Method for measuring glycated hemoglobin, method for reducing influence of plasma in sample, measurement reagent, and measurement kit

Abstract

Provided is a method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, the method including reacting glycated hemoglobin in a sample containing hemoglobin and plasma, a proteolytic enzyme, and a fructosyl peptide oxidase to generate hydrogen peroxide, and measuring the generated hydrogen peroxide, wherein the reaction is performed in the presence of ferrocyanide and a ferrocene derivative.

Description

Method for measuring glycated hemoglobin, method for reducing the influence of plasma in the sample, measurement reagents, and measurement kits.

[0001] The present invention relates to a method for measuring glycated hemoglobin, a method for reducing the influence of plasma in a sample, a measuring reagent, and a measuring kit.

[0002] Glycated proteins are found in bodily fluids such as blood and in biological samples such as hair. The concentration of glycated proteins in the blood depends on the concentration of sugars such as glucose dissolved in the serum. In the field of clinical diagnosis, the measurement of the concentration of hemoglobin A1c (hereinafter referred to as HbA1c), a glycated protein in the blood, is used for the diagnosis and monitoring of diabetes (see Non-Patent Literature 1). Hemoglobin is a heme protein with a molecular weight of 64,000, having two α-chains and two β-chains. HbA1c is specifically defined as hemoglobin in which the N-terminal valine residue of the β-chain has been glycated. Methods for measuring HbA1c include instrumental analysis using high-performance liquid chromatography (HPLC) (see Non-Patent Literature 2) and immunoassay methods using antigen-antibody reactions (Non-Patent Literature 3).

[0003] In recent years, the development of enzymatic methods for measuring HbA1c that can be applied to general-purpose automated analyzers and are easy to operate has progressed, and various methods have been reported. The enzymatic methods for measuring HbA1c reported so far mainly use proteases and glycated peptide oxidases. One known method using proteases and glycated peptide oxidases involves reacting HbA1c in blood cells with a protease to produce a glycated peptide called fructosyl dipeptide (Fru-Val-His), reacting the produced fructosyl dipeptide with fructosyl peptide oxidase to produce hydrogen peroxide, and measuring the produced hydrogen peroxide to measure the HbA1c in the sample (see Patent Document 1).

[0004] Japanese Patent Publication No. 2001-095598, Japanese Patent Publication No. 2008-245657, Japanese Patent Publication No. 49-050991

[0005] Clin Chem Lab Med, Vol. 36, pp. 299-308 (1998). Diabetes, Vol. 27, No. 2, pp. 102-107 (1978). Journal of the Japanese Society for Clinical Laboratory Automation, Vol. 18, No. 4, p. 620 (1993).

[0006] In enzymatic methods for measuring HbA1c, whole blood and blood cell fractions obtained by centrifugation of whole blood to remove plasma components are often used as samples. To measure HbA1c more simply, it is preferable to use whole blood as the sample, as centrifugation is not required. On the other hand, when whole blood is used as the sample, the influence of plasma components in the whole blood often makes it difficult to accurately measure glycated hemoglobin in the sample. To solve this problem, a method for measuring the amount of hemoglobin glycation using a specific protease has been known (see Patent Document 2). However, even with these methods, the influence of plasma components is not always avoided, and there is a need for a more accurate method for measuring glycated hemoglobin in a sample that is not affected by plasma components.

[0007] Furthermore, colorimetric analysis, which involves reacting the generated hydrogen peroxide with an oxidative colorimetric chromogen in the presence of peroxidase to produce a dye, and then measuring the absorbance of the colored reaction solution containing the produced dye, is routinely used in clinical testing to measure the target component in a biological sample. In this colorimetric analysis based on hydrogen peroxide measurement, the influence of interfering substances such as bilirubin contained in the biological sample often becomes a problem. In particular, bilirubin affects the reaction between hydrogen peroxide and an oxidative colorimetric chromogen in the presence of peroxidase, and its reducing effect has a negative impact. A method using ferrocyanide ions has been known to solve this problem (see Patent Document 3).

[0008] However, when the inventors performed enzymatic measurement of HbA1c in the presence of ferrocyanide in order to reduce the influence of bilirubin in the sample, they found that the influence of plasma was further amplified by the ferrocyanide.

[0009] The present invention aims to provide a measurement method, a measurement reagent, and a measurement kit for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, in the presence of ferrocyanides, in a simple and more accurate manner.

[0010] This disclosure provides, in one aspect, the following: [1] A method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising reacting glycated hemoglobin in the sample containing hemoglobin and plasma with a protease and a fructosyl peptide oxidase to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, wherein the reaction is carried out in the presence of a ferrocyanide and a ferrocene derivative. [2] The method according to [1], wherein the ferrocyanide is potassium ferrocyanide. [3] The method according to [1] or [2], wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocene carboxyaldehyde. [4] The method according to any one of [1] to [3], wherein the sample containing hemoglobin and plasma is whole blood. [5] A method for measuring hydrogen peroxide according to any one of [1] to [4], wherein the hydrogen peroxide is measured using a hydrogen peroxide measuring reagent containing peroxidase and an oxidative color-developing chromogen. [6] A method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising reacting glycated hemoglobin in the sample containing hemoglobin and plasma with a protease and a fructosyl peptide oxidase to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, wherein the reaction is carried out in the presence of a ferrocyanide and a ferrocene derivative to reduce the influence of plasma in the sample. [7] The method according to [6], wherein the ferrocyanide is potassium ferrocyanide. [8] The method according to [6] or [7], wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocene carboxyaldehyde. [9] The method according to any one of [6] to [8], wherein the sample containing hemoglobin and plasma is whole blood.

[10] The method according to any one of [6] to [9], wherein the hydrogen peroxide is measured using a hydrogen peroxide measuring reagent containing peroxidase and an oxidative colorimetric chromogen.

[11] A reagent for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising a protease, a fructosyl peptide oxidase, a ferrocyanide, and a ferrocene derivative.

[12] The reagent according to

[11] , wherein the ferrocyanide is potassium ferrocyanide.

[13] The reagent according to

[11] or

[12] , wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocene carboxyaldehyde.

[14] The reagent according to any one of

[11] to

[13] , wherein the sample containing hemoglobin and plasma is whole blood.

[15] The reagent according to any one of

[11] to

[14] , comprising a hydrogen peroxide measurement reagent containing peroxidase and an oxidative colorimetric chromogen.

[16] A kit for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising a first reagent containing a protease and a second reagent containing a fructosyl peptide oxidase, wherein the first reagent and / or the second reagent contains a ferrocyanide and the first reagent and / or the second reagent contains a ferrocene derivative.

[17] A kit for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising a first reagent containing a fructosyl peptide oxidase and a second reagent containing a protease, wherein the first reagent and / or the second reagent contains a ferrocyanide and the first reagent and / or the second reagent contains a ferrocene derivative.

[18] The kit according to

[16] or

[17] , wherein the ferrocyanide is potassium ferrocyanide.

[19] The kit according to any one of

[16] to

[18] , wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocene carboxyaldehyde.

[20] The kit according to any one of

[16] to

[19] , wherein the sample containing hemoglobin and plasma is whole blood.

[21] The kit according to any one of

[16] to

[20] , wherein the first reagent and / or the second reagent comprises a hydrogen peroxide measurement reagent containing peroxidase and an oxidative colorimetric chromogen.

[0011] The present invention provides a measurement method, a measurement reagent, and a measurement kit for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, in the presence of ferrocyanides, in a simple and more accurate manner.

[0012] The embodiments for implementing this disclosure will be described in detail below. The embodiments described below are merely examples of typical embodiments of this disclosure and should not be interpreted as narrowing the scope of the disclosure. Numerical ranges indicated using "~" represent a range that includes the numbers before and after "~" as the minimum and maximum values, respectively. Furthermore, the amount of each component in the solution, unless otherwise specified, refers to the total amount of multiple substances present in the reaction or reagent when multiple substances corresponding to each component are present in the solution.

[0013] [Measurement Method] One embodiment of the present invention is a method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising reacting glycated hemoglobin in a sample containing hemoglobin and plasma with a proteolytic enzyme and a fructosyl peptide oxidase to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, wherein the above reaction is carried out in the presence of ferrocyanide and ferrocene derivatives.

[0014] The inventors have found that in the measurement of glycated hemoglobin, the presence of ferrocyanide, which is used to mitigate the influence of bilirubin in the sample, increases the measurement error due to plasma components. The measurement method according to this embodiment suppresses measurement errors caused by plasma and ferrocyanide by carrying out the reaction in the presence of a ferrocene derivative. By using the measurement method according to this embodiment, even when using whole blood that has not been centrifuged as a sample, for example, measurement errors due to the presence of plasma can be reduced, and glycated hemoglobin can be measured more simply and accurately.

[0015] The sample used in the measurement method according to this embodiment is a sample containing hemoglobin and plasma. The sample may be, for example, whole blood or a sample in which plasma is mixed with blood cells, or a sample that has been hemolyzed. The sample may or may not have been centrifuged. Examples of hemolyzation methods include physical methods, chemical methods, and biological methods. Examples of physical methods include methods using hypotonic solutions such as distilled water and methods using ultrasound. Examples of chemical methods include methods using organic solvents such as methanol, ethanol, and acetone, and methods using polyoxyethylene surfactants. Examples of biological methods include methods using antibodies and complement.

[0016] In the measurement method according to this embodiment, the plasma concentration in the sample may be, for example, a plasma concentration such that, when the total of blood cell components and plasma components is set to 100% by mass, the proportion of plasma components is 5 to 70% by mass, 20 to 70% by mass, or 40 to 70% by mass.

[0017] The sample to be measured may contain bilirubin. In the measurement method according to this embodiment, a ferrocyanide is included to reduce the effect of bilirubin, so even if the sample contains bilirubin, its effect can be reduced. The sample to be measured may not contain bilirubin. In the measurement method according to this embodiment, the concentration of bilirubin in the sample may be, for example, 0.5 to 40 mg / dL, 1 to 30 mg / dL, or 3 to 20 mg / dL.

[0018] Glycated hemoglobin is produced when sugars such as glucose bind to hemoglobin. Examples of glycated hemoglobin that can be measured by the measurement method according to this embodiment include hemoglobin A1a (HbA1a), hemoglobin A1b (HbA1b), hemoglobin A1c (HbA1c), etc., with hemoglobin A1c (HbA1c) being preferred.

[0019] The ferrocyanide used in the measurement method according to this embodiment may be, for example, a potassium salt or a sodium salt, and preferably potassium ferrocyanide.

[0020] In the measurement method according to this embodiment, the concentration of ferrocyanide in the reaction solution may be, for example, 0.01 to 0.5 mg / L, 0.05 to 0.3 mg / L, or 0.1 to 0.2 mg / L.

[0021] The ferrocene derivative used in the measurement method according to this embodiment may be, for example, at least one ferrocene derivative selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocene carboxaldehyde. The ferrocene derivative is preferably (ferrocenylmethyl)trimethylammonium chloride.

[0022] In the measurement method according to this embodiment, the concentration of the ferrocene derivative in the reaction solution may be, for example, 0.01 to 20 mg / L, 0.1 to 15 mg / L, or 0.5 to 10 mg / L.

[0023] In the measurement method according to this embodiment, the reaction between glycated hemoglobin, protease, and fructosyl peptide oxidase does not require them to be present simultaneously. For example, glycated hemoglobin may be reacted with protease first, and then the reaction product (e.g., fructosyl dipeptide) may be reacted with fructosyl peptide oxidase. Alternatively, for example, a sample containing glycated hemoglobin may be mixed with fructosyl peptide oxidase first, and then the mixed solution may be mixed with protease to react glycated hemoglobin, protease, and fructosyl peptide oxidase.

[0024] The measurement method according to this embodiment may specifically include the following steps: (1) A step of reacting a proteolytic enzyme with glycated hemoglobin in a hemoglobin and plasma-containing sample. (2) A step of reacting a fructosyl peptide oxidase with the reaction product obtained in step (1) in the presence of a ferrocyanide and a ferrocene derivative to generate hydrogen peroxide. (3) A step of measuring the hydrogen peroxide generated in step (2). (4) A step of determining the glycated hemoglobin concentration in the hemoglobin-containing sample from the amount of hydrogen peroxide measured in step (3), based on a calibration curve that has been prepared in advance using glycated hemoglobin of known concentration and represents the relationship between the amount of hydrogen peroxide and the glycated hemoglobin concentration.

[0025] In the measurement method according to this embodiment, the ferrocyanide and ferrocene derivative are present together at least in step (2). The ferrocyanide and ferrocene derivative may also be present together from step (1).

[0026] The method for measuring glycated hemoglobin according to this embodiment may include a step of calculating the ratio of the amount of glycated hemoglobin in a hemoglobin-containing sample to the amount of total hemoglobin (i.e., total hemoglobin, which is the sum of hemoglobin and glycated hemoglobin). In this case, the measurement method according to this embodiment may specifically include, for example, the following steps.

[0027] (1) A step to determine the total amount of hemoglobin (i.e., total hemoglobin including hemoglobin and glycated hemoglobin) in a hemoglobin-containing sample. (2) A step to react the glycated hemoglobin in the hemoglobin-containing sample with a proteolytic enzyme. (3) A step to generate hydrogen peroxide by reacting the reaction product obtained in step (2) with fructosyl peptide oxidase in the presence of ferrocyanide and ferrocene derivatives. (4) A step to measure the hydrogen peroxide generated in step (3). (5) A step to determine the amount of glycated hemoglobin in the hemoglobin-containing sample from the amount of hydrogen peroxide measured in step (4), based on a calibration curve that shows the relationship between the amount of hydrogen peroxide and the amount of glycated hemoglobin, which was prepared in advance using a known amount of glycated hemoglobin. (6) A step of calculating the ratio of the amount of glycated hemoglobin in the hemoglobin-containing sample to the total amount of hemoglobin, based on the total amount of hemoglobin determined in step (1) and the amount of glycated hemoglobin determined in step (5).

[0028] The determination of the total hemoglobin amount in step (1) can also be performed after step (2).

[0029] The total hemoglobin amount can be determined by known methods, such as the cyanmethemoglobin method, the oxyhemoglobin method, the SLS-hemoglobin method, etc.

[0030] When measuring the concentration of HbA1c as glycated hemoglobin, the HbA1c measurement value is calculated as an NGSP value, which is an international standard value, based on the total hemoglobin concentration and HbA1c concentration in the sample, using the following formula.

[0031]

[0032] The reaction between glycated hemoglobin in a hemoglobin-containing sample and proteolytic enzymes is preferably carried out in an aqueous medium. Examples of aqueous mediums include those described later.

[0033] The reaction temperature in the reaction between glycated hemoglobin in a hemoglobin-containing sample and a proteolytic enzyme may be, for example, 10 to 50°C, and may also be 20 to 40°C, 30 to 40°C, 35 to 40°C, or 37°C. The reaction time may be, for example, 1 minute to 3 hours, and may also be 1 minute to 1 hour, 1 to 15 minutes, 1 to 10 minutes, 3 to 7 minutes, or 5 minutes.

[0034] The concentration of the proteolytic enzyme in the reaction solution in the measurement method may be, for example, 50 to 25000 kU / L, and may also be 250 to 15000 kU / L, 1000 to 10000 kU / L, or 3000 to 8000 kU / L.

[0035] As the proteolytic enzyme, an enzyme that acts on glycated hemoglobin in a hemoglobin-containing sample and generates glycated peptides from glycated hemoglobin is used. Examples of the proteolytic enzyme include serine protease (such as chymotrypsin, subtilisin, etc.), cysteine protease (such as papain, caspase, etc.), aspartic acid protease (such as pepsin, cathepsin D, etc.), metalloprotease (such as thermolysin, etc.), N-terminal threonine protease, glutamic acid protease, and the like. In the present embodiment, commercially available proteolytic enzymes can also be used. Examples of commercially available products include protease P "Amano" 3G, protease K "Amano" (both manufactured by Amano Enzyme Co., Ltd.), actinase AS, actinase E (both manufactured by Kaken Pharmaceutical Co., Ltd.), thermolysin (manufactured by Daiwa Kasei Co., Ltd.), Sumiteam MP (manufactured by Shin Nippon Chemical Industry Co., Ltd.), and the like.

[0036] In the reaction in which the above proteolytic enzyme acts, a hemoglobin denaturant such as nitrite, surfactant, etc. may coexist. Examples of the surfactant include cationic surfactant, anionic surfactant, amphoteric surfactant, nonionic surfactant, and the like.

[0037] Examples of the cationic surfactant include quaternary ammonium salt, pyridinium salt, phosphonium salt, imidazolium salt, isoquinolinium salt, etc., and quaternary ammonium salt, pyridinium salt, phosphonium salt are preferred.

[0038] As the pyridinium salt, the pyridinium salt represented by the following general formula (hereinafter referred to as compound (I)) can be used.

[0039] In the formula, R １ is a substituted or unsubstituted alkyl, or a substituted or unsubstituted alkenyl; Ra is a hydrogen atom, a substituted or unsubstituted alkyl, or a substituted or unsubstituted alkenyl; n is an integer from 1 to 5; and X is a monovalent anion.

[0040] R １ In this context, examples of alkyl groups in substituted or unsubstituted alkyl groups include linear alkyl groups having 1 to 20 carbon atoms, branched alkyl groups having 3 to 20 carbon atoms, etc., with linear alkyl groups having 8 to 20 carbon atoms and branched alkyl groups having 8 to 20 carbon atoms being preferred. Examples of linear alkyl groups having 1 to 20 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, eicosyl, etc. Examples of branched alkyl groups having 3 to 20 carbon atoms include isopropyl, isobutyl, isopentyl, isohexyl, isoheptyl, isooctyl, isononyl, isodecyl, isoundecyl, isododecyl, isotridecyl, isotetradecyl, isopentadecyl, isohexadecyl, isoheptadecyl, isooctacyl, isononadecyl, isoicosyl, and octyldodecyl. Examples of linear alkyl groups having 8 to 20 carbon atoms include the aforementioned linear alkyl groups having 8 to 20 carbon atoms. Examples of branched alkyl groups having 8 to 20 carbon atoms include isooctyl, isononyl, isodecyl, isoundecyl, isododecyl, isotridecyl, isotetradecyl, isopentadecyl, isohexadecyl, isoheptadecyl, isooctacyl, isononadecyl, isoicosyl, and octyldodecyl.

[0041] R １In this context, examples of substituted or unsubstituted alkenyls include alkenyls having 2 to 20 carbon atoms, with alkenyls having 8 to 20 carbon atoms being preferred. Examples of alkenyls having 2 to 20 carbon atoms include vinylpropenyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, oleyl, nonadecenyl, and icosenyl. Examples of alkenyls having 8 to 20 carbon atoms include octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, oleyl, nonadecenyl, and icosenyl.

[0042] R １ Examples of substituents in substituted alkyl and substituted alkenyl compounds include phenyl groups, hydroxyl groups, sulfo groups, cyano groups, and halogen atoms. Examples of halogen atoms include chlorine atoms, bromine atoms, and iodine atoms.

[0043] In Ra, examples of alkyl groups in substituted or unsubstituted alkyl groups include linear alkyl groups having 1 to 20 carbon atoms and branched alkyl groups having 3 to 20 carbon atoms. Examples of linear alkyl groups having 1 to 20 carbon atoms include the aforementioned linear alkyl groups having 1 to 20 carbon atoms. Examples of branched alkyl groups having 3 to 20 carbon atoms include the aforementioned branched alkyl groups having 3 to 20 carbon atoms.

[0044] In Ra, examples of substituted or unsubstituted alkenyls include alkenyls having 2 to 20 carbon atoms. Examples of alkenyls having 2 to 20 carbon atoms include the aforementioned linear alkenyls having 2 to 20 carbon atoms.

[0045] Examples of substituents in Ra include phenyl groups, hydroxyl groups, sulfo groups, cyano groups, and halogen atoms. Examples of phenyl-substituted alkyl groups include benzyl and 1-phenylethyl. Examples of halogen atoms include chlorine atoms, bromine atoms, and iodine atoms.

[0046] When there are two or more substituents on the pyridine ring, the substituents may be the same or different. X in the compound (I) represents a monovalent anion. Examples of the monovalent anion include, for example, halogen ions, OH － , PF ６ － , BF ４ － , CH ３ CH ２ OSO ３ － , (CF ３ SO ２ ) ２ N － and other anions. Examples of the halogen ion include, for example, CL － , Br － , I － and the like.

[0047] Specific examples (products) of the compound (I) include, for example, 1-dodecylpyridinium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), 1-cetylpyridinium chloride, 1-cetyl-4-methylpyridinium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), N-octadecyl-4-stilbazole bromide (manufactured by Tokyo Chemical Industry Co., Ltd.) and the like.

[0048] When the reaction solution contains a surfactant, the concentration of the surfactant may be any concentration at which the reaction between the glycated hemoglobin in the hemoglobin-containing sample and the proteolytic enzyme proceeds. For example, it may be 0.001 to 100 g / L, 0.01 to 10 g / L, 0.1 to 8 g / L, 0.5 to 5 g / L, or 1 to 3 g / L.

[0049] By the reaction between the glycated hemoglobin in the hemoglobin-containing sample and the proteolytic enzyme, a reaction product containing a glycated peptide is produced. Then, the glycated peptide in this reaction product reacts with fructosyl peptide oxidase to generate hydrogen peroxide. The reaction between the glycated peptide and fructosyl peptide oxidase is preferably carried out in an aqueous medium. Examples of the aqueous medium include the aqueous media described below.

[0050] The reaction temperature in the reaction between glycated peptide and fructosyl peptide oxidase may be, for example, 10 to 50°C, 20 to 40°C, 30 to 40°C, 35 to 40°C, or 37°C. The reaction time may be, for example, 1 minute to 3 hours, 1 minute to 1 hour, 1 to 15 minutes, 1 to 10 minutes, 3 to 7 minutes, or 5 minutes. The concentration of fructosyl peptide oxidase may be any concentration at which the reaction between glycated hemoglobin and fructosyl peptide oxidase proceeds. The concentration of fructosyl peptide oxidase in the reaction solution may be, for example, 0.1 to 30 kU / L, 0.2 to 15 kU / L, 0.3 to 10 kU / L, 0.5 to 8 kU / L, or 0.8 to 5 kU / L.

[0051] As the fructosyl peptide oxidase, an enzyme that acts on glycated peptides to produce hydrogen peroxide is used. The fructosyl peptide oxidase may be derived from, for example, filamentous fungi, yeast, actinomycetes, bacteria, or archaea. Commercially available fructosyl peptide oxidases may also be used. Examples of commercially available products include FPOX-CE (manufactured by Kikkoman Corporation), FPOX-EE (manufactured by Kikkoman Corporation), and FPOX-CET (manufactured by Kikkoman Corporation).

[0052] Methods for measuring the generated hydrogen peroxide include, for example, methods using electrodes and methods using hydrogen peroxide measuring reagents, with the method using hydrogen peroxide measuring reagents being preferred. Hydrogen peroxide measuring reagents are reagents for converting hydrogen peroxide into a detectable substance. Detectable substances include, for example, dyes, light (luminescence), and fluorescence, with dyes being preferred.

[0053] When the detectable substance is a dye, the reagent for measuring hydrogen peroxide includes a reagent containing a peroxidizing active substance such as peroxidase and an oxidative color-developing chromophor. Examples of oxidative color-developing chromophores include oxidative coupling chromophores and leuco-type chromophores, with leuco-type chromophores being preferred. In this specification, a leuco-type chromophor refers to a substance that is converted into a dye on its own in the presence of hydrogen peroxide and a peroxidizing active substance.

[0054] 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.

[0055] 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.

[0056] Examples of triphenylmethane-based chromogens include N,N,N'N'N''N''-hexa(3-sulfopropyl)-4,4'4''-triaminotriphenylmethane (TPM-PS). 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).

[0057] When the detectable substance is light (luminescence), reagents for measuring hydrogen peroxide include reagents containing peroxidizing active substances such as peroxidase and chemiluminescent substances. Examples of chemiluminescent substances include luminol, isolminol, lucigenin, and acridinium esters.

[0058] When the detectable substance is fluorescent, reagents for measuring hydrogen peroxide include reagents containing a peroxidizing active substance such as peroxidase and a fluorescent substance. Examples of fluorescent substances include 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, and coumarin.

[0059] When using peroxidase as a hydrogen peroxide measurement reagent, the concentration of peroxidase in the reaction solution may be, for example, 10 to 500 kU / L, 20 to 300 kU / L, or 30 to 200 kU / L.

[0060] [Method to reduce the influence of plasma in the sample] The measurement method described above can reduce measurement errors caused by the presence of plasma in the sample. Therefore, the measurement method according to this embodiment can also be described as a method to reduce the influence of plasma in the sample.

[0061] [Measuring Reagent] Another embodiment of the present invention is a reagent for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising a protease, a fructosyl peptide oxidase, a ferrocyanide, and a ferrocene derivative. This measuring reagent can be used in the measurement method described above.

[0062] Since the measurement reagent according to this embodiment contains a ferrocene derivative, when used to measure glycated hemoglobin in a sample containing hemoglobin and plasma, the influence of plasma in the sample can be reduced even in the presence of ferrocyanides, allowing for simpler and more accurate measurement of glycated hemoglobin.

[0063] The concentration of the ferrocyanide in the measurement reagent according to this embodiment may be, for example, 0.01 to 1.0 mg / L, 0.05 to 0.5 mg / L, or 0.1 to 0.3 mg / L.

[0064] The concentration of the ferrocene derivative in the measurement reagent according to this embodiment may be, for example, 0.01 to 30 mg / L, 0.1 to 20 mg / L, or 0.5 to 15 mg / L.

[0065] The concentration of the protease in the measurement reagent according to this embodiment may be, for example, 50 to 25,000 kU / L, 250 to 15,000 kU / L, 1,000 to 12,000 kU / L, or 3,000 to 10,000 kU / L.

[0066] The concentration of fructosyl peptide oxidase in the measurement reagent according to this embodiment may be, for example, 0.1 to 30 kU / L, 0.2 to 15 kU / L, 0.5 to 12 kU / L, or 1 to 10 kU / L.

[0067] The measurement reagent according to this embodiment may further include a surfactant, a hydrogen peroxide measurement reagent, and the like.

[0068] If the measuring reagent contains a surfactant, the concentration of the surfactant in the measuring reagent may be, for example, 0.001 to 100 g / L, or 0.01 to 20 g / L, 0.1 to 15 g / L, 0.5 to 10 g / L, or 1 to 5 g / L.

[0069] If the measuring reagent contains peroxidase as a hydrogen peroxide measuring reagent, the concentration of peroxidase in the measuring reagent may be, for example, 10 to 500 kU / L, 20 to 300 kU / L, or 50 to 200 kU / L.

[0070] The measurement reagent may be in a lyophilized state or dissolved in a medium such as an aqueous medium. When measuring glycated hemoglobin using a lyophilized reagent, the reagent should be dissolved in the medium before use.

[0071] In the measurement reagent according to this embodiment, the sample, proteolytic enzyme, fructosyl peptide oxidase, ferrocyanide, ferrocene derivative, hydrogen peroxide measurement reagent, surfactant, etc., can be adapted to the embodiments of the measurement method described above.

[0072] The measurement reagent according to this embodiment may optionally contain an aqueous medium, stabilizers, preservatives, salts, interfering substance scavengers, organic solvents, etc. Examples of aqueous mediums include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred.

[0073] The pH of the aqueous medium may be, for example, pH 4 to 10. When using a buffer solution as the aqueous medium, it is desirable to use a buffer agent appropriate to the set pH. Examples of buffer agents that can be used in buffer solutions include tris(hydroxymethyl)aminomethane buffer, phosphate buffer, borate buffer, and Good's buffer.

[0074] Examples of buffering agents for Good include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamide)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), and 3-morpholino-2-hydroxypropanesulfonic acid (MOPS). O), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), N-[ Tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid (HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid [(H)EPPS], N-[Tris(hydroxymethyl] Examples include tris(hydroxymethyl)glycine, N,N-bis(2-hydroxyethyl)glycine, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).

[0075] The concentration of the buffer solution may be, for example, 0.001 to 2.0 mol / L, and preferably 0.005 to 1.0 mol / L.

[0076] Examples of stabilizers include ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride, calcium acetate, calcium nitrate, potassium ferrocyanide, bovine serum albumin (BSA), and polyoxyethylene surfactants such as polyoxyethylene alkylphenyl ethers.

[0077] Examples of preservatives include sodium azide and antibiotics. Examples of salts include sodium chloride, sodium nitrate, sodium sulfate, sodium carbonate, sodium formate, sodium acetate, potassium chloride, potassium nitrate, potassium sulfate, potassium carbonate, potassium formate, potassium acetate, etc.

[0078] Examples of interfering substance scavenging agents include ascorbate oxidase, which is used to neutralize the effects of ascorbic acid.

[0079] Examples of organic solvents include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, acetone, methanol, and ethanol, which are used as solubilizers for leuco-type chromogenic compounds in aqueous media.

[0080] [Measurement Kit] The above-mentioned glycated hemoglobin measurement reagent may take the form of a kit consisting of multiple reagents, and may be stored, distributed, and / or used in kit form, for example. The measurement kit may be, for example, a two-reagent system or a three-reagent system, with a two-reagent system being preferred. The protease and fructosyl peptide oxidase may be contained in separate reagents, for example.

[0081] Another embodiment of the present invention is a kit for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising a first reagent containing a protease and a second reagent containing a fructosyl peptide oxidase, wherein the first reagent and / or the second reagent contains a ferrocyanide, and the first reagent and / or the second reagent contains a ferrocene derivative.

[0082] Ferrocyanides and ferrocene derivatives may be included independently in the first reagent, the second reagent, or both. In the case of a kit using a leuco-type chromogenic agent, it is preferable that the leuco-type chromogenic agent and the ferrocyanides or ferrocene derivatives be included in separate reagents to enhance the stability of the leuco-type chromogenic agent. Specifically, if the first reagent contains a leuco-type chromogenic agent, it is preferable that the ferrocyanides and ferrocene derivatives be included in the second reagent, and if the second reagent contains a leuco-type chromogenic agent, it is preferable that the ferrocyanides and ferrocene derivatives be included in the first reagent.

[0083] The measurement kit may contain a hydrogen peroxide measurement reagent in either the first reagent, the second reagent, or both. When using a reagent containing peroxidase and a leuco-type chromogenic agent as the hydrogen peroxide measurement reagent, it is preferable that the peroxidase and the leuco-type chromogenic agent are contained in separate reagents. That is, it is preferable that the peroxidase and the leuco-type chromogenic agent are contained in the first reagent and the second reagent, or in the second reagent and the first reagent, respectively.

[0084] The concentration of the protease in the reagent constituting the measurement kit according to this embodiment may be, for example, 50 to 25,000 kU / L, 250 to 15,000 kU / L, 1,000 to 12,000 kU / L, or 3,000 to 10,000 kU / L.

[0085] The concentration of fructosyl peptide oxidase in the reagents constituting the measurement kit according to this embodiment may be, for example, 0.1 to 30 kU / L, preferably 0.2 to 15 kU / L, 0.5 to 12 kU / L, or 1 to 10 kU / L.

[0086] The measurement kit may contain a surfactant in the first reagent, the second reagent, or both. If the measurement kit contains a surfactant, the concentration of the surfactant in the reagent may be, for example, 0.001 to 100 g / L, 0.01 to 20 g / L, 0.1 to 15 g / L, 0.5 to 10 g / L, or 1 to 5 g / L.

[0087] When measuring glycated hemoglobin using a two-reagent kit, for example, a hemoglobin and plasma-containing sample and the first reagent are added to a reaction cell, the reaction is carried out at a constant temperature for a certain period of time, then the second reagent is added, and the generated hydrogen peroxide is measured to measure the glycated hemoglobin. The reaction conditions can be the same as those of the embodiments of the measurement method described above.

[0088] The measurement kit may further contain, in addition to the first and / or second reagents, a denaturant, an aqueous medium, a stabilizer, a preservative, salts, an interfering substance scavenger, an organic solvent, and the like.

[0089] The glycated hemoglobin measurement kit may be in a lyophilized state or dissolved in a medium such as an aqueous medium. Examples of aqueous mediums include the aforementioned aqueous mediums. When measuring glycated hemoglobin in a hemoglobin-containing sample using a lyophilized kit, the reagents constituting the kit are dissolved in the medium before use.

[0090] In the measurement kit according to this embodiment, the sample, proteolytic enzyme, fructosyl peptide oxidase, ferrocyanide, ferrocene derivative, hydrogen peroxide measurement reagent, surfactant, and other components can be those of the measurement method or measurement reagent described above.

[0091] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. In the following examples, reagents from the following manufacturers were used.

[0092] MES (2-morpholinoethanesulfonic acid monohydrate: manufactured by Dojin Chemical Laboratories), 1-dodecylpyridinium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), potassium ferrocyanide (manufactured by Kanto Chemical Co., Ltd.), (ferrocenylmethyl)trimethylammonium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), thermolysin (proteinase: manufactured by Yamato Chemical Co., Ltd.), peroxidase (manufactured by Toyobo Co., Ltd.), ADA (N-(2-acetamide)iminodiacetic acid: manufactured by Dojin Chemical Laboratories), DA-67 (10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), dimethyl The following substances were used: ruhoxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), FPOX-CET (fructosyl peptide oxidase: manufactured by Kikkoman Corporation), ferrocenyl PEG (ferrocenyl polyethylene glycol: a compound in which polyethylene glycol is bonded to ferrocene: manufactured by Dojin Chemical Laboratories Co., Ltd.), ferrocene carboxaldehyde (manufactured by Tokyo Chemical Industry Co., Ltd.), Metaboreed HbA1c (HbA1c measurement kit: manufactured by Minaris Medical Co., Ltd.), Metaboreed Calibrator for HbA1c measurement (a calibration standard used in combination with Metaboreed HbA1c: manufactured by Minaris Medical Co., Ltd.), and physiological saline (Otsuka Saline Solution: manufactured by Otsuka Pharmaceutical Co., Ltd.).

[0093] [Example 1] Preparation of HbA1c Measurement Kit A measurement kit A was prepared containing the following reagents: (Reagent 1) MES 4.26 g / L 1-Dodecylpyridinium chloride 2.5 g / L Potassium ferrocyanide 0.2 mg / L (Ferrocenylmethyl)trimethylammonium chloride 1 mg / L Thermolysin 7200 kU / L Peroxidase 96 kU / L The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Reagent 2) ADA 5.7 g / L DA-67 26.0 mg / L Dimethyl sulfoxide 1.032 g / L FPOX-CET 5 kU / L The pH was adjusted to 7.0 with an appropriate amount of sodium hydroxide. Dimethyl sulfoxide is used as a solvent to dissolve DA-67, and the solution of DA-67 dissolved in dimethyl sulfoxide was added to the second reagent.

[0094] [Example 2] Preparation of HbA1c measurement kits Kits B, C, D, and E were prepared in the same manner as Kit A, except that the 1 mg / L of (ferrocenylmethyl)trimethylammonium chloride in Kit A of Example 1 was replaced with 10 mg / L of (ferrocenylmethyl)trimethylammonium chloride, 1 mg / L of ferrocenyl PEG, 10 mg / L of ferrocenyl PEG, and 1 mg / L of ferrocenecarboxaldehyde, respectively.

[0095] [Comparative Example 1] Preparation of HbA1c measurement kit Kit a was prepared in the same manner as Kit A of Example 1, except that 1 mg / L of (ferrocenylmethyl)trimethylammonium chloride was not added.

[0096] [Comparative Example 2] Preparation of HbA1c Measurement Kit Kit b was prepared in the same manner as Kit a in Comparative Example 1, except that 0.2 mg / L of potassium ferrocyanide was not added.

[0097] [Comparative Example 3] Preparation of HbA1c measurement kits Kits c and d were prepared in the same manner as kits B and D in Example 2, except that 0.2 mg / L of potassium ferrocyanide was not added.

[0098] [Example 3] Confirmation of correlation between HbA1c (%) measured values ​​in each HbA1c measurement kit and HbA1c (%) values ​​determined by the HbA1c reference method. An automated analyzer JCA-BM9130 (manufactured by JEOL Ltd.) was used as the measurement instrument, and measurement kit A from Example 1 was used as the HbA1c measurement kit. Six concentrations of whole blood, with HbA1c concentrations of 5.09%, 5.57%, 6.15%, 7.10%, 8.23%, and 10.2% respectively, were used as samples. The following procedure was used to calculate the ratio of the HbA1c concentration (amount) to the total hemoglobin concentration (amount) in each sample (HbA1c (%) measured value), and the correlation with the HbA1c (%) value determined using the KO500 method was confirmed.

[0099] (1) Preparation of a calibration curve for determining total hemoglobin concentration Using the first reagent of the commercially available HbA1c measurement kit, Metaboread HbA1c, as the hemoglobin concentration measurement kit, and using Metaboread Calibrator HbA1c measurement (calibration standard; two samples with hemoglobin concentrations of 83.7 μmol / L and 122.1 μmol / L) as the samples, measurements were performed according to the following procedure, and a calibration curve showing the relationship between hemoglobin concentration and absorbance was prepared.

[0100] Each standard (4.8 μL) and the first reagent of Metabolide HbA1c (60 μL) were added to a reaction cell, and the reaction was allowed to proceed at 37°C for 5 minutes. The absorbance of the reaction solution was measured at a primary wavelength of 571 nm and a secondary wavelength of 694 nm, and this was recorded as the absorbance relative to each standard. The same method was used, except that physiological saline was used instead of the standard, and this was recorded as the absorbance relative to physiological saline. The value calculated by subtracting the absorbance relative to physiological saline from the absorbance relative to each standard was recorded as the blank-corrected absorbance for each standard. A calibration curve showing the relationship between total hemoglobin concentration (μmol / L) and absorbance was prepared from the blank-corrected absorbance for each standard and the blank-corrected absorbance relative to physiological saline (0 Abs).

[0101] (2) Preparation of a calibration curve for determining HbA1c concentration Using the measurement kit A from Example 1 as the HbA1c measurement kit, and using two blood cell fractions as samples, the HbA1c concentrations were determined to be 3.84 μmol / L and 11.2 μmol / L, respectively, based on the KO500 method and the total hemoglobin value of the blood cell fraction. Measurements were performed according to the following procedure, and a calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance was created.

[0102] Three μL of each blood cell fraction was mixed with 120 μL of purified water to obtain hemolyzed blood cell fractions. Four μL of the hemolyzed blood cell fraction and five μL of the first reagent from the measurement kit A of Example 1 were added to a reaction cell and reacted at 37°C for five minutes. Next, eight μL of the second reagent from the measurement kit A of Example 1 was added to this reaction solution, and the absorbance of the reaction solution (E1) was measured at a primary wavelength of 658 nm and a secondary wavelength of 805 nm. Next, the reaction was continued at 37°C for five minutes, and the absorbance of the reaction solution (E2) was measured at a primary wavelength of 658 nm and a secondary wavelength of 805 nm. The absorbance difference ΔE was calculated by subtracting E1 from E2 and was taken as the absorbance for each blood cell fraction. The absorbance difference ΔE' was calculated in the same manner as above, except that physiological saline was used instead of each blood cell fraction, and was taken as the absorbance for physiological saline. The blank-corrected absorbance difference for each blood cell fraction was calculated by subtracting the absorbance ΔE' for physiological saline from the absorbance difference ΔE for each blood cell fraction. A calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance was created from the blank-corrected absorbance difference for each blood cell fraction and the blank-corrected absorbance difference for physiological saline (0 Abs).

[0103] (3) Calculation of total hemoglobin concentration in each blood cell fraction One of the six whole blood samples, each with HbA1c concentrations determined to be 5.09%, 5.57%, 6.15%, 7.10%, 8.23%, and 10.2% using the KO500 method, which is the HbA1c reference method, was centrifuged at 25°C and 3000 rpm (1500 × G) for 5 minutes to obtain blood cell fractions. 3 μL of each blood cell fraction was mixed with 120 μL of purified water to obtain hemolyzed blood cell fractions. Each hemolyzed blood cell fraction was measured using the first reagent of Metabolide HbA1c in the same manner as in (1) above, and the total hemoglobin concentration (μmol / L) in each blood cell fraction was calculated from the obtained measurement values ​​and the calibration curve in (1) above.

[0104] (4) Calculation of HbA1c concentration in each blood cell fraction For each hemolyzed blood cell fraction obtained in (3) above, measurements were performed using the measurement kit A of Example 1 in the same manner as in (2) above, and the HbA1c concentration (μmol / L) in each blood cell fraction was calculated from the obtained measurement values ​​and the calibration curve in (2) above.

[0105] (5) Calculation of HbA1c (%) measurement value The HbA1c (%) measurement value in each blood cell fraction was calculated as the NGSP value (International Normalized Value) using the following formula (I) from the total hemoglobin concentration (μmol / L) in each blood cell fraction calculated in (3) above and the HbA1c concentration (μmol / L) in each blood cell fraction calculated in (4) above.

[0106]

[0107] (6) Confirmation of correlation between HbA1c (%) measured value and value obtained by HbA1c reference method The correlation between the measurement method using measurement kit A and the KO500 method was verified from the HbA1c (%) measured value calculated in (5) above using measurement kit A of Example 1 and the HbA1c (%) value determined in advance by the KO500 method, which is the HbA1c reference method, and the correlation coefficient was calculated. The results are shown in Table 1.

[0108] [Example 4] Confirmation of correlation between HbA1c (%) measured values ​​in each HbA1c measurement kit and HbA1c (%) values ​​determined by the HbA1c reference method. The HbA1c (%) measured values ​​in each sample were calculated in the same manner as in Example 3, except that measurement kits B, C, D, and E from Example 2 were used instead of measurement kit A from Example 1. The correlation between the measurement method using measurement kits B, C, D, and E and the KO500 method was verified from the calculated HbA1c (%) measured values ​​and the HbA1c (%) values ​​determined in advance by the KO500 method, which is the HbA1c reference method, and the correlation coefficient was calculated. The results are shown in Table 1.

[0109] [Comparative Example 4] Confirmation of the correlation between HbA1c (%) measured values ​​in each HbA1c measurement kit and HbA1c (%) values ​​determined by the HbA1c reference method. The HbA1c (%) measured values ​​in each sample were calculated in the same manner as in Example 3, except that the measurement kit a of Comparative Example 1, the measurement kit b of Comparative Example 2, and the measurement kits c and d of Comparative Example 3 were used instead of the measurement kit A of Example 1. The correlation between the measurement method using measurement kits a, b, c and d and the KO500 method was verified from the calculated HbA1c (%) measured values ​​and the HbA1c (%) values ​​determined in advance by the KO500 method, which is the HbA1c reference method, and the correlation coefficient was calculated. The results are shown in Table 1.

[0110]

[0111] As shown in Table 1, the correlation coefficient between the HbA1c (%) measured value and the HbA1c (%) value obtained by the HbA1c reference method was 0.8 or higher for each measurement kit used: Measurement Kit A for Example 1, Measurement Kits B, C, D, and E for Example 2, Measurement Kit a for Comparative Example 1, Measurement Kit b for Comparative Example 2, and Measurement Kits c and d for Comparative Example 3. A good correlation was observed between the HbA1c (%) measured value and the HbA1c (%) value obtained by the HbA1c reference method using each measurement kit. Therefore, it became clear that the HbA1c (%) in a sample can be measured using any of the kits: Measurement Kit A for Example 1, Measurement Kits B, C, D, and E for Example 2, Measurement Kit a for Comparative Example 1, Measurement Kit b for Comparative Example 2, and Measurement Kits c and d for Comparative Example 3, as long as the blood cell fraction obtained by centrifugation of whole blood, i.e., a sample from which the plasma component has been removed from whole blood by centrifugation.

[0112] [Example 5] Evaluation of the effect of plasma in each HbA1c measurement kit An automated analyzer JCA-BM9130 (manufactured by JEOL Ltd.) was used as the measurement instrument, and measurement kits A from Example 1 and measurement kits B, C, D, and E from Example 2 were used as the HbA1c measurement kits. Whole blood with an HbA1c concentration of 7.10% determined by the KO500 method, an HbA1c reference method, was used as the sample, and the effect of plasma in each measurement kit was evaluated according to the following procedure.

[0113] (1) Preparation of a calibration curve for determining total hemoglobin concentration A calibration curve showing the relationship between total hemoglobin concentration (μmol / L) and absorbance was prepared using the same method as in (1) of Example 3.

[0114] (2) Preparation of a calibration curve for determining HbA1c concentration A calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance was prepared using the same method as in (2) of Example 3.

[0115] (3) Preparation of samples for evaluation of plasma effects A whole blood sample with an HbA1c concentration of 7.10% determined by the KO500 method, which is the HbA1c reference method, was centrifuged at 25°C and 3000 rpm (1500 × G) for 5 minutes to obtain a blood cell fraction. 3 μL of the blood cell fraction obtained by centrifugation was mixed with 123 μL of purified water to obtain a hemolyzed blood cell fraction (without plasma contamination). In addition, 3 μL of the blood cell fraction obtained by centrifugation was mixed with 3 μL of plasma from a healthy person and 120 μL of purified water to obtain a hemolyzed blood cell fraction (with plasma contamination).

[0116] Note that the total hemoglobin concentration (μmol / L) and HbA1c concentration (μmol / L) in the hemolyzed blood cell fraction (without plasma contamination) are the same, and the only difference is the presence or absence of plasma (the same amount of plasma as in the blood cell fraction). Therefore, if the measurement using each measurement kit is not affected by plasma, the HbA1c (%) measured values ​​will be similar when using the hemolyzed blood cell fraction (without plasma contamination) and the hemolyzed blood cell fraction (with plasma contamination). However, if the measurement using each measurement kit is affected by plasma, the HbA1c (%) measured values ​​will be different when using the hemolyzed blood cell fraction (without plasma contamination) and the hemolyzed blood cell fraction (with plasma contamination).

[0117] (4) Calculation of total hemoglobin concentration in blood cell fractions The total hemoglobin concentration (μmol / L) in each blood cell fraction was calculated in the same manner as in (3) of Example 3, except that the hemolyzed blood cell fraction (without plasma contamination) and the hemolyzed blood cell fraction (with plasma contamination) described in (3) above were used as samples.

[0118] (5) Calculation of HbA1c concentration in each blood cell fraction The HbA1c concentration (μmol / L) in each blood cell fraction was calculated in the same manner as in (4) of Example 3, except that the hemolyzed blood cell fraction (without plasma contamination) and the hemolyzed blood cell fraction (with plasma contamination) described in (3) above were used as samples.

[0119] (6) Calculation of HbA1c (%) measurement value The HbA1c (%) measurement value in each blood cell fraction was calculated as the NGSP value (International Normalized Value) using the formula (I) above, based on the total hemoglobin concentration (μmol / L) in each blood cell fraction calculated in (4) above and the HbA1c concentration (μmol / L) in each blood cell fraction calculated in (5) above.

[0120] (7) Evaluation of the effect of plasma in each HbA1c measurement kit The difference in measurement values ​​when plasma is mixed was calculated for each HbA1c measurement kit by subtracting the HbA1c (%) measurement value when hemolyzed blood cell fraction (with plasma contamination) was used as a sample, calculated in (6) above, from the HbA1c (%) measurement value when hemolyzed blood cell fraction (with plasma contamination) was used as a sample, calculated in (6) above. The results are shown in Table 2.

[0121] [Comparative Example 5] Evaluation of the effect of plasma in each HbA1c measurement kit. The difference in measured values ​​when plasma was mixed in each HbA1c measurement kit was calculated in the same manner as in Example 5, except that the measurement kit a of Comparative Example 1, the measurement kit b of Comparative Example 2, and the measurement kits c and d of Comparative Example 3 were used instead of the measurement kit A of Example 1 and the measurement kits B, C, D, and E of Example 2. The results are shown in Table 2.

[0122]

[0123] If measurements using each testing kit are not affected by plasma, the HbA1c (%) values ​​obtained from hemolyzed blood cell fractions (without plasma contamination) and hemolyzed blood cell fractions (with plasma contamination) will be similar. In other words, the difference between HbA1c (%) values ​​when plasma contamination is present will be close to zero. On the other hand, if measurements using each testing kit are affected by plasma, the HbA1c (%) values ​​obtained from hemolyzed blood cell fractions (without plasma contamination) and hemolyzed blood cell fractions (with plasma contamination) will be different. In other words, the difference between HbA1c (%) values ​​when plasma contamination is present will be far from zero. Therefore, the closer the difference between HbA1c (%) values ​​when plasma contamination is present, the less affected the measurements are by plasma.

[0124] As shown in Table 2, the difference in measured values ​​when plasma contamination occurred was smaller (2.9 to 4.7) when using measurement kits A, B, C, D, and E, which contain both potassium ferrocyanide and ferrocene derivatives, compared to the difference in measured values ​​when plasma contamination occurred when using measurement kit a, which contains potassium ferrocyanide but does not contain ferrocene derivatives (7.5). Therefore, it became clear that in the measurement of glycated hemoglobin, the presence of ferrocene derivatives reduces the influence of plasma when potassium ferrocyanide is present.

[0125] Furthermore, the results in Table 2 show that the difference in measured values ​​when plasma contamination occurred was greater when using measurement kit a, which contains potassium ferrocyanide but not ferrocene derivatives, compared to the difference in measured values ​​when plasma contamination occurred when using measurement kit b, which does not contain either potassium ferrocyanide or ferrocene derivatives (4.0) (7.5). This indicates that the presence of potassium ferrocyanide makes the measurement of glycated hemoglobin more susceptible to the influence of plasma. Also, the results in Table 2 show that the difference in measured values ​​when plasma contamination occurred was greater when using measurement kits c and d, which do not contain potassium ferrocyanide and contain ferrocene derivatives, compared to the difference in measured values ​​when plasma contamination occurred when using measurement kit b, which does not contain either potassium ferrocyanide or ferrocene derivatives (4.0) (4.3-6.4). This indicates that, in the absence of potassium ferrocyanide, ferrocene derivatives do not have the effect of reducing the influence of plasma in the measurement of glycated hemoglobin.

[0126] In summary, it was found that in the measurement of glycated hemoglobin, ferrocene derivatives do not have the effect of reducing the influence of plasma in the absence of potassium ferrocyanide. On the other hand, in the presence of potassium ferrocyanide, the influence of plasma is reduced when ferrocene derivatives are present. Therefore, it was found that by measuring glycated hemoglobin in the presence of potassium ferrocyanide and ferrocene derivatives, the influence of plasma in the sample can be reduced, thereby enabling accurate measurement.

Claims

1. A method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising: reacting glycated hemoglobin in the sample containing hemoglobin and plasma with a proteolytic enzyme and a fructosyl peptide oxidase to produce hydrogen peroxide; and measuring the produced hydrogen peroxide, wherein the reaction is carried out in the presence of a ferrocyanide and a ferrocene derivative.

2. The measurement method according to claim 1, wherein the ferrocyanide is potassium ferrocyanide.

3. The measurement method according to claim 1 or 2, wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocenecarboxaldehyde.

4. The measurement method according to claim 1 or 2, wherein the sample containing hemoglobin and plasma is whole blood.

5. The measurement method according to claim 1 or 2, wherein the measurement of hydrogen peroxide is performed using a hydrogen peroxide measuring reagent containing peroxidase and an oxidative colorimetric chromogen.

6. A method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, comprising reacting glycated hemoglobin in the sample containing hemoglobin and plasma with a proteolytic enzyme and a fructosyl peptide oxidase to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, wherein the reaction is carried out in the presence of a ferrocyanide and a ferrocene derivative to reduce the influence of plasma in the sample.

7. A reagent for measuring glycated hemoglobin in samples containing hemoglobin and plasma, comprising a protease, a fructosyl peptide oxidase, a ferrocyanide, and a ferrocene derivative.

8. The reagent according to claim 7, wherein the ferrocyanide is potassium ferrocyanide.

9. The reagent according to claim 7 or 8, wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocene carboxaldehyde.

10. The reagent according to claim 7 or 8, wherein the sample containing hemoglobin and plasma is whole blood.

11. The reagent according to claim 7 or 8, comprising a hydrogen peroxide measurement reagent containing peroxidase and an oxidative colorimetric chromogen.

12. A kit for measuring glycated hemoglobin in samples containing hemoglobin and plasma, comprising a first reagent containing a protease and a second reagent containing a fructosyl peptide oxidase, wherein the first reagent and / or the second reagent contains a ferrocyanide, and the first reagent and / or the second reagent contains a ferrocene derivative.

13. A kit for measuring glycated hemoglobin in samples containing hemoglobin and plasma, comprising a first reagent containing fructosyl peptide oxidase and a second reagent containing protease, wherein the first reagent and / or the second reagent contains a ferrocyanide, and the first reagent and / or the second reagent contains a ferrocene derivative.

14. The kit according to claim 12 or 13, wherein the ferrocyanide is potassium ferrocyanide.

15. The kit according to claim 12 or 13, wherein the ferrocene derivative is at least one selected from the group consisting of (ferrocenylmethyl)trimethylammonium chloride, ferrocenyl polyethylene glycol, and ferrocenecarboxaldehyde.

16. The kit according to claim 12 or 13, wherein the sample containing hemoglobin and plasma is whole blood.

17. The kit according to claim 12 or 13, wherein the first reagent and / or the second reagent comprises a hydrogen peroxide measurement reagent containing peroxidase and an oxidative colorimetric chromophor.

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