Method for reducing measurement errors

Abstract

The present invention addresses the problem of providing a method by which the positive influence of a peroxide originating from a sample can be suppressed in the case of measuring a measurement target in the sample by an enzymatic method. More specifically, the present invention addresses the problem of providing a measurement method, by which an increase in a measured value can be suppressed regardless of the presence or absence of catalase in a sample, and a measurement reagent.　Provided is a measurement method that comprises contacting a sample with an enzyme in the presence of one or more compounds selected from the group consisting of a compound represented by general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position and histidine. Thus, hydrogen peroxide originating from a measurement target can be correctly quantified without being affected by the sample. In formula (I), R1 and R2 may be either the same or different and represent hydrogen, an optionally substituted linear or branched alkyl group having 1-6 carbon atoms, an optionally substituted aryl group, or an alkyloxy group having 1-6 carbon atoms.

Description

Technical Field

[0001] The present invention relates to a method for measuring a measurement target in a specimen based on an enzymatic method. More specifically, it relates to a measurement method for quantifying a measurement target by quantifying hydrogen peroxide generated by reacting the measurement target in a specimen with an enzyme.

Background Art

[0002] In clinical diagnosis, measurement methods for detecting the amount of components in a specimen derived from a biological sample such as whole blood, serum, plasma, urine, etc. using an enzymatic reaction are known. This measurement method is a measurement method called the so-called enzymatic method, in which the measurement target component in the specimen is reacted with an enzyme having it as a substrate to generate hydrogen peroxide, and a color developer is allowed to act on the generated hydrogen peroxide in the presence of peroxidase (POD), and the measurement target component is quantified by detecting the color change.

[0003] The measurement of the measurement target component in a specimen derived from a biological sample based on such an enzymatic method is frequently used for continuous measurement using an automatic analyzer for reasons such as the simplicity of operation. However, the enzymatic method is easily affected by components other than the measurement target derived from the biological sample or the reagent, and it has been a problem that the measured value is affected by a positive effect of becoming higher than the theoretical value or a negative effect of becoming lower.

[0004] For example, as a method for solving the problem that the measured value becomes higher due to hydrogen peroxide derived from components other than the measurement target component, a method of eliminating hydrogen peroxide derived from components other than the measurement target component by adding catalase earlier than the "main reaction" in which the measurement target component and the enzyme react is known (Patent Document 1). However, in this method, when the first reagent contains azide or protease, catalase may be destabilized by these.

[0005] Furthermore, while surfactants are often used in enzyme-based measurement reagents for controlling enzyme reactivity and sample pretreatment, depending on the type of surfactant, peroxides can easily form from the surfactant during reagent storage. In such cases, these peroxides can affect the main reaction, leading to inflated measurement values. To address this problem, a method is known in which the enzyme reaction is performed in an aqueous solution containing α-keto acid, thereby eliminating peroxides contained in the reagent components and preventing their influence on the color change caused by the quantitative determination of hydrogen peroxide generated from the sample being measured (Patent Document 2).

[0006] Incidentally, acatalasia (or acatalasemia), or Takahara's disease, is a constitutional disorder caused by an autosomal recessive gene that is almost completely deficient in catalase, discovered by Takahara in 1946 (Non-Patent Literature 1, 2). Biological samples from patients with acatalasia (Non-Patent Literature 1, 2) do not contain catalase. In contrast, biological samples from patients without acatalasia contain endogenous catalase, so if endogenous peroxides are present in the patient's biological sample, the peroxides are usually eliminated beforehand by the patient's own endogenous catalase, and do not affect the color change caused by the quantitative determination of hydrogen peroxide produced from the sample being measured. However, since biological samples from patients with acatalasia do not contain catalase, it is not possible to eliminate endogenous peroxides contained in the biological sample beforehand. Therefore, if endogenous peroxides originating from the patient's biological sample are present, they positively affect the color change caused by hydrogen peroxide other than that originating from the sample being measured in the enzymatic method, leading to the problem of inflated measurement values.

[0007] The inventors of this application investigated the positive effect of endogenous peroxides derived from such patient biological samples using pyruvate, an example of an α-keto acid disclosed in Patent Document 2. They found that while it was possible to suppress the elevated values ​​in samples derived from biological samples of patients with acatalasemia (hereinafter sometimes simply referred to as acatalase samples), the measured absorbance values ​​decreased in samples derived from biological samples of healthy individuals and in calibrators (comparative examples described in the examples later in this specification). The reason for this is unclear, but it is likely that the α-keto acid interfered with the main reaction itself, in which hydrogen peroxide, which is present in the sample being measured, reacts with peroxidase and a chromogenic agent. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2007-236234 [Patent Document 2] International Open Brochure WO2012 / 081539 [Non-patent literature]

[0009] [Non-Patent Document 1] Kawasaki Medical and Welfare Society Journal, Vol. 5, No. 1, 1995, 19-29 [Non-Patent Document 2] Acta Med. Okayama, 2008,Vol.62, No.6, 345-361 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a measurement method for measuring a target substance in a sample using an enzymatic method, which can suppress the positive influence of peroxides derived from the sample. More specifically, the invention aims to provide a measurement method and a measurement reagent that can suppress elevated levels in catalase-free samples without affecting the color development of samples from healthy individuals or calibrators. [Means for solving the problem]

[0011] The present invention aims to solve the above problems, and in a method for measuring a target component by reacting the target component in a sample with an enzyme and quantifying the hydrogen peroxide produced, we have diligently searched for a compound that does not affect the color development of samples from healthy individuals or calibrators, and can suppress the increase in "catalase-free samples". As a result, we have found that by contacting the sample with an enzyme 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, it is possible to correctly quantify hydrogen peroxide originating from the target component without being affected by the sample, and thus completed the present invention. [ka] However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms. In other words, the present invention has the following configuration. <1> A method for measuring a target component in a sample by reacting the target component with an enzyme and quantifying the hydrogen peroxide produced, the method comprising the following steps; (A) In the presence of one or more compounds selected from the group consisting of compounds represented by the following general formula (I), benzimidazole derivatives having an electron-donating substituent at the 2-position, and histidine, A process for generating hydrogen peroxide by bringing a sample into contact with an enzyme. (B) A step in which the generated hydrogen peroxide is reacted with a colorant in the presence of peroxidase. (C) Process for detecting color changes [ka] However, in formula (I), R1 and R2 may be the same or different, and each represents 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. <2> The measuring method according to <1>, wherein R1 and R2 in formula (I) are any one of hydrogen, a linear alkyl group having 1 to 4 carbon atoms, a phenyl group, or an alkyloxy group having 1 to 4 carbon atoms. <3> The measuring method according to <1>, wherein either one of R1 or R2 in formula (I) is hydrogen or a methyl group, and the other is any one selected from the group consisting of 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 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. <7> A measuring reagent comprising one or more compounds selected from the group consisting of an enzyme that acts on the component to be measured to generate hydrogen peroxide, a chromogenic agent, peroxidase, a compound represented by the following general formula (I), a benzimidazole derivative having an electron - donating substituent at the 2 - position, and histidine.

Chemical formula

Chemical formula

[0012] The present invention provides an enzymatic measurement method for measuring a target component in a sample by reacting the target component with an enzyme to produce hydrogen peroxide, which is then reacted with a chromogenic agent. By performing the enzymatic reaction in the presence of a specific compound, the present invention enables accurate quantification of hydrogen peroxide derived from the target component without being affected by other components of the sample. In particular, it provides a measurement method and reagent that can suppress the proliferation of high values ​​derived from components other than the target component in non-catalase samples, without affecting the color development of the target component in non-catalase samples or calibrators. In other words, the present invention enables accurate measurement by enzymatic method without being affected by other components of the sample, regardless of whether the sample is a non-catalase sample or not. [Modes for carrying out the invention]

[0013] (Measurement method) The present invention provides a method for measuring a target component in a sample by quantitatively determining the amount of hydrogen peroxide produced by reacting the target component with an enzyme, and the method includes the following steps. (A) A step of generating hydrogen peroxide by contacting a sample with a reagent containing an enzyme in the presence of one or more specific compounds selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2 position, and histidine. (B) A step in which the generated hydrogen peroxide is reacted with a colorant in the presence of peroxidase. (C) Process for detecting color changes [ka] However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms.

[0014] In step (A), the reaction between the sample and the enzyme, which acts on the target component to produce hydrogen peroxide in the presence of a specific compound, can be carried out under any reaction conditions that produce hydrogen peroxide, for example, 10 to 50°C, preferably 20 to 40°C, with a reaction time of 5 seconds to 60 minutes, preferably 30 seconds to 15 minutes, more preferably 1 minute to 10 minutes, and most preferably 3 to 5 minutes. The concentration of the specific compound in this reaction should be such that it can avoid the influence of hydrogen peroxide originating from components other than the target component in the sample, thereby reducing measurement errors. Specifically, the concentration should be 0.001 to 1 v / v%, preferably 0.01 to 0.5 v / v%, and more preferably 0.05 to 0.3% in the solution in which the reaction takes place. Furthermore, the concentration of the specific compound when formulated in the measurement reagent should be such that it reaches the above concentration in the solution in which the reaction takes place.

[0015] The colorant in step (B) can be any colorant that reacts with hydrogen peroxide in the presence of peroxidase to produce a pigment. Examples of colorants include oxidation coupling type chromogens and leuco type chromogens.

[0016] Leuco-type chromogens are substances that are converted into pigments on their own in the presence of hydrogen peroxide and peroxidase.

[0017] Examples of leuco-type chromophores include phenothiazine-based chromophores, triphenylmethane-based chromophores, diphenylamine-based chromophores, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, tetramethylbenzidine, etc., with phenothiazine-based chromophores being preferred.

[0018] Examples of phenothiazine-based chromogens include 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), and 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67). Among the phenothiazine-based chromogens, 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67) is particularly preferred.

[0019] Examples of triphenylmethane-based chromogens include N,N,N',N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triaminotriphenylmethane (TPM-PS).

[0020] Examples of diphenylamine-based chromogens include N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 4,4'-bis(dimethylamino)diphenylamine, and bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA).

[0021] Oxidative coupling chromogens are substances that produce pigments through oxidative coupling of two compounds in the presence of hydrogen peroxide and a peroxidizing agent. Examples of combinations of two compounds include a coupler and anilines (Trinder's reagent), and a coupler and phenols.

[0022] Examples of couplers include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine.

[0023] Anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N,N-bis(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N- Examples include ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS).

[0024] Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

[0025] In process (C), the change in the color tone of the generated pigment can be detected by measuring its absorbance. Absorbance can be measured, for example, using a spectrophotometer. The color change can be analyzed by applying the absorbance measured in step (C) to a calibration curve, which is created using known concentrations of the target component and represents the relationship between the concentration of the target component and its absorbance.

[0026] The measurement method of the present invention allows for the accurate quantification of hydrogen peroxide originating from the target component while avoiding positive influences from components other than the target component in the sample. Examples of components other than the target component in the sample include peroxides other than the target component in the biological sample, peroxides contained in the measurement reagent, and peroxides generated by the pretreatment of the biological sample. Since biological samples from patients with acatalasemia do not contain catalase, the presence of endogenous peroxides in the biological sample cannot be eliminated beforehand, resulting in a positive influence. Therefore, the present invention is particularly preferable because it can avoid the influence of such endogenous peroxides.

[0027] (Sample, specimen) The sample used in the method of the present invention may be any sample of biological origin, including whole blood, serum, plasma, urine, red blood cells separated from whole blood, and red blood cells separated from whole blood and further washed. Biological samples are used as specimens for this measurement method either directly or after undergoing certain pretreatment and dilution. Pretreatment includes any pretreatment necessary for measuring the target substance in the biological sample, such as filtration, centrifugation, treatment with anticoagulants, treatment with preservatives, heating, or cooling.

[0028] (Specific compound) Examples of specific compounds of the present invention include the following compounds 1 to 3. 1. Compounds represented by the following general formula (I) [ka]

[0029] Here, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms.

[0030] Examples of linear or branched alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, and hexyl groups. Specifically, examples include methylglyoxal (R1=hydrogen, R2=methyl group, CAS number [78-98-8]), diacetyl (R1=methyl group, R2=methyl group, CAS number [431-03-8]), 2,3-pentanedione (R1=methyl group, R2=ethyl group, CAS number [600-14-6]), 3,4-hexanedione (R1=ethyl group, R2=ethyl group, CAS number [4437-51-8]), 2,3-heptanedione (R1=methyl group, R2=n-butyl group, CAS number [96-04-8]), and 5-methyl-2,3-hexanedione (R1=methyl group, R2=tert-butyl group, CAS number [13706-86-0]). Among these, compounds in which R1 and R2 are the same or different, and which are hydrogen, a methyl group, or an ethyl group, are preferred. Examples of substituents include halogen atoms, methyl groups, amino groups, sulfo groups, and carboxyl groups.

[0031] Examples of aryl groups include phenyl groups and naphthyl groups, and examples of substituents include halogen atoms, methyl groups, amino groups, sulfo groups, and carboxyl groups. Specifically, examples include phenylglyoxal (R1=hydrogen, R2=phenyl group, CAS number [1075-06-5]), 1-phenyl-1,2-propanedione (R1=methyl group, R2=phenyl group, CAS number [579-07-7]), 1-(4-chlorophenyl)propane-1,2-dione (CAS number [10557-21-8]), 1-[4-(trifluoromethyl)phenyl]propane-1,2-dione (CAS number [10557-13-8]), and 1-(4-nitrophenyl)propane-1,2-dione (CAS number [6159-25-7]). Among these, compounds in which R1 is hydrogen or a methyl group and R2 is a phenyl group are preferred.

[0032] Examples of alkoxy groups having 1 to 6 carbon atoms include methoxy, ethoxy, propiooxy, butoxy, pentoxy, and hexoxy groups. Specifically, examples include methyl pyruvate (R1=methyl group, R2=methoxy group, CAS number [600-22-6]), methyl 2-oxobutyrate (R1=ethyl group, R2=methoxy group, CAS number [3952-66-7]), ethyl pyruvate (R1=methyl group, R2=ethoxy group, CAS number [617-35-6]), dimethyl oxalate (R1=R2=methoxy group, CAS number [553-90-2]), methyl 2-oxovalerate (R1=propyl group, R2=methoxy group, CAS number [6376-59-6]), diethyl oxalate (R1=R2=ethoxy group, CAS number [95-92-1]), and methyl benzoylmate (R1=phenyl group, R2=methoxy group, CAS number [15206-55-0]). Of these, compounds that are readily soluble in water are preferred, and among these, compounds in which one of R1 or R2 is a hydrogen or methyl group and the other is a methoxy or ethoxy group are preferred.

[0033] The compound represented by formula (I) above is more preferably one in which R1 and R2 are hydrogen, a linear or branched alkyl group having 1 to 4 carbon atoms, a phenyl group which may have substituents, or an alkoxy group having 1 to 4 carbon atoms. 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. More specifically, phenylglyoxal (R1=hydrogen, R2=phenyl group, CAS number [1075-06-5]), glyoxal (R1=hydrogen, R2=hydrogen, CAS number [107-22-2]), diacetyl (R1=methyl group, R2=methyl group, CAS number [431-03-8]), 2,3-pentanedione (R1=methyl group, R2=ethyl group, CAS number [600-14-6]), 1-phenyl-1,2-propanedione (R1=methyl group, R2=phenyl group, CAS number [579-07-7]), methyl pyruvate (R1=methyl group, R2=methoxy group, CAS number [600-22-6]), or ethyl pyruvate (R1=methyl group, R2=ethoxy group, CAS number [617-35-6]) are more preferred, and phenylglyoxal is even more preferred.

[0034] Benzimidazole derivatives having an electron-donating substituent at the 2.2 position The benzimidazole derivative having an electron-donating substituent at the 2-position of the present invention can be any compound having a benzimidazole skeleton with a substituent substituted at the 2-position, which has the effect of suppressing the positive influence of peroxides originating from the sample in a method for measuring a target substance in a sample by enzymatic method. Examples of electron-donating substituents bonded to the 2-position of the derivative having the aforementioned effect include amino groups (-NH2, -NR2: where R is an alkyl group), alkoxy groups, alkyl groups, and aryl groups. Examples of such compounds include 2-aminobenzimidazole (CAS number [934-32-7]), in which the 2-position is substituted with an amino group, and 2-ethyl-1H-benzimidazole (CAS number [1848-84-6]), in which the 2-position is substituted with an ethyl group. Of these, 2-aminobenzimidazole is preferred.

[0035] 3. Histidine The histidine used in this invention is the compound specified by CAS number [71-00-1].

[0036] (Components to be measured) The enzymatic method in the present invention is a measurement method that involves reacting a target component in a sample with an enzyme that uses it as a substrate to produce hydrogen peroxide, and then reacting the produced hydrogen peroxide with a chromogenic agent in the presence of peroxidase (POD) to detect a change in color and quantify the target component. Therefore, the target component is not particularly limited, and any component that can be measured by quantifying the hydrogen peroxide produced by the enzymatic reaction can be measured. Examples of enzymes that react with the target component to produce hydrogen peroxide include enzymes that directly convert the target component to hydrogen peroxide, enzymes that indirectly convert the target component to hydrogen peroxide, enzymes that directly produce hydrogen peroxide from the target component, and enzymes that indirectly produce hydrogen peroxide from the target component. Therefore, any component that can be measured by such an enzymatic reaction can be the target of measurement in the present invention.

[0037] Specifically, these include hemoglobin A1c (HbA1c), glycated albumin (GA), uric acid, creatinine, cholesterol, triglycerides, polyamines, bile acids, 1,5-anhydroglucitol, pyruvate, lactic acid, phospholipids, urea, glucose, choline, creatine, and free fatty acids. Furthermore, the following are listed as oxidizing enzymes specific to the above-mentioned target components or their derivatives, in the format of "Target Component (Enzyme)". Examples include hemoglobin A1c (protease, fructosyl amino acid oxidase or fructosyl peptide oxidase), GA (protease, ketoamine oxidase), uric acid (uricase), creatinine (creatininase, creatinase, sarcosine oxidase), cholesterol (cholesterol oxidase), triglycerides (lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase), polyamines (polyamine amide hydrolase, polyamine oxidase, putresin oxidase), bile acids (3-α-hydroxysteroid dehydrogenase, diaphorase), 1,5-anhydroglucitol (1,5-anhydroglucitol oxidase, pyranose oxidase), pyruvate (pyruvate oxidase), lactic acid (lactic acid oxidase), phospholipids (phospholipase D, choline oxidase), urea (urea amide lyase, pyruvate kinase, pyruvate oxidase), etc.

[0038] In the measurement method of the present invention, the reaction between the component to be measured and the enzyme that acts on the component to be measured to produce hydrogen peroxide is preferably carried out in an aqueous medium. Examples of aqueous mediums include deionized water, distilled water, and buffer solutions. The pH of the buffer solution is preferably 4.0 to 10.0, with a pH of 6.0 to 8.0 being preferred. Examples of buffering agents used in the buffer solution include phosphate buffers, borate buffers, and Good's buffers. Furthermore, the reaction between the target component and the enzyme can also be carried out in the presence of stabilizers, preservatives, interfering substance scavengers, reaction accelerators, etc.

[0039] (Measuring reagents) Examples of reagent configurations for carrying out the measurement method of the present invention include two-reagent kits and three-reagent kits. The three-reagent kit includes a pretreatment solution for pretreatment of the sample, a first reagent, and a second reagent, while the two-reagent kit includes a first reagent and a second reagent. The colorant and POD are contained in separate reagents, and the specific compound component of the present invention is contained in the pretreatment solution or the first reagent. The specific reagent formulation is shown below. (1) Hemoglobin A1c measurement reagent kit First reagent: protease, color developer, specific compound of the present invention Second reagent: Fructosyl peptide oxidase, POD (2) Glycoalbumin measurement reagent kit First reagent: Protease, POD, specific compound of the present invention Second reagent: Ketoamine oxidase, colorant The measurement reagent of the present invention can be applied to various automated analyzers, as in the conventional method. By applying it to various automated analyzers, it is possible to provide a method for measuring each target component that eliminates the positive influence from components other than the target component in the sample.

[0040] (Method and reagents for measuring hemoglobin A1c) The components to be measured in this invention are not particularly limited; any component that can be measured by quantifying hydrogen peroxide produced by an enzymatic reaction can be measured. However, the case of measuring hemoglobin A1c will be described in particular. An example of an HbA1c measurement method according to the present invention is a method using the following hemoglobin A1c measurement reagent kit. First reagent: protease, color developer, specific compound of the present invention Second reagent: Fructosyl peptide oxidase, POD First, the first reagent is added to the sample, and the glycated dipeptide at the N-terminus of the HbA1cβ chain is digested and cleaved by a protease. Next, the second reagent is added, and an oxidase specific to the glycated dipeptide (fructosyl peptide oxidase) is reacted with it. The resulting hydrogen peroxide is then reacted with a chromogenic agent in the presence of peroxidase, and the result is quantified colorimetrically. The enzyme reaction that acts on the target component to produce hydrogen peroxide is carried out in the presence of the specific compound of the present invention contained in the first reagent, thereby suppressing positive influences originating from the target component.

[0041] Hemoglobin A1c is generally expressed as HbA1c%. "HbA1c%" refers to the ratio (%) of the A1c concentration to the hemoglobin concentration in a sample, as is commonly used in clinical practice. The method for measuring HbA1c% basically includes a first step of optically measuring the hemoglobin concentration in a sample, a second step of optically measuring the HbA1c concentration in the sample, and a step of calculating the ratio of the HbA1c concentration to the hemoglobin concentration in the sample (HbA1c%). The aforementioned measurement of hemoglobin A1c corresponds to the second step of the HbA1c% measurement method. In the first step of the A1c% measurement method, the optical measurement of the sample is performed by measuring the absorbance using light of a first wavelength and light of a second wavelength, respectively. The hemoglobin concentration measured at the first wavelength is corrected using the measurement value at the second wavelength. The corrected hemoglobin concentration is used in the HbA1c% calculation step.

[0042] When measuring hemoglobin concentration, measures may be taken to stabilize the absorbance of hemoglobin by establishing a fixed structure using known methods such as metmyopropyl alcohol synthesis. Furthermore, when measuring HbA1c concentration, measures may be taken to facilitate the digestion and cleavage of glycated dipeptides from the HbA1c β-chain by introducing surfactants or the like during protease digestion and cleavage.

[0043] In this specification, "first wavelength" means the wavelength for measuring hemoglobin, and preferably the wavelength of the first light used for the 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.

[0044] In this specification, "second wavelength" means the wavelength used to correct the apparent hemoglobin concentration measured at the first wavelength to the true hemoglobin concentration, and is preferably the wavelength of the second light used for optical measurement in the first step of the method of the present invention. The second wavelength can be appropriately selected from the range of 690 nm to 900 nm.

[0045] (Method for reducing measurement errors) In other words, the measurement method of the present invention can be described as a method for reducing measurement errors in a method for measuring a target component by avoiding the influence of hydrogen peroxide originating from components other than the target component contained in the sample and quantifying the hydrogen peroxide originating from the target component by an enzymatic method. The measurement error reduction method of the present invention is characterized by a step of contacting the sample with one or more compounds selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine, and each component is as described above. [ka] However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms. [Examples]

[0046] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0047] [Reference Example 1] Preparation of a catalase-free model sample Blood samples A and B from two healthy individuals were collected using EDTA blood collection tubes and centrifuged to obtain blood cells A and B. An aqueous solution of sodium azide, which is known to inhibit catalase activity, was added to these blood cells to create catalase-free model samples A and B.

[0048] [Example 1] Study using a catalase-free model sample 1. Measurement reagents and measurement samples: <Protease-containing substrate reagent (R1)> 50mM MES pH6.0 1.0% Emal 20C (Kao Corporation) Protin PC10F (Yamato Chemical Industries Co., Ltd.) DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium, Wako Pure Chemical Industries, Ltd.) Additives listed in Tables 1-4 The manufacturers and distributors of the additives listed in Tables 1-4 are as follows: TCI: Tokyo Chemical Industry Co., Ltd. Kishida: Kishida Chemical Co., Ltd. Fuji: Fujifilm Wako Pure Chemical Corporation

[0049] <Colorimetric reagent (R2)> 50 mM citric acid, pH 6.0 Fructosyl peptide oxidase (Kikkoman Corporation) Peroxidase (Toyobo Co., Ltd.)

[0050] <Measurement Sample> (1) Nordia NHbA1c Calibrator (Sekisui Medical Co., Ltd.) 1, 2 (2) Catalase-free model samples A and B prepared in Reference Example 1

[0051] 2.Operation Using an automated analyzer JCA-9130 (JEOL Ltd.), the absorbance of HbA1c (%) of the sample was measured using reagents R1 and R2, which contain various additives shown in Tables 1-4, with the following measurement parameters. The time settings below assume that R2 is added approximately 5 minutes after R1 is added and the first reaction begins. Parameters: [HbA1c] Analysis method: EPA Calculation method: MSTD Measurement wavelength (secondary / main): HbA1c 805 / 658 Main DET.Pl-Pm-Pn:HbA1c 0-95-98 Deputy DET.Pp-Pr:HbA1c 44-47 Reaction time: 10 minutes No sample dilution Sample volume: 6.4 μL First reagent volume (R1): 60 μL, Second reagent volume: 0 μL Reagent volume 3 (R2): 20 μL, Reagent volume 4: 0 μL

[0052] 3.Results The relative values ​​(%) of the absorbance of HbA1c (%) measured under each condition, compared to the absorbance obtained under condition 1 (no additives), were calculated and are shown in Tables 1-4. It is preferable that the relative values ​​of the measured absorbances of calibrator 1 and calibrator 2 are close to 100% of the value under condition 1. Furthermore, it is preferable that the relative values ​​of the measured absorbances of non-catalase model samples A and B are lower than those under condition 1.

[0053] [Table 1]

[0054] [Table 2]

[0055] [Table 3] [Table 4]

[0056] 3-1. Compounds represented by general formula (I) First, under condition 2, in which 0.10% pyruvate, an α-keto acid described in Patent Document 2, was added, the relative absorbance of the non-catalase model sample decreased to 20.1-22.1%, but the relative absorbance of calibrators 1 and 2 also decreased to 70.1-87.1%, indicating an effect on the main reaction. Furthermore, under condition 3, in which 1.0% pyruvate was added, the relative absorbance of the non-catalase model sample decreased to 86.2-97.7%, but the relative absorbance of the calibrator increased to 204.3-593.2%, suggesting an abnormality in the color reaction. In addition, under conditions 4 and 5, in which 2-ketoglutaric acid, a compound similar to α-keto acid, was added, the behavior was similar to that of pyruvate. Under condition 6, in which levulinic acid (3-acetylpropionic acid) was added, the relative absorbance of the calibrator decreased while the relative absorbance of the non-catalase model sample increased, suggesting an abnormality in the color reaction.

[0057] On the other hand, when using condition 8: 0.10% phenylglyoxal, the compound discovered in this application, the relative absorbance of the catalase-free model sample decreased to 21.0-21.6%, but the relative absorbance of calibrators 1 and 2 was maintained at 92.4-110.4%. Furthermore, when using condition 9: 0.20% phenylglyoxal, the relative absorbance of the catalase-free model sample decreased to 17.1-17.5%, but the relative absorbance of calibrators 1 and 2 was maintained at 80.6-109.0%. Even under condition 7, where 0.01% phenylglyoxal was added, the absorbance of the calibrator did not decrease, and only the absorbance of the catalase-free model sample decreased. In particular, the relative absorbance of the catalase-free model sample under conditions 8 and 9 was similar to that under condition 2, but conditions 8 and 9 were superior to condition 2 in that the relative absorbance of calibrators 1 and 2 was maintained.

[0058] Similarly, for glyoxal (condition 10), diacetyl (conditions 11, 12), 2,3-pentanedione (conditions 30, 31, 32), 1-phenyl-1,2-propanedione (condition 33), methyl pyruvate (conditions 34, 35), and ethyl pyruvate (conditions 36, 37), which have the chemical structure represented by general formula (I), the relative absorbance values ​​of the non-catalase model samples decreased, while the relative absorbance values ​​of calibrators 1 and 2 were maintained. Thus, the group of compounds having the structure of general formula (I) were found to specifically eliminate endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the substance being measured, and thus possess the function desired by the present invention.

[0059] 3-2. Benzimidazole derivatives having electron-donating substituents at the 2-position Conditions 13-21 and 38-40 were examined. When imidazole was added, at 0.01% (condition 13), no change in absorbance occurred for either the calibrator or the catalase-free model sample. When 0.1-0.2% was added (conditions 14, 15), the decrease in the relative absorbance of the calibrator was greater than the decrease in the relative absorbance of the catalase-free model sample, and it was considered unsuitable for measurement. When 0.01-0.2% of benzimidazole was added (conditions 16-18), the relative absorbance of the catalase-free model sample decreased at all concentrations, but the relative absorbance of the calibrator decreased even more, showing similar behavior to imidazole. On the other hand, when 0.01% to 0.2% of 2-aminobenzimidazole, as discovered in this invention, was added (conditions 19 to 21), the relative absorbance of the non-catalase model sample decreased significantly compared to the relative absorbance of the calibrator at all concentrations. Furthermore, when 0.01% of 2-ethyl-1H-benzimidazole was added (condition 38), the relative absorbance of the sample decreased while the relative absorbance of the calibrator remained at 100.6 to 101.4%, indicating that it is a compound with the desired function of the present invention. In addition, since the relative absorbance of the sample did not decrease with 2-aminoimidazole sulfate (conditions 39 and 40), it was considered that the benzimidazole skeleton is necessary for the compound to have the function of the present invention. Thus, it was found that benzimidazole derivatives having an electron-donating substituent at the 2-position specifically eliminate endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the substance being measured, thus possessing the function desired by the present invention.

[0060] 3-3. About Histidine Conditions 22-25 were examined. In condition 22, with the addition of 0.1% methionine, and in condition 23, with the addition of 0.1% tryptophan, the relative absorbance values ​​of the non-catalase model samples remained almost unchanged. On the other hand, in conditions 24 and 25, with the addition of L-histidine, the relative absorbance values ​​of the non-catalase model samples decreased, and the magnitude of this decrease was greater than the decrease in the relative absorbance values ​​of the calibrator. Thus, it was found that histidine, among the amino acids, is a compound that possesses the function desired by the present invention.

[0061] 4. Judgment result Furthermore, the results of the relative value determination shown in Tables 1-4 are shown in Table 5. The evaluation criteria are as follows: 4-1. Evaluation Criteria for Calibrators A: The relative absorbance values ​​measured by calibrators 1 and 2 are both between 90% and 110%. B: The relative values ​​of the measured absorbances of calibrators 1 and 2 are both between 80% and 120%. C: One of the relative values ​​of the measured absorbances of calibrators 1 and 2 is less than 80% or greater than 120%. Since the relative values ​​of the measured absorbances of calibrator 1 and calibrator 2 are preferably close to 100% compared to the case without additives, A was determined to be the most preferred, followed by B, and C was deemed undesirable.

[0062] 4-2. Criteria for evaluating specimens Under the condition that the calibrator's determination is A or B, the following was assumed: A: The relative values ​​of both samples A and B are 60% or less. B: Furthermore, the relative values ​​of both samples A and B are 90% or less. C: The relative values ​​of both samples A and B are 95% or less. D: The relative values ​​of both samples A and B are 95% or higher. The relative absorbance values ​​of the non-catalase model samples A and B were lower than those of condition 1, making A the most preferred, followed by B and C. Sample D was judged not to possess the desired performance. Samples with a calibrator rating of "C" were excluded from the sample evaluation (indicated as "-" in the table).

[0063] 4-3. Criteria for Overall Evaluation Based on the evaluation of the calibrator and the samples, the following was determined: A: Both the calibrator and the sample are rated A. B: Calibrator is A and sample is B, or calibrator is B and sample is A or B. C: Calibrator is A or B and sample is C. D: Calibrator is C or sample is D The overall evaluation was that A was the most preferable, followed by B and C, while D was judged to have shown no effect. Note that condition 14 would normally receive an overall evaluation of C according to this standard, but because the relative values ​​of samples A and B were higher than those of calibrator 2, it received an overall evaluation of D.

[0064] 4-4. Discussion Table 5 shows that phenylglyoxal, methyl pyruvate, and ethyl pyruvate, which contain an overall rating of A, are the most preferred compounds. Furthermore, glyoxal, diacetyl, 2-aminobenzimidazole, histidine, and 2,3-pentanedione, which contain a rating of B, are also preferred compounds. From the above, it has been found that the compound represented by general formula (I) found in the present invention, the benzimidazole derivative having an electron-donating substituent at the 2-position, or histidine, when present in the measurement system of a method for measuring target components in a sample by enzymatic method, all specifically eliminate endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the target components. [Table 5]

[0065] [Reference Example 2] Measurement of healthy individual samples and acatalasemia samples Blood samples C and D from two healthy individuals, collected using EDTA blood collection tubes, and a catalaseosis sample E were centrifuged to obtain blood cells C, D, and E. Furthermore, blood cell counts C, D, and E were measured using the HPLC method (Tosoh G11), which is unaffected by the presence or absence of catalase. HbA1c (%) was 4.95% for healthy sample C, 6.50% for healthy sample D, and 6.20% for catalase-free sample E.

[0066] [Example 2] Study using catalase-free samples 1. Measurement reagents <Protease-containing substrate reagent (R1-A)> 50mM MES pH6.0 1.0% Emal 20C (Kao Corporation) Protin PC10F (Yamato Chemical Industries Co., Ltd.) DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium, Wako Pure Chemical Industries, Ltd.)

[0067] <Protease-containing substrate reagent (R1-B)> 50mM MES pH6.0 1.0% Emal 20C (Kao Corporation) Protin PC10F (Yamato Chemical Industries Co., Ltd.) DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium, Wako Pure Chemical Industries, Ltd.) 0.10% Phenylglyoxal

[0068] <Colorimetric reagent (R2)> 50 mM citric acid, pH 6.0 Fructosyl peptide oxidase (Kikkoman Corporation) Peroxidase (Toyobo Co., Ltd.)

[0069] 2. Test sample <Calibrator> A Nordia NHbA1c calibrator was used. <Diluent> Purified water was used. <Measurement Sample> Normal samples C and D, and catalase-free sample E, prepared in Reference Example 2, were diluted 26-fold with purified water and measured.

[0070] 2.Operation Using an automated analyzer JCA-9130 (JEOL Ltd.), test samples were measured using the following measurement parameters for combinations of reagent R1-A and reagent R2, and reagent R1-B and reagent R2, and HbA1c (%) was calculated. Parameters: [HbA1c, Hb] Analysis method: EPA Calculation method: MSTD Measurement wavelength (secondary / main): HbA1c 805 / 658, Hb 805 / 478 Main DET.Pl-Pm-Pn:HbA1c 0-95-98, Hb 0-44-47 Vice DET.Pp-Pr:HbA1c 44-47, Hb 0-0 Reaction time: 10 minutes No sample dilution Sample volume: 6.4 μL First reagent volume (R1): 60 μL, Second reagent volume: 0 μL Reagent volume 3 (R2): 20 μL, Reagent volume 4: 0 μL

[0071] 3.Results Table 6 shows the results of measurements using R1-A (no additive) and R1-B (phenylglyoxal added) as the first reagent. When using reagent R1-A, healthy samples C and D were measured accurately, while the catalase-free sample E showed a significantly higher value of 15.46% compared to the HPLC measurement value of 6.20%. On the other hand, when using reagent R1-B, which contains 0.10% of the phenylglyoxal discovered in this invention, the measured value of the catalase-free sample was 6.84% compared to the HPLC value of 6.20%, demonstrating much higher accuracy compared to reagent R1-A without additives. When using reagent R1-B, healthy sample C showed a slightly higher value of 5.29% compared to the HPLC value of 4.95%, and healthy sample D showed a slightly higher value of 6.65% compared to the HPLC value of 6.50%. This is presumed to be a measurement error caused by using a calibrator optimized for reagent R1-A when performing calibration with reagent R1-B. Therefore, it is believed that even more accurate measurements could be achieved by separately optimizing the calibrator for the R1-B reagent.

[0072] [Table 6] [Industrial applicability]

[0073] According to the present invention, it is possible to provide a measurement method and reagent that can accurately quantify hydrogen peroxide derived from a sample other than the target substance, in a measurement method based on an enzymatic method, without being affected by components derived from the sample other than the target substance. In particular, accurate measurement of the target substance is possible by an enzymatic method, regardless of whether the sample is catalase-free or not, without being affected by components derived from the sample other than the target substance.

Claims

1. A method for measuring hydrogen peroxide produced by reacting HbA1c, a target component in a sample, with an enzyme, comprising the following steps: (A) One or more compounds selected from the group consisting of compounds represented by the following general formula (I), benzimidazole derivatives having an electron-donating substituent at the 2-position, and histidine, which specifically eliminate endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the target component, A process for generating hydrogen peroxide by bringing a sample into contact with an enzyme. (B) A step in which the generated hydrogen peroxide is reacted with a color developer in the presence of peroxidase. (C) Process for detecting color changes 【Chemistry 1】 However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms.

2. The measurement method according to claim 1, wherein R1 and R2 in 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.

3. The measurement method according to claim 1, wherein either R1 or R2 of formula (I) is hydrogen or a methyl group, and the other is selected from the group consisting of hydrogen, a methyl group, an ethyl group, a phenyl group, a methoxy group, and an ethoxy group.

4. Claim 1, wherein the compound represented by formula (I) is phenylglyoxal, glyoxal, diacetyl, 2,3-pentanedione, methyl pyruvate, or ethyl pyruvate. Measurement method.

5. The measurement method according to claim 1, wherein the benzimidazole derivative having an electron-donating substituent at the 2-position is 2-aminobenzoimidazole.

6. A measurement reagent comprising an enzyme that acts on HbA1c, the component to be measured, to produce hydrogen peroxide; a chromogenic agent; a peroxidase; and one or more compounds selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine, which specifically eliminates endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the component to be measured. 【Chemistry 1】 However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms.

7. The measuring reagent according to claim 6, wherein R1 and R2 of formula (I) are any of hydrogen, a linear alkyl group having 1 to 4 carbon atoms, a phenyl group, or an alkyloxy group having 1 to 4 carbon atoms.

8. The measuring reagent according to claim 6, wherein either R1 or R2 of formula (I) is hydrogen or a methyl group, and the other is selected from the group consisting of hydrogen, a methyl group, an ethyl group, a phenyl group, a methoxy group, and an ethoxy group.

9. The measurement reagent according to claim 6, wherein the compound represented by formula (I) is phenylglyoxal, glyoxal, diacetyl, 2,3-pentanedione, methyl pyruvate, or ethyl pyruvate.

10. The measurement reagent according to claim 6, wherein the benzimidazole derivative having an electron-donating substituent at the 2-position is 2-aminobenzimidazole.

11. The measurement reagent according to any one of claims 6 to 10, wherein the enzyme that acts on the component to be measured and produces hydrogen peroxide is a protease and a fructosyl peptide oxidase.

12. A measurement reagent kit comprising an enzyme that acts on HbA1c, the component to be measured, to produce hydrogen peroxide, a chromogenic agent, a peroxidase, and one or more compounds selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine, wherein the compound specifically eliminates endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the component to be measured, The first reagent contains one or more compounds selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine. The second reagent contains an enzyme. The aforementioned measurement reagent kit. 【Chemistry 1】 However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms.

13. A method for reducing measurement errors in a method for measuring a target component, which involves avoiding the influence of hydrogen peroxide derived from components other than HbA1c, the target component, contained in a sample, and quantifying the hydrogen peroxide derived from HbA1c, the target component, by an enzymatic method, A method for reducing measurement errors, characterized by including the step of contacting a sample with one or more compounds selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at the 2-position, and histidine, which specifically eliminates endogenous peroxides in the sample while not affecting the main reaction caused by hydrogen peroxide generated from the component to be measured. 【Chemistry 1】 However, in formula (I), R1 and R2 may be the same or different, and represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms which may have substituents, an aryl group which may have substituents, or an alkyloxy group having 1 to 6 carbon atoms.

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