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

Abstract

To provide a measurement method, reagents, and kit for measuring glycated hemoglobin in samples containing hemoglobin and plasma, which is simple, reduces the influence of plasma, and enables accurate measurement. [Solution] 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 above reaction is carried out in the presence of a dithio compound.

Description

[Technical Field]

[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. [Background technology]

[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 analytical methods using high-performance liquid chromatography (HPLC) (see Non-Patent Literature 2) and immunoassay methods using antigen-antibody reactions (see Non-Patent Literature 3).

[0003] In recent years, the development of enzymatic methods for the measurement of 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 the measurement of 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] Furthermore, in a method for measuring glycated hemoglobin using protease and glycated peptide oxidase, a method is known in which thiol chelating agents such as N-ethylmaleimide and 2,2'-dithiodipyridine are used to reduce the influence of thiol compounds, which are hemoglobin stabilizers added to the sample for glycated hemoglobin measurement (see Patent Document 2). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2001-095598 [Patent Document 2] Japanese Patent Publication No. 2010-187604 [Patent Document 3] Japanese Patent Publication No. 2008-245657 [Non-patent literature]

[0006] [Non-Patent Document 1] Clin Chem Lab Med, Vol. 36, pp. 299-308 (1998). [Non-Patent Document 2] Diabetes, Vol. 27, No. 2, pp. 102-107 (1978). [Non-Patent Document 3] Journal of the Japanese Society for Clinical Laboratory Automation, Vol. 18, No. 4, p. 620 (1993). [Overview of the project] [Problems that the invention aims to solve]

[0007] Whole blood and blood cell fractions obtained by centrifuging whole blood to remove plasma components are often used as samples for the enzymatic measurement of HbA1c. To measure HbA1c more simply, it is preferable to use whole blood as the sample, as this does not require centrifugation. 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 3). 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.

[0008] 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 a simple manner and with reduced influence from plasma, for accurate measurement. [Means for solving the problem]

[0009] This disclosure provides, in part, 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 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 a dithio compound. [2] The measurement method according to [1], wherein the dithio compound is dithiodipyridine. [3] The measurement method according to [2], wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine. [4] The measurement method according to any one of [1] to [3], wherein the sample containing hemoglobin and plasma is whole blood. [5] 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 above reaction is carried out in the presence of a dithio compound, thereby reducing the influence of plasma in the sample. [6] The method according to [5], wherein the dithio compound is dithiodipyridine. [7] The method according to [6], wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine. [8] The method according to any one of [5] to [7], wherein the sample containing hemoglobin and plasma is whole blood. [9] Reagents for measuring glycated hemoglobin in samples containing hemoglobin and plasma, comprising a protease, fructosyl peptide oxidase, and dithio compounds.

[10] The reagent according to [9], wherein the dithio compound is dithiodipyridine.

[11] The reagent according to

[10] , wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine.

[12] The reagent according to any one of [9] to

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

[13] 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 contain a dithio compound.

[14] 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 contain a dithio compound.

[15] The kit according to

[13] or

[14] , wherein the dithio compound is dithiodipyridine.

[16] The kit according to

[15] , wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'dithiodipyridine and 4-4'dithiodipyridine.

[17] The kit according to any one of

[13] to

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

Advantages of the Invention

[0010] According to the present invention, there are provided a measurement method, a measurement reagent, and a measurement kit for simply and accurately measuring glycated hemoglobin in a sample containing hemoglobin and plasma while reducing the influence of plasma.

Embodiments for Carrying Out the Invention

[0011] Hereinafter, embodiments for carrying out the present disclosure will be described in detail. The embodiments described below show an example of typical embodiments of the present disclosure, and the scope of the disclosure is not construed narrowly thereby. The numerical range indicated by "~" indicates a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In addition, the amount of each component in the solution means the total amount of the plurality of substances present in the reaction or reagent when there are a plurality of substances corresponding to each component in the solution, unless otherwise specified.

[0012] [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 fructosylpeptide oxidase to generate hydrogen peroxide, and measuring the generated hydrogen peroxide, wherein the reaction is carried out in the presence of a dithio compound.

[0013] The measurement method according to this embodiment allows for the suppression of measurement errors caused by plasma by carrying out the reaction in the presence of dithio compounds. By using the measurement method according to this embodiment, even when using whole blood that has not been centrifuged as a sample, measurement errors due to the presence of plasma can be reduced, and glycated hemoglobin can be measured more simply and accurately.

[0014] 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.

[0015] 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 sum of blood cell components and plasma components is set to 100%, the proportion of plasma components is 5-70% by mass, 20-70% by mass, or 40-70% by mass.

[0016] 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.

[0017] The dithio compound used in the measurement method according to this embodiment may be, for example, dithiodipyridine. The dithiodipyridine may be, for example, at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine. The dithio compound is preferably 2-2'-dithiodipyridine.

[0018] In the measurement method according to this embodiment, the concentration of the dithio compound in the reaction solution may be, for example, 0.01 to 30 mg / L, 0.1 to 25 mg / L, 1 to 15 mg / L, or 5 to 10 mg / L.

[0019] 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.

[0020] The measurement method according to this embodiment may specifically include, for example, the following steps. (1) A step of reacting a proteolytic enzyme with glycated hemoglobin in a hemoglobin and plasma-containing sample. (2) A step in which the reaction product obtained in step (1) is reacted with fructosyl peptide oxidase in the presence of a dithio compound to generate hydrogen peroxide. (3) A step to measure the hydrogen peroxide generated in step (2). (4) A step to determine the concentration of glycated hemoglobin in a hemoglobin-containing sample from the amount of hydrogen peroxide measured in step (3), based on a calibration curve that was prepared in advance using glycated hemoglobin of known concentrations and shows the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin.

[0021] In the measurement method according to this embodiment, the dithio compound is present in the presence of at least step (2). The dithio compound may also be present from step (1).

[0022] 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 total amount of hemoglobin (i.e., the 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.

[0023] (1) A step to determine the total amount of hemoglobin (i.e., total hemoglobin, which is the sum of hemoglobin and glycated hemoglobin) in a hemoglobin-containing sample. (2) A step of reacting glycated hemoglobin in a hemoglobin-containing sample with a proteolytic enzyme. (3) A step in which the reaction product obtained in step (2) is reacted with fructosyl peptide oxidase in the presence of a dithio compound to produce hydrogen peroxide. (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 was prepared in advance using a known amount of glycated hemoglobin and shows the relationship between the amount of hydrogen peroxide and the amount of glycated hemoglobin. (6) A step to calculate 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).

[0024] The determination of the total hemoglobin amount in step (1) can also be performed after step (2).

[0025] The total hemoglobin amount can be determined by known methods, such as the cyanmethemoglobin method, the oxyhemoglobin method, or the SLS-hemoglobin method.

[0026] When measuring the concentration of HbA1c as glycated hemoglobin, the HbA1c measurement value is calculated as an international standard value (NGSP value) based on the total hemoglobin concentration and HbA1c concentration in the sample using the following formula.

[0027]

number

[0028] 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.

[0029] The reaction temperature in the reaction between glycated hemoglobin in a hemoglobin-containing sample and proteolytic enzymes may be, for example, 10 to 50°C, but 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, but may also be 1 minute to 1 hour, 1 to 15 minutes, 1 to 10 minutes, 3 to 7 minutes, or 5 minutes.

[0030] The concentration of proteolytic enzyme in the reaction solution in the measurement method may be, for example, 50 to 25,000 kU / L, 250 to 15,000 kU / L, 1,000 to 10,000 kU / L, or 3,000 to 8,000 kU / L.

[0031] As the proteolytic enzyme, an enzyme is used that acts on glycated hemoglobin in a hemoglobin-containing sample to produce glycated peptides from glycated hemoglobin. Examples of proteolytic enzymes include serine proteases (chymotrypsin, subtilisin, etc.), cysteine ​​proteases (papain, caspase, etc.), aspartate proteases (pepsin, cathepsin D, etc.), metalloproteases (thermolysin, etc.), N-terminal threonine proteases, glutamate proteases, etc. In this 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 Pharma Co., Ltd.), Thermolysin (manufactured by Yamato Kasei Co., Ltd.), Sumizyme MP (manufactured by Shin Nippon Chemical Industries, Ltd.), etc.

[0032] In the reaction in which the above-mentioned proteolytic enzyme is applied, hemoglobin denaturants such as nitrites and surfactants may be present. Examples of surfactants include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants.

[0033] Examples of cationic surfactants include quaternary ammonium salts, pyridinium salts, phosphonium salts, imidazolium salts, and isoquinolinium salts, with quaternary ammonium salts, pyridinium salts, and phosphonium salts being preferred.

[0034] As the pyridinium salt, the pyridinium salt represented by the following general formula (hereinafter referred to as compound (I)) can be used. [ka]

[0035] 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.

[0036] 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, isoheftadecyl, isooctdecyl, isononadecyl, isoicosyl, and octyldodecyl. Examples of linear alkyl groups having 8 to 20 carbon atoms include the linear alkyl groups with 8 to 20 carbon atoms mentioned above. Examples of branched alkyl groups having 8 to 20 carbon atoms include isooctyl, isononyl, isodecyl, isoundecyl, isododecyl, isotridecyl, isotetradecyl, isopentadecyl, isohexadecyl, isoheptadecyl, isooctdecyl, isononadecyl, isoicosyl, and octyldodecyl.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In Ra, examples of the substituents in the substituted alkyl and substituted alkenyl include a phenyl group, a hydroxyl group, a sulfo group, a cyano group, a halogen atom, etc. Examples of the phenyl group-substituted alkyl include benzyl, 1-phenylethyl, etc. Examples of the halogen atom include a chlorine atom, a bromine atom, an iodine atom, etc.

[0042] 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 a halogen ion, OH - , PF6 - , BF4 - , CH3CH2OSO3 - , (CF3S02)2N - and other anions. Examples of the halogen ion include CL - , Br - , I - etc.

[0043] Specific examples (products) of the compound (I) include, for example, 1-dodecylpyridinium chloride (hereinafter referred to as C12py, manufactured by Tokyo Chemical Industry Co., Ltd.), 1-hexadecylpyridinium chloride, 1-hexadecyl-4-methylpyridinium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), N-octadecyl-4-stilbazole bromide (manufactured by Tokyo Chemical Industry Co., Ltd.), etc.

[0044] 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.

[0045] The reaction between glycated hemoglobin in a hemoglobin-containing sample and a proteolytic enzyme produces a reaction product containing glycated peptides. Subsequently, the glycated peptides in this reaction product react with fructosyl peptide oxidase to produce hydrogen peroxide. The reaction between glycated peptides and fructosyl peptide oxidase is preferably carried out in an aqueous medium. Examples of aqueous mediums include those described later.

[0046] The reaction temperature in the reaction between glycated peptide and fructosyl peptide oxidase may be, for example, 10-50°C, 20-40°C, 30-40°C, 35-40°C, or 37°C. The reaction time may be, for example, 1 minute to 3 hours, 1 minute to 1 hour, 1-15 minutes, 1-10 minutes, 3-7 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, 0.1-30 kU / L, 0.2-15 kU / L, 0.3-10 kU / L, 0.5-8 kU / L, or 0.8-5 kU / L.

[0047] 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).

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] 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.

[0054] 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.

[0055] When using peroxidase as a hydrogen peroxide measurement reagent, the concentration of peroxidase in the reaction solution may be, for example, 10-500 kU / L, 20-300 kU / L, or 30-200 kU / L.

[0056] [Methods to mitigate the effects of plasma in a 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 for reducing the influence of plasma in the sample.

[0057] [Method reagents] 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, and a dithio compound. This measuring reagent can be used in the measurement method described above.

[0058] Since the measurement reagent according to this embodiment contains a dithio compound, when used to measure glycated hemoglobin in a sample containing hemoglobin and plasma, the influence of plasma present in the sample can be reduced, allowing for simpler and more accurate measurement of glycated hemoglobin.

[0059] The concentration of the dithio compound in the measurement reagent according to this embodiment may be, for example, 0.01 to 30 mg / L, 0.1 to 25 mg / L, 1 to 20 mg / L, or 5 to 15 mg / L.

[0060] 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.

[0061] 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.

[0062] The measurement reagent according to this embodiment may further include a surfactant, a hydrogen peroxide measurement reagent, and the like.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] In the measurement reagent according to this embodiment, the sample, protease, fructosyl peptide oxidase, dithio compound, hydrogen peroxide measurement reagent, surfactant, etc., can be adapted to the embodiments of the measurement method described above.

[0067] 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.

[0068] 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.

[0069] 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 (TAPS0), 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Examples of interfering substance scavenging agents include ascorbate oxidase, which is used to neutralize the effects of ascorbic acid.

[0074] 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.

[0075] [Measurement kit] The glycated hemoglobin measurement reagent described above 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.

[0076] 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 contain a dithio compound.

[0077] The dithio compound may be included in the first reagent, the second reagent, or both, and is preferably included in at least the first reagent.

[0078] The measurement kit may contain the 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.

[0079] The concentration of the protease in the reagents 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.

[0080] 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.

[0081] 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.

[0082] 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 glycated hemoglobin. The reaction conditions can be the same as those of the embodiments of the measurement method described above.

[0083] 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.

[0084] 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.

[0085] In the measurement kit according to this embodiment, the sample, protease, fructosyl peptide oxidase, dithio compound, hydrogen peroxide measurement reagent, surfactant, and other components can be those of the measurement method or measurement reagent described above. [Examples]

[0086] 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.

[0087] MES (2-morpholinoethanesulfonic acid monohydrate; manufactured by Dojin Chemical Laboratories), 1-dodecylpyridinium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), 2,2'-dithiodipyridine (manufactured by Fujifilm Wako Pure Chemical Industries, 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 Co., Ltd.), DA-67 (10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine) The following substances were used: sodium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), FPOX-CET (fructosyl peptide oxidase; manufactured by Kikkoman Corporation), N-ethylmaleimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Metabolid HbA1c (HbA1c measurement kit; manufactured by Minaris Medical Co., Ltd.), Metabolid Calibrator for HbA1c measurement (calibration standard used in combination with Metabolid HbA1c; manufactured by Minaris Medical Co., Ltd.), and physiological saline (Otsuka Saline Solution; manufactured by Otsuka Pharmaceutical Co., Ltd.).

[0088] [Example 1] Preparation of an HbA1c measurement kit A measurement kit A was prepared containing reagents 1 and 2 with the following compositions. (First reagent) MES 4.26g / L 1-Dodecylpyridinium chloride 2.5g / L 2,2'-Dithiodipyridine 10 mg / L Thermolysin 7200kU / L Peroxidase 96 kU / L The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Second reagent) ADA 5.7g / L DA-67 26.0 mg / L Dimethyl sulfoxide 1.032 g / L FPOX-CET 5kU / L The pH was adjusted to 7.0 with an appropriate amount of sodium hydroxide. Dimethyl sulfoxide was used as a solvent to dissolve DA-67, and the solution of DA-67 dissolved in dimethyl sulfoxide was added to the second reagent.

[0089] [Comparative Example 1] Preparation of an HbA1c measurement kit Kit a was prepared using the same method as Kit A in Example 1, except that 10 mg / L of 2,2'-dithiodipyridine was not added.

[0090] [Comparative Example 2] Preparation of an HbA1c measurement kit Kit b was prepared in the same manner as Kit A in Example 1, except that the 2,2'-dithiodipyridine 10 mg / L was replaced with N-ethylmaleimide 10 mg / L.

[0091] [Example 2] Confirmation of correlation between HbA1c (%) measured values ​​in each HbA1c measurement kit and HbA1c (%) values ​​determined by the HbA1c reference method. Using the JCA-BM9130 automated analyzer (manufactured by JEOL Ltd.) as the measuring instrument, and the measurement kit A from Example 1 as the HbA1c measurement kit, we used whole blood samples with six HbA1c concentrations determined to be 5.09%, 5.57%, 6.15%, 7.10%, 8.23%, and 10.2% respectively by the KO500 method, which is the HbA1c reference method. Following the procedure below, we calculated the ratio of the HbA1c concentration (amount) to the total hemoglobin concentration (amount) in each sample (HbA1c (%) measured value) and confirmed its correlation with the HbA1c (%) value determined using the KO500 method.

[0092] (1) Creation 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 created. 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).

[0093] (2) Creation 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.

[0094] 3 μL of each blood cell fraction was mixed with 120 μL of purified water to obtain hemolyzed blood cell fractions. The hemolyzed blood cell fractions (4.8 μL) and the first reagent (54 μL) of the measurement kit A from Example 1 were added to a reaction cell and reacted at 37°C for 5 minutes. Next, the second reagent (18 μL) of the measurement kit A from 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 5 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, 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).

[0095] (3) Calculation of total hemoglobin concentration in each blood cell fraction Using the KO500 method, which is the HbA1c reference method, six whole blood samples with HbA1c concentrations determined to be 5.09%, 5.57%, 6.15%, 7.10%, 8.23%, and 10.2% were 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.

[0096] (4) Calculation of HbA1c concentration in each blood cell fraction Each hemolyzed blood cell fraction obtained in (3) above was measured using the same method as in (2) above with the measurement kit A from Example 1, 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.

[0097] (5) Calculation of HbA1c (%) measurement value Based on 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, the HbA1c (%) measured value in each blood cell fraction was calculated as the NGSP value (International Normalized Value) using the following formula (I).

[0098]

number

[0099] (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 by comparing the HbA1c (%) measured value calculated in (5) above using measurement kit A in Example 1 with the HbA1c (%) value determined in advance by the KO500 method, which is an HbA1c reference method, and the correlation coefficient was calculated. The results are shown in Table 1.

[0100] [Comparative Example 3] Confirmation of the correlation between HbA1c (%) measured values ​​in each HbA1c measurement kit and HbA1c (%) values ​​determined by the HbA1c reference method. The HbA1c (%) values ​​in each sample were calculated using the same method as in Example 2, except that the measurement kit a from Comparative Example 1 and the measurement kit b from Comparative Example 2 were used instead of the measurement kit A from Example 1. The correlation between the measurement method using measurement kits a and b and the KO500 method was verified using the calculated HbA1c (%) values ​​and the HbA1c (%) values ​​determined in advance by the KO500 method, which is an HbA1c reference method, and the correlation coefficient was calculated. The results are shown in Table 1.

[0101] [Table 1]

[0102] As shown in Table 1, the correlation coefficient between the HbA1c (%) measured value and the HbA1c (%) value obtained by the HbA1c reference method was 0.9 or higher for each measurement kit used in Example 1 (Kit A), Comparative Example 1 (Kit a), and Comparative Example 2 (Kit b), indicating a good correlation between the HbA1c (%) measured value and the HbA1c (%) value obtained by the HbA1c reference method. Therefore, it became clear that the HbA1c (%) in a sample can be measured using any of the kits: Measurement Kit A in Example 1, Measurement Kit a in Comparative Example 1, and Measurement Kit b in Comparative Example 2, as long as the blood cell fraction obtained by centrifugation of whole blood is used, i.e., the sample from which the plasma component has been removed by centrifugation.

[0103] [Example 3] Evaluation of the effect of plasma on each HbA1c measurement kit The effect of plasma on each measurement kit was evaluated using the following procedure, with the automated analyzer JCA-BM9130 (manufactured by JEOL Ltd.) as the measuring instrument, measurement kit A from Example 1 as the HbA1c measurement kit, and whole blood with an HbA1c concentration of 7.10% determined by the KO500 method, which is the HbA1c reference method, as the sample.

[0104] (1) Creation 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 Example 2 (1).

[0105] (2) Creation of a calibration curve for determining HbA1c concentration A calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance was created using the same method as in (2) of Example 2.

[0106] (3) Preparation of samples for evaluation of plasma effects Whole blood samples with an HbA1c concentration of 7.10% determined by the KO500 method, an HbA1c reference method, were 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). Additionally, 3 μL of the blood cell fraction obtained by centrifugation was mixed with 3 μL of healthy human plasma and 120 μL of purified water to obtain a hemolyzed blood cell fraction (with plasma contamination). 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).

[0107] (4) Calculation of total hemoglobin concentration in blood cell fractions The total hemoglobin concentration (μmol / L) in each blood cell fraction was calculated using the same method as in Example 2 (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.

[0108] (5) Calculation of HbA1c concentration in each blood cell fraction The HbA1c concentration (μmol / L) in each blood cell fraction was calculated using the same method as in (4) of Example 2, 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.

[0109] (6) Calculation of HbA1c (%) measurement value 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, the HbA1c (%) measurement value in each blood cell fraction was calculated as the NGSP value (International Normalized Value) using the above formula (I).

[0110] (7) Evaluation of the effect of plasma on each HbA1c measurement kit The difference in measured values ​​when plasma is present in each HbA1c measurement kit was calculated by subtracting the HbA1c (%) measured value when hemolyzed blood cell fraction (with plasma contamination) was used as a sample (calculated in (6) above) from the HbA1c (%) measured 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.

[0111] [Comparative Example 4] Evaluation of the effect of plasma in each HbA1c measurement kit The difference in measured values ​​when plasma was added to each HbA1c measurement kit was calculated using the same method as in Example 3, except that the measurement kits from Comparative Example 1 (kit a) and Comparative Example 2 (kit b) were used instead of the measurement kit A from Example 1. The results are shown in Table 2.

[0112] [Table 2]

[0113] 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 results are by plasma.

[0114] As shown in Table 2, the difference in measured values ​​when plasma contamination occurred was smaller when using measurement kit A containing dithio compounds (1.3) compared to when using measurement kit a without dithio compounds (4.0). Therefore, it was found that the presence of dithio compounds reduces the influence of plasma on the measurement of glycated hemoglobin.

[0115] Furthermore, the results in Table 2 show that the difference in measured values ​​when plasma contamination occurred was greater when using measurement kit b containing N-ethylmaleimide (6.3) compared to when using measurement kit a which does not contain dithio compounds or N-ethylmaleimide (4.0). This clearly indicates that N-ethylmaleimide does not have the effect of reducing the influence of plasma on the measurement of glycated hemoglobin.

[0116] From the above findings, it was revealed that the presence of dithio compounds reduces the influence of plasma on the measurement of glycated hemoglobin. Therefore, it was found that measuring glycated hemoglobin in the presence of dithio compounds can reduce the influence of plasma in the sample, enabling more accurate measurements.

Claims

1. The process involves reacting glycated hemoglobin in a sample containing hemoglobin and plasma with a proteolytic enzyme and a fructosyl peptide oxidase to produce hydrogen peroxide, This includes measuring the hydrogen peroxide produced. A method for measuring glycated hemoglobin in a sample containing hemoglobin and plasma, wherein the reaction is carried out in the presence of a dithio compound.

2. The measurement method according to claim 1, wherein the dithio compound is dithiodipyridine.

3. The measurement method according to claim 2, wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine.

4. The measurement method according to any one of claims 1 to 3, wherein the sample containing hemoglobin and plasma is whole blood.

5. In measuring glycated hemoglobin in a sample containing hemoglobin and plasma, The process involves reacting glycated hemoglobin in a sample containing hemoglobin and plasma with a proteolytic enzyme and a fructosyl peptide oxidase to produce hydrogen peroxide, This includes measuring the hydrogen peroxide produced. A method for carrying out the above reaction in the presence of a dithio compound to reduce the influence of plasma in the sample.

6. A reagent comprising a protease, a fructosyl peptide oxidase, and a dithio compound for measuring glycated hemoglobin in samples containing hemoglobin and plasma.

7. The reagent according to claim 6, wherein the dithio compound is dithiodipyridine.

8. The reagent according to claim 7, wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine.

9. The reagent according to any one of claims 6 to 8, wherein the sample containing hemoglobin and plasma is whole blood.

10. 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 contain a dithio compound.

11. 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 contain a dithio compound.

12. The kit according to claim 10 or 11, wherein the dithio compound is dithiodipyridine.

13. The kit according to claim 12, wherein the dithiodipyridine is at least one selected from the group consisting of 2-2'-dithiodipyridine and 4-4'-dithiodipyridine.

14. The kit according to claim 10 or 11, wherein the sample containing hemoglobin and plasma is whole blood.

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