Method for measuring glycated hemoglobin in a sample containing abnormal hemoglobin

Abstract

Provided is a method for measuring glycated hemoglobin in a sample containing abnormal hemoglobin, said method involving generating hydrogen peroxide by reacting, in the presence of at least one type of anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acids, glycated hemoglobin in a sample containing abnormal hemoglobin with an enzyme that catalyzes a reaction that generates hydrogen peroxide by oxidizing the glycated hemoglobin, and measuring the generated hydrogen peroxide.

Description

[Technical field]

[0001] The present invention relates to a method for measuring glycated hemoglobin in a sample containing abnormal hemoglobin, a measuring reagent, a measuring kit and a composition. This application claims priority based on Japanese Patent Application No. 2020-55732, filed on March 26, 2020, the contents of which are incorporated herein by reference. [Background technology]

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

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

[0004] [Patent Document 1] JP 2001-95598 A [Patent Document 2] International Publication No. 2008 / 108385 [Patent Document 3] International Publication No. 2013 / 162035 [Patent Document 4] International Publication No. 2015 / 005258 [Patent Document 5] International Publication No. 2015 / 005257 [Patent Document 6] International Publication No. 2015 / 060429 [Patent Document 7] International Publication No. 2018 / 021530 [Non-patent literature]

[0005] [Non-Patent Document 1] Clin Chem Lab Med, Vol. 36, p. 299-308 (1998). [Non-Patent Document 2] Diabetes, Vol. 27, No. 2, pp. 102-107 (1978). [Non-Patent Document 3] Journal of the Japanese Society for Clinical Laboratory Automation, Vol. 18, No. 4, p. 620 (1993). [Non-Patent Document 4] Scientific Reports (2019) 9:942 Summary of the Invention [Problem to be solved by the invention]

[0006] In addition to normal hemoglobin such as hemoglobin A, abnormal hemoglobin is also known to exist. Examples of abnormal hemoglobin include hemoglobin C (HbC) in which the sixth glutamic acid from the N-terminus of the hemoglobin β-chain is replaced with lysine, and hemoglobin S (HbS) in which the sixth glutamic acid from the N-terminus of the hemoglobin β-chain is replaced with valine. HbS is also known as a cause of sickle cell disease. Abnormal hemoglobin is inherited genetically, and may be contained in a sample.

[0007] However, in a method for measuring glycated hemoglobin that uses an enzyme that catalyzes a reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide without using a protease, the accuracy of measuring the concentration of glycated hemoglobin in a sample containing abnormal hemoglobin may be inferior to that in a sample not containing abnormal hemoglobin. This is thought to be because in the method of measuring glycated hemoglobin using an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide without using protease, the abnormal hemoglobin molecule itself, rather than the short glycated peptides generated by protease, serves as the substrate for the enzyme and is therefore easily affected by abnormal hemoglobin.

[0008] The present invention has been made to solve the problems described above, and aims to provide a method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample that uses an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide without using a protease, which has excellent accuracy in measuring glycated hemoglobin in the abnormal hemoglobin-containing sample, a reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, a kit for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, and a composition. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the inventors have discovered that 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, the accuracy of measuring glycated hemoglobin in a sample containing abnormal hemoglobin can be improved by using a specific anionic surfactant, and have thus completed the present invention. That is, the present invention relates to the following [1] to

[30] .

[0010] [1] A method for measuring glycated hemoglobin in a sample containing abnormal hemoglobin, comprising reacting glycated hemoglobin in the sample containing abnormal hemoglobin with an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide in the presence of at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid to produce hydrogen peroxide, and measuring the hydrogen peroxide produced. [2] The method for measuring glycated hemoglobin according to [1], wherein the sulfonic acid anionic surfactant is at least one anionic surfactant selected from the group consisting of alkenyl sulfonic acids, alkyl sulfonic acids, N-acyltaurines in which the hydrogen atom of the amino group may be substituted with a substituent, alkyl sulfoacetic acids, alkyl benzene sulfonic acids, and alkyl polyoxyethylene sulfosuccinates. [3] The method for measuring glycated hemoglobin described in [1] or [2], wherein the abnormal hemoglobin is one in which one or more amino acids have been deleted, substituted or added in at least the amino acid sequence 1 to 6 from the N-terminus of the β chain of human hemoglobin A. [4] The method for measuring glycated hemoglobin according to any one of [1] to [3], wherein the abnormal hemoglobin is human hemoglobin C or hemoglobin S. [5] The method for measuring glycated hemoglobin according to any one of [1] to [4], wherein the content of the abnormal hemoglobin relative to the total hemoglobin in the hemoglobin-containing sample is 20% or more. [6] The method for measuring glycated hemoglobin according to any one of [1] to [5], wherein the measurement of hydrogen peroxide is carried out using a reagent for measuring hydrogen peroxide. [7] The method for measuring glycated hemoglobin according to [6], wherein the hydrogen peroxide measurement reagent is a reagent containing peroxidase and a leuco chromogen. [8] The method for measuring glycated hemoglobin according to [7], wherein the leuco chromogen is a phenothiazine chromogen. [9] The method for measuring glycated hemoglobin according to [8], wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

[0011]

[10] An enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide; A reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, comprising at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid.

[11] The measurement reagent described in

[10] , wherein the sulfonic acid anionic surfactant is at least one anionic surfactant selected from the group consisting of alkenyl sulfonic acids, alkyl sulfonic acids, N-acyltaurines in which the hydrogen atoms of the amino groups may be substituted with substituents, alkyl sulfoacetic acids, alkyl benzene sulfonic acids, and alkyl polyoxyethylene sulfosuccinates.

[12] The measurement reagent according to

[10] or

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

[13] The hydrogen peroxide measurement reagent according to

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

[14] The measurement reagent according to

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

[15] The measurement reagent according to

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

[0012]

[16] A kit for measuring glycated hemoglobin in a sample containing abnormal hemoglobin, comprising: a first reagent containing at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and a second reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide.

[17] The measurement kit described in

[16] , wherein the sulfonic acid anionic surfactant is at least one anionic surfactant selected from the group consisting of alkenyl sulfonic acids, alkyl sulfonic acids, N-acyltaurines in which the hydrogen atoms of the amino groups may be substituted with substituents, alkyl sulfoacetic acids, alkyl benzene sulfonic acids, and alkyl polyoxyethylene sulfosuccinates.

[18] The measurement kit according to

[16] or

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

[19] The measurement kit according to

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

[20] The measurement kit according to

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

[0013]

[21] Glycated hemoglobin, including abnormal hemoglobin, and at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acids.

[22] The composition described in

[21] , wherein the sulfonic acid anionic surfactant is at least one anionic surfactant selected from the group consisting of alkenyl sulfonic acids, alkyl sulfonic acids, N-acyltaurines in which the hydrogen atoms of the amino groups may be substituted with substituents, alkyl sulfoacetic acids, alkylbenzene sulfonic acids, polyoxyethylene alkyl sulfosuccinates, and polyoxyethylene alkyl ether acetic acids.

[23] The composition according to

[21] or

[22] , further comprising an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide.

[24] The composition according to any one of

[21] to

[23] , further comprising a reagent for measuring hydrogen peroxide.

[25] The composition described in

[24] , wherein the hydrogen peroxide measurement reagent is at least one selected from the group consisting of peroxidase and leuco chromogen.

[26] The composition described in

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

[27] The composition described in

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

[28] The composition described in any one of

[21] to

[27] , wherein the abnormal hemoglobin is one in which one or more amino acids have been deleted, substituted or added in at least the amino acid sequence 1 to 6 from the N-terminus of the β chain of human hemoglobin A.

[29] The composition according to any one of

[21] to

[28] , wherein the abnormal hemoglobin is human hemoglobin C or hemoglobin S.

[30] The composition according to any one of

[21] to

[29] , wherein the content of the abnormal hemoglobin relative to the total hemoglobin in the composition is 20% or more. Effect of the Invention

[0014] According to the present invention, there are provided a method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, which has excellent accuracy in measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, a reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, a kit for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, and a composition. [Brief description of the drawings]

[0015] [Figure 1] 1 is a graph showing the relationship between HbA1c concentration and absorbance difference ΔE in the measurement of HbA1c in a hemoglobin-containing sample using Kit A containing FPOX-47Δ3 as the second reagent. The vertical axis represents absorbance difference ΔE (Abs×10000), and the horizontal axis represents HbA1c concentration (μmol / L). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, embodiments of the method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, the reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, the kit for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, and the composition according to the present invention will be described.

[0017] (1) Method for measuring glycated hemoglobin in a sample containing abnormal hemoglobin A method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample of an embodiment (hereinafter referred to as the measurement method of the embodiment) is a method comprising reacting glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid to produce hydrogen peroxide, and measuring the hydrogen peroxide produced. Specifically, the measurement method may include the following steps.

[0018] (i) a step of reacting glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acids to generate hydrogen peroxide; (ii) measuring the hydrogen peroxide produced in step (i); and (iii) A step of carrying out measurements according to the above steps (i) and (ii) using glycated hemoglobin of known concentration, and determining the concentration of glycated hemoglobin in the abnormal hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (ii) with a calibration curve, which has been prepared in advance and which shows the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin.

[0019] In the above step (i), the step of reacting glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid, to generate hydrogen peroxide, comprises: The method may include a step of mixing at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid with a sample containing abnormal hemoglobin, and then adding an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide, thereby reacting the glycated hemoglobin in the sample containing abnormal hemoglobin with the enzyme that catalyzes the reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide. Specifically, the measurement method may include the following steps.

[0020] (i-1) reacting hemoglobin in a sample containing abnormal hemoglobin with at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acids in an aqueous medium; (i-2) a step of adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium of the step (i-1) to generate hydrogen peroxide; (ii) measuring the hydrogen peroxide produced in the step (i-2); and (iii) A step of carrying out measurements according to the above steps (i-1), (i-2) and (ii) using glycated hemoglobin of known concentration, and determining the concentration of glycated hemoglobin in the abnormal hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (ii) with a calibration curve, which has been prepared in advance and which shows the relationship between the amount of hydrogen peroxide and the concentration of glycated hemoglobin.

[0021] The method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample of the embodiment also includes a method for 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 an abnormal hemoglobin-containing sample of the embodiment may specifically be a measurement method including the following steps:

[0022] (i) measuring the total hemoglobin amount in a sample containing abnormal hemoglobin; (ii) a step of reacting glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid to generate hydrogen peroxide; (iii) measuring the hydrogen peroxide produced in step (ii); (iv) performing the measurements according to the above (ii) and (iii) using a known amount of glycated hemoglobin, and measuring the amount of glycated hemoglobin in the abnormal hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iii) with a calibration curve that has been prepared in advance and shows the relationship between the amount of hydrogen peroxide and the amount of glycated hemoglobin; and (v) calculating the ratio of the amount of glycated hemoglobin to the amount of total hemoglobin in the abnormal 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).

[0023] In the above step (ii), the step of generating hydrogen peroxide by reacting glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid, comprises: The method may include a step of mixing at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid with a sample containing abnormal hemoglobin, and then adding an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide, thereby reacting the glycated hemoglobin in the sample containing abnormal hemoglobin with the enzyme that catalyzes the reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide. Specifically, the measurement method may include the following steps.

[0024] (i) measuring the total hemoglobin amount in a sample containing abnormal hemoglobin; (ii) a step of reacting glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid to generate hydrogen peroxide; (ii-1) reacting hemoglobin in a sample containing abnormal hemoglobin with at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid in an aqueous medium; (ii-2) a step of adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide to the aqueous medium of the step (ii-1) to generate hydrogen peroxide; (iii) measuring the hydrogen peroxide produced in the step (ii-2); (iv) performing measurements according to the above (ii-1), (ii-2) and (iii) using a known amount of glycated hemoglobin, and measuring the amount of glycated hemoglobin in the abnormal hemoglobin-containing sample by comparing the amount of hydrogen peroxide measured in the step (iii) with a calibration curve that has been prepared in advance and shows the relationship between the amount of hydrogen peroxide and the amount of glycated hemoglobin; and (v) calculating the ratio of the amount of glycated hemoglobin to the amount of total hemoglobin in the abnormal 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).

[0025] The measurement of the total hemoglobin amount in step (i) can also be performed after adding at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid to the abnormal hemoglobin-containing sample to denature the hemoglobin and glycated hemoglobin in the abnormal hemoglobin-containing sample.

[0026] The abnormal hemoglobin-containing sample in the measurement method of the embodiment is not particularly limited as long as it contains abnormal hemoglobin and is a sample to which the measurement method of glycated hemoglobin of the embodiment can be applied, and may contain normal hemoglobin that does not fall under abnormal hemoglobin in addition to abnormal hemoglobin. Examples of the above sample include whole blood, blood cells, samples in which blood cells are mixed with plasma, and samples obtained by hemolyzing these samples. The hemolyzing treatment is not particularly limited as long as it is a treatment for hemolyzing whole blood, blood cells, and samples in which blood cells are mixed with plasma, and examples of the hemolyzing treatment include physical methods, chemical methods, biological methods, and the like. Examples of the physical methods include a method using a hypotonic solution such as distilled water, and a method using ultrasound. Examples of the chemical methods include a method using an organic solvent such as methanol, ethanol, and acetone, and a method using a polyoxyethylene surfactant. Examples of the biological methods include a method using an antibody or a complement, and the like.

[0027] As used herein, the term "abnormal hemoglobin" refers to a hemoglobin molecule that has a mutation such as partial amino acid substitution in the amino acid sequence of the amino acid chain that constitutes the hemoglobin molecule, and preferably has a mutation such as partial amino acid substitution in the amino acid sequence of the amino acid chain that constitutes the β chain of hemoglobin. The mutation can be based on, for example, the amino acid sequence of the globin chain that constitutes hemoglobin A, or the amino acid sequence that constitutes the β chain of hemoglobin A.

[0028] Examples of normal hemoglobin that does not fall under the category of abnormal hemoglobin include hemoglobin A (HbA0, HbA1, HbA2) and hemoglobin F.

[0029] The hemoglobin is preferably human (Homo sapiens) hemoglobin. That is, the abnormal hemoglobin-containing sample in the measurement method of the embodiment preferably contains human abnormal hemoglobin.

[0030] 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, the accuracy of measurement of the glycated hemoglobin contained in a sample containing abnormal amino acids may be lower than that in a sample not containing abnormal amino acids. This is probably because the structure and charge of the hemoglobin molecule change due to a mutation, such as a substitution, in the amino acid sequence of the globin chain that constitutes the hemoglobin molecule, resulting in a decrease in reactivity with the enzyme that catalyzes the reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide.

[0031] On the other hand, in the conventional method of measuring HbA1c by reacting protease and fructosyl peptide oxidase with HbA1c and measuring hydrogen peroxide derived from fructosyl dipeptide (Fru-Val-His), a glycosylated peptide, such a problem of decreased reactivity is unlikely to occur. For example, since fructosyl peptide oxidase acts on fructosyl dipeptide (Fru-Val-His) generated by cleavage with protease, even if there are mutations in amino acids other than the part corresponding to the dipeptide in the hemoglobin β chain (the first valine and the second histidine from the N-terminus) like abnormal hemoglobin HbC or HbS, it can be measured in the same way as normal hemoglobin.

[0032] The enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide is presumed to exert its catalytic effect by interacting with the vicinity of the N-terminus of the hemoglobin β-chain. Therefore, it is considered that the decrease in the reactivity of the enzyme is more likely to be affected when there is a mutation such as a substitution in the amino acid sequence of the 1st to 6th amino acids from the N-terminus of the hemoglobin β-chain, which is presumed to be important for the enzymatic reaction.

[0033] From the above viewpoint, the abnormal hemoglobin in the abnormal hemoglobin-containing sample may be at least human hemoglobin A (more specifically, hemoglobin A0) in which one or more amino acids have been deleted, substituted or added in the 1st to 6th amino acid sequence from the N-terminus of the β-chain, The abnormal hemoglobin in the abnormal hemoglobin-containing sample may be one in which one or more amino acids have been deleted, substituted or added in at least the amino acid sequence of the 3rd to 6th amino acids from the N-terminus of the β-chain of human hemoglobin A. The term "one or more" may mean 1 to 3, or may mean 1 or 2.

[0034] Hemoglobin C (HbC), known as an abnormal human hemoglobin, is a hemoglobin in which the sixth glutamic acid from the N-terminus of the hemoglobin β-chain is replaced with lysine, as compared with hemoglobin A. Moreover, in hemoglobin S (HbS), the sixth glutamic acid from the N-terminus of the hemoglobin β-chain is replaced with valine, as compared with hemoglobin A. These HbC and HbS are highly likely to be contained in a sample, and have a substitution in the amino acid sequence of the first to sixth amino acids from the N-terminus of the β-chain, and are therefore considered to have a large effect on the accuracy of the measurement.

[0035] From the above viewpoint, it is more preferable that the abnormal hemoglobin in the abnormal hemoglobin-containing sample is human hemoglobin C or hemoglobin S.

[0036] SEQ ID NO: 11 shows the amino acid sequence of the β-chain of human hemoglobin, which is the subject of comparison.

[0037] The abnormal hemoglobin-containing sample may contain both normal hemoglobin and abnormal hemoglobin. The abnormal hemoglobin content in the abnormal hemoglobin-containing sample, i.e., the ratio of the abnormal hemoglobin amount to the total hemoglobin amount in the sample, is not particularly limited, and for example, on a number basis, may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. There is no particular upper limit for the ratio of abnormal hemoglobin to total hemoglobin in a sample, and the upper limit may be 100% or less, or on a number basis 99% or less, or 90% or less, or 80% or less, or 70% or less, or 60% or less, or 50% or less, or 40% or less. The above numerical ranges can be freely combined. For example, the ratio of the amount of abnormal hemoglobin to the amount of total hemoglobin in the sample may be 5 to 100%, 5 to 90%, 10 to 80%, 10 to 70%, 20 to 60%, 20 to 50%, or 30 to 40% based on the number of pieces. The number standard can be in moles.

[0038] The abnormal hemoglobin-containing sample preferably contains human hemoglobin C or hemoglobin S as the abnormal hemoglobin and human hemoglobin A as the normal hemoglobin.

[0039] In this specification, glycated hemoglobin is produced by binding a sugar such as glucose to hemoglobin, and may be produced by binding a sugar such as glucose to the β chain of hemoglobin, and may be hemoglobin A1, and examples thereof include hemoglobin A1a, hemoglobin A1b, HbA1c, etc., with HbA1c being preferred. In the measurement of HbA1c in the Examples described below, glycated hemoglobin of abnormal hemoglobin HbC, which is not HbA, is also glycated hemoglobin produced by the binding of sugar to the corresponding position of the hemoglobin β chain corresponding to hemoglobin A1, so for convenience, these are collectively expressed as the amount of HbA1c.

[0040] In the present specification, the term "glycated hemoglobin" is a concept that includes glycosylated hemoglobin of abnormal hemoglobin. The glycosylated hemoglobin contained in the abnormal hemoglobin-containing sample can include glycosylated hemoglobin of abnormal hemoglobin.

[0041] In the measurement method of the embodiment, the total hemoglobin concentration (μmol / L) in the reaction solution is not particularly limited, but is preferably 0.5 to 100 μmol / L, more preferably 3 to 20 μmol / L, and examples of such concentrations include 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, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 μmol / L.

[0042] Here, the reaction liquid in the measurement method of the embodiment means a component of the reaction system in the step of generating hydrogen peroxide. In the examples described later, an example of the reaction liquid is a mixture of a sample, a first reagent, and a second reagent. The term "in the presence of an anionic surfactant" means that the reaction liquid in the step of generating hydrogen peroxide can contain an anionic surfactant.

[0043] In the measurement method of the embodiment, at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acids is used.

[0044] In this specification, the term "sulfonic acid anionic surfactant" refers to an anionic surfactant, which is a compound having a sulfonic acid group. Examples of sulfonic acid-based anionic surfactants include at least one anionic surfactant selected from the group consisting of alkenyl sulfonic acids, alkyl sulfonic acids, N-acyltaurines in which the hydrogen atom of the amino group may be substituted with a substituent, alkyl sulfoacetic acids, alkyl benzene sulfonic acids, and alkyl polyoxyethylene sulfosuccinates.

[0045] In this specification, the alkenyl in the alkenylsulfonic acid includes, for example, an alkenyl group having 8 to 20 carbon atoms, and preferably an alkenyl group having 10 to 18 carbon atoms. Examples of the alkenyl having 8 to 20 carbon atoms include octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, and the like. In the present specification, the alkenylsulfonic acid may be in the form of a salt, such as a sodium salt, a potassium salt, an ammonium salt, a magnesium salt, a calcium salt, or a monoethanolamine salt. Specific examples (products) of alkenylsulfonic acids or salts thereof include K-Liporan PJ-400J (sodium tetradecenesulfonate; manufactured by Lion Corporation). In the measurement method of the embodiment, the concentration of the alkenylsulfonic acid or a salt thereof in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0046] In the present specification, the alkyl in the alkylsulfonic acid includes, for example, alkyl having 8 to 20 carbon atoms, and preferably alkyl having 10 to 18 carbon atoms. Examples of the alkyl having 8 to 20 carbon atoms include octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, and icosyl. Examples of the alkyl having 10 to 18 carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, and octadecyl (stearyl). In the present specification, the alkylsulfonic acid may be in the form of a salt, such as a sodium salt, a potassium salt, an ammonium salt, a magnesium salt, a calcium salt, or a monoethanolamine salt. A specific example (product) of alkylsulfonic acid or a salt thereof is sodium lauryl sulfonate (manufactured by Tokyo Chemical Industry Co., Ltd.). In the measurement method of the embodiment, the concentration of the alkylsulfonic acid or a salt thereof in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0047] In the present specification, examples of the substituent in the N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent include, for example, an alkyl group, a halogenated alkyl group, and a phenyl group. Examples of the halogenated alkyl group include, for example, a fluoroalkyl group, a chloroalkyl group, a bromoalkyl group, and an iodoalkyl group. Examples of the alkyl in the alkyl group and the halogenated alkyl group include, for example, an alkyl having 1 to 6 carbon atoms. Examples of the alkyl having 1 to 6 carbon atoms include, for example, methyl, ethyl, propyl, butyl, pentyl, and hexyl. As used herein, examples of N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent include N-acyltaurine, N-acyl-N-alkyltaurine, etc., and N-acyl-N-alkyltaurine, etc. are preferred.

[0048] Examples of the acyl in N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent include acyl having 8 to 20 carbon atoms, and preferably acyl having 10 to 18 carbon atoms. Examples of the acyl having 8 to 20 carbon atoms include octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl (lauroyl), tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl (palmitoyl), heptadecanoyl, octadecanoyl (stearoyl), oleoyl, vaccenoyl, linoleoyl, nonadecanoyl, and eicosanoyl. Examples of acyl having 10 to 18 carbon atoms include decanoyl, undecanoyl, dodecanoyl (lauroyl), tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl (palmitoyl), heptadecanoyl, octadecanoyl (stearoyl), oleoyl, vaccenoyl, and linoleoyl. The alkyl in the N-acyl-N-alkyltaurine includes, for example, alkyl having 1 to 6 carbon atoms. Examples of the alkyl having 1 to 6 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, and hexyl.

[0049] In the present specification, the N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent may be a salt, such as a sodium salt, a potassium salt, an ammonium salt, a magnesium salt, a calcium salt, or a monoethanolamine salt. Specific examples (products) of N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent, or a salt thereof, include, for example, NIKKOL LMT (sodium N-lauroyl-N-methyl taurine; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL PMT (sodium N-palmitoyl-N-methyl taurine; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL MMT (sodium N-myristoyl-N-methyl taurine; manufactured by Nikko Chemicals Co., Ltd.), and NIKKOL SMT (sodium N-stearoyl-N-methyl taurine; manufactured by Nikko Chemicals Co., Ltd.). In the measurement method of the embodiment, the concentration of the N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent, or a salt thereof, in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even more preferably 0.5 to 5 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0050] In the present specification, the alkyl in the alkylsulfoacetic acid includes, for example, alkyl having 8 to 20 carbon atoms, and preferably alkyl having 10 to 18 carbon atoms. Examples of the alkyl having 8 to 20 carbon atoms include octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, and icosyl. Examples of the alkyl having 10 to 18 carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, and octadecyl (stearyl).

[0051] In the present specification, the alkylsulfoacetic acid may be in the form of a salt, such as a sodium salt, a potassium salt, an ammonium salt, a magnesium salt, a calcium salt, or a monoethanolamine salt. Specific examples (products) of alkylsulfoacetic acid or salts thereof include NIKKOL LSA-F (sodium laurylsulfoacetate; manufactured by Nikko Chemicals Co., Ltd.). In the measurement method of the embodiment, the concentration of the alkylsulfoacetic acid or a salt thereof in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0052] In the present specification, the alkyl in the alkylbenzenesulfonic acid includes, for example, alkyl having 8 to 20 carbon atoms, and preferably alkyl having 10 to 18 carbon atoms. Examples of the alkyl having 8 to 20 carbon atoms include octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, and icosyl. Examples of the alkyl having 10 to 18 carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, and octadecyl (stearyl). In the present specification, the alkylbenzenesulfonic acid may be in the form of a salt, such as a sodium salt, a potassium salt, an ammonium salt, a magnesium salt, a calcium salt, or a monoethanolamine salt. Specific examples (products) of alkylbenzenesulfonic acid or its salts include LAS-C8 (sodium pn-octylbenzenesulfonate; manufactured by Nacalai Tesque). In the measurement method of the embodiment, the concentration of alkylbenzenesulfonic acid or a salt thereof in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0053] In the present specification, the alkyl in the polyoxyethylene alkyl sulfosuccinate includes, for example, alkyl having 8 to 20 carbon atoms, and preferably alkyl having 10 to 18 carbon atoms. Examples of the alkyl having 8 to 20 carbon atoms include octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, and icosyl. Examples of the alkyl having 10 to 18 carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, and octadecyl (stearyl). In the present specification, the polyoxyethylene alkyl sulfosuccinate may be in the form of a salt, such as a sodium salt, a potassium salt, an ammonium salt, a magnesium salt, a calcium salt, or a monoethanolamine salt. Specific examples (products) of alkyl polyoxyethylene sulfosuccinate or salts thereof include Neohitenor L-30 (disodium polyoxyethylene lauryl sulfosuccinate; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.). In the measurement method of the embodiment, the concentration of the polyoxyethylene alkyl sulfosuccinate or a salt thereof in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even more preferably 0.5 to 5 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0054] In the present specification, the alkyl in the polyoxyethylene alkyl ether acetic acid may be, for example, an alkyl having 8 to 20 carbon atoms, and preferably an alkyl having 10 to 18 carbon atoms. Examples of the alkyl having 8 to 20 carbon atoms include octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, and icosyl. Examples of the alkyl having 10 to 18 carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, and octadecyl (stearyl). In the present specification, the polyoxyethylene alkyl ether acetic acid may be a salt. Examples of the polyoxyethylene alkyl ether acetate salt include sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, and monoethanolamine salt. Specific examples (products) of polyoxyethylene alkyl ether acetic acid or a salt thereof include, for example, NIKKOL AKYPO RLM 45 NV (polyoxyethylene lauryl ether sodium acetate; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL AKYPO RLM 45 (polyoxyethylene lauryl ether acetic acid; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL AKYPO RLM 100 (polyoxyethylene lauryl ether acetic acid; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL ECT-3NEX (polyoxyethylene tridecyl ether sodium acetate; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL ECTD-3NEX (polyoxyethylene tridecyl ether sodium acetate; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL ECTD-6NEX (polyoxyethylene tridecyl ether sodium acetate; manufactured by Nikko Chemicals Co., Ltd.), and Neohytenol ECL-45 (polyoxyethylene lauryl ether sodium acetate; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.). In the measurement method of the embodiment, the concentration of the polyoxyethylene alkyl ether acetic acid or a salt thereof in the reaction solution is preferably 0.01 to 50 g / L, more preferably 0.1 to 5 g / L, and even 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, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L.

[0055] 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 abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid to the abnormal hemoglobin-containing sample.

[0056] The reaction between glycated hemoglobin in an abnormal 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid can be carried out under any conditions as long as the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide can act on the glycated hemoglobin.

[0057] The reaction between glycated hemoglobin in an abnormal hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate 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 measurement method of the embodiment, and examples thereof include deionized water, distilled water, and buffer solutions, and buffer solutions are preferred. The pH of the aqueous medium is not particularly limited as long as it allows the measurement method of the embodiment, and may be, for example, pH 4 to 10, such as 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, and Good's buffering agent.

[0058] Examples of Good's buffers include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPS), and O), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), N-[ Tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid (HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid [(H)EPPS], N-[tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid N,N-bis(2-hydroxyethyl)glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).The concentration of the buffer solution is not particularly limited as long as it is a concentration that enables the measurement method of the embodiment, and is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, 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.

[0059] The reaction temperature in the reaction between glycated hemoglobin in an abnormal hemoglobin-containing sample and an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide is not particularly limited as long as it is a reaction temperature that enables the measurement method of the embodiment, and 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 is a reaction time that enables the measurement method of the embodiment, and is preferably 1 minute to 3 hours, and more preferably 2.5 minutes to 1 hour, for example, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours. The concentration of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide is not particularly limited as long as it is a concentration that enables the measurement method of the embodiment, and 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.

[0060] In the present specification, the enzyme activity (U) of the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide was calculated by the following method. <Reagents for measuring enzyme activity> First Reagent Phosphate buffer (pH8.0) 0.1mmol / L N-Ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE) 0.3g / L Peroxidase 3kU / L 4-Aminoantipyrine 0.1g / L

[0061] Second Reagent Fructosyl valine aqueous solution 1.7mmol / L The above-mentioned aqueous fructosyl valine solution was prepared by the method described in J. Agric. Food Chem., Vol. 24, No. 1, pp. 70-73 (1976).

[0062] <Specimen diluent> Phosphate buffer (pH8.0) 10mmol / L Bovine serum albumin 0.15% <Sample> FPOX-47△3 (an enzyme that catalyzes the reaction of oxidizing glycosylated hemoglobin to produce hydrogen peroxide) diluted with the above sample dilution solution

[0063] <Measurement> The above sample (2 μL) and the first reagent (150 μL) of the enzyme activity measuring reagent were added to the reaction cell, and the reaction (first reaction) was carried out at 37° C. for 3 minutes. Then, the second reagent (20 μL) of the enzyme activity measuring reagent was added, and the absorbance [Abs 酵素(3分後) ], and the absorbance [Abs 酵素(5分後) ] were measured at a main wavelength of 546 nm and a sub-wavelength of 700 nm, and Abs 酵素(5分後) From Abs 酵素(3分後) Subtract the absorbance difference △Abs' 酵素 Calculate the absorbance difference △Abs' 酵素Divide by 2 (minutes) to get the change in absorbance per minute, △Abs' 酵素 / min was calculated. The absorbance difference ΔAbs' was measured in the same manner, except that the above sample dilution solution was used instead of the above sample. ブランク / min was calculated. Calculated △Abs' 酵素 / min to △Abs' ブランク / min is subtracted, and the absorbance difference for the enzyme is △Abs 酵素 / min was determined.

[0064] <Enzyme activity> The above determined △Abs 酵素 Based on the number of times per minute, the enzyme activity was calculated according to the following formula (I).

[0065]

number

[0066] 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 sample containing abnormal hemoglobin and generates hydrogen peroxide from glycated hemoglobin, and examples of the enzyme include enzymes described in International Publication No. 2015 / 005258 and amadoriase described in International Publication No. 2015 / 060429, the contents of which are incorporated herein by reference. Specific examples of the enzyme include FPOX-18A, FPOX-18B, FPOX-18C, FPOX-18D, and FPOX19-46 described in International Publication No. 2015 / 005258. Specific examples of modified forms of the enzyme include FPOX-47△3, which is produced in Experiment 1 described below.

[0067] The method for measuring the generated hydrogen peroxide is not particularly limited as long as it is a method capable of measuring hydrogen peroxide, and examples thereof include a method using an electrode and a method using a reagent for measuring hydrogen peroxide, and a method using a reagent for measuring hydrogen peroxide is preferred. The reagent for measuring hydrogen peroxide is a reagent for converting hydrogen peroxide into a detectable substance. Examples of the detectable substance include dyes, light (luminescence), and fluorescence, and dyes are preferred.

[0068] When the detectable substance is a dye, the reagent for measuring hydrogen peroxide may be a reagent containing a peroxidatively active substance such as peroxidase and an oxidative color-developing chromogen. Examples of the oxidative color-developing chromogen include an oxidative coupling chromogen and a leuco chromogen, with the leuco chromogen being preferred.

[0069] In this specification, the term "leuco chromogen" refers to a substance that is converted into a dye by itself in the presence of hydrogen peroxide and a peroxidative substance, such as peroxidase. Examples of the leuco chromogen include phenothiazine chromogens, triphenylmethane chromogens, diphenylamine chromogens, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, and tetramethylbenzidine, with phenothiazine chromogens being preferred. Examples of phenothiazine chromogens include 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67), etc. Among phenothiazine chromogens, 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67) is particularly preferred. Examples of triphenylmethane chromogens include N,N,N',N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triaminotriphenylmethane (TPM-PS). Examples of diphenylamine chromogens include N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 4,4'-bis(dimethylamino)diphenylamine, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA), etc.

[0070] In this specification, the oxidative coupling type chromogen refers to two compounds that generate a dye by oxidative coupling in the presence of hydrogen peroxide and a peroxidative active substance. Examples of the combination of two compounds include a combination of a coupler and an aniline, a combination of a coupler and a phenol, etc. Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine. Anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N,N-bis(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N- Ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS), and the like. Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

[0071] When the detectable substance is light (luminescence), the hydrogen peroxide measuring reagent may be a reagent containing a peroxidatively active substance such as peroxidase and a chemiluminescent substance. Examples of chemiluminescent substances include luminol, isoluminol, lucigenin, acridinium ester, etc. When the detectable substance is fluorescence, the hydrogen peroxide measuring reagent may be a reagent containing a peroxidatively active substance such as peroxidase and a fluorescent substance. Examples of fluorescent substances include 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, coumarin, etc.

[0072] (2) Reagent for measuring glycated hemoglobin in samples containing abnormal hemoglobin The embodiment of the measuring reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample (hereinafter referred to as the measuring reagent of the embodiment) is a reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, and at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid. The measuring reagent of the embodiment is used in the embodiment of the measuring method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample. The measuring reagent of the embodiment may further contain a measuring reagent for hydrogen peroxide.

[0073] In the measurement reagent of the embodiment, examples of the enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; and the hydrogen peroxide measurement reagent include, for example, an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; and a hydrogen peroxide measurement reagent, which are exemplified in the measurement method of the embodiment described above. When the measurement reagent of the embodiment contains a sulfonic acid anionic surfactant, the sulfonic acid anionic surfactant is preferably at least one anionic surfactant selected from the group consisting of alkenylsulfonic acid, alkylsulfonic acid, N-acyltaurine in which the hydrogen atom of the amino group may be substituted with a substituent, alkylsulfoacetic acid, alkylbenzenesulfonic acid, and alkyl polyoxyethylenesulfosuccinate.

[0074] The measurement reagent of the embodiment may be in a freeze-dried state or in a state dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous media. When measuring glycated hemoglobin in an abnormal hemoglobin-containing sample using a freeze-dried reagent, the reagent can be 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 embodiment is preferably a content such that the concentration when dissolved in an aqueous medium is 0.1 to 30 kU / L, more preferably a content of 0.2 to 15 kU / L, 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.

[0075] In the glycosylated hemoglobin measurement reagent of the embodiment, the content of at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid is preferably such that the concentration in a state dissolved in an aqueous medium is 0.001 to 10 g / L, more preferably 0.01 to 5 g / L. 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, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / L.

[0076] The measurement reagent of the embodiment 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, pyruvic acid, 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.

[0077] (3) Glycated hemoglobin measurement kit for samples containing abnormal hemoglobin The reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample of the embodiment may be stored, distributed, and used in the form of a kit. The kit for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample of the embodiment (hereinafter referred to as the kit of the embodiment) is used in the method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample of the embodiment. Examples of the measuring kit of the embodiment include a two-reagent kit and a three-reagent kit, and a two-reagent kit is preferred.

[0078] In the kit of the embodiment, examples of the enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide; at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and a hydrogen peroxide measurement reagent include, for example, an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide; at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and a hydrogen peroxide measurement reagent, which are exemplified in the measurement method of the embodiment described above. When the kit of the embodiment contains a sulfonic acid anionic surfactant, the sulfonic acid anionic surfactant is preferably at least one anionic surfactant selected from the group consisting of alkenylsulfonic acids, alkylsulfonic acids, N-acyltaurines in which the hydrogen atom of the amino group may be substituted with a substituent, alkylsulfoacetic acids, alkylbenzenesulfonic acids, and alkyl polyoxyethylenesulfosuccinates.

[0079] In this specification, when measuring glycated hemoglobin in a sample containing abnormal hemoglobin using a two-reagent kit, for example, the sample containing abnormal hemoglobin and the first reagent are added to a reaction cell, and a reaction is carried out at a constant temperature for a certain period of time. Thereafter, the second reagent is added, and the generated hydrogen peroxide is measured, thereby measuring glycated hemoglobin.

[0080] Kit 1 A first reagent containing at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and The kit further comprises a second reagent comprising an enzyme that catalyzes a reaction for oxidizing glycated hemoglobin to produce hydrogen peroxide.

[0081] Kit 2 A first reagent containing at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and A kit comprising a second reagent containing an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide, and comprising a hydrogen peroxide measurement reagent in either or both of the first reagent and the second reagent.

[0082] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0083] Kit 3 a first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide; and a second reagent containing at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; Kit including:

[0084] Kit 4 a first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide; and a second reagent containing at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and a hydrogen peroxide measurement reagent is included in either or both of the first and second reagents.

[0085] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0086] Kit 5 A first reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, and at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; and Secondary reagent containing hydrogen peroxide measuring reagent Kit including:

[0087] Kit 6 a first reagent comprising an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid, and a hydrogen peroxide measurement reagent; and Secondary reagent containing hydrogen peroxide measuring reagent Kit including:

[0088] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0089] Kit 7 a first reagent containing a hydrogen peroxide measurement reagent; and a second reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, and at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid; Kit including:

[0090] 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 anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid, and a second reagent comprising a hydrogen peroxide measurement reagent.

[0091] When a reagent containing peroxidase and a leuco chromogen is used as a reagent for measuring hydrogen peroxide, it is preferable that the peroxidase and the leuco chromogen are contained in separate reagents, i.e., the peroxidase and the leuco chromogen are preferably contained in the first and second reagents, or the second and first reagents, respectively.

[0092] The kit of the embodiment may be in a freeze-dried state or in a state dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous media. When measuring glycated hemoglobin in an abnormal hemoglobin-containing sample using a freeze-dried kit, the reagents constituting the kit can be 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 kit of the embodiment is preferably a content such that the concentration when dissolved in an aqueous medium is 0.2 to 60 kU / L, more preferably a content of 0.4 to 30 kU / L, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 kU / L.

[0093] In the reagent constituting the kit of the embodiment, the content of at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acids is preferably such that the concentration in a state dissolved in an aqueous medium is 0.002 to 20 g / L, more preferably 0.02 to 10 g / L, and even more preferably 0.1 to 10 g / L. For example, the contents are 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, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / L.

[0094] (4) Composition The composition of the embodiment contains glycated hemoglobin including abnormal hemoglobin, and at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid.

[0095] The composition of the embodiment may further contain an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide.

[0096] The composition of the embodiment may further contain a hydrogen peroxide measuring reagent. As the hydrogen peroxide measuring reagent, at least one selected from the group consisting of peroxidase and leuco chromogen can be used. The composition of the embodiment may contain, for example, either one of peroxidase and leuco chromogen, or both of peroxidase and leuco chromogen.

[0097] The composition of the embodiment can be used as a reaction liquid in the method of the embodiment for measuring glycated hemoglobin in a sample containing abnormal hemoglobin, or as a liquid for adjusting the reaction liquid.

[0098] As an example of the composition of the embodiment, the following composition can be exemplified.

[0099] ·Composition 1 Glycated hemoglobin including abnormal hemoglobin; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; A composition comprising:

[0100] ·Composition 2 Glycated hemoglobin including abnormal hemoglobin; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; A composition comprising:

[0101] ·Composition 3 Glycated hemoglobin including abnormal hemoglobin; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; and an enzyme that catalyzes a reaction that oxidizes glycated hemoglobin to produce hydrogen peroxide.

[0102] ·Composition 4 Glycated hemoglobin including abnormal hemoglobin; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; An enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide; A composition comprising:

[0103] ·Composition 5 Glycated hemoglobin including abnormal hemoglobin; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; An enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide; A composition comprising:

[0104] ·Composition 6 Glycated hemoglobin including abnormal hemoglobin; at least one anionic surfactant selected from the group consisting of sulfonic acid-based anionic surfactants and polyoxyethylene alkyl ether acetic acid; An enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide; A leuco chromogen; A composition comprising:

[0105] Here, glycated hemoglobin including abnormal hemoglobin means that the glycated hemoglobin includes glycated hemoglobin of abnormal hemoglobin. Examples of glycated hemoglobin of abnormal hemoglobin include glycated hemoglobin of abnormal hemoglobin in which one or more amino acids are deleted, substituted or added at least in the 1st to 6th amino acid sequence from the N-terminus of the β-chain of human hemoglobin A; glycated hemoglobin C; glycated hemoglobin S, etc.

[0106] In the composition of the embodiment, examples of the abnormal hemoglobin, the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide, the at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid, and the hydrogen peroxide measurement reagent include, for example, the abnormal hemoglobin, the enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide, the at least one anionic surfactant selected from the group consisting of sulfonic acid anionic surfactants and polyoxyethylene alkyl ether acetic acid, and the hydrogen peroxide measurement reagent, which are exemplified in the measurement method of the embodiment described above. When the composition of the embodiment contains a sulfonic acid anionic surfactant, the sulfonic acid anionic surfactant is preferably at least one anionic surfactant selected from the group consisting of alkenylsulfonic acids, alkylsulfonic acids, N-acyltaurines in which the hydrogen atom of the amino group may be substituted with a substituent, alkylsulfoacetic acids, alkylbenzenesulfonic acids, and alkyl polyoxyethylenesulfosuccinates.

[0107] The content of abnormal hemoglobin in the composition of the embodiment may be any of the values ​​exemplified as the amount in the sample containing abnormal hemoglobin in the measurement method of the embodiment described above. The total hemoglobin content in the composition of the embodiment may be any of the values ​​exemplified as the amount in the reaction solution in the measurement method of the above embodiment. EXAMPLES

[0108] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0109] Information on the reagents used is shown below. Bis-Tris (manufactured by Dojindo Chemical Research Institute) DA-67 (Wako Pure Chemical Industries, Ltd.) Peroxidase (Toyobo)

[0110] K Liporan PJ-400J [Sodium tetradecene sulfonate; Lion Corporation] NIKKOL LMT [Sodium N-lauroyl-N-methyl taurate; manufactured by Nikko Chemicals] NIKKOL LSA-F [Sodium lauryl sulfoacetate; manufactured by Nikko Chemicals] LAS-C8 [Na p-octylbenzenesulfonate; Nacalai Tesque] Neo Hytenol L-30 [polyoxyethylene lauryl sulfosuccinate disodium; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.] NIKKOL ECTD-3NEX [Polyoxyethylene tridecyl ether acetate (Sodium) Manufactured by Nikko Chemicals]

[0111] NIKKOL Alaninate LN-30 [Sodium Lauroyl Methylalanine; manufactured by Nikko Chemicals] NIKKOL Sarcosinate LN-30 [N-Lauroyl Sarcosine Sodium; manufactured by Nikko Chemicals]

[0112] [Experiment 1] Creation of FPOX-47△3 (1) Construction of FPOX-47 expression vector pTrc-FPOX-47 Using the expression plasmid pTrc-FPOX-42 containing DNA consisting of the base sequence represented by SEQ ID NO: 1 as template DNA, and using FPOX-42 F342V-F consisting of the base sequence represented by SEQ ID NO: 2 and FPOX-42 F342V-R consisting of the base sequence represented by SEQ ID NO: 3 as forward and reverse primers, respectively, PCR was performed with the following reagent composition and PCR conditions to obtain a PCR product. pTrc-FPOX-42 is an expression plasmid containing DNA encoding FPOX-42, an enzyme that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide, and can be constructed according to the method disclosed in International Publication No. 2015 / 005258. PCR was performed based on the protocol of the PCR kit, DNA polymerase "KOD-Plus-" manufactured by Toyobo Co., Ltd.

[0113] (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 50 μL.

[0114] (PCR conditions) 1. 94℃ 2 minutes 2. 98℃ 15 seconds 3. 60℃ 30 seconds 4. 68℃ 6 minutes 5. Repeat steps 2 to 4 (total 30 cycles) 6. 68℃ 10 minutes

[0115] 1 μL of "restriction enzyme Dpn I" (New England Biolabs) was added to 50 μL of the obtained PCR product, and the template DNA was degraded by incubating at 37° C. for 1 hour. The restriction enzyme-treated PCR product was purified using Promega's "Wizard SV Gel and PCR Clean-Up System" according to the kit's protocol to obtain a purified PCR product sample. The purified PCR product was then used to transform E. coli competent cells "Competent high DH5α" manufactured by Toyobo Co., Ltd. Colonies grown on LB agar medium containing 50 mg / L ampicillin were selected, and plasmids were extracted using "Wizard Plus SV Minipreps DNA Purification" manufactured by Promega Co., Ltd. according to the kit's protocol. The extracted plasmid was subjected to sequence analysis using a DNA sequencer to confirm the construction of pTrc-FPOX-47, a plasmid containing DNA encoding FPOX-47 and having the base sequence shown in SEQ ID NO: 4. For sequence analysis, pTrc-F consisting of the base sequence shown in SEQ ID NO: 5, which reflects the base sequences immediately before and after the multicloning site of the pTrc99a vector (GE Healthcare Biosciences), and pTrc-R consisting of the base sequence shown in SEQ ID NO: 6 were used as the forward and reverse primers, respectively.

[0116] (2) Construction of FPOX-47△3 expression vector pTrc-FPOX-47△3 We designed and chemically synthesized FPOX-47△3-R, which consists of the base sequence represented by SEQ ID NO: 7, in which a stop codon and a Bam HI restriction enzyme recognition site have been added, with the 9 bases located immediately before the stop codon on the 3'-end side of the base sequence encoding FPOX-47 contained in plasmid pTrc-FPOX-47 removed, and pTrc-F, which consists of the base sequence represented by SEQ ID NO: 5, which reflects the base sequence immediately before the multicloning site of the pTrc99a vector. Using pTrc-F and FPOX-47△3-R as a primer pair and pTrc-FPOX-47 as a template, PCR was performed with the following reagent composition and PCR conditions to obtain a PCR product containing a DNA fragment containing a base sequence encoding a deletion form FPOX-47△3 in which three amino acids at the C-terminus of FPOX-47 were deleted. PCR was performed based on the protocol of the PCR kit and DNA polymerase "KOD-Plus-" manufactured by Toyobo Co., Ltd.

[0117] (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 50 μL.

[0118] (PCR conditions) 1. 94℃ 2 minutes 2. 98℃ 15 seconds 3. 60℃ 30 seconds 4. 68℃ 2 minutes 5. Repeat steps 2 to 4 (total 30 cycles) 6. 68℃ 5 minutes

[0119] A portion of the resulting PCR product was electrophoresed on a 1% agarose TAE gel to confirm that a DNA fragment of the expected size had been obtained. The resulting PCR product was purified using Promega's Wizard SV Gel and PCR Clean-Up system according to the kit's protocol to obtain a DNA fragment containing DNA encoding FPOX-47△3. The resulting DNA fragment was treated with two types of restriction enzymes, Nco I and Bam HI (both manufactured by Takara Bio Inc.), at 37°C for 1 hour, and the resulting reaction mixture was purified using Promega's Wizard SV Gel and PCR Clean-Up system according to the kit's protocol to obtain a restriction enzyme-treated fragment.

[0120] In addition, pTrc-FPOX-15, which expresses the glycopeptide oxidase FPOX-15 that does not have the activity of oxidizing fructosyl hexapeptide and is described in International Publication No. 2010 / 041715, was similarly treated with the same restriction enzymes and electrophoresed on a 1% agarose TAE gel to excise a DNA fragment corresponding to the vector. The DNA fragment was purified using the Wizard SV Gel and PCR Clean-Up system to obtain a plasmid fragment from which the DNA encoding FPOX-15 had been removed. The resulting restriction enzyme-treated fragment was mixed with a plasmid fragment from which the DNA encoding FPOX-15 had been removed, and ligated using a DNA Ligation Kit (Takara Bio Inc.). The ligation product was then introduced into Escherichia coli competent cells "Competent high DH5α" (Toyobo Co., Ltd.). The E. coli into which the ligation product had been introduced was inoculated on LB agar medium containing 50 mg / L ampicillin, cultured at 37°C for 14 hours, and the colonies that emerged were selected. The selected colonies were cultured in LB liquid medium containing 50 mg / L ampicillin at 37°C for 14 hours, and a plasmid was prepared using Promega's Wizard Plus SV Minipreps DNA Purification kit according to the kit's protocol. PCR was performed using pTrc-F consisting of the base sequence represented by SEQ ID NO: 5 and pTrc-R consisting of the base sequence represented by SEQ ID NO: 6 as forward and reverse primers, respectively, to confirm the base sequence of the prepared plasmid, and it was confirmed that the plasmid contained the base sequence represented by SEQ ID NO: 8, in which the 3'-side 9 bases excluding the stop codon of the base sequence of FPOX-47 were deleted. This plasmid is called pTrc-FPOX-47△3.

[0121] (3) Acquisition of FPOX-47△3 The E. coli containing pTrc-FPOX-47△3 obtained in (2) above was cultured in LB medium containing 50 mg / L ampicillin at 37°C for 14 hours with shaking. The resulting culture solution was purified according to the purification method for glycopeptide oxidases FPOX-9 and FPOX-15 described in WO 2010 / 041715 to obtain a solution of purified FPOX-47△3. The amino acid sequence of FPOX-47 is shown in SEQ ID NO:9, and the amino acid sequence of FPOX-47△3 is shown in SEQ ID NO:10.

[0122] (4) Confirmation of the enzyme activity of FPOX-47△3 that catalyzes the reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide (hereinafter referred to as glycated hemoglobin oxidase activity) Using the purified FPOX-47Δ3 solution obtained in (3) above, the glycated hemoglobin oxidase activity of FPOX-47Δ3 against HbA1c was confirmed by the following measurement kit and measurement procedure.

[0123] <Measurement Kit A> First Reagent Bis-Tris (pH 6.5) 50mmol / L DA-67 40μmol / L NIKKOL LMT 1g / L Second Reagent Phosphate buffer (pH6.5) 10mmol / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0124] <Measurement kit a> First Reagent Bis-Tris (pH 6.5) 50mmol / L DA-67 40μmol / L Second Reagent Phosphate buffer (pH6.5) 10mmol / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0125] Measurement kit a is the same as measurement kit A except that the first reagent does not contain NIKKOL LMT.

[0126] <Sample> The samples used were hemolyzed samples prepared by hemolysis after diluting blood cells obtained by centrifuging human blood with deionized water, and the HbA1c concentrations were determined to be 2.6 μmol / L, 4.5 μmol / L, 6.4 μmol / L, and 8.4 μmol / L using the KO500 method, which is the standard method for measuring HbA1c, and the SLS-hemoglobin method, which is a method for measuring total hemoglobin.

[0127] <Measurement procedure> Each sample (10 μL) and the first reagent (100 μL) of the measurement kit A were added to a reaction cell and reacted at 37° C. for 5 minutes (first reaction), and the absorbance (E1) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. Next, the second reagent (40 μL) of the measurement kit A was added to this reaction solution and reacted at 37° C. for another 5 minutes (second reaction), and the absorbance (E2) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. E1 was subtracted from E2 to obtain the absorbance difference ΔE'. A was calculated. Each of the above samples (10 μL) and the first reagent (100 μL) of the above measurement kit a were added to a reaction cell, and reacted at 37° C. for 5 minutes (first reaction), and the absorbance (E3) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. Next, the second reagent (40 μL) of the above measurement kit a was added to this reaction solution, and reacted at 37° C. for another 5 minutes (second reaction), and the absorbance (E4) of the reaction solution was measured at a main wavelength of 660 nm and a secondary wavelength of 800 nm. E3 was subtracted from E4 to obtain the absorbance difference ΔE'. a was calculated. △E' A From △E' a The absorbance difference for each sample was calculated as ΔE. The relationship between the HbA1c concentration and ΔE for each sample is shown in Figure 1.

[0128] As shown in Figure 1, a quantitative relationship was observed between HbA1c concentration and absorbance in the measurement using a kit (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.

[0129] [Experiment 2] Next, whether HbA1c could be measured using the HbA1c measurement kit containing the various anionic surfactants listed below for samples that did not contain abnormal hemoglobin was confirmed by correlation with the standard method, the KO500 method.

[0130] HbA1c measurement kits (Kits 1A to 1F) consisting of the following first and second reagents were prepared.

[0131] (Kit 1A~1F) First Reagent Bis-Tris (pH 7.0) 50mmol / L Sodium pyruvate 2g / L DA-67 40μmol / L Anionic surfactants A to F (see Table 2) Second Reagent Bis-Tris (pH 7.0) 50mmol / L Potassium ferrocyanide 1.5mg / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0132] In addition, kits for measuring HbA1c (kits 1a to 1b) consisting of the following first and second reagents were prepared.

[0133] (Kit 1a~1b) First Reagent Bis-Tris (pH 7.0) 50mmol / L Sodium pyruvate 2g / L DA-67 40μmol / L Anionic surfactants a-b (see Table 2) Second Reagent Bis-Tris (pH 7.0) 50mmol / L Potassium ferrocyanide 1.5mg / L FPOX-47△3 1.5kU / L Peroxidase 200kU / L

[0134] Kit 1A consisting of the first and second reagents described above was used as the HbA1c measurement kit, and six concentrations of whole blood (not containing abnormal hemoglobin) with HbA1c concentrations of 5.11%, 5.73%, 6.40%, 7.45%, 8.55% and 10.56%, respectively, determined 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.

[0135] (1) Preparation of a calibration curve for determining total hemoglobin concentration Measurements were performed according to the following procedure using a total hemoglobin measurement kit, Hemoglobin B-Test Wako (SLS-hemoglobin method) (manufactured by Wako Pure Chemical Industries, Ltd.), and the standard provided with the Hemoglobin B-Test Wako (total hemoglobin concentration: 15.0 mg / ml, no abnormal hemoglobin), and the absorbance for the standard was measured. The standard (10 μL) and Hemoglobin B-Test Wako (250 μL) were added to the reaction cell and reacted at 37° C. for 10 minutes. The absorbance (E) of the reaction solution was measured at a main wavelength of 546 nm and a sub-wavelength of 660 nm, and was taken as the absorbance for the standard. The same method was used except that saline was used instead of the standard, and the absorbance for saline was taken as the absorbance for the standard. The value calculated by subtracting the absorbance for saline from the absorbance for the standard was taken as the blank-corrected absorbance for the standard. A calibration curve showing the relationship between total hemoglobin concentration (μmol / L) and absorbance was created from the blank-corrected absorbance for the standard and the blank-corrected absorbance for saline (0 Abs).

[0136] (2) Preparation of a calibration curve for determining HbA1c concentration Two hemolyzed blood cell fractions, the HbA1c concentrations of which were estimated to be 3.72 μmol / L and 10.38 μmol / L by the K0500 method, were measured using the above-mentioned HbA1c measurement kit 1A 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 above HbA1c measurement kit 1A were added to the 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 above HbA1c measurement kit 1A 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 obtain the absorbance difference ΔE'. 1A The absorbance difference ΔE' was calculated as the absorbance for each blood cell fraction. 生理食塩水 was calculated and used as the absorbance for physiological saline. The blank-corrected absorbance for the blood cell fraction was calculated by subtracting the absorbance for physiological saline from the absorbance for each blood cell fraction. A calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance was created from the blank-corrected absorbance for the blood cell fraction and the blank-corrected absorbance for physiological saline (0 Abs).

[0137] (3) Determination of total hemoglobin concentration in each blood cell fraction Each of the six concentrations of whole blood samples was centrifuged at 25°C, 3000 rpm (1500 x G) for 5 minutes to obtain blood cell fractions. 4 mL of purified water was added to 100 μL of each blood cell fraction to obtain hemolyzed blood cell fractions. Each hemolyzed blood cell fraction was measured using a Hemoglobin B-Test Wako in the same manner as in (1), and the total hemoglobin concentration (μmol / L) in each blood cell fraction was determined from the obtained measurements and the calibration curve in (1).

[0138] (4) Determination of HbA1c concentration in each blood cell fraction Each of the hemolyzed blood cell fractions obtained in (3) above was measured in the same manner as in (2) using the HbA1c measurement kit 1A described above, and the HbA1c concentration (μmol / L) in each blood cell fraction was determined from the obtained measurements and the calibration curve in (2).

[0139] (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).

[0140]

number

[0141] (6) Correlation between the measurement method using the HbA1c measurement kit and the KO500 method Using the measurement method using HbA1c Measurement Kit 1A, the correlation between the measurement method using HbA1c Measurement Kit 1A and the K0500 method was verified based on the HbA1c (%) determined in (5) above and the HbA1c (%) previously determined using the KO500 method, and the correlation coefficient was determined.

[0142] In addition, HbA1c (%) in each sample was determined by the same procedure as above, except that the above-mentioned measurement kits 1B, 1C, 1D, 1E, 1F, 1a and 1b were used instead of the above-mentioned HbA1c measurement kit 1A, and the correlation between the measurement method using each kit and the KO500 method was verified and the correlation coefficient was determined.

[0143] The results are shown in Table 2. The correlation coefficient between the two measurement methods was 0.98 or more, and a good correlation was observed between the two measurement methods. Therefore, it was revealed that the above HbA1c measurement kits 1A, 1B, 1C, 1D, 1E, 1F, 1a and 1b can accurately measure HbA1c in a sample if the sample does not contain abnormal hemoglobin.

[0144] [Experiment 3] Next, it was confirmed whether HbA1c could be measured for abnormal hemoglobin-containing samples containing abnormal hemoglobin HbC using the HbA1c measurement kits containing the various anionic surfactants described above. In this experiment, the measurement results of glycated hemoglobin, which includes abnormal hemoglobin HbC other than HbA, are also expressed as the amount of HbA1c.

[0145] (1) Preparation of hemolyzed samples Hemolyzed samples were prepared by adding 4 mL of purified water to 100 μL each of three blood cell samples (samples 1 to 3: see Table 1) purchased from Discovery Life Science, whose HbC content and HbA1c (%) had already been determined by HPLC.

[0146] [Table 1]

[0147] (2) Determination of total hemoglobin concentration in each hemolyzed sample For each hemolyzed sample prepared in (1) above, a measurement similar to that in Experiment 2 above was performed using a total hemoglobin measurement reagent, Hemoglobin B-Test Wako, and the total hemoglobin concentration in each hemolyzed sample was determined from the obtained absorbance and the calibration curve showing the relationship between the total hemoglobin concentration (μmol / L) and absorbance prepared in Experiment 2 above.

[0148] (3) Determination of HbA1c concentration in each hemolyzed sample Each hemolyzed sample prepared in (1) above was measured using the HbA1c measurement kit 1A in the same manner as in Experiment 2 above, and the HbA1c concentration (μmol / L) in each hemolyzed sample was determined from the obtained measurement values ​​and the calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance prepared in Experiment 2 above.

[0149] (4) Determination of HbA1c (%) (= ratio 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 hemolyzed sample measured in (2) above and the HbA1c concentration (μmol / L) in each hemolyzed sample measured in (3) above, according to the following formula (II).

[0150]

number

[0151] (5) Comparison of the value displayed at the time of purchase with the HbA1c (%) determined in (4) above For each sample, the HbA1c concentration (%) priced at the time of purchase from Discovery Life Sciences was set as the reference value of 0, and the difference between this reference value and the HbA1c (%) determined in (4) above [△HbA1c concentration (%)] was calculated.

[0152] In addition, the same measurement as above was performed except that the above HbA1c measurement kits 1B, 1C, 1D, 1E, 1F, 1a and 1b were used instead of the above HbA1c measurement kit 1A, and the HbA1c concentration (%) in each hemolyzed sample was measured for each kit. For each sample, the HbA1c concentration (%) priced at the time of purchase from Discovery Life Science was set as standard 0, and the difference between the standard value and the measured HbA1c (%) [△HbA1c concentration (%)] was calculated.

[0153] The measurements using HbA1c measurement kits 1A, 1B, 1C, 1D, 1E and 1F in Experiment 3 correspond to examples to which the present invention is applied.

[0154] The results are shown in Table 2. When the HbA1c measurement kits 1a and 1b were used for the measurement of samples containing abnormal hemoglobin, the ΔHbA1c concentration (%) was less than 0 and was smaller than the ΔHbA1c concentration (%) value in the Examples, and the accuracy of the measurement of HbA1c in samples containing abnormal hemoglobin tended to be inferior to that in the Examples in which the present invention was applied.

[0155] On the other hand, when samples containing abnormal hemoglobin were measured using HbA1c measurement kits 1A, 1B, 1C, 1D, 1E, and 1F, the △HbA1c concentration (%) values ​​were close to 0, demonstrating that HbA1c in samples containing abnormal hemoglobin can be measured more accurately.

[0156] [Table 2]

[0157] The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited to each embodiment, but is limited only by the scope of the claims.

Claims

1. A method for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, comprising reacting glycated hemoglobin in the abnormal 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 anionic surfactant selected from the group consisting of alkenylsulfonic acid, N-acyltaurine, alkylsulfoacetic acid, alkylbenzenesulfonic acid, alkyl polyoxyethylenesulfosuccinate, and polyoxyethylene alkyl ether acetic acid to generate hydrogen peroxide, and measuring the generated hydrogen peroxide.

2. The method for measuring glycated hemoglobin according to claim 1, wherein the abnormal hemoglobin is one in which one or more amino acids have been deleted, substituted or added in at least the first to sixth amino acid sequence from the N-terminus of the β-chain of human hemoglobin A.

3. 3. The method for measuring glycated hemoglobin according to claim 1, wherein the abnormal hemoglobin is human hemoglobin C or hemoglobin S.

4. 4. The method for measuring glycated hemoglobin according to claim 1, wherein the content of the abnormal hemoglobin is 20% or more relative to the total hemoglobin in the hemoglobin-containing sample.

5. The method for measuring glycated hemoglobin according to any one of claims 1 to 4, wherein the measurement of hydrogen peroxide is carried out using a hydrogen peroxide measuring reagent.

6. The method for measuring glycated hemoglobin according to claim 5 , wherein the hydrogen peroxide measuring reagent contains peroxidase and a leuco chromogen.

7. The method for measuring glycated hemoglobin according to claim 6 , wherein the leuco chromogen is a phenothiazine chromogen.

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

9. An enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide; A reagent for measuring glycated hemoglobin in an abnormal hemoglobin-containing sample, comprising at least one anionic surfactant selected from the group consisting of alkenylsulfonic acid, N-acyltaurine, alkylsulfoacetic acid, alkylbenzenesulfonic acid, alkyl polyoxyethylenesulfosuccinate, and polyoxyethylene alkyl ether acetic acid.

10. The measuring reagent according to claim 9 , further comprising a hydrogen peroxide measuring reagent.

11. The hydrogen peroxide measuring reagent according to claim 10, which comprises peroxidase and a leuco chromogen.

12. The measurement reagent according to claim 11 , wherein the leuco chromogen is a phenothiazine chromogen.

13. The measuring reagent according to claim 12, wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

14. A first reagent containing at least one anionic surfactant selected from the group consisting of alkenylsulfonic acid, N-acyltaurine, alkylsulfoacetic acid, alkylbenzenesulfonic acid, polyoxyethylene alkyl sulfosuccinate, and polyoxyethylene alkyl ether acetic acid; and a second reagent containing an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to produce hydrogen peroxide.

15. The measuring kit according to claim 14 , further comprising a peroxidase and a leuco chromogen in the first and second reagents, or in the second and first reagents, respectively.

16. The measurement kit according to claim 15 , wherein the leuco chromogen is a phenothiazine chromogen.

17. The measuring kit according to claim 16, wherein the phenothiazine chromogen is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or a salt thereof.

18. Glycated hemoglobin including abnormal hemoglobin; and at least one anionic surfactant selected from the group consisting of alkenyl sulfonic acids, N-acyltaurine, alkyl sulfoacetic acids, alkylbenzene sulfonic acids, polyoxyethylene alkyl sulfosuccinates, and polyoxyethylene alkyl ether acetic acids.

19. The composition according to claim 18, further comprising an enzyme that catalyzes a reaction in which glycated hemoglobin is oxidized to produce hydrogen peroxide.

20. The composition according to claim 18 or 19, further comprising a hydrogen peroxide measuring reagent.

21. The composition according to claim 20, wherein the hydrogen peroxide measurement reagent is at least one selected from the group consisting of peroxidase and leuco chromogen.

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

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

24. The composition according to any one of claims 18 to 23, wherein the abnormal hemoglobin is one in which one or more amino acids have been deleted, substituted or added in at least the first to sixth amino acid sequence from the N-terminus of the β-chain of human hemoglobin A.

25. The composition according to any one of claims 18 to 24, wherein the abnormal hemoglobin is human hemoglobin C or hemoglobin S.

26. The composition according to any one of claims 18 to 25, wherein the content of the abnormal hemoglobin is 20% or more relative to the total hemoglobin in the composition.

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