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.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Organic Chemistry (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Food Science & Technology (AREA)
• Cell Biology (AREA)
• Medicinal Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• General Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biophysics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=80632169

Family Applications (1)

Country Status (7)

Citations (6)

Family Cites Families (5)

• 2021
  • 2021-09-10 JP JP2022548342A patent/JP7845609B2/ja active Active
  • 2021-09-10 TW TW110133776A patent/TW202227639A/zh unknown
  • 2021-09-10 CN CN202180061542.8A patent/CN116075594A/zh active Pending
  • 2021-09-10 KR KR1020237011784A patent/KR20230066051A/ko active Pending
  • 2021-09-10 US US18/025,813 patent/US20230358767A1/en active Pending
  • 2021-09-10 WO PCT/JP2021/033235 patent/WO2022054890A1/ja not_active Ceased
  • 2021-09-10 EP EP21866847.3A patent/EP4212627A4/en active Pending

Patent Citations (6)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events