Measurement method for glycated hemoglobin

Abstract

Disclosed is a method for measuring glycated hemoglobin in a sample, the method including: a step for mixing a sample containing glycated hemoglobin with a first reagent to obtain a reaction solution containing a fructosyl peptide; a step for adding a second reagent to the reaction solution, thereby causing a leuco-type chromogen to develop a color; a step for calculating the change in absorbance of the mixed solution of the reaction solution and the second reagent due to the color development of the leuco-type chromogen, where the change in absorbance is the difference between a first absorbance immediately after the addition of the second reagent and a second absorbance after a prescribed time has elapsed from the measurement of the first absorbance; and a step for determining the concentration of glycated hemoglobin in the sample on the basis of the change in absorbance.

Description

Method for measuring glycated hemoglobin

[0001] This invention relates to a method for measuring glycated hemoglobin.

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

[0003] In recent years, the development of enzymatic measurement methods for HbA1c that are applicable to general-purpose automatic analyzers and are also easy to operate has advanced, and various methods have been reported. The enzymatic measurement methods for HbA1c reported so far are mainly methods using protease (protein-degrading enzyme) and glycated peptide oxidase. As a method using protease and glycated peptide oxidase, protease is allowed to act on HbA1c in a sample to generate fructosyl-dipeptide (Fru-Val-His), which is a glycated peptide. Then, fructosyl peptide oxidase (fructosyl peptide oxidizing enzyme) is allowed to act on the generated fructosyl-dipeptide to generate hydrogen peroxide, and the HbA1c in the sample is measured by measuring the generated hydrogen peroxide (see Patent Document 1). In addition, HbA1c measurement kits are sold by multiple manufacturers. For example, there is the "Metabolead HbA1c", which is an HbA1c measurement kit composed of a first reagent containing a phenothiazine-based leuco-type chromogen that develops color upon reaction with hydrogen peroxide and a second reagent containing fructosyl peptide oxidase (Non-Patent Document 4). When the first reagent of "Metabolead HbA1c" and then the second reagent are added to a hemolyzed sample, the leuco-type chromogen develops color, and the HbA1c concentration is calculated based on the change in absorbance due to this color development. Here, the change in absorbance due to color development is calculated from the absorbance measured immediately before the addition of the second reagent containing fructosyl peptide oxidase and the absorbance measured approximately 5 minutes after the addition of the second reagent (see "Instructions for Use (Operation Method)" 3. Operation Method (2) of Non-Patent Document 4).

[0004] Japanese Patent Application Laid-Open No. 2001-095598

[0005] Clin Chem Lab Med, Vol. 36, p. 299-308 (1998). Diabetes, Vol. 27, No. 2, p. 102-107 (1978). Journal of the Japanese Society for Clinical Laboratory Automation, Vol. 18, No. 4, p. 620 (1993). Attachment Document of Glycohemoglobin A1c Kit "Metabolead HbA1c", Revised in April 2024 (9th Edition)

[0006] The inventors prepared an HbA1c measurement kit containing a first reagent containing a protease and a second reagent containing fructosyl peptide oxidase and a leuco-type chromophor. When they attempted to measure HbA1c using this kit in the same procedure as "Metaboread HbA1c," they found that the absorbance of the second reagent itself (reagent blank absorbance) increased over time due to the degradation of the leuco-type chromophor in the second reagent. This increased absorbance caused the apparent measurement value to rise, making accurate measurement of HbA1c impossible. Therefore, the object of the present invention is to provide a measurement method that enables accurate measurement of glycated hemoglobin when using a glycated hemoglobin measurement kit containing a first reagent containing a protease and a second reagent containing fructosyl peptide oxidase and a leuco-type chromophor.

[0007] Furthermore, the deterioration of the leuco-type chromophor in the second reagent causes the absorbance of the second reagent itself (reagent blank absorbance) to increase over time. This phenomenon, where the apparent measurement value increases by the amount of this increase in reagent blank absorbance, has a significant impact on the measurement value, especially when calibration with standards is performed infrequently. Therefore, an object of the present invention is to provide a measurement method that enables accurate measurement of glycated hemoglobin when using a kit for measuring glycated hemoglobin comprising a first reagent containing a proteolytic enzyme and a second reagent containing fructosyl peptide oxidase and a leuco-type chromophor, especially when calibration with standards is performed infrequently.

[0008] As a result of diligent research, the inventors discovered that instead of measuring the absorbance immediately before adding the second reagent, they could measure the absorbance (first absorbance) immediately after adding the second reagent, and calculate the change in absorbance due to color development from this first absorbance and the second absorbance measured after a predetermined time has elapsed since the measurement of the first absorbance. This allowed for accurate measurement of HbA1c without frequent calibration, thus completing the present invention.

[0009] The present invention includes the following embodiments: Item 1. (1) A step of mixing a sample containing glycated hemoglobin with a first reagent containing a protease, thereby reacting the glycated hemoglobin with the protease to obtain a reaction solution containing fructosyl peptide; (2) A step of adding a second reagent containing fructosyl peptide oxidase and a leuco-type chromogen to the reaction solution, thereby reacting the fructosyl peptide with the fructosyl peptide oxidase to produce hydrogen peroxide, wherein the leuco-type chromogen develops color due to the hydrogen peroxide; (3) A step of calculating the change in absorbance of a mixture of the reaction solution from step (1) and the second reagent due to the color development of the leuco-type chromogen, wherein the change in absorbance is the difference between a first absorbance immediately after adding the second reagent and a second absorbance after a predetermined time has elapsed since the measurement of the first absorbance; and (4) A method for measuring glycated hemoglobin in a sample, comprising the step of determining the concentration of glycated hemoglobin in the sample based on the absorbance change. Item 2. The method according to Item 1, wherein the first absorbance is the absorbance 1 second to 60 seconds after the addition of the second reagent. Item 3. The method according to Item 2, wherein the first absorbance is the absorbance 1 second to 30 seconds after the addition of the second reagent. Item 4. The method according to Item 3, wherein the first absorbance is the absorbance 1 second to 15 seconds after the addition of the second reagent. Item 5. The method according to any one of Items 1 to 4, wherein the predetermined time is 3 to 5 minutes. Item 6. The method according to any one of Items 1 to 5, wherein the first reagent further contains a hemoglobin denaturing agent. Item 7. The method according to Item 6, wherein the hemoglobin denaturing agent is a surfactant. Item 8. The method according to Item 7, wherein the surfactant is a cationic surfactant. Item 9. The method according to any one of claims 1 to 8, wherein step (4) is a step of determining the concentration of glycated hemoglobin in the sample by comparing the absorbance change calculated in step (3) with a calibration curve that shows the relationship between glycated hemoglobin concentration and absorbance change, which has been prepared in advance using a standard sample containing glycated hemoglobin of known concentration, and the calibration curve prepared in advance is a calibration curve that was prepared 1 to 30 days before steps (1) to (3) are performed.Item 10. The method according to Item 9, wherein the pre-prepared calibration curve is a calibration curve prepared 14 to 30 days before performing steps (1) to (3).

[0010] According to the present invention, a measurement method is provided that enables accurate measurement of glycated hemoglobin even with a low frequency of calibration using standards, when using a kit for measuring glycated hemoglobin comprising a first reagent containing a proteolytic enzyme and a second reagent containing fructosyl peptide oxidase and a leuco-type chromogen.

[0011] A method for measuring glycated hemoglobin in a sample according to one aspect of the present invention is: (1) a step of mixing a sample containing glycated hemoglobin with a first reagent containing a protease, thereby reacting the glycated hemoglobin with the protease to obtain a reaction solution containing fructosyl peptide; (2) a step of adding a second reagent containing fructosyl peptide oxidase and a leuco-type chromogen to the reaction solution, thereby reacting the fructosyl peptide with the fructosyl peptide oxidase to produce hydrogen peroxide, wherein the leuco-type chromogen develops color due to the hydrogen peroxide; (3) a step of calculating the change in absorbance of the mixture of the reaction solution and the second reagent from step (1) due to the color development of the leuco-type chromogen, wherein the change in absorbance is the difference between the first absorbance immediately after adding the second reagent and the second absorbance after a predetermined time has elapsed since the measurement of the first absorbance; and (4) The process includes determining the concentration of the glycated hemoglobin in the sample based on the absorbance change described above.

[0012] In step (1), a sample containing glycated hemoglobin is mixed with a first reagent containing a proteolytic enzyme, causing the glycated hemoglobin and the proteolytic enzyme to react and produce fructosyl peptide.

[0013] Glycated hemoglobin may be hemoglobin A1c (HbA1c).

[0014] The sample containing glycated hemoglobin is not particularly limited and may be a biological sample such as whole blood, blood cells, or a mixture of blood cells and plasma. Whole blood and blood cells may be hemolyzed. The method for hemolyzing whole blood and blood cells is not particularly limited and may be a physical, chemical, or biological method. Examples of physical methods include using a hypotonic solution such as water, or using ultrasound. Examples of chemical methods include using an organic solvent such as methanol, ethanol, or acetone, or using a polyoxyethylene-based surfactant. Examples of biological methods include using an antibody or complement.

[0015] The proteolytic enzyme is not particularly limited as long as it is an enzyme that breaks down glycated hemoglobin into fructosyl peptides. The fructosyl peptide produced may be, for example, a fructosyl dipeptide (e.g., fructosylvalylhistidine) or a fructosyl hexapeptide (e.g., fructosylvalylhistidylleucylthreonylprolylglutamic acid). Examples of proteolytic enzymes include serine proteases (chymotrypsin, subtilisin, etc.), cysteine ​​proteases (papain, caspase, etc.), aspartate proteases (pepsin, cathepsin D, etc.), metalloproteases (thermolysin, etc.), N-terminal threonine proteases, or glutamate proteases.

[0016] The first reagent preferably further contains a hemoglobin denaturant. The hemoglobin denaturant is not particularly limited as long as it is a substance that denatures hemoglobin so that it can react more readily with proteolytic enzymes, and examples include surfactants or nitrites. The surfactant may be a cationic surfactant, anionic surfactant, amphoteric surfactant, or nonionic surfactant. Examples of cationic surfactants include quaternary ammonium salts, pyridinium salts, phosphonium salts, imidazolium salts, or isoquinolinium salts, with pyridinium salts being preferred. Examples of pyridinium salts include 1-dodecylpyridinium chloride, 1-cetylpyridinium chloride, 1-cetyl-4-methylpyridinium chloride, and N-octadecyl-4-stilbazole bromide. Examples of anionic surfactants include N-acyl taurine, alkyl sulfoacetic acid, polyoxyethylene alkyl ether acetate, N-acyl amino acid, polyoxyethylene alkyl ether phosphate, polyoxyethylene polycyclic phenyl ether phosphate, alkyl phosphate, linear alkylbenzene sulfonate, alkyl sulfate, or polyoxyethylene alkyl ether sulfate. Examples of amphoteric surfactants include alkyl carboxybetaine or alkyldimethylamine oxide. Examples of nonionic surfactants include alkyl glucoside, fatty acid sorbitan ester, fatty acid diethanolamide, polyoxyethylene alkyl ether, or alkyl monoglyceryl ether.

[0017] The first reagent may further contain peroxidase. From the viewpoint of developing the color of the chromogen, it is preferable that at least one of the first reagent and the second reagent contains peroxidase. Furthermore, if the second reagent contains a leuco-type chromogen, coexisting peroxidase and the leuco-type chromogen in the second reagent may promote the spontaneous color development (destabilization) of the leuco-type chromogen by the peroxidase; therefore, it is more preferable that only the first reagent contains peroxidase.

[0018] The first reagent may further contain a buffer. Examples of buffers include lactic acid buffers, citrate buffers, acetate buffers, succinate buffers, phthalate buffers, phosphate buffers, triethanolamine buffers, diethanolamine buffers, lysine buffers, barbiturate buffers, imidazole buffers, malic acid buffers, oxalate buffers, glycine buffers, borate buffers, carbonate buffers, Tris buffers, or Good's buffers. Good buffering agents include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamide)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[N-(2-acetamide)amino]ethanesulfonic acid (ACES), and 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO). ), 2-[N,N-bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), 2-{N-[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), N-(2-hydroxyethyl)-N'-(2-sulfoethyl)piperazine (HEPES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 2 -Hydroxy-3-{[N-Tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropane-3-sulfonic acid) (POPSO), N-(2-hydroxyethyl)-N'-(2-hydroxy-3-sulfopropyl)piperazine (HEPPSO), N-(2-hydroxyethyl)-N'-(3-sulfopropyl)piperazine (EPPS), N-Tris(hydroxymethyl) Examples include methylglycine (tricine), N,N-bis(2-hydroxyethyl)glycine (bicine), 3-[N-tris(hydroxymethyl)methyl]aminopropanesulfonic acid (TAPS), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 3-(N-cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO), or 3-(N-cyclohexylamino)propanesulfonic acid (CAPS).

[0019] The first reagent may contain other components such as stabilizers, preservatives, salts, interfering substance scavengers, and solubilizers, as long as they do not interfere with the various reactions in steps (1) and (2). Examples of stabilizers include ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride, calcium acetate, calcium nitrate, potassium ferrocyanide, or bovine serum albumin (BSA). Examples of preservatives include antibiotics. Examples of salts include sodium chloride, sodium nitrate, sodium sulfate, sodium carbonate, sodium formate, sodium acetate, potassium chloride, potassium nitrate, potassium sulfate, potassium carbonate, potassium formate, or potassium acetate. Examples of interfering substance scavengers include ascorbate oxidase to eliminate the effect of ascorbic acid. Examples of solubilizers include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, acetone, methanol, or ethanol.

[0020] The first reagent may be a liquid or a solid, and is preferably a liquid, more specifically an aqueous solution. If the first reagent is an aqueous solution, the components contained in the first reagent are dissolved in an aqueous medium. The aqueous medium may be water, or a mixed solvent of water and a water-soluble solvent, and the water may be deionized water or distilled water. The pH of the first reagent can be adjusted as appropriate according to the optimal pH of the enzyme, and may be, for example, 4 to 11, 5 to 10, or 6 to 9.

[0021] The concentration of the protease in the first reagent may be, for example, 250 kU / L to 15,000 kU / L, 1,000 kU / L to 12,000 kU / L, or 3,000 kU / L to 10,000 kU / L. The concentration of the hemoglobin denaturant in the first reagent may be, for example, 0.01 g / L to 20 g / L, 0.1 g / L to 15 g / L, or 0.5 g / L to 10 g / L. The concentration of the peroxidase in the first reagent may be, for example, 10 kU / L to 500 kU / L, 20 kU / L to 300 kU / L, or 50 kU / L to 200 kU / L. The concentration of the buffer in the first reagent may be, for example, 0.1 g / L to 50 g / L, 0.5 g / L to 30 g / L, or 1 g / L to 20 g / L.

[0022] The amount of the first reagent can be adjusted as appropriate depending on the sample. For example, the amount of the first reagent may be 3 to 60 volumes, 6 to 30 volumes, or 10 to 20 volumes per 1 volume of sample.

[0023] The length of step (1), that is, the length from the start of mixing of the sample and the first reagent to the addition of the second reagent, can be appropriately adjusted according to the sample, the concentration of each component, the reaction conditions, etc., so that the reaction between glycated hemoglobin and proteolytic enzyme proceeds sufficiently. The length of step (1) may be, for example, 1 to 20 minutes, 1 to 10 minutes, 3 to 6 minutes, or 5 minutes. The temperature of step (1) can be appropriately adjusted according to the optimal temperature of the enzyme, etc., and may be, for example, 10 to 50°C, 15 to 45°C, 20 to 40°C, or 37°C.

[0024] In step (2), a second reagent containing fructosyl peptide oxidase and a leuco-type chromopropyl alcohol is added to the reaction solution obtained in step (1), causing the fructosyl peptide and fructosyl peptide oxidase to react and produce hydrogen peroxide. This hydrogen peroxide reacts with the leuco-type chromopropyl alcohol, generating a dye and producing color.

[0025] Fructosyl peptide oxidase is an oxidase that uses fructosyl peptides as a substrate to produce glucosone, peptides, and hydrogen peroxide. The origin of fructosyl peptide oxidase is not particularly limited; for example, it may be wild-type fructosyl peptide oxidase derived from microorganisms such as fungi and bacteria, or a recombinant thereof.

[0026] Leuco-type chromogens are substances that are converted into dyes on their own in the presence of hydrogen peroxide and peroxidizing agents such as peroxidase. Examples of leuco-type chromogens include phenothiazine-based chromogens, triphenylmethane-based chromogens, diphenylamine-based chromogens, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, or tetramethylbenzidine. Leuco-type chromogens are preferably phenothiazine-based chromogens.

[0027] Examples of phenothiazine-based colorants include 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), or 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67). The phenothiazine-based colorant is preferably 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67).

[0028] Examples of triphenylmethane-based chromogens include N,N,N',N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triaminotriphenylmethane (TPM-PS). Examples of diphenylamine-based chromogens include N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 4,4'-bis(dimethylamino)diphenylamine, or bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA).

[0029] The stability of the leuco-type chromogen may decrease when it is in the presence of metal ions that may be contained in the protease, or the hemoglobin denaturant, etc. Therefore, the leuco-type chromogen is included in the second reagent, not in the first reagent which contains the protease and optionally the hemoglobin denaturant.

[0030] The second reagent may further contain peroxidase.

[0031] The second reagent preferably further contains a stabilizer for the leuco-type chromogen. The stabilizer for the leuco-type chromogen may be, for example, a polyoxyethylene alkylamine. Examples of polyoxyethylene alkylamines include polyoxyethylene dodecylamine, polyoxyethylene octadecylamine, and polyoxyethylene alkyl(beef tallow)amine.

[0032] The second reagent may further contain a buffer. Examples of buffers include those exemplified for the first reagent.

[0033] The second reagent may contain other components, as long as they do not interfere with the various reactions in step (2). Examples of other components include those exemplified for the first reagent.

[0034] The second reagent may be a liquid or a solid, and is preferably a liquid, more specifically an aqueous solution. If the second reagent is an aqueous solution, the components contained in the second reagent are dissolved in an aqueous medium. Examples of aqueous mediums include those exemplified for the first reagent. The pH of the second reagent can be adjusted as appropriate according to the optimal pH of the enzyme, and may be, for example, 4 to 11, 5 to 10, 6 to 9, or 7 to 8.

[0035] The concentration of fructosyl peptide oxidase in the second reagent may be, for example, 0.01 kU / L to 30 kU / L, 0.1 to 15 kU / L, 0.5 to 12 kU / L, or 1 to 10 kU / L.

[0036] The concentration of the leuco-type chromogen in the second reagent may be, for example, 0.1 mg / L or more, 0.5 mg / L or more, 1 mg / L or more, 2 mg / L or more, 3 mg / L or more, 4 mg / L or more, 5 mg / L or more, 6 mg / L or more, 7 mg / L or more, 8 mg / L or more, 9 mg / L or more, 10 mg / L or more, 15 mg / L or more, or 20 mg / L or more. Alternatively, the concentration of the leuco-type chromogen in the second reagent may be, for example, 200 mg / L or less, 150 mg / L or less, 100 mg / L or less, 50 mg / L or less, 40 mg / L or less, 30 mg / L or less, or 25 mg / L or less. The above values ​​can be freely combined. For example, the concentration of the leuco-type chromogen in the second reagent may be 1 mg / L to 50 mg / L, 5 mg / L to 40 mg / L, or 15 mg / L to 30 mg / L.

[0037] The concentration of peroxidase in the second reagent may be, for example, 3 kU / L to 150 kU / L, 7 kU / L to 100 kU / L, or 15 kU / L to 70 kU / L. The concentration of the leuco-type chromogen stabilizer in the second reagent may be, for example, 0.001 g / L to 100 g / L, 0.005 g / L to 50 g / L, or 0.01 g / L to 10 g / L. The concentration of the buffer in the second reagent may be, for example, 0.1 g / L to 50 g / L, 0.5 g / L to 30 g / L, or 1 g / L to 20 g / L.

[0038] The amount of the second reagent can be adjusted as appropriate depending on the sample used in step (1). For example, the amount of the second reagent may be 1 to 20 volumes, 2 to 10 volumes, or 3 to 7 volumes per 1 volume of the sample used in step (1).

[0039] The length of step (2), that is, the time for which an environment is maintained that allows for the enzymatic reaction by fructosyl peptide oxidase and the color development reaction of the chromogen, can be appropriately adjusted according to the sample, the concentration of each component, the reaction conditions, etc., so that these reactions proceed sufficiently. The length of step (2) may be, for example, 1 to 20 minutes, 1 to 10 minutes, 3 to 6 minutes, or 5 minutes. The temperature of step (2) can be appropriately adjusted according to the optimal temperature of the enzyme, etc., and may be, for example, 10 to 50°C, 15 to 45°C, 20 to 40°C, or 37°C.

[0040] In step (3), the change in absorbance due to the color development of the leuco-type chromogen is calculated for the mixture produced in step (2), i.e., the mixture of the reaction solution obtained in step (1) and the second reagent. Here, the change in absorbance is the difference between the first absorbance immediately after the addition of the second reagent and the second absorbance after a predetermined time has elapsed since the measurement of the first absorbance.

[0041] Generally, in an enzymatic method for measuring glycated hemoglobin in a sample by performing a two-step reaction using two types of reagents and causing a chromogenic agent to develop color, the difference between the absorbance before the chromogenic agent's color development reaction progresses (first absorbance) and the absorbance after the color development reaction is completed (second absorbance) is calculated. These differences indicate the change in absorbance due to the color development reaction, and the concentration of glycated hemoglobin is determined based on this. Since the color development reaction of the chromogenic agent begins when the second reagent is added to the reaction solution obtained in the first step, it was conventionally thought that the absorbance of the mixture of the reaction solution and the second reagent would increase immediately after the addition of the second reagent. Therefore, it was thought that the first absorbance needed to be measured immediately before adding the second reagent. In contrast, the inventors have found that immediately after adding the second reagent, the color development reaction has not progressed to the extent that it would affect the measurement value, and the absorbance measured at this timing can be used as the first absorbance. In other words, the inventors discovered that the difference between the first absorbance immediately after adding the second reagent and the second absorbance measured after a predetermined time has elapsed since the measurement of the first absorbance can be used as the change in absorbance due to the color development of the chromopropyl alcohol. If a portion of the chromopropyl alcohol develops color before the second reaction due to deterioration of the chromopropyl alcohol, the increase in absorbance due to such color development occurs in both the first and second absorbances, and therefore does not affect the final calculated absorbance difference. Thus, by using such an absorbance difference as the change in absorbance due to the color development of the chromopropyl alcohol, glycated hemoglobin can be accurately measured.

[0042] The wavelength of light used to measure absorbance is not limited to the absorption wavelength of the dye produced from the leuco-type chromophor, but preferably it is the maximum absorption wavelength of the dye produced from the leuco-type chromophor. The wavelength of light used to measure absorbance can be appropriately selected depending on the dye, as it is suitable for the quantitative determination of the produced dye. For example, if the leuco-type chromophor is 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt, light of 600 nm to 700 nm or 658 nm can be used. Absorbance can also be measured using the two-wavelength method. In the two-wavelength method, the calculated absorbance obtained by subtracting the absorbance at the secondary wavelength from the absorbance at the primary wavelength is used for various subsequent analyses. The primary and secondary wavelengths can be selected as conventionally known. For example, if the leuco-type chromophor is 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt, the dominant wavelength may be the wavelength described above, and the secondary wavelength may be 750 nm to 850 nm, or 805 nm.

[0043] Regarding the timing of the measurement of the first absorbance, "immediately after adding the second reagent" means within 60 seconds after adding the second reagent. The first absorbance may be, for example, the absorbance within 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, or 10 seconds after adding the second reagent, and more specifically, the absorbance 1 to 60 seconds, 1 to 55 seconds, 1 to 50 seconds, 1 to 45 seconds, 1 to 40 seconds, 1 to 35 seconds, 1 to 30 seconds, 1 to 25 seconds, 1 to 20 seconds, 1 to 15 seconds, or 1 to 10 seconds after adding the second reagent.

[0044] Also, a step of stirring the mixture obtained by adding the second reagent may be interposed between the addition of the second reagent and the measurement of the first absorbance. In this case, it can be said that "immediately after adding the second reagent" means "immediately after adding the second reagent and stirring the mixture". The above stirring may be performed, for example, by inserting a rod or spatula-shaped stirring member into the container containing the mixture and then moving the stirring member back and forth or left and right, or by applying ultrasonic waves from the outside to the container containing the mixture.

[0045] Regarding the timing of measuring the second absorbance, the "predetermined time" in the expression "after a predetermined time has elapsed since the measurement of the first absorbance" is the time required for the enzyme reaction and the color development reaction in step (2) to proceed sufficiently. The specific value of the "predetermined time" depends on the sample, the concentration of each component, and the reaction conditions, but may be, for example, 1 minute to 20 minutes, 1 minute to 10 minutes, or 3 to 5 minutes.

[0046] The difference between the first absorbance and the second absorbance is more specifically a value obtained by subtracting the first absorbance from the second absorbance. The difference between the first absorbance and the second absorbance may be a value corrected for blank or a value not corrected for blank. Blank correction can be performed by a conventionally known method. For example, the above steps (1) to (3) are performed using physiological saline instead of the sample, and the difference between the first absorbance and the second absorbance obtained (the absorbance difference of physiological saline) is subtracted from the difference between the first absorbance and the second absorbance obtained in step (3) (the absorbance difference of the sample) to obtain a blank-corrected absorbance difference. When performing steps (1) to (3) using physiological saline, the various reactions in steps (1) and (2) do not proceed.

[0047] In step (4), based on the absorbance change calculated in step (3), the concentration of glycated hemoglobin in the sample is determined. Specifically, the absorbance change calculated in step (3) is compared with the absorbance change calculated by performing the above steps (1) to (3) using a standard sample containing glycated hemoglobin with a known concentration as the sample, whereby the concentration of glycated hemoglobin in the sample can be determined. The comparison of the absorbance changes may be performed using a calibration curve showing the relationship between the glycated hemoglobin concentration and the absorbance change, which is prepared in advance using a standard sample containing glycated hemoglobin with a known concentration. That is, in step (4), the absorbance change calculated in step (3) may be compared with a calibration curve showing the relationship between the glycated hemoglobin concentration and the absorbance change, which is prepared in advance using a standard sample containing glycated hemoglobin with a known concentration, to determine the concentration of glycated hemoglobin in the sample. Further, according to the present invention, even when there is a gap (for example, one day or more) between the calculation of the absorbance change using such a standard sample and the preparation (i.e., calibration) of the calibration curve and the calculation of the absorbance change using a sample containing glycated hemoglobin with an unknown concentration in steps (1) to (3), the influence on the calculated value due to the deterioration of the chromogen can be reduced, and glycated hemoglobin can be accurately measured. That is, the calibration curve showing the relationship between the glycated hemoglobin concentration and the absorbance change, which is prepared in advance, may be, for example, a calibration curve prepared 1 to 30 days, 3 to 30 days, 3 to 14 days, or 14 to 30 days before performing steps (1) to (3).

[0048] When the sample contains hemoglobin (i.e., non-glycated hemoglobin), the method for measuring glycated hemoglobin in the sample according to this aspect of the present invention may further include a step (step (5)) of calculating the ratio (%) of the amount of glycated hemoglobin to the amount of total hemoglobin in the sample based on the concentration of glycated hemoglobin determined in step (4) and the total hemoglobin concentration in the sample.

[0049] Total hemoglobin concentration refers to the concentration of all forms of hemoglobin, including glycated hemoglobin. The total hemoglobin concentration in a sample can be measured by known methods such as the cyanmethemoglobin method, the oxyhemoglobin method, and the SLS-hemoglobin method. If the first reagent contains a hemoglobin denaturant, the measurement of the total hemoglobin concentration in the sample can be performed in parallel with the measurement of the glycated hemoglobin concentration. In this case, the total hemoglobin concentration in the sample can be determined, for example, based on the absorbance (third absorbance) at the absorption wavelength of the denatured hemoglobin in the reaction solution obtained in step (1). The measurement of the third absorbance can be performed after step (1) and before step (2).

[0050] The wavelength of light used to measure the third absorbance is preferably the maximum absorption wavelength of denatured hemoglobin. For example, light of 500 nm to 600 nm or 571 nm can be used. The third absorbance can also be measured using a two-wavelength method. The primary and secondary wavelengths can be selected as conventionally known. For example, the primary wavelength may be the wavelengths mentioned above, and the secondary wavelength may be 650 nm to 750 nm or 694 nm.

[0051] The third absorbance may be a blank-corrected or unblank-corrected value. Blank correction can be performed by conventionally known methods. For example, the above-described step (1) is performed using physiological saline instead of the sample, and the absorbance of the resulting reaction solution is measured at the same wavelength as when the third absorbance was measured. Then, by subtracting the measured absorbance (absorbance of physiological saline) from the measured value of the third absorbance, a blank-corrected third absorbance can be obtained. Note that when performing step (1) using physiological saline, the enzymatic reaction in step (1) does not proceed.

[0052] The determination of the total hemoglobin concentration in a sample based on the third absorbance can be performed by carrying out the above-described step (1) using a standard sample containing a known concentration of total hemoglobin, measuring the absorbance of the resulting reaction solution at the same wavelength as when measuring the third absorbance, and comparing the obtained measurement value with the third absorbance. The comparison of absorbances may also be performed using a calibration curve that shows the relationship between total hemoglobin concentration and absorbance, which has been prepared in advance using a standard sample containing a known concentration of total hemoglobin. That is, the concentration of total hemoglobin in the sample may be determined by comparing the third absorbance with a calibration curve that shows the relationship between total hemoglobin concentration and absorbance, which has been prepared in advance using a standard sample containing a known concentration of total hemoglobin.

[0053] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[0054] In the following examples, reagents from the following manufacturers were used: MES (2-morpholinoethanesulfonic acid monohydrate; manufactured by Dojin Chemical Laboratories), 1-dodecylpyridinium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), thermolysin (proteinase; manufactured by Yamato Chemical Co., Ltd.), peroxidase (manufactured by Toyobo Co., Ltd.), sodium hydroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), ADA (N-(2-acetamide)iminodiacetic acid; manufactured by Dojin Chemical Laboratories), DA-67 (10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazinesodium The following substances were used: ferric acid salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), dimethyl sulfoxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Naimine S-220 (polyoxyethylene octadecylamine; manufactured by NOF Corporation), FPOX-CET (fructosyl peptide oxidase; manufactured by Kikkoman Corporation), Metaboreed HbA1c (HbA1c measurement kit; manufactured by Minaris Medical Co., Ltd.), Metaboreed Calibrator for HbA1c measurement (calibration standard; manufactured by Minaris Medical Co., Ltd.), and physiological saline (Otsuka Saline Injection; manufactured by Otsuka Pharmaceutical Co., Ltd.).

[0055] [Experimental Example 1] Preparation of HbA1c Measurement Kit A measurement kit A was prepared containing the following reagents, a first reagent and a second reagent. Water was used as the solvent for the first reagent and the second reagent. (First Reagent) MES 4.26 g / L 1-Dodecylpyridinium chloride 2.5 g / L Thermolysin 7200 kU / L Peroxidase 96 kU / L The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Second Reagent) ADA 5.7 g / L DA-67 26.0 mg / L Dimethyl sulfoxide 1.032 g / L Naimine S-220 0.5 g / L FPOX-CET 5 kU / L The pH was adjusted to 7.0 with an appropriate amount of sodium hydroxide. Dimethyl sulfoxide is used as a solvent to dissolve DA-67, and the second reagent was prepared using a solution of DA-67 dissolved in dimethyl sulfoxide.

[0056] [Example 1] Calculation of HbA1c (%) measurement value An automated analyzer JCA-BM9130 (manufactured by JEOL Ltd.) was used as the measuring instrument, and the measurement kit A from Experimental Example 1 was used as the HbA1c measurement kit. A pooled sample (hereinafter referred to as the evaluation sample) was used, which was a mixture of multiple blood cell fractions prepared from the whole blood of multiple people. The ratio of the HbA1c concentration (amount) in the sample to the total hemoglobin concentration (amount) (HbA1c (%) measurement value) was calculated according to the following procedure. The pooled sample (evaluation sample) was prepared by mixing multiple blood cell fractions obtained by centrifuging the whole blood of multiple people at 25°C and 3000 rpm (1500 × G) for 5 minutes, and was used after being frozen and stored at -80°C. Furthermore, when glycated hemoglobin in the evaluation sample was measured using Metaboread HbA1c, a commercially available HbA1c measurement kit, according to the method specified in the Metaboread HbA1c package insert, the measured HbA1c (%) value was 5.61%.

[0057] (1) Preparation of a calibration curve for determining total hemoglobin concentration Using the first reagent of the measurement kit A from Experimental Example 1, and using the "Metabolid Calibrator for HbA1c measurement" instead of a sample, measurements were performed according to the following procedure to create a calibration curve showing the relationship between total hemoglobin concentration and absorbance. The "Metabolid Calibrator for HbA1c measurement" is a calibration standard and comes as a set of two calibrators: Calibrator Low and Calibrator High. Calibrator Low was determined to have a hemoglobin concentration of 83.7 μmol / L and an HbA1c concentration of 2.52 μmol / L, while Calibrator High was determined to have a hemoglobin concentration of 122.1 μmol / L and an HbA1c concentration of 9.93 μmol / L. To create a calibration curve showing the relationship between total hemoglobin concentration and absorbance, both calibrator Low and calibrator High referenced hemoglobin concentration.

[0058] Each standard (4.8 μL) and the first reagent (54 μL) from measurement kit A were added to the reaction cell, and the reaction was allowed to proceed at 37°C for 5 minutes. The absorbance of the reaction solution was measured at a primary wavelength of 571 nm and a secondary wavelength of 694 nm, and the obtained absorbances were taken as the absorbance of each standard. The same method was used for measurements, except that physiological saline was used instead of the standard, and the obtained absorbances were taken as the absorbance of physiological saline. The value calculated by subtracting the absorbance of physiological saline from the absorbance of each standard was taken as the blank-corrected absorbance of each standard. A calibration curve showing the relationship between total hemoglobin concentration (μmol / L) and absorbance was created from the blank-corrected absorbance of each standard and the blank-corrected absorbance of physiological saline (0 Abs).

[0059] (2) Preparation of a calibration curve for determining HbA1c concentration Using the measurement kit A from Experimental Example 1 as the HbA1c measurement kit, and using the "Metabolid Calibrator for HbA1c Measurement" instead of a sample, measurements were performed according to the following procedure to create a calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance difference. The "Metabolid Calibrator for HbA1c Measurement" is a calibration standard and consists of two calibrators: Calibrator Low and Calibrator High. Calibrator Low was determined to have a hemoglobin concentration of 83.7 μmol / L and an HbA1c concentration of 2.52 μmol / L, while Calibrator High was determined to have a hemoglobin concentration of 122.1 μmol / L and an HbA1c concentration of 9.93 μmol / L. To create a calibration curve showing the relationship between HbA1c concentration and absorbance difference, both calibrator Low and calibrator High were referenced for HbA1c concentration.

[0060] Each standard (4.8 μL) and the first reagent (54 μL) from measurement kit A were added to a reaction cell, and the mixture was reacted at 37°C for 5 minutes. Next, the second reagent (18 μL) from measurement kit A was added to this reaction solution, and the absorbance of the mixture (first absorbance) was measured at a primary wavelength of 658 nm and a secondary wavelength of 805 nm. Next, this mixture was reacted at 37°C for 5 minutes, and the absorbance of the reaction solution (second absorbance) was measured at a primary wavelength of 658 nm and a secondary wavelength of 805 nm. The absorbance difference ΔE was calculated by subtracting the first absorbance from the second absorbance, and this was taken as the absorbance difference for each standard. The absorbance difference ΔE' was calculated using the same method, except that physiological saline was used instead of each standard, and this was taken as the absorbance difference for physiological saline. The blank-corrected absorbance difference for each standard was calculated by subtracting the absorbance difference ΔE' of physiological saline from the absorbance difference ΔE of each standard. A calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance difference was created from the blank-corrected absorbance difference of each standard and the blank-corrected absorbance difference of physiological saline (0 Abs). In the above process, in order to calculate the absorbance change due to the color development caused by the reaction between hydrogen peroxide, which is generated by the action of HbA1c in the sample and each component in the measurement kit A, and the chromogenic agent DA-67, the measurement of the first absorbance, which is to be subtracted from the second absorbance (the absorbance after the reaction), was performed within 30 seconds after the addition of the second reagent of the measurement kit A containing the chromogenic agent DA-67.

[0061] (3) Calculation of total hemoglobin concentration in the evaluation sample Three μL of the evaluation sample and 120 μL of purified water were mixed to obtain a hemolyzed blood cell fraction. The hemolyzed blood cell fraction was measured using the first reagent of the measurement kit A of Experimental Example 1 in the same manner as in (1) above, and the total hemoglobin concentration (μmol / L) in the evaluation sample was calculated from the obtained measurement value and the calibration curve in (1) above.

[0062] (4) Calculation of HbA1c concentration in the evaluation sample The hemolyzed blood cell fraction obtained in (3) above was measured using the measurement kit A from Experimental Example 1 in the same manner as in (2) above, and the HbA1c concentration (μmol / L) in the evaluation sample was calculated from the obtained measurement values ​​and the calibration curve in (2) above.

[0063] (5) Calculation of HbA1c (%) measurement value The HbA1c (%) measurement value in the evaluation sample was calculated as the NGSP value (International Standard Value) using the following formula (I) from the total hemoglobin concentration (μmol / L) in the evaluation sample calculated in (3) above and the HbA1c concentration (μmol / L) in the evaluation sample calculated in (4) above.

[0064]

[0065] (6) Calculation of measurement values ​​(day 0) of evaluation samples and storage of the measurement kit used for measurement The HbA1c (%) measurement value calculated in (5) above was used as the measurement value (day 0) of the evaluation sample. Next, the measurement kit A used in Experimental Example 1 was stored at 4°C.

[0066] [Example 2] Calculation of HbA1c (%) measurement value Using the measurement kit A stored at 4°C in (6) of Example 1, the HbA1c (%) measurement value of the evaluation sample was calculated in the same manner as in (3), (4), and (5) of Example 1 after storage for 1 day, 2 days, 3 days, 8 days, 10 days, 14 days, 21 days, and 30 days, respectively. These values ​​were recorded as the measurement value (day 1), measurement value (day 2), measurement value (day 3), measurement value (day 8), measurement value (day 10), measurement value (day 14), measurement value (day 21), and measurement value (day 30) of the evaluation sample. Next, the ratio of the measurement value on day n to the measurement value on day 0 was calculated as a percentage (%) using the following formula (II) from the measured values ​​of the evaluation sample calculated here (n = 1, 2, 3, 8, 10, 14, 21, 30) and the measured values ​​of the evaluation sample calculated in Example 1 (day 0). The results are shown in Table 1.

[0067]

[0068] In Example 2, when calculating the measured value (day n) of the evaluation sample, the calibration curves for determining the total hemoglobin concentration and the calibration curve for determining the HbA1c concentration were the same ones created when the measurements for calculating the measured value (day 0) of the evaluation sample were performed in Example 1. In other words, in calculating the measured value (day n) of the evaluation sample in Example 2 and the ratio (%) of that value to the subsequent measured value on day 0, no calibration using a standard was performed.

[0069] [Example 3] Calculation of HbA1c (%) measurement value In calculating the measurement value (day n) of the evaluation sample in Example 2, the measurement value (day n) of the evaluation sample was calculated in the same manner as in Example 2, except that calibration curves for determining total hemoglobin concentration and calibration curves for determining HbA1c concentration were created for each measurement day using the methods of (1) and (2) in Example 1. The ratio (%) with the measurement value on day 0 was then calculated. The results are shown in Table 1. Note that in the calculation of the measurement value (day n) of the evaluation sample in Example 3 and the subsequent calculation of the ratio (%) with the measurement value on day 0, calibration was performed using a standard product for each measurement day.

[0070] [Comparative Example 1] Calculation of HbA1c (%) measurement value An automated analyzer JCA-BM9130 (manufactured by JEOL Ltd.) was used as the measuring instrument, measurement kit A from Experimental Example 1 was used as the HbA1c measurement kit, and the evaluation sample used in Example 1 was used as the sample. The ratio of the HbA1c concentration (amount) in the sample to the total hemoglobin concentration (amount) (HbA1c (%) measurement value) was calculated according to the following procedure.

[0071] (1) Preparation of a calibration curve for determining total hemoglobin concentration A calibration curve showing the relationship between total hemoglobin concentration and absorbance was prepared using the same method as in (1) of Example 1.

[0072] (2) Preparation of a calibration curve for determining HbA1c concentration Using the measurement kit A from Experimental Example 1 as the HbA1c measurement kit, and using the "Metabolid Calibrator for HbA1c Measurement" as the sample, measurements were performed according to the following procedure to create a calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance difference. The "Metabolid Calibrator for HbA1c Measurement" is a calibration standard and is a set of two calibrators: Calibrator Low and Calibrator High. Calibrator Low was determined to have a hemoglobin concentration of 83.7 μmol / L and an HbA1c concentration of 2.52 μmol / L, while Calibrator High was determined to have a hemoglobin concentration of 122.1 μmol / L and an HbA1c concentration of 9.93 μmol / L. To create a calibration curve showing the relationship between HbA1c concentration and absorbance difference, both calibrator Low and calibrator High were referenced for HbA1c concentration.

[0073] Each standard (4.8 μL) and the first reagent (54 μL) from measurement kit A were added to the reaction cell, and the reaction was allowed to proceed at 37°C for 5 minutes. The absorbance of the reaction solution (first absorbance) was measured at a primary wavelength of 658 nm and a secondary wavelength of 805 nm. Next, the second reagent (18 μL) from measurement kit A was added to this reaction solution, and the reaction was allowed to proceed at 37°C for 5 minutes. The absorbance of the reaction solution (second absorbance) was measured at a primary wavelength of 658 nm and a secondary wavelength of 805 nm. The absorbance difference ΔE was calculated by subtracting the first absorbance from the second absorbance, and this was taken as the absorbance difference for each standard. The absorbance difference ΔE' was calculated using the same method, except that physiological saline was used instead of each standard, and this was taken as the absorbance difference for physiological saline. The value calculated by subtracting the absorbance difference ΔE' for physiological saline from the absorbance difference ΔE for each standard was taken as the blank-corrected absorbance difference for each standard. A calibration curve showing the relationship between HbA1c concentration (μmol / L) and absorbance difference was created from the blank-corrected absorbance difference of each standard and the blank-corrected absorbance difference of physiological saline (0 Abs). In the above process, in order to calculate the absorbance change due to the color development caused by the reaction between hydrogen peroxide, which is generated by the action of HbA1c in the sample and each component in the measurement kit A, and the chromogenic agent DA-67, the measurement of the first absorbance, which is to be subtracted from the second absorbance (the absorbance after the reaction), was performed within 30 seconds before adding the second reagent of the measurement kit A containing the chromogenic agent DA-67.

[0074] (3) Calculation of total hemoglobin concentration in the evaluation sample Three μL of the evaluation sample described above was mixed with 120 μL of purified water to obtain a hemolyzed blood cell fraction. The hemolyzed blood cell fraction was used as a sample, and the total hemoglobin concentration (μmol / L) in the evaluation sample was calculated in the same manner as in (3) of Example 1.

[0075] (4) Calculation of HbA1c concentration in the evaluation sample The hemolyzed blood cell fraction obtained in (3) above was measured using the measurement kit A from Experimental Example 1 in the same manner as in (2) above, and the HbA1c concentration (μmol / L) in the evaluation sample was calculated from the obtained measurement values ​​and the calibration curve in (2) above.

[0076] (5) Calculation of HbA1c (%) measurement value The HbA1c (%) measurement value in the evaluation sample was calculated as the NGSP value (International Standard Value) using the formula (I) above, based on the total hemoglobin concentration (μmol / L) in the evaluation sample calculated in (3) above and the HbA1c concentration (μmol / L) in the evaluation sample calculated in (4) above.

[0077] (6) Calculation of measurement values ​​(day 0) of evaluation samples and storage of the measurement kit used for measurement The HbA1c (%) measurement value calculated in (5) above was used as the measurement value (day 0) of the evaluation sample. Next, the measurement kit A used in Experimental Example 1 was stored at 4°C.

[0078] The only difference between Example 1 and Comparative Example 1 is that, in calculating the change in absorbance due to the color development caused by the reaction of hydrogen peroxide, which is generated by the action of HbA1c in the sample and each component in the measurement kit A, with the colorant DA-67 in steps (2) and (4), the measurement of the first absorbance, which is subtracted from the second absorbance (the absorbance after the reaction), is performed either within 30 seconds after the addition of the second reagent of the measurement kit A containing the colorant DA-67, or within 30 seconds before the addition.

[0079] [Comparative Example 2] Calculation of HbA1c (%) measurement value Using the measurement kit A stored at 4°C as in (6) of Comparative Example 1, the HbA1c (%) measurement value in the evaluation sample was calculated in the same manner as in (3), (4), and (5) of Comparative Example 1 after storage for 1 day, 2 days, 3 days, 8 days, 10 days, 14 days, 21 days, and 30 days, respectively. These values ​​were recorded as the measurement value (day 1), measurement value (day 2), measurement value (day 3), measurement value (day 8), measurement value (day 10), measurement value (day 14), measurement value (day 21), and measurement value (day 30) of the evaluation sample. Next, the ratio of the measurement value on day n to the measurement value on day 0 was calculated as a percentage (%) using the above formula (II) from the measured values ​​of the evaluation sample calculated here (n = 1, 2, 3, 8, 10, 14, 21, 30) and the measured values ​​of the evaluation sample calculated in Comparative Example 1 (day 0). The results are shown in Table 1.

[0080] In Comparative Example 2, when calculating the measured value (day n) of the evaluation sample, the calibration curves for determining the total hemoglobin concentration and the calibration curves for determining the HbA1c concentration were the same ones created when the measurements for calculating the measured value (day 0) of the evaluation sample were performed in Comparative Example 1. In other words, in the calculation of the measured value (day n) of the evaluation sample in Comparative Example 2 and the subsequent calculation of the ratio (%) to the measured value on day 0, no calibration using a standard was performed.

[0081] [Comparative Example 3] Calculation of HbA1c (%) measurement value In calculating the measurement value (day n) of the evaluation sample in Comparative Example 2, the measurement value (day n) of the evaluation sample was calculated in the same manner as in Comparative Example 2, except that calibration curves for determining total hemoglobin concentration and calibration curves for determining HbA1c concentration were created for each measurement day using the methods of (1) and (2) of Comparative Example 1. The ratio (%) with the measurement value on day 0 was then calculated. The results are shown in Table 1. In Comparative Example 3, calibration using a standard was performed for each measurement day in the calculation of the measurement value (day n) of the evaluation sample and the subsequent calculation of the ratio (%) with the measurement value on day 0.

[0082]

[0083] The ratio (%) of the measurement value on day n to the measurement value on day 0 should preferably remain close to 100% from day 0 to day 30, since the same evaluation sample is being measured. Therefore, if the ratio (%) of the measurement value on day n to the measurement value on day 0 in each measurement method deviates from 100%, that measurement method can be evaluated as inaccurate. On the other hand, if the ratio (%) of the measurement value on day n to the measurement value on day 0 in each measurement method remains close to 100%, that measurement method can be evaluated as accurate.

[0084] As shown in Table 1, in the measurement methods of Comparative Examples 1, 2, and 3, in which the first absorbance, which is to be subtracted from the second absorbance (the absorbance after the reaction), was measured before adding the second reagent of the measurement kit A containing the color agent DA-67, it became clear that, as in Comparative Examples 1 and 2, the measured values ​​gradually increased as the number of days passed, making accurate measurement impossible, and that calibration using a standard product was necessary each day of measurement to obtain accurate measurements, as in Comparative Example 3. In other words, in the measurement method in which the first absorbance is measured before adding the reagent containing the leuco-type color agent, it became clear that the measured values ​​gradually increased as the number of days passed, making accurate measurement impossible, and that calibration using a standard product was necessary each day of measurement to obtain accurate measurements. Furthermore, due to the cost required for standard samples and the complexity of the calibration process, it is preferable to minimize the frequency of calibration using standard samples. In addition, in the measurement methods of Comparative Examples 1 and 2, the reason why the measured values ​​gradually increase over time is that the absorbance of the second reagent itself (reagent blank absorbance) gradually increases due to the deterioration of DA-67 in the second reagent over time, and the apparent measured values ​​increase by the amount of this increase in reagent blank absorbance.

[0085] Furthermore, as shown in Table 1, in the measurement methods of Examples 1, 2, and 3, in calculating the absorbance change due to the color development caused by the reaction of hydrogen peroxide, which is generated by the action of HbA1c in the sample and each component in the measurement kit A, with the chromogenic agent DA-67, the measurement of the first absorbance, which is subtracted from the second absorbance (the absorbance after the reaction), was performed after the addition of the second reagent of the measurement kit A containing the chromogenic agent DA-67. In these methods, even when calibration with a standard was performed only on day 0, the measured values ​​did not change even after several days had passed, just as when calibration with a standard was performed on each measurement day, and accurate measurements were possible. In other words, it was revealed that the measurement method in which the first absorbance is measured immediately after the addition of the reagent containing the leuco-type chromogenic agent is an accurate measurement method.

[0086] From the above, it has become clear that in the measurement of glycated hemoglobin, accurate measurement of glycated hemoglobin is possible by calculating the difference between the first absorbance immediately after adding the second reagent and the second absorbance after a predetermined time has elapsed since the measurement of the first absorbance, as this is the change in absorbance due to the color development of the leuco-type chromogen.

Claims

1. (1) A step of mixing a sample containing glycated hemoglobin with a first reagent containing a proteolytic enzyme, thereby reacting the glycated hemoglobin with the proteolytic enzyme to obtain a reaction solution containing fructosyl peptide; (2) A step of adding a second reagent containing fructosyl peptide oxidase and a leuco-type chromogen to the reaction solution, thereby reacting the fructosyl peptide with the fructosyl peptide oxidase to produce hydrogen peroxide, wherein the leuco-type chromogen develops color due to the hydrogen peroxide; (3) A step of calculating the change in absorbance of the mixture of the reaction solution from step (1) and the second reagent due to the color development of the leuco-type chromogen, wherein the change in absorbance is the difference between the first absorbance immediately after adding the second reagent and the second absorbance after a predetermined time has elapsed since the measurement of the first absorbance; and (4) A method for measuring glycated hemoglobin in a sample, comprising the step of determining the concentration of glycated hemoglobin in the sample based on the absorbance change.

2. The method according to claim 1, wherein the first absorbance is the absorbance 1 second to 60 seconds after the addition of the second reagent.

3. The method according to claim 2, wherein the first absorbance is the absorbance 1 second to 30 seconds after the addition of the second reagent.

4. The method according to claim 3, wherein the first absorbance is the absorbance 1 to 15 seconds after the addition of the second reagent.

5. The method according to any one of claims 1 to 4, wherein the predetermined time is 3 to 5 minutes.

6. The method according to any one of claims 1 to 4, wherein the first reagent further contains a hemoglobin denaturant.

7. The method according to claim 6, wherein the hemoglobin denaturant is a surfactant.

8. The method according to claim 7, wherein the surfactant is a cationic surfactant.

9. The method according to any one of claims 1 to 4, wherein step (4) is a step of determining the concentration of glycated hemoglobin in the sample by comparing the absorbance change calculated in step (3) with a calibration curve that shows the relationship between glycated hemoglobin concentration and absorbance change, which has been prepared in advance using a standard sample containing glycated hemoglobin of known concentration, and the calibration curve prepared in advance is a calibration curve that was prepared 1 to 30 days before steps (1) to (3) are performed.

10. The method according to claim 9, wherein the pre-prepared calibration curve is a calibration curve prepared 14 to 30 days before performing steps (1) to (3).

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