し、2位が置換基で置換されたものであって、酵素法により検体中の測定対象を測定する方法において、検体に由来する過酸化物の正の影響を抑制する作用を有する化合物であればよい。
前記作用を有する誘導体の2位に結合している電子供与性置換基は、アミノ基(-NH2、-NR2:但しRはアルキル基)、アルコキシ基、アルキル基、アリール基などが挙げられる。
このような化合物としては、2位がアミノ基で置換された2-アミノベンゾイミダゾール(CAS番号[934-32-7])、エチル基で置換された2-エチル-1H-ベンゾイミダゾール(CAS番号[1848-84-6])が挙げられる。中でも、2-アミノベンゾイミダゾールが好ましい。
本発明に用いられるヒスチジンは、CAS番号[71-00-1]で規定される化合物である。
本発明における酵素法は、検体中の測定対象成分を、それを基質とする酵素と反応させて過酸化水素を生成させ、生成した過酸化水素をパーオキシダーゼ(POD)存在下で発色剤を作用させ、呈色変化を検出することで測定対象成分の定量を行う測定方法である。したがって、測定対象成分としては、特に限定されるものではなく、酵素反応により生成する過酸化水素を定量することによって測定が可能な成分は全て測定可能である。測定対象成分と反応して過酸化水素を生成させる酵素には、例えば測定対象成分を直接過酸化水素に変換する酵素、測定対象成分を間接的に過酸化水素に変換する酵素、測定対象成分から直接過酸化水素を生成させる酵素、測定対象成分から間接的に過酸化水素を生成させる酵素等が挙げられる。したがって、このような酵素反応によって測定可能な成分が全て本発明の測定対象となり得る。
また、測定対象成分と前記酵素との反応は、安定化剤、防腐剤、干渉物質消去剤、反応促進剤等を共存させて行うこともできる。
本発明の測定方法を実施するための測定試薬構成としては、2試薬系、3試薬系のキットが挙げられる。3試薬としては、試料を前処理するための前処理液、第一試薬、第二試薬からなる構成が挙げられ、2試薬としては、第一試薬、第二試薬からなる構成が挙げられる。
発色剤とPODはそれぞれ別の試薬に含まれ、本発明の特定の化合物成分は、前処理液又は、第一試薬に含まれる。
具体的な試薬処方の態様を以下に示す。
(1)ヘモグロビンA1c測定試薬キット
第一試薬 プロテアーゼ、発色剤、本発明の特定化合物
第二試薬 フルクトシルペプチドオキシダーゼ、POD
(2)グリコアルブミン測定試薬キット
第一試薬 プロテアーゼ、POD、本発明の特定化合物
第二試薬 ケトアミン酸化酵素、発色剤
本発明の測定試薬は、従来どおり、各種の自動分析装置に適用することができる。各種の自動分析装置に適用することにより、検体中の測定対象成分以外の成分に由来する正の影響を排除した各測定対象成分の測定方法を提供することができる。
本発明の測定対象成分としては、特に限定されるものではなく、酵素反応により生成する過酸化水素を定量することによって測定が可能な成分は全て測定可能であるが、そのうちでも特にヘモグロビンA1cを測定する場合について説明する。
本発明によるHbA1c測定方法としては、例えば、以下のヘモグロビンA1c測定試薬キットを用いる方法が挙げられる。
第一試薬 プロテアーゼ、発色剤、本発明の特定化合物
第二試薬 フルクトシルペプチドオキシダーゼ、POD
まず、検体に第一試薬を添加し、HbA1cβ鎖N末端の糖化ジペプチドをプロテアーゼにより消化、切断する。次に、第二試薬を添加し、前記糖化ジペプチドに特異的な酸化酵素(フルクトシルペプチドオキシダーゼ)を作用させ、生成した過酸化水素に対してパーオキシダーゼの存在下で発色剤を作用させ、比色定量する方法が挙げられる。測定対象成分に作用し過酸化水素を生成する酵素の反応は、第一試薬に含まれる本発明の特定化合物の存在下で行われることにより、測定対象成分に由来する正の影響を抑制することができる。
HbA1c%の測定方法は、基本的には、試料中のヘモグロビン濃度を光学的に測定する第一工程と、該試料中のHbA1c濃度を光学的に測定する第二工程と、該試料中のヘモグロビン濃度に対するHbA1c濃度の比率(HbA1c%)を算出する工程を含む。前述のヘモグロビンA1cの測定は、HbA1c%の測定方法の第二工程に相当することになる。
A1c%の測定方法は、第一工程において、該試料の光学的測定は、第1の波長の光と第2の波長の光をそれぞれ用いて、吸光度を測定することにより行われる。該第1の波長で測定されたヘモグロビン濃度は、該第2の波長での測定値を用いて補正される。該補正されたヘモグロビン濃度が、該HbA1c%算出工程で用いられる。
本発明の測定方法は、換言すれば、検体中に含まれる測定対象成分以外の成分に由来する過酸化水素の影響を回避して、測定対象成分に由来する過酸化水素を酵素法により定量することにより、前記測定対象成分を測定する方法における測定誤差低減方法とも言える。本発明の測定誤差低減方法は、検体と、以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を接触させる工程を含むことを特徴とする方法であり、各構成は前述のとおりである。
EDTA採血管を用いて採血した健常人2名の血液A、Bを遠心分離し、血球A、Bを得た。この血球に対してカタラーゼ活性を阻害することが知られているアジ化ナトリウムの水溶液を添加し、無カタラーゼモデル検体A、Bとした。
1.測定試薬と測定サンプル:
<プロテアーゼ含有基質試薬(R1)>
50mM MES pH6.0
1.0% エマール20C(花王社)
プロチンPC10F(大和化成工業社)
DA-67(10-(カルボキシメチルアミノカルボニル)-3,7-ビス(ジメチルアミノ)フェノチアジンナトリウム、和光純薬工業社)
表1~4記載の添加剤
なお、表1~4記載の添加剤の製造販売元は、以下を示す。
TCI:東京化成工業株式会社
キシダ:キシダ化学株式会社
富士:富士フイルム和光純薬株式会社
50mM クエン酸 pH6.0
フルクトシルペプチドオキシダーゼ(キッコーマン社)
パーオキシダーゼ(東洋紡社)
(1)ノルディアNHbA1cキャリブレーター(積水メディカル株式会社)1、2
(2)参考例1で調製した無カタラーゼモデル検体A、B
自動分析装置JCA-9130(日本電子社)を用い、表1~4に示した各種添加剤を含む試薬R1と試薬R2を組み合わせて、以下の測定パラメータにより測定サンプルのHbA1c(%)の吸光度を測定した。なお下記の時間設定は、R1が添加され第一反応が開始して約5分後にR2が添加される設定である。
パラメータ:
[HbA1c]
分析方法:EPA
計算方法:MSTD
測定波長(副/主):HbA1c 805/658
主DET.Pl-P.m-P.n:HbA1c 0-95-98
副DET.P.p-P.r:HbA1c 44-47
反応時間:10分
検体希釈なし
反応検体量(Sample量):6.4μL
第1試薬量(R1):60μL、第2試薬量:0μL、
第3試薬量(R2):20μL、第4試薬量:0μL
各条件で測定した際のHbA1c(%)の吸光度の、条件1(添加剤なし)で得られた吸光度に対する相対値(%)を算出し、表1~4に示した。キャリブレーター1、キャリブレーター2の測定吸光度の相対値は、条件1に対して100%に近い値であることが好ましい。また、無カタラーゼモデル検体A、Bの測定吸光度の相対値は、条件1に対して低下していることが好ましい。
まず、特許文献2に記載されたα-ケト酸であるピルビン酸を0.10%添加した条件2では、無カタラーゼモデル検体の吸光度相対値は20.1~22.1%と低下したが、キャリブレーター1、2の吸光度相対値も70.1~87.1%へ低下し、主反応への影響が認められた。またピルビン酸を1.0%添加した条件3では、無カタラーゼモデル検体の吸光度相対値は86.2~97.7%へ低下したが、キャリブレーターの吸光度相対値は204.3~593.2%と、発色反応に異常が生じていると考えられた。さらに、α-ケト酸の類似化合物である2-ケトグルタル酸を添加した条件4、5は、ピルビン酸と同様の挙動を示し、レブリン酸(3-アセチルプロピオン酸)を添加した条件6では、キャリブレーターの吸光度相対値が低下した一方で無カタラーゼモデル検体の吸光度相対値は増加し、発色反応に異常が生じていると考えられた。
このように一般式(I)の構造を有する化合物群は、検体中の内因性過酸化物を特異的に消去する一方、測定対象物質から発生した過酸化水素に起因する主反応に影響を及ぼさず、本発明の目的の機能を有する化合物であることがわかった。
条件13~21、38~40について検証する。イミダゾールを添加した場合、0.01%(条件13)では、キャリブレーター、無カタラーゼモデル検体のいずれに対しても吸光度変動を引き起こさなかった。0.1~0.2%添加した場合(条件14,15)は、無カタラーゼモデル検体の吸光度相対値が低下するよりも、キャリブレーターの吸光度相対値の低下幅の方が大きく、測定に適さないと考えられた。ベンゾイミダゾールを0.01%~0.2%添加した場合(条件16~18)、いずれの濃度においても無カタラーゼモデル検体の吸光度相対値は低下したが、それ以上にキャリブレーターの吸光度相対値が低下し、イミダゾールと同様の挙動を示した。一方で、本願で見いだされた2-アミノベンゾイミダゾールを0.01%~0.2%添加した場合(条件19~21)、いずれの濃度においても、キャリブレーターの吸光度相対値と比較して、無カタラーゼモデル検体の吸光度相対値が有意に低下した。さらに、2-エチル-1H-ベンゾイミダゾールを0.01%添加した場合(条件38)も、キャリブレーターの吸光度相対値が100.6~101.4%と維持されたのに対して、検体の吸光度相対値が低下し、本発明の目的の機能を有する化合物であることがわかった。なお、2-アミノイミダゾール硫酸塩(条件39、40)は、検体の吸光度相対値が低下しなかったことから、本発明の機能を有する化合物としてベンゾイミダゾール骨格が必要であると考えられた。
このように2位に電子供与性置換基を有するベンゾイミダゾール誘導体は、検体中の内因性過酸化物を特異的に消去する一方、測定対象物質から発生した過酸化水素に起因する主反応に影響を及ぼさず、本発明の目的の機能を有する化合物であることがわかった。
条件22~25について検証する。メチオニンを0.1%添加した条件22、トリプトファンを0.1%添加した条件23では、無カタラーゼモデル検体の吸光度相対値はほぼ変動しなかった。一方で、L-ヒスチジンを添加した条件24、25では、無カタラーゼモデル検体の吸光度相対値は低下し、その幅はキャリブレーターの吸光度相対値の低下幅よりも大きかった。
このように、アミノ酸のうちでもヒスチジンは、本発明の目的の機能を有する化合物であることがわかった。
さらに、表1~4に示した相対値の判定結果を表5に示した。
評価基準は以下のとおりである。
4-1.キャリブレーターの評価基準
A:キャリブレーター1、2の測定吸光度の相対値が双方とも90~110%である
B:キャリブレーター1、2の測定吸光度の相対値が双方とも80~120%である
C:キャリブレーター1、2の測定吸光度の相対値の片方が80%未満または120%より大きい
キャリブレーター1、キャリブレーター2の測定吸光度の相対値は、添加剤なしに対して100%に近い値であることが好ましいため、Aが最も好ましく、次にBの順で好ましく、Cは好ましくないと判断した。
キャリブレーターの判定がAまたはBである条件において、以下の通りとした。
A:検体A、Bの双方の相対値が60%以下である
B:さらに検体A、Bの双方の相対値が90%以下である
C:検体A、Bの双方の相対値が95%以下である
D:検体A、Bの双方の相対値が95%以上である
無カタラーゼモデル検体A、Bの測定吸光度の相対値は、条件1に対して低下していることが好ましいためAが最も好ましく、次にB、Cの順で好ましく、Dは求める性能を有しないと判断した。なお、キャリブレーターの判定が「C」の場合は検体評価の対象外とした(表中、「ー」と示す)。
キャリブレーターおよび検体の評価に基づき、以下の通りとした。
A:キャリブレーターと検体の双方が評価Aである
B:キャリブレーターがAで検体がB、またはキャリブレーターがBで検体がAまたはBである
C:キャリブレーターがAまたはBで検体がCである
D:キャリブレーターがCまたは検体がDである
総合評価は、Aが最も好ましく、次にB、Cの順で好ましく、Dは効果を示さなかったと判断した。なお、条件14はこの基準によると総合評価Cだが、検体A、Bの相対値がキャリブレーター2より高かったため、総合評価Dとした。
表5より、総合評価Aが含まれるフェニルグリオキサール、ピルビン酸メチル、ピルビン酸エチルが最も好ましい化合物である。さらに評価Bが含まれるグリオキサール、ジアセチル、2-アミノベンゾイミダゾール、ヒスチジン、2,3-ペンタンジオンが好ましい化合物である。
以上より、本発明で見いだされた一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、またはヒスチジンは、検体中の測定対象成分を酵素法により測定する方法において、測定系内に存在させることにより、いずれも検体中の内因性過酸化物を特異的に消去する一方、測定対象成分から発生した過酸化水素に起因する主反応に影響を及ぼさないことがわかった。
EDTA採血管を用いて採血した健常人2名の血液C、D、および無カタラーゼ症検体Eを遠心分離し、血球C、D、Eを得た。
さらに、血球C、D、Eを、カタラーゼ有無の影響を受けない測定法であるHPLC法で測定(東ソーG11)した。HbA1c(%)は、健常検体C:4.95%、健常検体D:6.50%、無カタラーゼ検体E:6.20%であった。
1.測定試薬
<プロテアーゼ含有基質試薬(R1-A)>
50mM MES pH6.0
1.0% エマール20C(花王社)
プロチンPC10F(大和化成工業社)
DA-67(10-(カルボキシメチルアミノカルボニル)-3,7-ビス(ジメチルアミノ)フェノチアジンナトリウム、和光純薬工業社)
50mM MES pH6.0
1.0% エマール20C(花王社)
プロチンPC10F(大和化成工業社)
DA-67(10-(カルボキシメチルアミノカルボニル)-3,7-ビス(ジメチルアミノ)フェノチアジンナトリウム、和光純薬工業社)
0.10% フェニルグリオキサール
50mM クエン酸 pH6.0
フルクトシルペプチドオキシダーゼ(キッコーマン社)
パーオキシダーゼ(東洋紡社)
<キャリブレーター>
ノルディアNHbA1cキャリブレーターを使用した。
<希釈液>
精製水を使用した。
<測定サンプル>
参考例2で調製した正常検体C、D、無カタラーゼ検体Eを精製水で26倍希釈して測定した。
自動分析装置JCA-9130(日本電子社)を用い、試薬R1-Aと試薬R2、試薬R1-Bと試薬R2の組み合わせにおいて、以下の測定パラメータにより試験試料を測定し、HbA1c(%)を算出した。
パラメータ:
[HbA1c,Hb]
分析方法:EPA
計算方法:MSTD
測定波長(副/主):HbA1c 805/658、Hb 805/478
主DET.Pl-P.m-P.n:HbA1c 0-95-98、Hb 0-44-47
副DET.P.p-P.r:HbA1c 44-47、Hb 0-0
反応時間:10分
検体希釈なし
反応検体量(Sample量):6.4μL
第1試薬量(R1):60μL、第2試薬量:0μL、
第3試薬量(R2):20μL、第4試薬量:0μL
第一試薬としてR1-A(添加剤なし)、R1-B(フェニルグリオキサール添加)それぞれを用いて測定した結果を表6に示した。R1-A試薬を使用した場合、健常検体C、Dは精度良く測定できた一方で、無カタラーゼ検体Eは、HPLC測定値6.20%に対して15.46%と、大幅に高値化した。一方で、本発明で見いだされたフェニルグリオキサールを0.10%添加したR1-B試薬を用いた場合、無カタラーゼ検体の測定値は、HPLC値6.20%に対して6.84%と、添加剤を使用していないR1-A試薬と比較し、はるかに高い精度で測定することができた。なお、R1-B試薬を使用した場合、健常検体CはHPLC値4.95%に対して5.29%、健常検体DはHPLC値6.50%に対して6.65%とやや高値化した。これは、R1-B試薬でキャリブレーションをおこなう際に、R1-A試薬用に至適化されたキャリブレーターを使用したために生じた測定誤差であると推測している。したがってR1-B試薬用に別途キャリブレーターの至適化をおこなえば、さらに高精度な測定が可能と考えられる。
Claims (14)
- 検体中の測定対象成分を酵素と反応させて生成される過酸化水素を定量することにより測定対象成分を測定する方法であって、以下の工程を含む前記測定方法;
(A)以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物の存在下、
検体と酵素を接触させることにより過酸化水素を生成する工程
(B)生成した過酸化水素をパーオキシダーゼ存在下、発色剤と反応させる工程
(C)色調変化を検出する工程
- 式(I)のR1、R2が、水素、炭素数1~4の直鎖アルキル基、フェニル基又は炭素数1~4のアルキルオキシ基のいずれかである、請求項1に記載の測定方法。
- 式(I)のR1またはR2のいずれか片方が水素又はメチル基であって、もう片方が水素、メチル基、エチル基、フェニル基、メトキシ基及びエトキシ基からなる群から選択されるいずれかである、請求項1に記載の測定方法。
- 式(I)で示される化合物が、フェニルグリオキサール、グリオキサール、ジアセチル、2,3-ペンタンジオン、ピルビン酸メチル又はピルビン酸エチルである請求項1に記載の測定方法。
- 2位に電子供与性置換基を有するベンゾイミダゾール誘導体が、2-アミノベンゾイミダゾールである請求項1に記載の測定方法。
- 測定対象成分がHbA1cである請求項1~5のいずれかに記載の測定方法。
- 式(I)のR1、R2が、水素、炭素数1~4の直鎖アルキル基、フェニル基又は炭素数1~4のアルキルオキシ基のいずれかである請求項7に記載の測定試薬。
- 式(I)のR1又はR2のいずれか片方が水素又はメチル基であって、もう片方が水素、メチル基、エチル基、フェニル基、メトキシ基及びエトキシ基からなる群から選択されるいずれかである、請求項7に記載の測定試薬。
- 式(I)で示される化合物が、フェニルグリオキサール、グリオキサール、ジアセチル、2,3-ペンタンジオン、ピルビン酸メチル又はピルビン酸エチルである請求項5に記載の測定試薬。
- 2位に電子供与性置換基を有するベンゾイミダゾール誘導体が、2-アミノベンゾイミダゾールである請求項7に記載の測定試薬。
- 測定対象成分がHbA1cであり、測定対象成分に作用し過酸化水素を生成する酵素が、プロテアーゼ及びフルクトシルペプチドオキシダーゼである請求項7~11のいずれかに記載の測定試薬。
- 測定対象成分に作用し過酸化水素を生成する酵素、発色剤、パーオキシダーゼ並びに以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を含む測定試薬キットであって、
第1試薬に以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を含み、
第2試薬に酵素を含む、
前記測定試薬キット。
- 検体中に含まれる測定対象成分以外の成分に由来する過酸化水素の影響を回避して、測定対象成分に由来する過酸化水素を酵素法により定量することにより、前記測定対象成分を測定する方法における測定誤差低減方法であって、
検体と、以下の一般式(I)で示される化合物、2位に電子供与性置換基を有するベンゾイミダゾール誘導体、及びヒスチジンからなる群から選択される1以上の化合物を接触させる工程を含むことを特徴とする測定誤差低減方法。
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JPS59183698A (ja) * | 1983-04-02 | 1984-10-18 | Wako Pure Chem Ind Ltd | 基質又は酵素活性の定量方法 |
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