JP7645238B2 - Method for reducing the effects of hemoglobin, method for measuring glycated hemoglobin, reagent for measuring glycated hemoglobin, and kit for measuring glycated hemoglobin - Google Patents

Description

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.

Glycated proteins are contained in body fluids such as blood and biological samples such as hair. The concentration of glycated proteins in blood depends on the concentration of sugars such as glucose dissolved in serum, and in the field of clinical diagnosis, 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 subunits each of two types of α chains and β chains. HbA1c is defined as a hemoprotein in which the N-terminal valine residue of the β chain is specifically 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).

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. Specifically, there is a method for measuring HbA1c in a specimen by allowing a protease to act on HbA1c in blood cells to produce a fructosyl dipeptide (Fru-Val-His), which is a glycosylated peptide, and then allowing fructosyl peptide oxidase to act on the fructosyl dipeptide to produce hydrogen peroxide, and measuring the hydrogen peroxide produced (see Patent Document 1); and there is a method for measuring HbA1c in a specimen by allowing a protease to act on HbA1c in blood cells to produce a fructosyl hexapeptide (Fru-Val-His-Leu-Thr-Pro-Glu), which is a glycosylated peptide, and then allowing fructosyl hexapeptide oxidase to act on the fructosyl hexapeptide produced 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).

In addition, 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).

JP 2001-95598 A WO 2008/108385 International Publication No. 2013/162035 International Publication No. 2015/005258 International Publication No. 2015/005257 International Publication No. 2015/060429 International Publication No. 2018/021530 JP 2010-187604 A

Clin Chem Lab Med, Vol. 36, p. 299-308 (1998). Diabetes, Vol. 27, No. 2, pp. 102-107 (1978). Journal of the Japanese Society for Clinical Laboratory Automation, Vol. 18, No. 4, p. 620 (1993). Scientific Reports (2019) 9:942

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.

The present inventors conducted intensive research to solve this problem, and discovered that in a method for measuring glycated hemoglobin in a hemoglobin-containing sample using an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized 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 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, 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.

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] The method for reducing the effect of hemoglobin described in [1], comprising reacting glycated hemoglobin in the hemoglobin-containing sample with an enzyme that catalyzes a reaction of oxidizing the glycated hemoglobin to produce hydrogen peroxide in the presence of a denaturing agent for the 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 according to [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 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.
[10] The method for measuring glycated hemoglobin according to [9], 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.
[11] The method for measuring glycated hemoglobin according to [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 hydrogen peroxide measurement reagent.
[14] The method for measuring glycated hemoglobin according to [13], wherein the hydrogen peroxide measuring 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 measuring 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 of oxidizing glycated hemoglobin to produce hydrogen peroxide.
[24] The measuring kit according to [23], wherein a denaturant for glycated hemoglobin is contained in either or both of the first reagent and the second reagent.
[25] The measuring 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.

The present invention provides a method for measuring glycated hemoglobin in a hemoglobin-containing sample using an enzyme that catalyzes a reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide without using a protease, a method for reducing the influence of hemoglobin in the hemoglobin-containing sample, 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.

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).

(1) Method for reducing the influence of hemoglobin in a method for measuring glycated hemoglobin The method for reducing the influence of hemoglobin in a 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 of oxidizing glycated hemoglobin to produce hydrogen peroxide to produce hydrogen peroxide, and measuring the produced hydrogen peroxide, characterized in that the glycated hemoglobin in the hemoglobin-containing sample is reacted with the 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.
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.

(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 the 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 according to the steps (i) and (ii) above using glycated hemoglobin of known concentrations.

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, thereby reacting the 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.

(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 the step (i) to generate hydrogen peroxide;
(iii) measuring the hydrogen peroxide produced in step (ii); and
(iv) determining the concentration 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 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.

Furthermore, the method for measuring glycated hemoglobin in a hemoglobin-containing sample in the method for reducing the effects 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 effects of hemoglobin of the present invention is specifically a measurement method including the following steps:

(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 measurements according to 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).

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, thereby reacting the 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.

(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 the 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 steps (ii) to (iv) above 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).

The measurement of the total hemoglobin amount in 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 the hemoglobin-containing sample.

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 denaturant for glycated hemoglobin 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.

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 of the hemolysis treatment include whole blood, blood cells, blood cells mixed with plasma, and samples obtained by hemolyzing these samples. The hemolysis treatment is not particularly limited as long as it is a treatment that hemolyzes whole blood, blood cells, and blood cells mixed with plasma, and examples of the hemolysis treatment include physical methods, chemical methods, biological methods, and the like. 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.

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.

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

In the method for reducing the effects 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, 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.

The dithio compounds are 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.

The maleimide-based compound is not particularly limited, and examples thereof include 6-maleimidohexanoic acid, N-ethylmaleimide, N-methylmaleimide, N-benzylmaleimide, and N-cyclohexylmaleimide, with 6-maleimidohexanoic acid (6-MH) or a derivative thereof being 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.

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 a hemoglobin-containing sample.

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 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 under which the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide can act on glycated hemoglobin.

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 pH to be set. 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.

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), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and the like.
The concentration of the buffer solution is not particularly limited as long as it is a concentration that enables the measurement of glycated hemoglobin according to 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.

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 produce hydrogen peroxide is not particularly limited as long as it is a reaction temperature that enables the measurement method of glycated hemoglobin of the present invention, but is preferably 10 to 50°C, more preferably 20 to 40°C, 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, and 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.

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 (pH 8.0) 0.1 mmol/L
N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE) 0.3g/L
Peroxidase 3 kU/L
4-Aminoantipyrine 0.1g/L

Second reagent: Fructosyl valine aqueous solution 1.7 mmol/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).

<Sample diluent>
Phosphate buffer (pH 8.0) 10 mmol/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

<Measurement>
The above sample (2 μL) and the first reagent (150 μL) of the enzyme activity measuring reagent were added to a reaction cell, and reacted at 37° C. for 3 minutes (first reaction), and then the second reagent (20 μL) of the enzyme activity measuring reagent was added. The absorbance of the reaction solution 3 minutes after the addition of the second reagent [Abs _{enzyme (after 3 minutes)} ] and the absorbance of the reaction solution 5 minutes after the addition of the second reagent [Abs _{enzyme (after 5 minutes)} ] were both measured at a dominant wavelength of 546 nm and a sub-wavelength of 700 nm. Abs _{enzyme (after 3 minutes)} was subtracted from Abs _{enzyme (after 5 minutes)} to calculate the absorbance difference ΔAbs enzyme. The calculated absorbance difference ΔAbs _enzyme was further divided by 2 (minutes) to calculate the amount of absorbance change per minute ΔAbs _enzyme /min.
The absorbance difference ΔAbs′ _blank /min was calculated in the same manner, except that the above specimen diluent was used instead of the above sample.
The ΔAbs' _blank /min was subtracted from the calculated ΔAbs' _enzyme /min to determine the absorbance difference ΔAbs _enzyme /min for the enzyme.

<Enzyme activity>
Based on the ΔAbs _enzyme /min determined above, the enzyme activity was calculated according to the following formula (I).

上記式（Ｉ）において、３３．８は、反応により生成されるキノイミン色素のミリモル吸光係数［（ｍｍｏｌ／Ｌ）^－１・ｃｍ^－１］である。
上記式（Ｉ）において、０．５は、１ｍｏｌの過酸化水素により生成されるキノイミン色素のｍｏｌ数である。

In the above formula (I), 33.8 is the millimolar absorption coefficient [(mmol/L) ^-1 ·cm ^-1 ] of the quinoimine dye produced by the reaction.
In the above formula (I), 0.5 is the number of moles of the quinoimine dye produced by 1 mol of hydrogen peroxide.

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 such enzymes include the 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 such enzymes 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.

There are no particular limitations on the method for measuring the generated hydrogen peroxide, so long as it is a method capable of measuring hydrogen peroxide. Examples include a method using an electrode, a method using a reagent for measuring hydrogen peroxide, etc., with a method using a reagent for measuring hydrogen peroxide being preferred. A reagent for measuring hydrogen peroxide is a reagent for converting hydrogen peroxide into a detectable substance. Examples of detectable substances include dyes, light (luminescence), and fluorescence, with dyes being preferred.

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 oxidative coupling chromogens and leuco chromogens, with leuco chromogens being preferred.

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 type 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.

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 couplers include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine.
Examples of anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N,N-bis(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N- Examples thereof include ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS).
Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

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 reagent for measuring hydrogen peroxide 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.

(2) Method for measuring glycated hemoglobin in a hemoglobin-containing sample The method for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention 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 generate hydrogen peroxide in the presence of at least one compound selected from the group consisting of dithio compounds and maleimide compounds to generate hydrogen peroxide, and measuring the generated hydrogen peroxide.
Specifically, the measurement method includes the following steps.

(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 the 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 according to the steps (i) and (ii) above using glycated hemoglobin of known concentrations.

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 the 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, thereby reacting the 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.

(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 the step (i) to generate hydrogen peroxide;
(iii) measuring the hydrogen peroxide produced in step (ii); and
(iv) determining the concentration 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 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.

The method of measuring glycated hemoglobin in a hemoglobin-containing sample 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 of measuring glycated hemoglobin in a hemoglobin-containing sample is specifically a measurement method including the following steps:

(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 measurements according to 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).

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, thereby reacting the 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.

(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 the 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 steps (ii) to (iv) above 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).

The measurement of the total hemoglobin amount in 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 the hemoglobin-containing sample.

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.

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 for measuring glycated hemoglobin in a hemoglobin-containing sample of the present invention include the above-mentioned dithio compounds and maleimide compounds.

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.

Examples of 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 the hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide described above. Also, examples of 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.

(3) Reagent for measuring glycated hemoglobin in a hemoglobin-containing sample 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 measurement reagent of the present invention may further contain a reagent for measuring hydrogen peroxide.

In the measurement reagent of the present invention, examples of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce 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 produce hydrogen peroxide; at least one compound selected from the group consisting of dithio compounds and maleimide compounds; and the hydrogen peroxide measurement reagent.

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 aqueous media described above. 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 measurement method of glycated hemoglobin of the present invention, but a content such that the concentration when dissolved in an aqueous medium is 0.1 to 30 kU/L is preferable, and a content of 0.2 to 15 kU/L is more preferable, and for example, a content of 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.

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 preferred, and a content in which the concentration is 0.1 to 1 g/L is more preferred, 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.

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 denaturing agent 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 such that the concentration when dissolved in an aqueous medium is 0.01 to 50 g/L is preferable, and a content of 0.1 to 5 g/L is more preferable, 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.
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. 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.

(4) Kit for measuring glycated hemoglobin in a hemoglobin-containing sample 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.

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 for a certain period of time at a certain temperature. After that, the second reagent is added and the hydrogen peroxide produced is measured, thereby measuring glycated hemoglobin.

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.

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 of oxidizing glycated hemoglobin to produce hydrogen peroxide, and comprising a hydrogen peroxide measurement reagent in either or both of the first reagent and the second reagent.

When a reagent containing peroxidase and a leuco chromogen is used as a hydrogen peroxide measurement reagent, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents. That is, it is preferable that the peroxidase and the leuco chromogen are contained in the first reagent and the second reagent, or the second reagent and the first reagent, respectively.

Kit 3
a first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide; and
A kit comprising a second reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds.

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

When a reagent containing peroxidase and a leuco chromogen is used as a hydrogen peroxide measurement reagent, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents. That is, it is preferable that the peroxidase and the leuco chromogen are contained in the first reagent and the second reagent, or the second reagent and the first reagent, respectively.

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
A kit including a second reagent containing a hydrogen peroxide measurement reagent.

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
A kit including a second reagent containing a hydrogen peroxide measurement reagent.

When a reagent containing peroxidase and a leuco chromogen is used as a hydrogen peroxide measurement reagent, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents. That is, it is preferable that the peroxidase and the leuco chromogen are contained in the first reagent and the second reagent, or the second reagent and the first reagent, respectively.

Kit 7
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, and a second reagent containing at least one compound selected from the group consisting of dithio compounds and maleimide compounds.

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 containing a hydrogen peroxide measurement reagent.

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 is the concentration of the above-mentioned glycated hemoglobin denaturant when dissolved in an aqueous medium.

When a reagent containing peroxidase and a leuco chromogen is used as a hydrogen peroxide measurement reagent, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents. That is, it is preferable that the peroxidase and the leuco chromogen are contained in the first reagent and the second reagent, or the second reagent and the first reagent, respectively.

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 such that the concentration when dissolved in an aqueous medium is 0.1 to 60 kU/L is preferable, and a content of 0.4 to 30 kU/L is more preferable, for example, a content of 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.

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 preferred, and a content in which the concentration is 0.1 to 1 g/L is more preferred, 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.

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

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 Co., Ltd.]
NIKKOL ECTD-3NEX [Polyoxyethylene alkyl ether acetic acid (sodium polyoxyethylene tridecyl ether acetate); manufactured by Nikko Chemicals Co., Ltd.]
K Liporan PJ-400J [alkenylsulfonic acid (sodium tetradecene sulfonate); manufactured by Kao Corporation]

Reference Example 1: Construction 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.

(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
・_MgSO4 1 mmol/L
・DNA polymerase (KOD-Plus-) 0.02 U/μL
- Add sterile water to make the volume 50 μL.

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

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 a plasmid, pTrc-FPOX-47, 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 forward and reverse primers, respectively.

(2) Construction of FPOX-47△3 expression vector pTrc-FPOX-47△3 FPOX-47△3-R, consisting of the base sequence represented by SEQ ID NO: 7, was designed by removing the 9 bases located immediately before the stop codon on the 3' end of the base sequence encoding FPOX-47 contained in the plasmid pTrc-FPOX-47 and adding a stop codon and a Bam HI restriction enzyme recognition site, and pTrc-F, consisting of the base sequence represented by SEQ ID NO: 5, which reflects the base sequence immediately before the multicloning site of the pTrc99a vector, were designed and chemically synthesized.
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.

(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
・_MgSO4 1 mmol/L
・DNA polymerase (KOD-Plus-) 0.02 U/μL
- Add sterile water to make the volume 50 μL.

(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 min

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 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 DNA fragment was treated with two restriction enzymes, Nco I and Bam HI (both from 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.

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 E. 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 termination codon of the base sequence of FPOX-47 were deleted. This plasmid is called pTrc-FPOX-47△3.

(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℃ for 14 hours with shaking. The resulting culture solution was purified according to the purification method of 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.

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

<Measurement kit A>
First reagent: Bis-Tris (pH 6.5) 50 mmol/L
DA-67 40μmol/L
NIKKOL LMT 1g/L
Second reagent: Phosphate buffer (pH 6.5) 10 mmol/L
FPOX-47△3 1.5kU/L
Peroxidase 200 kU/L

<Measurement kit a>
First reagent: Bis-Tris (pH 6.5) 50 mmol/L
DA-67 40μmol/L
Second reagent: Phosphate buffer (pH 6.5) 10 mmol/L
FPOX-47△3 1.5kU/L
Peroxidase 200 kU/L

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

<Sample>
The hemolyzed samples used were prepared by hemolyzing blood cells obtained by centrifuging human blood and diluting them with deionized water. The hemolyzed samples had HbA1c concentrations of 2.6 μmol/L, 4.5 μmol/L, 6.4 μmol/L, and 8.4 μmol/L according to the KO500 method, which is the standard method for measuring HbA1c, and the SLS-hemoglobin method, which is a method for measuring total hemoglobin.

<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 the 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 calculate the absorbance difference _ΔE'A .
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 (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 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 calculate the absorbance difference _ΔE'a .
The absorbance difference ΔE for each sample was determined by subtracting ΔE' _a from ΔE' _A. The relationship between the HbA1c concentration and ΔE in each sample is shown in FIG.

As is clear from 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.

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

First Reagent (1-A) Reagent Bis-Tris (pH 7.0) 50 mmol/L
NIKKOL LMT 3g/L
DA-67 80μmol/L
(1-B) Reagent Bis-Tris (pH 7.0) 50 mmol/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 a first reagent.
Second reagent: Bis-Tris (pH 7.0) 50 mmol/L
FPOX-47△3 1.5kU/L
Peroxidase 200 kU/L

[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.

[Example 2]
Kit 1A of Example 1 was used as the HbA1c measurement kit, and six concentrations of whole blood, the HbA1c concentrations of which were determined to be 5.11%, 5.73%, 6.40%, 7.45%, 8.55%, and 10.56% by the KO500 HPLC method, 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.

(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 a standard specimen (total hemoglobin concentration: 15.0 mg/ml) attached to the Hemoglobin B-Test Wako, and the absorbance for the standard specimen 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. Measurement was performed in the same manner except that saline was used instead of the standard, and was taken as the absorbance for saline. 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).

(2) Preparation of a calibration curve for determining HbA1c concentration Two hemolyzed blood cell fractions, the HbA1c concentrations of which were determined 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 sub-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 5 minutes (second reaction), and the absorbance (E2) of the reaction solution was measured at a main wavelength of 660 nm and a sub-wavelength of 800 nm. E1 was subtracted from E2 to calculate the absorbance difference ΔE' 1A} , which was used as the absorbance for each blood cell fraction. The absorbance difference ΔE' _saline was calculated by the same method except that saline was used instead of each blood cell fraction, and used as the absorbance for saline. The blank-corrected absorbance for each blood cell fraction was calculated by subtracting the absorbance for 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 each blood cell fraction and the blank-corrected absorbance for saline (0 Abs).

(3) Determination of total hemoglobin concentration in each blood cell fraction For each of the six concentrations of whole blood samples, centrifugation was performed 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. For each hemolyzed blood cell fraction, measurement was performed 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 measurement value and the calibration curve in (1).

(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).

(5) Determination of HbA1c (%) (proportion of HbA1c concentration to total hemoglobin concentration) HbA1c (%) was calculated as the NGSP value (international standard value) using 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, according to the following formula (II).

(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 measurement method of the present invention and the KO500 method was verified from the HbA1c (%) determined in (5) above and the HbA1c (%) previously determined using the KO500 method, and the correlation coefficient was determined. The results are shown in Table 1.

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. 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.

[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.

As shown in Table 1, in measurements using 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, indicating a good correlation between the two measurement methods. Therefore, it has become clear that the measurement method of the present invention using kits 1A to 1O of Example 1 can accurately measure HbA1c in a sample.

[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 subjected to the same measurement 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.

(2) Determination of HbA1c concentration in each hemolyzed sample Each hemolyzed sample prepared in (1) was measured 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.

(3) Determination of HbA1c (%) (= ratio of HbA1c concentration to total hemoglobin concentration) HbA1c (%) was calculated as an NGSP value (international standard value) using 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, according to formula (II) above.

(4) Evaluation of the effect of total hemoglobin concentration 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.

Instead of HbA1c measurement kit 1A of Example 1, similar measurements were performed using kits 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N and 1O of Example 1 as HbA1c measurement kits, 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 [ΔHbA1c concentration (%)] was calculated. The results are shown in Table 2.

[Comparative Example 3]
The HbA1c concentration (%) in each hemolyzed sample was measured for kit 1a in the same manner as in Example 3, except that kit 1a in Comparative Example 1 was used instead of kit 1A in Example 1. 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 2.

As mentioned above, the hemolyzed samples used in the measurements 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 the ΔHbA1c concentration (%) is to 0, the less the influence of hemoglobin is. As is clear from Table 2, the method of measuring glycated hemoglobin of the present invention using each of Kits 1A to 1O of the present invention was found to reduce the influence of hemoglobin compared to the method of measuring glycated hemoglobin using Kit 1a of Comparative Example 1.

[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) 50 mmol/L
DA-67 40μmol/L
6,6-dithiodinicotinic acid (DTDN) 0.3g/L
Surfactants (see Table 3)
Second reagent: Bis-Tris (pH 7.0) 50 mmol/L
FPOX-47△3 1.5kU/L
Peroxidase 200 kU/L

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

[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.

In addition, measurements were performed in the same manner as in Example 3, 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 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 HbA1c concentration (%) of each hemolyzed sample from the standard [ΔHbA1c concentration (%)] was calculated. The results are shown in Table 3.

[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.

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 demonstrates that even when the type of denaturing agent for glycated hemoglobin is changed, the method for measuring glycated hemoglobin of the present invention using 6,6-dithiodinicotinic acid, a dithio compound, is less affected by hemoglobin.

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 glycated hemoglobin measurement reagent and a glycated hemoglobin measurement kit.

Claims (23)

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 . The method for reducing the effects of hemoglobin according to claim 1, wherein the glycated hemoglobin in the hemoglobin-containing sample is reacted with an enzyme that catalyzes a reaction in which the glycated hemoglobin is oxidized to produce hydrogen peroxide in the presence of a denaturing agent for the glycated hemoglobin. 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. 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. The method for reducing the effect of hemoglobin according to claim 4 , wherein the leuco chromogen is a phenothiazine chromogen. 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. 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 produced hydrogen peroxide. 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. 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. The method for measuring glycated hemoglobin according to claim 9 , wherein the hydrogen peroxide measuring reagent contains peroxidase and a leuco chromogen. The method for measuring glycated hemoglobin according to claim 10 , wherein the leuco chromogen is a phenothiazine chromogen. 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. 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 . The measurement reagent according to claim 13 , further comprising a denaturant for glycated hemoglobin. The reagent for measuring hydrogen peroxide according to claim 13 or 14 , further comprising a reagent for measuring hydrogen peroxide. The hydrogen peroxide measuring reagent according to claim 15 , which comprises peroxidase and a leuco chromogen. The measurement reagent according to claim 16 , wherein the leuco chromogen is a phenothiazine chromogen. The measuring reagent according to claim 17 , wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof. 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 in which glycated hemoglobin is oxidized to produce hydrogen peroxide. 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. 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. The measurement kit according to claim 21 , wherein the leuco chromogen is a phenothiazine chromogen. 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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