Method for reducing the effects of hemoglobin, method for measuring glycated hemoglobin, reagent for measuring glycated hemoglobin, and kit for measuring glycated hemoglobin

Abstract

Provided is a method for measuring glycated hemoglobin in a hemoglobin-containing sample, said method involving generating hydrogen peroxide by reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction for generating hydrogen peroxide by oxidizing the glycated hemoglobin, and measuring the generated hydrogen peroxide, wherein a method for mitigating the impact of hemoglobin in the method for measuring glycated hemoglobin is characterized by reacting, in the presence of at least one type of compound selected from the group consisting of dithio-based compounds and maleimide-based compounds, glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction for generating hydrogen peroxide by oxidizing glycated hemoglobin.

Description

[Technical field]

[0001] The present invention relates to a method for reducing the effect of hemoglobin, a method for measuring glycated hemoglobin, a reagent for measuring glycated hemoglobin, and a kit for measuring glycated hemoglobin. This application claims priority based on Japanese Patent Application No. 2020-55731, filed on March 26, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Glycated proteins are contained in body fluids such as blood in living organisms and biological samples such as hair. The concentration of glycated proteins present in blood depends on the concentration of sugars such as glucose dissolved in serum, and in the field of clinical diagnosis, the measurement of the concentration of hemoglobin A1c (hereinafter referred to as HbA1c), a glycated protein in blood, is used for diagnosing and monitoring diabetes (see Non-Patent Document 1). Hemoglobin is a hemoprotein with a molecular weight of 64,000, which has two types of subunits, α chain and β chain. HbA1c is particularly defined as a β chain in which the N-terminal valine residue is glycated. Methods for measuring this HbA1c include an instrumental analysis method using high performance liquid chromatography (HPLC) (see Non-Patent Document 2) and an immunoassay method using an antigen-antibody reaction (Non-Patent Document 3).

[0003] In recent years, enzymatic methods for measuring HbA1c that can be applied to general-purpose automated analyzers and are easy to operate have been developed, and various methods have been reported. The enzymatic methods for measuring HbA1c that have been reported so far mainly use proteases and glycopeptide oxidases. That is, there is a method of measuring HbA1c in a sample by acting a protease on HbA1c in blood cells to produce a glycated peptide, fructosyl dipeptide (Fru-Val-His), acting fructosyl peptide oxidase on the fructosyl dipeptide to produce hydrogen peroxide, and measuring the hydrogen peroxide produced (see Patent Document 1); and there is a method of measuring HbA1c in a sample by acting a protease on HbA1c in blood cells to produce a glycated peptide, fructosyl hexapeptide (Fru-Val-His-Leu-Thr-Pro-Glu), acting fructosyl hexapeptide oxidase on the fructosyl hexapeptide to produce hydrogen peroxide, and measuring the hydrogen peroxide produced (see Patent Documents 2 to 4). On the other hand, a method for measuring glycated hemoglobin in a sample is also known that does not use a protease but uses an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to generate hydrogen peroxide (see Patent Documents 4 to 7 and Non-Patent Document 4).

[0004] Furthermore, in a method for measuring glycated hemoglobin using protease and glycated peptide oxidase, a method has been reported in which a thiol blocking agent is used to reduce the influence of a thiol compound, which is a hemoglobin stabilizer added to a sample for measuring glycated hemoglobin (see Patent Document 8). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2001-95598 A [Patent Document 2] International Publication No. 2008 / 108385 [Patent Document 3] International Publication No. 2013 / 162035 [Patent Document 4] International Publication No. 2015 / 005258 [Patent Document 5] International Publication No. 2015 / 005257 [Patent Document 6] International Publication No. 2015 / 060429 [Patent Document 7] International Publication No. 2018 / 021530 [Patent Document 8] JP 2010-187604 A [Non-patent literature]

[0006] [Non-Patent Document 1] Clin Chem Lab Med, Vol. 36, p. 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). [Non-Patent Document 4] Scientific Reports (2019) 9:942 Summary of the Invention [Problem to be solved by the invention]

[0007] However, in a method for measuring glycated hemoglobin using an enzyme that catalyzes a reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide without using a protease, there is a problem that the concentration of glycated hemoglobin in a specimen cannot be accurately measured due to the influence of hemoglobin, which is a reducing substance present in a hemoglobin-containing sample. Therefore, an object of the present invention is to provide a method for reducing the influence of hemoglobin present in a hemoglobin-containing sample in a method for measuring glycated hemoglobin using an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide without using a protease, a method for simply and accurately measuring glycated hemoglobin in a hemoglobin-containing sample, a glycated hemoglobin measurement reagent, and a glycated hemoglobin measurement kit. [Means for solving the problem]

[0008] The present inventors have conducted intensive research to solve the problem, and have found that in a method for measuring glycated hemoglobin in a hemoglobin-containing sample using an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide without using a protease, by reacting the glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of dithio compounds and maleimide compounds, the influence of hemoglobin present in the hemoglobin-containing sample is reduced, and glycated hemoglobin in the hemoglobin-containing sample can be measured simply and accurately, thereby completing the present invention.

[0009] That is, the present invention relates to the following [1] to

[27] . [1] A method for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, comprising: A method for reducing the influence of hemoglobin in a method for measuring glycated hemoglobin, comprising reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of dithio compounds and maleimide compounds. [2] A method for reducing the effects of hemoglobin described in [1], comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide in the presence of a denaturing agent for glycated hemoglobin. [3] The method for reducing the effect of hemoglobin according to [1] or [2], wherein the dithio compound is 6,6-dithiodinicotinic acid or a derivative thereof. [4] The method for reducing the effect of hemoglobin according to any one of [1] to [3], wherein the maleimide compound is 6-maleimidohexanoic acid or a derivative thereof. [5] The method for reducing the effect of hemoglobin according to any one of [1] to [4], wherein the measurement of hydrogen peroxide is carried out using a hydrogen peroxide measurement reagent. [6] The method for reducing the effect of hemoglobin described in [5], wherein the hydrogen peroxide measurement reagent is a reagent containing peroxidase and a leuco chromogen. [7] The method for reducing the effect of hemoglobin according to [6], wherein the leuco chromogen is a phenothiazine chromogen. [8] The method for reducing the effect of hemoglobin according to [7], wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof. [9] A method for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of dithio compounds and maleimide compounds to produce hydrogen peroxide, and measuring the hydrogen peroxide produced.

[10] A method for measuring glycated hemoglobin described in [9], comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of a denaturing agent for glycated hemoglobin.

[11] The method for measuring glycated hemoglobin described in [9] or

[10] , wherein the dithio compound is 6,6-dithiodinicotinic acid or a derivative thereof.

[12] The method for measuring glycated hemoglobin according to any one of [9] to

[11] , wherein the maleimide compound is 6-maleimidohexanoic acid or a derivative thereof.

[13] The method for measuring glycated hemoglobin according to any one of [9] to

[12] , wherein the measurement of hydrogen peroxide is carried out using a reagent for measuring hydrogen peroxide.

[14] The method for measuring glycated hemoglobin according to

[13] , wherein the hydrogen peroxide measurement reagent is a reagent containing peroxidase and a leuco chromogen.

[15] The method for measuring glycated hemoglobin according to

[14] , wherein the leuco chromogen is a phenothiazine chromogen.

[16] The method for measuring glycated hemoglobin according to

[15] , wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

[17] A reagent for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide, and at least one compound selected from the group consisting of dithio compounds and maleimide compounds.

[18] The measurement reagent according to

[17] , further comprising a denaturant for glycated hemoglobin.

[19] The measurement reagent according to

[17] or

[18] , further comprising a hydrogen peroxide measurement reagent.

[20] The hydrogen peroxide measurement reagent according to

[19] , which is a reagent containing peroxidase and a leuco chromogen.

[21] The measurement reagent according to

[20] , wherein the leuco chromogen is a phenothiazine chromogen.

[22] The measurement reagent according to

[21] , wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

[23] A kit for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising: a first reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and a second reagent containing an enzyme that catalyzes a reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide.

[24] The measurement kit described in

[23] , wherein a denaturant for glycated hemoglobin is contained in either or both of the first reagent and the second reagent.

[25] The measurement kit according to

[23] or

[24] , further comprising a peroxidase and a leuco chromogen in the first and second reagents, or in the second and first reagents, respectively.

[26] The measurement kit according to

[25] , wherein the leuco chromogen is a phenothiazine chromogen.

[27] The measurement kit according to

[26] , wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof. Effect of the Invention

[0010] The present invention provides a method for reducing the influence of hemoglobin in a hemoglobin-containing sample, a method for simply and accurately measuring glycated hemoglobin in a hemoglobin-containing sample, a reagent for measuring glycated hemoglobin, and a kit for measuring glycated hemoglobin, in a method for measuring glycated hemoglobin in a hemoglobin-containing sample using an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide, without using a protease. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing the relationship between HbA1c concentration and absorbance difference ΔE in the measurement of HbA1c in a hemoglobin-containing sample using Kit A containing FPOX-47Δ3 as the second reagent. The vertical axis represents absorbance difference ΔE (Abs×10000), and the horizontal axis represents HbA1c concentration (μmol / L). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] (1) Method for reducing the effect of hemoglobin in the measurement of glycated hemoglobin The method for reducing the influence of hemoglobin in the method for measuring glycated hemoglobin of the present invention is a method for measuring glycated hemoglobin in a hemoglobin-containing sample, which comprises reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide to generate hydrogen peroxide, and measuring the generated hydrogen peroxide, characterized in that the glycated hemoglobin in the hemoglobin-containing sample is reacted with the enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of dithio compounds and maleimide compounds. In the method for reducing the effect of hemoglobin of the present invention, the method for measuring glycated hemoglobin in a hemoglobin-containing sample specifically comprises the following steps.

[0013] (i) reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds, to generate hydrogen peroxide; (ii) measuring the hydrogen peroxide produced in step (i); and (iii) determining the concentration of glycated hemoglobin in the hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in step (ii) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out measurements in accordance with steps (i) and (ii) above using glycated hemoglobin of known concentrations.

[0014] In the above step (i), the step of reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds may be a step of reacting at least one compound selected from the group consisting of dithio compounds and maleimide compounds with hemoglobin in a hemoglobin-containing sample in an aqueous medium, and then adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium to react glycated hemoglobin in the hemoglobin-containing sample with the enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide. Specifically, the measurement method includes the following steps.

[0015] (i) reacting hemoglobin in a hemoglobin-containing sample with at least one compound selected from the group consisting of dithio compounds and maleimide compounds in an aqueous medium; (ii) adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium of step (i) to generate hydrogen peroxide; (iii) measuring the hydrogen peroxide produced in step (ii); and (iv) determining the concentration of glycated hemoglobin in a hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iii) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out measurements according to the steps (i) to (iii) above using glycated hemoglobin of known concentrations.

[0016] The method for measuring glycated hemoglobin in a hemoglobin-containing sample in the method for reducing the influence of hemoglobin of the present invention also includes a step of calculating the ratio of the amount of glycated hemoglobin in the hemoglobin-containing sample to the total amount of hemoglobin (i.e., the total amount of hemoglobin and glycated hemoglobin combined). In this case, the method for measuring glycated hemoglobin in a hemoglobin-containing sample in the method for reducing the influence of hemoglobin of the present invention is specifically a measurement method including the following steps:

[0017] (i) measuring the total hemoglobin amount in a hemoglobin-containing sample; (ii) reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds to generate hydrogen peroxide; (iii) measuring the hydrogen peroxide produced in step (ii); (iv) measuring the amount of glycated hemoglobin in the hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iii) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the amount of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out the measurements in the steps (ii) and (iii) above using known amounts of glycated hemoglobin; and (v) calculating the ratio of the amount of glycated hemoglobin to the amount of total hemoglobin in the hemoglobin-containing sample from the amount of total hemoglobin measured in the step (i) and the amount of glycated hemoglobin measured in the step (iv).

[0018] In the above step (ii), the step of reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds may be a step of reacting at least one compound selected from the group consisting of dithio compounds and maleimide compounds with hemoglobin in a hemoglobin-containing sample in an aqueous medium, and then adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium to react glycated hemoglobin in the hemoglobin-containing sample with the enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide. Specifically, the measurement method includes the following steps.

[0019] (i) measuring the total hemoglobin amount in a hemoglobin-containing sample; (ii) reacting hemoglobin in a hemoglobin-containing sample with at least one compound selected from the group consisting of dithio compounds and maleimide compounds in an aqueous medium; (iii) adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium of the step (ii) to generate hydrogen peroxide; (iv) measuring the hydrogen peroxide produced in step (iii); and (v) determining the concentration of glycated hemoglobin in a hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iv) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out measurements according to the above steps (ii) to (iv) using glycated hemoglobin of known concentrations. (vi) calculating the ratio of the amount of glycated hemoglobin to the amount of total hemoglobin in the hemoglobin-containing sample from the amount of total hemoglobin measured in the step (i) and the amount of glycated hemoglobin measured in the step (v).

[0020] The measurement of the total hemoglobin amount in the step (i) can also be carried out after adding at least one compound selected from the group consisting of dithio compounds and maleimide compounds to a hemoglobin-containing sample.

[0021] The reaction between the glycated hemoglobin in the hemoglobin-containing sample and the enzyme that catalyzes the reaction of oxidizing the glycated hemoglobin to generate hydrogen peroxide can also be carried out in the presence of a denaturant for glycated hemoglobin that changes the structure of the glycated hemoglobin in the hemoglobin-containing sample so as to promote the reaction with the enzyme that catalyzes the reaction of oxidizing the glycated hemoglobin to generate hydrogen peroxide. Examples of the denaturant for glycated hemoglobin include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Examples of the nonionic surfactants include alkyl glucosides, fatty acid sorbitan esters, fatty acid diethanolamides, polyoxyethylene alkyl ethers, and alkyl monoglyceryl ethers. Examples of the cationic surfactants include quaternary ammonium salts, pyridinium salts, phosphonium salts, imidazolium salts, and isoquinolinium salts. Examples of anionic surfactants include N-acyltaurine, alkylsulfoacetic acid, polyoxyethylene alkyl ether acetic acid, N-acylamino acid, polyoxyethylene alkyl ether phosphate, polyoxyethylene polycyclic phenyl ether phosphate, alkyl phosphoric acid, alkylsulfonic acid, alkenylsulfonic acid, linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl ether sulfate, etc. Examples of amphoteric surfactants include alkylcarboxybetaine, alkyldimethylamine oxide, etc. In the method for reducing the effect of hemoglobin of the present invention, the concentration of the glycated hemoglobin denaturant in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring glycated hemoglobin of the present invention, but is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0022] The hemoglobin-containing sample in the hemoglobin effect reduction method of the present invention is not particularly limited as long as it contains hemoglobin and is a sample to which the glycated hemoglobin measurement method of the present invention can be applied, and examples thereof include whole blood, blood cells, blood cells mixed with plasma, and samples obtained by hemolyzing these samples. The hemolyzing treatment is not particularly limited as long as it is a treatment for hemolyzing whole blood, blood cells, and blood cells mixed with plasma, and examples thereof include physical methods, chemical methods, and biological methods. Examples of physical methods include a method using a hypotonic solution such as distilled water, and a method using ultrasound. Examples of chemical methods include a method using an organic solvent such as methanol, ethanol, and acetone, and a method using a polyoxyethylene surfactant. Examples of biological methods include a method using an antibody or complement.

[0023] The glycated hemoglobin in the method for reducing the effects of hemoglobin of the present invention is produced by binding sugars such as glucose to hemoglobin, and examples of such glycated hemoglobin include hemoglobin A1a, hemoglobin A1b, and HbA1c, with HbA1c being preferred.

[0024] In the method for reducing the effect of hemoglobin of the present invention, at least one compound selected from the group consisting of dithio compounds and maleimide compounds is used.

[0025] In the method for reducing the effect of hemoglobin of the present invention, the concentration of at least one compound selected from the group consisting of dithio compounds and maleimide compounds in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring glycated hemoglobin of the present invention, but is preferably 0.01 to 10 g / L, more preferably 0.1 to 1 g / L, and for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L.

[0026] The dithio compound is not particularly limited, and examples thereof include 6,6-dithiodinicotinic acid, 2,2-dithiodibenzoic acid, 5,5-dithiobis-(2-nitrobenzoic acid), 4,4-dithiodibutyric acid, and 5,5-dithiobis(1-phenyl-1H-tetrazole), with 6,6-dithiodinicotinic acid (DTDN) or a derivative thereof being preferred. These may be used alone or in combination of two or more.

[0027] The maleimide-based compound is not particularly limited, and examples thereof include 6-maleimidohexanoic acid, N-ethylmaleimide, N-methylmaleimide, N-benzylmaleimide, and N-cyclohexylmaleimide, but 6-maleimidohexanoic acid (6-MH) or its derivatives are preferred. These may be used alone or in combination of two or more. The dithio-based compound and the maleimide-based compound may also be used in combination.

[0028] The total hemoglobin amount can be measured by known methods, such as the cyanmethemoglobin method, the oxyhemoglobin method, the SLS-hemoglobin method, etc. The total hemoglobin amount can be measured not only by the hemoglobin-containing sample itself, but also by applying the cyanmethemoglobin method, the oxyhemoglobin method, the SLS-hemoglobin method, etc. to a sample obtained by adding at least one compound selected from the group consisting of dithio compounds and maleimide compounds to the hemoglobin-containing sample.

[0029] The reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds can be carried out under any conditions as long as the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide can act on the glycated hemoglobin.

[0030] The reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide is preferably carried out in an aqueous medium. The aqueous medium is not particularly limited as long as it is an aqueous medium that enables the method of measuring glycated hemoglobin of the present invention. Examples of the aqueous medium include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred. The pH of the aqueous medium is not particularly limited as long as it allows the method for measuring glycated hemoglobin of the present invention, and is, for example, pH 4, 5, 6, 7, 8, 9, or 10. When a buffer solution is used as the aqueous medium, it is desirable to use a buffering agent appropriate for the set pH. Examples of the buffering agent used in the buffer solution include tris(hydroxymethyl)aminomethane buffering agent, phosphate buffering agent, borate buffering agent, Good's buffering agent, and the like.

[0031] Examples of Good's buffers include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPS), and 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)methyl]-2-hydroxy-3-aminopropanesulfonic acid N,N-bis(2-hydroxyethyl)glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), 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). The concentration of the buffer solution is not particularly limited as long as it enables the method for measuring glycated hemoglobin of the present invention, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and for example, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mol / L.

[0032] The reaction temperature in the reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide is not particularly limited as long as it is a reaction temperature that enables the method for measuring glycated hemoglobin of the present invention, but is preferably 10 to 50°C, more preferably 20 to 40°C, and is, for example, 10, 15, 20, 25, 30, 35, 37, 40, 45, or 50°C. Furthermore, the reaction time in the reaction is not particularly limited as long as it enables the glycated hemoglobin measurement method of the present invention, but is preferably 1 minute to 3 hours, and more preferably 2.5 minutes to 1 hour, for example, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours. The concentration of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide is not particularly limited as long as it is a concentration that enables the method for measuring glycated hemoglobin of the present invention, but is preferably 0.1 to 30 kU / L, more preferably 0.2 to 15 kU / L, and for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 kU / L.

[0033] In the present invention, the enzyme activity (U) of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide can be calculated by the following method. <Reagents for measuring enzyme activity> First Reagent Phosphate buffer (pH8.0) 0.1mmol / L N-Ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE) 0.3g / L Peroxidase 3kU / L 4-Aminoantipyrine 0.1g / L

[0034] Second Reagent Fructosyl valine aqueous solution 1.7mmol / L The above-mentioned aqueous fructosyl valine solution can be prepared by the method described in J. Agric. Food Chem., Vol. 24, No. 1, pp. 70-73 (1976).

[0035] <Specimen diluent> Phosphate buffer (pH8.0) 10mmol / L Bovine serum albumin 0.15% <Sample> FPOX-47△3 (an enzyme that catalyzes the reaction of oxidizing glycosylated hemoglobin to produce hydrogen peroxide) diluted with the above sample dilution solution

[0036] <Measurement> The above sample (2 μL) and the first reagent (150 μL) of the enzyme activity measuring reagent were added to the reaction cell, and the reaction (first reaction) was carried out at 37° C. for 3 minutes. Then, the second reagent (20 μL) of the enzyme activity measuring reagent was added, and the absorbance [Abs 酵素(3分後) ], and the absorbance [Abs 酵素(5分後) ] were measured at a main wavelength of 546 nm and a sub-wavelength of 700 nm, and Abs 酵素(5分後) From Abs 酵素(3分後) Subtract the absorbance difference △Abs' 酵素 Calculate the absorbance difference △Abs' 酵素Divide by 2 (minutes) to get the change in absorbance per minute, △Abs' 酵素 / min was calculated. The absorbance difference ΔAbs' was measured in the same manner, except that the above sample dilution solution was used instead of the above sample. ブランク / min was calculated. Calculated △Abs' 酵素 / min to △Abs' ブランク / min is subtracted, and the absorbance difference for the enzyme is △Abs 酵素 / min was determined.

[0037] <Enzyme activity> The △Abs determined above 酵素 Based on the number of times per minute, the enzyme activity was calculated according to the following formula (I).

[0038]

number

[0039] The enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide is not particularly limited as long as it acts on glycated hemoglobin in a hemoglobin-containing sample and generates hydrogen peroxide from glycated hemoglobin, and examples of the enzyme include enzymes described in WO 2015 / 005258 and amadoriase described in WO 2015 / 060429, the contents of which are incorporated herein by reference. Specific examples of the enzyme include FPOX-18A, FPOX-18B, FPOX-18C, FPOX-18D, and FPOX19-46 described in WO 2015 / 005258. Specific examples of modified forms of the enzyme include FPOX-47△3, which is produced in Reference Example 1 described below.

[0040] The method for measuring the generated hydrogen peroxide is not particularly limited as long as it is a method capable of measuring hydrogen peroxide, and examples thereof include a method using an electrode and a method using a reagent for measuring hydrogen peroxide, and a method using a reagent for measuring hydrogen peroxide is preferred. The reagent for measuring hydrogen peroxide is a reagent for converting hydrogen peroxide into a detectable substance. Examples of the detectable substance include dyes, light (luminescence), and fluorescence, and dyes are preferred.

[0041] When the detectable substance is a dye, the reagent for measuring hydrogen peroxide may be a reagent containing a peroxidatively active substance such as peroxidase and an oxidative color-developing chromogen. Examples of the oxidative color-developing chromogen include an oxidative coupling chromogen and a leuco chromogen, with the leuco chromogen being preferred.

[0042] In the present invention, the leuco chromogen refers to a substance that is converted into a dye by itself in the presence of hydrogen peroxide and a peroxidative substance, such as peroxidase. Examples of the leuco chromogen include phenothiazine chromogens, triphenylmethane chromogens, diphenylamine chromogens, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, and tetramethylbenzidine, with phenothiazine chromogens being preferred. Examples of phenothiazine chromogens include 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67), etc. Among phenothiazine chromogens, 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67) is particularly preferred. Examples of triphenylmethane chromogens include N,N,N',N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triaminotriphenylmethane (TPM-PS). Examples of diphenylamine chromogens include N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 4,4'-bis(dimethylamino)diphenylamine, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA), etc.

[0043] In the present invention, the oxidative coupling type chromogen refers to two compounds that generate a dye by oxidative coupling in the presence of hydrogen peroxide and a peroxidative active substance. Examples of the combination of two compounds include a combination of a coupler and an aniline, a combination of a coupler and a phenol, etc. Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine. Anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N,N-bis(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N- Ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS), and the like. Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

[0044] When the detectable substance is light (luminescence), the reagent for measuring hydrogen peroxide may be a reagent containing a peroxidatively active substance such as peroxidase and a chemiluminescent substance, such as luminol, isoluminol, lucigenin, acridinium ester, etc. When the detectable substance is fluorescent, the hydrogen peroxide measuring reagent may be a reagent containing a peroxidatively active substance such as peroxidase and a fluorescent substance, for example, 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, coumarin, etc.

[0045] (2) Method for measuring glycated hemoglobin in a hemoglobin-containing sample The method of the present invention for measuring glycated hemoglobin in a hemoglobin-containing sample is a method characterized by reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of dithio compounds and maleimide compounds to produce hydrogen peroxide, and measuring the produced hydrogen peroxide. Specifically, the measurement method includes the following steps.

[0046] (i) reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds, to generate hydrogen peroxide; (ii) measuring the hydrogen peroxide produced in step (i); and (iii) determining the concentration of glycated hemoglobin in the hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in step (ii) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out measurements in accordance with steps (i) and (ii) above using glycated hemoglobin of known concentrations.

[0047] In the above step (i), the step of reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds may be a step of reacting at least one compound selected from the group consisting of dithio compounds and maleimide compounds with hemoglobin in a hemoglobin-containing sample in an aqueous medium, and then adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium to react glycated hemoglobin in the hemoglobin-containing sample with the enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide. Specifically, the measurement method includes the following steps.

[0048] (i) reacting hemoglobin in a hemoglobin-containing sample with at least one compound selected from the group consisting of dithio compounds and maleimide compounds in an aqueous medium; (ii) adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium of step (i) to generate hydrogen peroxide; (iii) measuring the hydrogen peroxide produced in step (ii); and (iv) determining the concentration of glycated hemoglobin in a hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iii) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out measurements according to the steps (i) to (iii) above using glycated hemoglobin of known concentrations.

[0049] The method of the present invention for measuring glycated hemoglobin in a hemoglobin-containing sample also includes a step of calculating the ratio of the amount of glycated hemoglobin in the hemoglobin-containing sample to the total amount of hemoglobin (i.e., the total amount of hemoglobin and glycated hemoglobin combined). In this case, the method of measuring glycated hemoglobin in a hemoglobin-containing sample specifically includes the following steps:

[0050] (i) measuring the total hemoglobin amount in a hemoglobin-containing sample; (ii) reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds to generate hydrogen peroxide; (iii) measuring the hydrogen peroxide produced in step (ii); (iv) measuring the amount of glycated hemoglobin in the hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iii) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the amount of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out the measurements in the steps (ii) and (iii) above using known amounts of glycated hemoglobin; and (v) calculating the ratio of the amount of glycated hemoglobin to the amount of total hemoglobin in the hemoglobin-containing sample from the amount of total hemoglobin measured in the step (i) and the amount of glycated hemoglobin measured in the step (iv).

[0051] In the above step (ii), the step of reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds may be a step of reacting at least one compound selected from the group consisting of dithio compounds and maleimide compounds with hemoglobin in a hemoglobin-containing sample in an aqueous medium, and then adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium to react glycated hemoglobin in the hemoglobin-containing sample with the enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide. Specifically, the measurement method includes the following steps.

[0052] (i) measuring the total hemoglobin amount in a hemoglobin-containing sample; (ii) reacting hemoglobin in a hemoglobin-containing sample with at least one compound selected from the group consisting of dithio compounds and maleimide compounds in an aqueous medium; (iii) adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium of the step (ii) to generate hydrogen peroxide; (iv) measuring the hydrogen peroxide produced in step (iii); and (v) determining the concentration of glycated hemoglobin in a hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iv) with a calibration curve showing the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin, the calibration curve having been prepared in advance by carrying out measurements according to the above steps (ii) to (iv) using glycated hemoglobin of known concentrations. (vi) calculating the ratio of the amount of glycated hemoglobin to the amount of total hemoglobin in the hemoglobin-containing sample from the amount of total hemoglobin measured in the step (i) and the amount of glycated hemoglobin measured in the step (v).

[0053] The measurement of the total hemoglobin amount in the step (i) can also be carried out after adding at least one compound selected from the group consisting of dithio compounds and maleimide compounds to a hemoglobin-containing sample.

[0054] The reaction between the glycated hemoglobin in the hemoglobin-containing sample and the enzyme that catalyzes the reaction of oxidizing the glycated hemoglobin to generate hydrogen peroxide can also be carried out in the presence of a denaturant for glycated hemoglobin that changes the structure of the glycated hemoglobin in the hemoglobin-containing sample so as to promote the reaction with the enzyme that catalyzes the reaction of oxidizing the glycated hemoglobin to generate hydrogen peroxide. Examples of the denaturant for glycated hemoglobin include the above-mentioned denaturants for glycated hemoglobin. In the method of measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention, the concentration of the glycated hemoglobin denaturant in the reaction solution is not particularly limited as long as it is a concentration that enables the method of measuring glycated hemoglobin of the present invention, and examples of the concentration include the concentrations of the glycated hemoglobin denaturant in the reaction solution described above.

[0055] In the method of measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention, the hemoglobin-containing sample and the glycated hemoglobin include the above-mentioned hemoglobin-containing sample and glycated hemoglobin. Furthermore, the dithio compounds and maleimide compounds in the method of the present invention for measuring glycated hemoglobin in a hemoglobin-containing sample include the above-mentioned dithio compounds and maleimide compounds.

[0056] The method for measuring the total hemoglobin amount includes the above-mentioned method for measuring the total hemoglobin amount. In addition, the reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds includes the reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in an aqueous medium containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds.

[0057] The reaction temperature and reaction time in the reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide include the reaction temperature and reaction time in the reaction between glycated hemoglobin in a hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide described above. In addition, the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, the concentration of the enzyme, and the method for measuring the generated hydrogen peroxide include the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide described above, the concentration of the enzyme, and the method for measuring hydrogen peroxide.

[0058] (3) Reagent for measuring glycated hemoglobin in hemoglobin-containing samples The reagent for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention is a reagent containing an enzyme that catalyzes a reaction for oxidizing glycated hemoglobin to generate hydrogen peroxide, and at least one compound selected from the group consisting of dithio compounds and maleimide compounds, and is used in the method for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention. The measuring reagent of the present invention may further contain a reagent for measuring hydrogen peroxide.

[0059] In the measurement reagent of the present invention, examples of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide; at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and the hydrogen peroxide measurement reagent include, for example, the above-mentioned enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide; at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and the hydrogen peroxide measurement reagent.

[0060] The glycated hemoglobin measurement reagent of the present invention may be in a lyophilized state or in a dissolved state in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous media. When the lyophilized reagent is used to measure glycated hemoglobin in a hemoglobin-containing sample, the reagent is dissolved in an aqueous medium before use. The content of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in the glycated hemoglobin measurement reagent of the present invention is not particularly limited as long as it is a content that enables the glycated hemoglobin measurement method of the present invention, but a content in which the concentration when dissolved in an aqueous medium is 0.1 to 30 kU / L is preferable, and a content in which the concentration is 0.2 to 15 kU / L is more preferable, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 kU / L.

[0061] The content of at least one compound selected from the group consisting of dithio compounds and maleimide compounds in the glycated hemoglobin measurement reagent of the present invention is not particularly limited as long as it is a content that enables the glycated hemoglobin measurement method of the present invention, but a content in which the concentration when dissolved in an aqueous medium is 0.01 to 10 g / L is preferable, and a content in which the concentration is 0.1 to 1 g / L is more preferable, and for example, a content of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L.

[0062] The glycosylated hemoglobin measurement reagent of the present invention may also contain a denaturant for glycosylated hemoglobin that changes the structure of glycosylated hemoglobin in a hemoglobin-containing sample so as to promote a reaction with an enzyme that catalyzes a reaction of oxidizing glycosylated hemoglobin to generate hydrogen peroxide. Examples of the denaturant for glycosylated hemoglobin include the above-mentioned denaturants for glycosylated hemoglobin. The content of the glycated hemoglobin denaturant in the glycated hemoglobin measurement reagent of the present invention is not particularly limited as long as it is a content that enables the glycated hemoglobin measurement method of the present invention, but a content in which the concentration when dissolved in an aqueous medium is 0.01 to 50 g / L is preferable, and a content in which the concentration is 0.1 to 5 g / L is more preferable, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L. The measurement reagent of the present invention may contain an aqueous medium, a stabilizer, a preservative, salts, an interference substance eliminator, an organic solvent, etc., as necessary. Examples of the aqueous medium include the above-mentioned aqueous medium, etc. Examples of the stabilizer include ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride, calcium acetate, calcium nitrate, potassium ferrocyanide, bovine serum albumin (BSA), polyoxyethylene-based nonionic surfactants such as polyoxyethylene alkylphenyl ether, glycerin, alkylene glycol, etc. Examples of the alkylene glycol include ethylene glycol, propylene glycol, etc. Examples of the preservative include sodium azide, antibiotics, etc. Examples of the salt 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. Examples of the interference substance eliminator include ascorbic acid oxidase for eliminating the effect of ascorbic acid, etc. Examples of the organic solvent include dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane, acetone, methanol, ethanol, etc., which act as solubilizers for dissolving leuco chromogens in aqueous media.

[0063] (4) Glycated hemoglobin measurement kit for hemoglobin-containing samples The reagent for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention may be stored, distributed, and used in the form of a kit. The kit for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention is used in the method for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention. The measurement kit of the present invention may be, for example, a two-reagent kit or a three-reagent kit, with a two-reagent kit being preferred.

[0064] In the present invention, when measuring glycated hemoglobin in a hemoglobin-containing sample using a two-reagent kit, for example, the hemoglobin-containing sample and the first reagent are added to a reaction cell, and a reaction is carried out at a constant temperature for a certain period of time. Thereafter, the second reagent is added, and the generated hydrogen peroxide is measured, thereby measuring glycated hemoglobin.

[0065] Kit 1 a first reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and The kit further comprises a second reagent comprising an enzyme that catalyzes a reaction for oxidizing glycated hemoglobin to produce hydrogen peroxide.

[0066] Kit 2 a first reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and A kit comprising a second reagent containing an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide, and comprising a hydrogen peroxide measurement reagent in either or both of the first reagent and the second reagent.

[0067] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0068] Kit 3 a first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide; and a second reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds; Kit including:

[0069] Kit 4 a first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide; and a second reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and a hydrogen peroxide measurement reagent is included in either or both of the first and second reagents.

[0070] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0071] Kit 5 A first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide, and at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and Secondary reagent containing hydrogen peroxide measuring reagent Kit including:

[0072] Kit 6 a first reagent comprising an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, at least one compound selected from the group consisting of dithio compounds and maleimide compounds, and a reagent for measuring hydrogen peroxide; and Secondary reagent containing hydrogen peroxide measuring reagent Kit including:

[0073] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0074] Kit 7 a first reagent containing a hydrogen peroxide measurement reagent; and a second reagent containing an enzyme that catalyzes a reaction for oxidizing glycated hemoglobin to produce hydrogen peroxide, and at least one compound selected from the group consisting of dithio compounds and maleimide compounds; Kit including:

[0075] Kit 8 a first reagent containing a hydrogen peroxide measurement reagent; and A kit comprising an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide, at least one compound selected from the group consisting of dithio compounds and maleimide compounds, and a second reagent comprising a hydrogen peroxide measurement reagent.

[0076] The reagent contained in the glycosylated hemoglobin measurement kit of the present invention may also contain a denaturant for glycosylated hemoglobin that changes the structure of glycosylated hemoglobin in a hemoglobin-containing sample so as to promote a reaction with an enzyme that catalyzes a reaction of oxidizing glycosylated hemoglobin to generate hydrogen peroxide. Examples of the denaturant for glycosylated hemoglobin include the above-mentioned denaturants for glycosylated hemoglobin. The content of the glycated hemoglobin denaturant in the reagent constituting the glycated hemoglobin measurement kit of the present invention is not particularly limited as long as it is a content that enables the glycated hemoglobin measurement method of the present invention, and examples of the content include a content that results in the concentration of the above-mentioned glycated hemoglobin denaturant when dissolved in an aqueous medium.

[0077] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0078] The glycated hemoglobin measurement kit of the present invention may be in a lyophilized state or in a dissolved state in an aqueous medium. Examples of the aqueous medium include the aqueous media described above. When measuring glycated hemoglobin in a hemoglobin-containing sample using a lyophilized kit, the reagents constituting the kit are dissolved in an aqueous medium before use. The content of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide in the reagent constituting the measurement kit of the present invention is not particularly limited as long as it is a content that enables the measurement method of glycated hemoglobin of the present invention, but a content in which the concentration when dissolved in an aqueous medium is 0.1 to 60 kU / L is preferable, and a content in which the concentration is 0.4 to 30 kU / L is more preferable, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 kU / L.

[0079] The content of at least one compound selected from the group consisting of dithio compounds and maleimide compounds in the reagent constituting the measurement kit of the present invention is not particularly limited as long as it is a content that enables the measurement method of glycated hemoglobin of the present invention, but a content in which the concentration when dissolved in an aqueous medium is 0.01 to 10 g / L is preferable, and a content of 0.1 to 1 g / L is more preferable, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L. EXAMPLES

[0080] The present invention will be described in more detail below with reference to examples, which are not intended to limit the scope of the present invention in any way. In the examples, comparative examples, and test examples, the following reagents and enzymes were used.

[0081] Bis-Tris (Dojindo Laboratories), DA-67 (Wako Pure Chemical Industries, Ltd.), 4-aminoantipyrine (Actec), EMSE (Dojindo Laboratories), peroxidase (Toyobo Co., Ltd.), fructosyl valine (prepared by the method described in J. Agric. Food Chem., Vol. 24, No. 1, p. 70-73 (1976)), 6,6-dithiodinicotinic acid (Tokyo Chemical Industry Co., Ltd.), 2,2-dithiodibenzoic acid (Tokyo Chemical Industry Co., Ltd.), 6-maleimidohexanoic acid (Tokyo Chemical Industry Co., Ltd.), N-ethylmaleimide (Tokyo Chemical Industry Co., Ltd.), N-methylmaleimide (Tokyo Chemical Industry Co., Ltd.), N-benzylmaleimide (Tokyo Chemical Industry Co., Ltd.), N-cyclohexylmaleimide (Tokyo Chemical Industry Co., Ltd.) NIKKOL LMT [N-acyltaurine salt (sodium N-lauroyl-N-methyltaurine); manufactured by Nikko Chemicals Co., Ltd.] NIKKOL Sarcosinate LN [N-Acylamino acid salt (N-lauroyl sarcosine sodium); manufactured by Nikko Chemicals] NIKKOL ECTD-3NEX [Polyoxyethylene alkyl ether acetate (sodium polyoxyethylene tridecyl ether acetate); manufactured by Nikko Chemicals Co., Ltd.] K Liporan PJ-400J [Alkenylsulfonic acid (sodium tetradecene sulfonate); manufactured by Kao Corporation]

[0082] [Reference Example 1] Creation of FPOX-47△3 (1) Construction of FPOX-47 expression vector pTrc-FPOX-47 Using the expression plasmid pTrc-FPOX-42 containing DNA consisting of the base sequence represented by SEQ ID NO: 1 as template DNA, and using FPOX-42 F342V-F consisting of the base sequence represented by SEQ ID NO: 2 and FPOX-42 F342V-R consisting of the base sequence represented by SEQ ID NO: 3 as forward and reverse primers, respectively, PCR was performed with the following reagent composition and PCR conditions to obtain a PCR product. pTrc-FPOX-42 is an expression plasmid containing DNA encoding FPOX-42, an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, and can be constructed according to the method disclosed in International Publication No. 2015 / 005258. PCR was performed based on the protocol of the PCR kit, DNA polymerase "KOD-Plus-" manufactured by Toyobo Co., Ltd.

[0083] (Reagent composition) KOD-Plus-Buffer Template DNA (pTrc-FPOX-42) 1-2 ng / μL Forward primer (FPOX-42 F342V-F) 0.3 μmol / L Reverse primer (FPOX-42 F342V-R) 0.3 μmol / L ·dNTP mixture 0.2 mmol / L each MgSO 4 1 mmol / L DNA polymerase (KOD-Plus-) 0.02 U / μL Add sterile water to make 50 μL.

[0084] (PCR conditions) 1. 94℃ 2 minutes 2. 98℃ 15 seconds 3. 60℃ 30 seconds 4. 68℃ 6 minutes 5. Repeat steps 2 to 4 (total 30 cycles) 6. 68℃ 10 minutes

[0085] 1 μL of "restriction enzyme Dpn I" (New England Biolabs) was added to 50 μL of the obtained PCR product, and the template DNA was degraded by incubating at 37° C. for 1 hour. The restriction enzyme-treated PCR product was purified using Promega's "Wizard SV Gel and PCR Clean-Up System" according to the kit's protocol to obtain a purified PCR product sample. The purified PCR product was then used to transform E. coli competent cells "Competent high DH5α" manufactured by Toyobo Co., Ltd. Colonies grown on LB agar medium containing 50 mg / L ampicillin were selected, and plasmids were extracted using "Wizard Plus SV Minipreps DNA Purification" manufactured by Promega Co., Ltd. according to the kit's protocol. The extracted plasmid was subjected to sequence analysis using a DNA sequencer to confirm the construction of pTrc-FPOX-47, a plasmid containing DNA encoding FPOX-47 and having the base sequence shown in SEQ ID NO: 4. For sequence analysis, pTrc-F consisting of the base sequence shown in SEQ ID NO: 5, which reflects the base sequences immediately before and after the multicloning site of the pTrc99a vector (GE Healthcare Biosciences), and pTrc-R consisting of the base sequence shown in SEQ ID NO: 6 were used as the forward and reverse primers, respectively.

[0086] (2) Construction of FPOX-47△3 expression vector pTrc-FPOX-47△3 We designed and chemically synthesized FPOX-47△3-R, which consists of the base sequence represented by SEQ ID NO: 7, in which a stop codon and a Bam HI restriction enzyme recognition site have been added, with the 9 bases located immediately before the stop codon on the 3'-end side of the base sequence encoding FPOX-47 contained in plasmid pTrc-FPOX-47 removed, and pTrc-F, which consists of the base sequence represented by SEQ ID NO: 5, which reflects the base sequence immediately before the multicloning site of the pTrc99a vector. Using pTrc-F and FPOX-47△3-R as a primer pair and pTrc-FPOX-47 as a template, PCR was performed with the following reagent composition and PCR conditions to obtain a PCR product containing a DNA fragment containing a base sequence encoding the deletion mutant FPOX-47△3, in which three amino acids are deleted from the C-terminus of FPOX-47. PCR was carried out based on the protocol of the PCR kit, DNA polymerase "KOD-Plus-" manufactured by Toyobo Co., Ltd.

[0087] (Reagent composition) KOD-Plus-Buffer Template DNA (pTrc-FPOX-47) 0.2-0.5 ng / μL Forward primer (pTrc-F) 0.3 μmol / L · Reverse primer (FPOX-47△3-R) 0.3 μmol / L ·dNTP mixture 0.2 mmol / L each MgSO 4 1 mmol / L DNA polymerase (KOD-Plus-) 0.02 U / μL Add sterile water to make 50 μL.

[0088] (PCR conditions) 1. 94℃ 2 minutes 2. 98℃ 15 seconds 3. 60℃ 30 seconds 4. 68℃ 2 minutes 5. Repeat steps 2 to 4 (total 30 cycles) 6. 68℃ 5 minutes

[0089] A portion of the resulting PCR product was electrophoresed on a 1% agarose TAE gel to confirm that a DNA fragment of the expected size had been obtained. The resulting PCR product was purified using Promega's Wizard SV Gel and PCR Clean-Up system according to the kit's protocol to obtain a DNA fragment containing DNA encoding FPOX-47△3. The resulting DNA fragment was treated with two types of restriction enzymes, Nco I and Bam HI (both manufactured by Takara Bio Inc.), at 37°C for 1 hour, and the resulting reaction mixture was purified using Promega's Wizard SV Gel and PCR Clean-Up system according to the kit's protocol to obtain a restriction enzyme-treated fragment.

[0090] In addition, pTrc-FPOX-15, which expresses the glycopeptide oxidase FPOX-15 that does not have the activity of oxidizing fructosyl hexapeptide and is described in International Publication No. 2010 / 041715, was similarly treated with the same restriction enzymes and electrophoresed on a 1% agarose TAE gel to excise a DNA fragment corresponding to the vector. The DNA fragment was purified using the Wizard SV Gel and PCR Clean-Up system to obtain a plasmid fragment from which the DNA encoding FPOX-15 had been removed. The resulting restriction enzyme-treated fragment was mixed with a plasmid fragment from which the DNA encoding FPOX-15 had been removed, and ligated using a DNA Ligation Kit (Takara Bio Inc.). The ligation product was then introduced into Escherichia coli competent cells "Competent high DH5α" (Toyobo Co., Ltd.). The E. coli into which the ligation product had been introduced was inoculated on LB agar medium containing 50 mg / L ampicillin, cultured at 37°C for 14 hours, and the colonies that emerged were selected. The selected colonies were cultured in LB liquid medium containing 50 mg / L ampicillin at 37°C for 14 hours, and a plasmid was prepared using Promega's Wizard Plus SV Minipreps DNA Purification kit according to the kit's protocol. PCR was performed using pTrc-F consisting of the base sequence represented by SEQ ID NO: 5 and pTrc-R consisting of the base sequence represented by SEQ ID NO: 6 as forward and reverse primers, respectively, to confirm the base sequence of the prepared plasmid, and it was confirmed that the plasmid contained the base sequence represented by SEQ ID NO: 8, in which the 3'-side 9 bases excluding the stop codon of the base sequence of FPOX-47 were deleted. This plasmid is called pTrc-FPOX-47△3.

[0091] (3) Acquisition of FPOX-47△3 The E. coli containing pTrc-FPOX-47△3 obtained in (2) above was cultured in LB medium containing 50 mg / L ampicillin at 37°C for 14 hours with shaking. The resulting culture solution was purified according to the purification method for glycopeptide oxidases FPOX-9 and FPOX-15 described in WO 2010 / 041715 to obtain a solution of purified FPOX-47△3. The amino acid sequence of FPOX-47 is shown in SEQ ID NO:9, and the amino acid sequence of FPOX-47△3 is shown in SEQ ID NO:10.

[0092] (4) Confirmation of the enzyme activity of FPOX-47△3 that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide (hereinafter referred to as glycated hemoglobin oxidase activity) Using the purified FPOX-47Δ3 solution obtained in (3) above, the glycated hemoglobin oxidase activity of FPOX-47Δ3 against HbA1c was confirmed by the following measurement kit and measurement procedure.

[0093] <Measurement Kit A> First Reagent Bis-Tris (pH 6.5) 50mmol / L DA-67 40μmol / L NIKKOL LMT 1g / L Second Reagent Phosphate buffer (pH6.5) 10mmol / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0094] <Measurement kit a> First Reagent Bis-Tris (pH 6.5) 50mmol / L DA-67 40μmol / L Second Reagent Phosphate buffer (pH6.5) 10mmol / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0095] Measurement kit a is the same as measurement kit A except that the first reagent does not contain NIKKOL LMT.

[0096] <Sample> The samples used were hemolyzed samples prepared by hemolysis after diluting blood cells obtained by centrifuging human blood with deionized water, and the HbA1c concentrations were determined to be 2.6 μmol / L, 4.5 μmol / L, 6.4 μmol / L, and 8.4 μmol / L using the KO500 method, which is the standard method for measuring HbA1c, and the SLS-hemoglobin method, which is a method for measuring total hemoglobin.

[0097] <Measurement procedure> Each sample (10 μL) and the first reagent (100 μL) of the measurement kit A were added to a reaction cell and reacted at 37° C. for 5 minutes (first reaction), and the absorbance (E1) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. Next, the second reagent (40 μL) of the measurement kit A was added to this reaction solution and reacted at 37° C. for another 5 minutes (second reaction), and the absorbance (E2) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. E1 was subtracted from E2 to obtain the absorbance difference ΔE'. A was calculated. Each of the above samples (10 μL) and the first reagent (100 μL) of the above measurement kit a were added to a reaction cell, and reacted at 37° C. for 5 minutes (first reaction), and the absorbance (E3) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. Next, the second reagent (40 μL) of the above measurement kit a was added to this reaction solution, and reacted at 37° C. for another 5 minutes (second reaction), and the absorbance (E4) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. E3 was subtracted from E4 to obtain the absorbance difference ΔE'. a was calculated. △E' A From △E' a The absorbance difference for each sample was calculated as ΔE. The relationship between the HbA1c concentration and ΔE for each sample is shown in Figure 1.

[0098] As shown in Figure 1, a quantitative relationship was observed between HbA1c concentration and absorbance in the measurement using the kit of the present invention (Kit A) containing the anionic surfactant N-acyltaurine. Therefore, it was confirmed that FPOX-47△3 is an enzyme that has the activity of catalyzing the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide.

[0099] [Example 1] Kits for measuring HbA1c (Kits 1A to 1O) consisting of the following first and second reagents were prepared.

[0100] First Reagent (1-A) Reagents Bis-Tris (pH 7.0) 50mmol / L NIKKOL LMT 3g / L DA-67 80μmol / L (1-B) Reagents Bis-Tris (pH 7.0) 50mmol / L Dithio compounds or maleimide compounds (see Table 1) The (1-B) reagent was stored at 4° C. or 30° C. for one week, and then mixed with the (1-A) reagent in a volume ratio of 1:1 to prepare the first reagent. Second Reagent Bis-Tris (pH 7.0) 50mmol / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0101] [Comparative Example 1] (1-B) A kit for measuring HbA1c (kit 1a) consisting of the first and second reagents was prepared in the same manner as in Example 1, except that the reagent did not contain a dithio compound or a maleimide compound.

[0102] [Example 2] Kit 1A of Example 1 was used as the HbA1c measurement kit, and six concentrations of whole blood with HbA1c concentrations determined by the KO500 HPLC method to be 5.11%, 5.73%, 6.40%, 7.45%, 8.55%, and 10.56%, respectively, were used as samples. The ratio of the HbA1c concentration (amount) to the total hemoglobin concentration (amount) in each sample [HbA1c (%)] was determined by the following procedure.

[0103] (1) Preparation of a calibration curve for determining total hemoglobin concentration Measurements were performed according to the following procedure using a total hemoglobin measurement kit, Hemoglobin B-Test Wako (SLS-hemoglobin method) (manufactured by Wako Pure Chemical Industries, Ltd.), and the standard provided with the Hemoglobin B-Test Wako (total hemoglobin concentration: 15.0 mg / ml), and the absorbance for the standard was measured. The standard (10 μL) and Hemoglobin B-Test Wako (250 μL) were added to the reaction cell and reacted at 37° C. for 10 minutes. The absorbance (E) of the reaction solution was measured at a main wavelength of 546 nm and a sub-wavelength of 660 nm, and was taken as the absorbance for the standard. The same method was used except that saline was used instead of the standard, and the absorbance for saline was taken as the absorbance for the standard. The value calculated by subtracting the absorbance for saline from the absorbance for the standard was taken as the blank-corrected absorbance for the standard. A calibration curve showing the relationship between total hemoglobin concentration (μmol / L) and absorbance was created from the blank-corrected absorbance for the standard and the blank-corrected absorbance for saline (0 Abs).

[0104] (2) Preparation of a calibration curve for determining HbA1c concentration Two hemolyzed blood cell fractions, the HbA1c concentrations of which were estimated to be 3.94 μmol / L and 10.82 μmol / L by the K0500 method, were measured using the HbA1c measurement kit 1A of Example 1 according to the following procedure, and the absorbance for each blood cell fraction was measured. The above hemolyzed blood cell fractions (9.6 μL) and the first reagent (120 μL) of the kit of Example 1 were added to the reaction cell, and the reaction was allowed to proceed at 37° C. for 5 minutes (first reaction), and the absorbance (E1) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. Next, the second reagent (40 μL) of the HbA1c measurement kit 1A of Example 1 was added to the reaction solution, and the reaction was allowed to proceed at 37° C. for another 5 minutes (second reaction), and the absorbance (E2) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. E1 was subtracted from E2 to obtain the absorbance difference ΔE' 1A The absorbance difference ΔE' was calculated as the absorbance for each blood cell fraction. 生理食塩水 was calculated and used as the absorbance for physiological saline. The blank-corrected absorbance for the blood cell fraction was calculated by subtracting the absorbance for physiological saline from the absorbance 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 for the blood cell fraction and the blank-corrected absorbance for physiological saline (0 Abs).

[0105] (3) Determination of total hemoglobin concentration in each blood cell fraction Each of the six concentrations of whole blood samples was centrifuged at 25°C, 3000 rpm (1500 x G) for 5 minutes to obtain blood cell fractions. 4 mL of purified water was added to 100 μL of each blood cell fraction to obtain hemolyzed blood cell fractions. Each hemolyzed blood cell fraction was measured using a Hemoglobin B-Test Wako in the same manner as in (1), and the total hemoglobin concentration (μmol / L) in each blood cell fraction was determined from the obtained measurements and the calibration curve in (1).

[0106] (4) Determination of HbA1c concentration in each blood cell fraction Each of the hemolyzed blood cell fractions obtained in (3) was measured in the same manner as in (2) using the measurement kit 1A of the present invention, and the HbA1c concentration (μmol / L) in each blood cell fraction was determined from the obtained measurement values ​​and the calibration curve in (2).

[0107] (5) Determination of HbA1c (%) (the ratio of HbA1c concentration to total hemoglobin concentration) From the total hemoglobin concentration (μmol / L) in each blood cell fraction determined in (3) above and the HbA1c concentration (μmol / L) in each blood cell fraction determined in (4) above, HbA1c (%) was calculated as the NGSP value (international standard value) according to the following formula (II).

[0108]

number

[0109] (6) Correlation between the measurement method of the present invention and the KO500 method Using the measurement method of the present invention, the correlation between the HbA1c (%) determined in (5) above and the HbA1c (%) previously determined using the KO500 method was verified, and the correlation coefficient was determined. The results are shown in Table 1.

[0110] The HbA1c (%) in each sample was determined by the same procedure as above, except that kits 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N and 1O of Example 1 were used instead of kit 1A of Example 1, and the correlation between the measurement method using kits 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N and 1O and the KO500 method was verified, and the correlation coefficient was determined. The results are shown in Table 1.

[0111] [Comparative Example 2] HbA1c (%) in each blood cell fraction was determined by the same procedure as in Example 2, except that Kit 1a of Comparative Example 1 was used instead of Kit 1A of Example 1, and the correlation between the measurement method using Kit 1a and the KO500 method was verified, and the correlation coefficient was determined. The results are shown in Table 1.

[0112] [Table 1]

[0113] As shown in Table 1, in the measurement using the kits 1A to 1O of the present invention, the correlation coefficient with the KO500 method was 0.998 to 1.000 regardless of the concentration of the dithio compound or maleimide compound, and whether the storage temperature of the (1-B) reagent during preparation of the first reagent was 4° C. or 30° C., and a good correlation was observed between the two measurement methods. Therefore, it was revealed that the measurement method of the present invention using the kits 1A to 1O of Example 1 can accurately measure HbA1c in a sample.

[0114] [Example 3] (1) Preparation of hemolyzed samples 4 mL of purified water was added to 100 μL of blood cell fraction obtained by centrifuging human blood, to obtain a hemolyzed blood cell fraction. The hemolyzed blood cell fraction was then measured in the same manner as in Example 2 (1) above using a total hemoglobin measurement reagent, Hemoglobin B-Test Wako, and the total hemoglobin concentration of the blood cell fraction was determined from the obtained absorbance and the calibration curve showing the relationship between the total hemoglobin concentration (μmol / L) and absorbance prepared in Example 2 (1). The valued blood cell fraction was then diluted with purified water to prepare hemolyzed samples with total hemoglobin concentrations of 4 mg / mL, 6 mg / mL, and 8 mg / mL.

[0115] (2) Determination of HbA1c concentration in each hemolyzed sample For each hemolyzed sample prepared in (1), measurement was performed in the same manner as in (2) of Example 2 above using the HbA1c measurement kit 1A of Example 1, and the HbA1c concentration (μmol / L) in each hemolyzed sample was determined from the obtained measurement value and the calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance prepared in (2) of Example 2 above.

[0116] (3) Determination of HbA1c (%) (= ratio of HbA1c concentration to total hemoglobin concentration) From the total hemoglobin concentration (μmol / L) in each hemolyzed sample prepared in (1) above and the HbA1c concentration (μmol / L) in each hemolyzed sample measured in (2) above, HbA1c (%) was calculated as the NGSP value (international standard value) according to the above formula (II).

[0117] (4) Evaluation of the effect of total hemoglobin concentration The HbA1c concentration (%) in the hemolyzed specimen with a total hemoglobin concentration of 6 mg / ml was set as the standard 0, and the difference in HbA1c concentration (%) of each hemolyzed specimen from the standard [△HbA1c concentration (%)] was calculated. The results are shown in Table 2.

[0118] Instead of the HbA1c measurement kit 1A of Example 1, the same measurement was performed using each of the kits 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N and 1O of Example 1 as the HbA1c measurement kit, and the HbA1c concentration (%) in each hemolyzed sample was measured for each kit. The HbA1c concentration (%) in a hemolyzed sample with a total hemoglobin concentration of 6 mg / ml was set as the standard 0, and the difference in the HbA1c concentration (%) of each hemolyzed sample from the standard [△HbA1c concentration (%)] was calculated. The results are shown in Table 2.

[0119] [Comparative Example 3] Except for using kit 1a of Comparative Example 1 instead of kit 1A of Example 1, the HbA1c concentration (%) in each hemolyzed sample was measured using kit 1a in the same manner as in Example 3. The HbA1c concentration (%) in a hemolyzed sample with a total hemoglobin concentration of 6 mg / mL was set as the standard 0, and the difference in HbA1c concentration (%) of each hemolyzed sample from the standard [ΔHbA1c concentration (%)] was calculated. The results are shown in Table 2.

[0120] [Table 2]

[0121] As described above, the hemolyzed samples used in the measurement were prepared from the same human blood, so the ratio (%) of HbA1c to total hemoglobin is constant regardless of the total hemoglobin concentration. Therefore, the closer ΔHbA1c concentration (%) is to 0, the less the influence of hemoglobin is. As is clear from Table 2, the method for measuring glycated hemoglobin of the present invention using each of the kits 1A to 1O of the present invention was found to reduce the influence of hemoglobin compared to the method for measuring glycated hemoglobin using the kit 1a of Comparative Example 1.

[0122] [Example 4] Kits for measuring HbA1c (Kits 2A to 2E) consisting of the following first and second reagents were prepared. First Reagent Bis-Tris (pH 7.0) 50mmol / L DA-67 40μmol / L 6,6-Dithiodinicotinic acid (DTDN) 0.3g / L Surfactants (see Table 3) Second Reagent Bis-Tris (pH 7.0) 50mmol / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0123] [Comparative Example 4] Kits for measuring HbA1c (kits 2a to 2e) each consisting of the first and second reagents were prepared in the same manner as in Example 4, except that the first reagent did not contain 6,6-dithiodinicotinic acid (DTDN).

[0124] [Example 5] HbA1c (%) in each sample was determined in the same manner as in Example 2, except that HbA1c measurement kits 2A, 2B, 2C, 2D and 2E of Example 4 were used instead of HbA1c measurement kit 1A of Example 1. The correlation between the measurement method using each of kits 2A, 2B, 2C, 2D and 2E and the KO500 method was verified, and the correlation coefficient was determined.

[0125] In addition, the same measurement as in Example 3 was performed except that the HbA1c measurement kits 2A, 2B, 2C, 2D, and 2E of Example 4 were used instead of the HbA1c measurement kit 1A of Example 1, and the HbA1c concentration (%) in each hemolyzed sample was measured for each kit. The HbA1c concentration (%) in a hemolyzed sample with a total hemoglobin concentration of 6 mg / ml was set as the standard 0, and the difference in HbA1c concentration (%) of each hemolyzed sample from the standard [△HbA1c concentration (%)] was calculated. The results are shown in Table 3.

[0126] [Comparative Example 5] The HbA1c concentration (%) in each hemolyzed sample was measured for each of the kits 2a, 2b, 2c, 2d, and 2e in Comparative Example 4 in the same manner as in Example 3, except that the kit 1A in Example 1 was replaced by the kit 2a, 2b, 2c, 2d, and 2e in Comparative Example 4. The HbA1c concentration (%) in a hemolyzed sample with a total hemoglobin concentration of 6 mg / mL was set as the standard 0, and the difference in the HbA1c concentration (%) of each hemolyzed sample from the standard [ΔHbA1c concentration (%)] was calculated. The results are shown in Table 3.

[0127] [Table 3]

[0128] As is clear from Table 3, the method for measuring glycated hemoglobin of the present invention using each of Kits 2A to 2E of the present invention was not affected by hemoglobin, compared to the method for measuring glycated hemoglobin using each of Kits 2a to 2e of Comparative Example 4. This demonstrated that even when the type of denaturing agent for glycated hemoglobin was changed, the method for measuring glycated hemoglobin of the present invention using 6,6-dithiodinicotinic acid, a dithio compound, was less affected by hemoglobin. [Industrial Applicability]

[0129] The present invention provides a method for reducing the effects of hemoglobin, which is useful for diagnosing diabetes and the like, a method for measuring glycated hemoglobin in a hemoglobin-containing sample, and a reagent and kit for measuring glycated hemoglobin.

Claims

1. A method for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, comprising: A method for reducing the influence of hemoglobin in a method for measuring glycated hemoglobin, comprising reacting glycated hemoglobin in a hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of 6,6-dithiodinicotinic acid, 6-maleimidohexanoic acid, N-ethylmaleimide, N-methylmaleimide, N-benzylmaleimide, and N-cyclohexylmaleimide.

2. 2. The method for reducing the effect of hemoglobin according to claim 1, wherein glycated hemoglobin in the hemoglobin-containing sample is reacted with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of a denaturing agent for glycated hemoglobin.

3. 3. The method for reducing the effect of hemoglobin according to claim 1, wherein the measurement of hydrogen peroxide is carried out using a hydrogen peroxide measurement reagent.

4. 4. The method for reducing the effect of hemoglobin according to claim 3, wherein the hydrogen peroxide measurement reagent is a reagent containing peroxidase and a leuco chromogen.

5. The method for reducing the effect of hemoglobin according to claim 4, wherein the leuco chromogen is a phenothiazine chromogen.

6. 6. The method for reducing the effect of hemoglobin according to claim 5, wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

7. A method for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of at least one compound selected from the group consisting of 6,6-dithiodinicotinic acid, 6-maleimidohexanoic acid, N-ethylmaleimide, N-methylmaleimide, N-benzylmaleimide, and N-cyclohexylmaleimide to produce hydrogen peroxide, and measuring the hydrogen peroxide produced.

8. 8. The method for measuring glycated hemoglobin according to claim 7, wherein glycated hemoglobin in the hemoglobin-containing sample is reacted with an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide in the presence of a denaturing agent for glycated hemoglobin.

9. The method for measuring glycated hemoglobin according to claim 7 or 8, wherein the measurement of hydrogen peroxide is carried out using a hydrogen peroxide measuring reagent.

10. The method for measuring glycated hemoglobin according to claim 9 , wherein the hydrogen peroxide measuring reagent contains peroxidase and a leuco chromogen.

11. The method for measuring glycated hemoglobin according to claim 10, wherein the leuco chromogen is a phenothiazine chromogen.

12. The method for measuring glycated hemoglobin according to claim 11, wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

13. A reagent for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising an enzyme that catalyzes a reaction for oxidizing glycated hemoglobin to produce hydrogen peroxide, and at least one compound selected from the group consisting of 6,6-dithiodinicotinic acid, 6-maleimidohexanoic acid, N-ethylmaleimide, N-methylmaleimide, N-benzylmaleimide, and N-cyclohexylmaleimide.

14. The measurement reagent according to claim 13 , further comprising a denaturant for glycated hemoglobin.

15. The measuring reagent according to claim 13 or 14, further comprising a hydrogen peroxide measuring reagent.

16. The hydrogen peroxide measuring reagent according to claim 15, which comprises peroxidase and a leuco chromogen.

17. The measurement reagent according to claim 16 , wherein the leuco chromogen is a phenothiazine chromogen.

18. The measuring reagent according to claim 17, wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

19. A kit for measuring glycated hemoglobin in a hemoglobin-containing sample, comprising: a first reagent containing at least one compound selected from the group consisting of 6,6-dithiodinicotinic acid, 6-maleimidohexanoic acid, N-ethylmaleimide, N-methylmaleimide, N-benzylmaleimide, and N-cyclohexylmaleimide; and a second reagent containing an enzyme that catalyzes a reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide.

20. The measuring kit according to claim 19 , wherein a denaturant for glycated hemoglobin is contained in either one or both of the first and second reagents.

21. The measuring kit according to claim 19 or 20, further comprising a peroxidase and a leuco chromogen in the first and second reagents, or in the second and first reagents, respectively.

22. The measurement kit according to claim 21 , wherein the leuco chromogen is a phenothiazine chromogen.

23. The measuring kit according to claim 22, wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

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