JP2019062890A - Measurement of glycated protein - Google Patents

Abstract

To provide measurement methods of glycated protein performed by an enzymatic method, in which steps are simplified.SOLUTION: The present invention relates to a measurement method of the glycated protein in a sample which comprises the following (1) to (3), in which a reagent to be mixed with a sample in order to perform the following (2) to (3) is one, in one aspect. (1) Modifying the protein in a sample. (2) Reacting the glycated protein in a sample with amadoriase to allow a color former to color. (3) Measuring the coloring signal of the sample after (1) and (2) to calculate the amount of the glycated protein.SELECTED DRAWING: None

Description

  The present disclosure relates to the measurement of glycated proteins, and more specifically to a method, apparatus, and system for measurement of glycated proteins using amadoriase.

When a protein and a sugar such as glucose coexist, an Amadori compound is formed, so the amount of sugar can be known by measuring the amount of the Amadori compound. The amount of sugar is measured in blood of humans and animals and in food.
In particular, measurement of the amount of glycated protein is important in the diagnosis and treatment of diabetes. As a method of measuring the amount of glycated protein, there are a method of measuring only the amount of glycated protein, a method of calculating the ratio by measuring the amount of glycated protein and the amount of protein, and the like. It may be limited to a specific protein or may be limited to a specific site of a specific protein.

Examples of such measurement of the amount of glycated protein include fructosamine, glycated albumin, glycated hemoglobin, and hemoglobin A1c.
For example, fructosamine measures the glycation amount of protein or albumin in serum or plasma, and glycated albumin measures the ratio of the glycation amount of albumin to albumin amount in serum or plasma. Glycated hemoglobin measures the amount of glycated hemoglobin in the blood or measures the ratio to the amount of hemoglobin. Hemoglobin A1c measures the β-chain N-terminal glycation amount (HbA1c amount) of hemoglobin in blood or the ratio to the amount of hemoglobin.
As a method of measuring these glycated proteins, an enzyme method using amadoriase which reacts with glycated amino acids and glycated peptides is generally used.

  The measurement method of fructosamine is patent 3034698 (patent document 1), the measurement method of glycated albumin is patent 4341809 (patent document 2) and JP-A 2008-295305 (patent document 3), the measurement method of only the amount of glycated hemoglobin is patent 5878096 (patent) Reference 4) and the like.

Among them, the measurement of the glycated hemoglobin ratio is important in the diagnosis and treatment of diabetes. The measurement of hemoglobin A1c (HbA1c), which is one of glycated hemoglobins, is particularly widely measured.
Methods of measuring the amount of glycated hemoglobin and the amount of hemoglobin are widely used to measure the glycated hemoglobin ratio.
As a method of measuring the amount of glycated hemoglobin, an enzyme method using amadoriase that reacts with glycated amino acids and glycated peptides is generally used. Examples of the amadoriase include fructosyl peptide oxidase that generates hydrogen peroxide and the like (patent 4231668 and patent document 5), and fructosyl peptide dehydrogenase (WO 2016-63984 and patent document 6) with improved dehydrogenase activity.

Amadoriase, which has been used in conventional enzymatic methods, has substrate specificity from glycated amino acid to glycated peptide. For this reason, when the amount of glycated hemoglobin is to be measured by amadoriase, it is necessary to treat glycated hemoglobin in advance with a protease or the like to decompose it into glycated amino acid or glycated peptide so that the amadoriase is easily acted (WO2006-120976, Patent Document 7). When glycated hemoglobin is degraded by protease, the amadoriase used for measurement needs to be prepared as a separate reagent in order not to be degraded by protease.
On the other hand, to measure the amount of hemoglobin after mixing the sample, amadoriase and protease, Hb reacts with the reagent (especially protease) and the color tone of Hb changes, and the action of amadoriase causes the concentration of glycated hemoglobin to It was difficult because the color forming pigment generated accordingly overlapped with the color tone of Hb and the glycated hemoglobin value was affected.

At present, a reagent of two-reagent system or three-reagent system is used as a reagent for measurement of the amount of glycated hemoglobin by the enzyme method. The following configurations 1 to 4 can be mentioned as the configuration of the reagent of the two-reagent system. Configuration 1
First reagent: amadoriase, buffer Second reagent: protease, color former, buffer Composition 2
First reagent: Protease, buffer Second reagent: amadoriase, color former, buffer Composition 3
First reagent: amadoriase, color former, buffer Second reagent: protease, buffer Composition 4
First reagent: Protease, color former, buffer Second reagent: amadoriase, buffer

In recent years, the amadoriase has been improved, and an amadoriase having substrate specificity for a glycated protein (that directly acts on the glycated protein) has been discovered (hereinafter, also referred to as "glycated protein direct type amadoriase") (WO2015-005257 (Patent Document 8) and WO2015-060429 (Patent Document 9)). As a method of measuring the amount of glycated hemoglobin using a glycated protein direct type amadoriase, for example, a measurement method including performing the following steps (i) to (ii) sequentially is disclosed.
(I) A step of causing glycated hemoglobin in a sample to act on glycated protein direct type amadoriase to oxidize glycated hemoglobin.
(Ii) measuring the substance produced in the step (i) or the substance consumed;

  In addition, the measurement of the amount of hemoglobin uses the method of measuring the red of the heme of hemoglobin. Hemoglobin has an oxidized form and a reduced form, and the charge state of heme changes depending on the type, and the spectrum changes, so it is necessary to use a denaturing agent to make a certain form.

Patent 3034698 Patent No. 4341809 JP 2008-295305 A Patent No. 5878096 Patent No. 4231668 gazette WO 2016-63984 WO2006-120976 WO2015-005257 WO2015-060429

  When the glycated protein ratio is measured with a two-reagent enzyme method reagent, there is a step of sequentially adding two solutions. That is, there are two steps, a step of mixing the sample and the first reagent, and a step of mixing the second reagent thereto. Therefore, the method of measuring the glycated protein ratio with the two-reagent system reagent is complicated in process. In addition, because there are two steps, it takes time for operations such as dispensing and mixing, so it is difficult to shorten the time.

  The present disclosure provides, in one aspect, a method for measuring a glycated protein performed by an enzymatic method, wherein the measuring method is simplified.

The present disclosure relates, in one aspect, to a method for measuring glycated protein in a sample, which comprises the following (1) to (3), wherein the reagent mixed with the sample to perform the following (2) and (3) is 1 One measurement method. Hereinafter, this measurement method is also referred to as “the measurement method according to the present disclosure”, and a reagent to be mixed with a sample to perform the following (2) and (3) is also referred to as “one reagent reagent”. In the present disclosure, one reagent-based reagent may be a reagent to be mixed with a sample in order to perform the following (1) to (3).
(1) Denature the protein in the sample.
(2) To develop a color former by the reaction of glycated protein in the sample with amadoriase. (3) To calculate the amount of glycated protein by measuring the chromogenic signal of the sample after (1) and (2).

  In another aspect, the present disclosure relates to a glycated protein measurement device or measurement system that implements the measurement method according to the present disclosure.

  According to the present disclosure, for example, the steps of the method of measuring a glycated protein performed by an enzyme method can be simplified. According to the present disclosure, for example, the step of adding 2 reagents or 3 reagents to a hemolyzed sample can be a step of mixing 1 reagent. Thereby, for example, the time taken to measure the hemoglobin A1c ratio (HbA1c%) can be shortened. Further, for example, since it is a mixture of one reagent, the measurement of the amount of glycated hemoglobin and the measurement of the amount of hemoglobin can be performed in the same solution, so that the accuracy and precision of the measurement can be improved.

FIG. 1 is an explanatory view of an example of a glycated protein measurement apparatus or measurement system that implements the measurement method according to the present disclosure.

In the present disclosure, the measurement of glycated protein includes, in one embodiment, measuring the amount of glycated protein, and in another embodiment, includes measuring the amount of glycated protein and the amount of protein, and the other one. In embodiments, it may include measuring glycated protein mass and protein mass to determine their ratio.
In the present disclosure, "protein" may include "glycated protein" unless otherwise stated.

In the present disclosure, “mixing with a sample” of one reagent-based reagent includes adding one reagent-based reagent to a sample and adding a sample to one reagent-based reagent.
The measurement method according to the present disclosure includes, in one or more embodiments, denaturation of protein, reaction of glycated protein with amadoriase, coloration of color former, measurement of absorbance of color former, and selectively denaturation of protein The measurement of absorbance can be performed in one mixing of a sample and one reagent-based reagent and in one container (for example, a photometric cell).

  In the present disclosure, to denature a protein in a sample with 1 reagent-based reagent means that 1 reagent-based reagent contains a denaturing agent (or a substance having a denaturing action) capable of denaturing a protein in a sample. As described later, the sample before mixing may contain a surfactant (denaturant) for hemolysis and the like.

  The glycated protein measured in the measurement method according to the present disclosure includes fructosamine, glycated albumin, glycated hemoglobin and the like, and in one embodiment is glycated albumin or glycated hemoglobin. Examples of glycated hemoglobin include hemoglobin A1c (HbA1c) in which the N-terminal end of the Hb β chain is glycated.

  The measurement target sample according to the present disclosure includes a sample containing a glycated protein. When the glycated protein is glycated hemoglobin, the sample to be measured includes a sample containing hemoglobin and glycated hemoglobin. The sample to be measured includes, in one or more non-limiting embodiments, a sample containing red blood cells such as whole blood and blood cells, and a sample obtained by hemolyzing the sample. When the sample to be measured is a sample containing red blood cells, hemolysis may be performed with one reagent-based reagent, or hemolysis sample may be used as hemolysis by means other than the one reagent-based reagent. Hemolysis of red blood cells can be performed by existing methods. For example, there are a method using osmotic pressure (eg, water), a method using a surfactant, a method of freezing, a method using ultrasonic waves, and the like. If a substance having a denatured action such as a surfactant is used, the protein can be denatured as well as the hemolysis.

  The calculation of the amount of glycated protein such as HbA1c and the like and the amount of protein such as hemoglobin and the like from the chromogenic signal of the sample can be performed by a known method in one or more embodiments.

Therefore, the present disclosure is, in one aspect, a method for measuring glycated hemoglobin in a sample, comprising the following (1) to (4), which is a reagent mixed with the sample to perform the following (1) to (4): Relating to the measurement method in which
(1) Denature the hemoglobin in the sample.
(2) To develop a color former by the reaction of glycated hemoglobin in the sample with amadoriase.
(3) To calculate the amount of glycated hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).
(4) To calculate the amount of hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).

In addition, when the measurement target sample is a sample containing red blood cells, hemolysis and denaturation of hemoglobin may be performed by means other than the one reagent-based reagent. That is, the present disclosure is, in one aspect, a method for measuring glycated hemoglobin in a sample, which comprises the following (0) to (4), and a reagent mixed with the sample to perform the following (2) to (4) Relating to the measurement method in which
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) To develop a color former by the reaction of glycated hemoglobin in the sample with amadoriase.
(3) To calculate the amount of glycated hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).
(4) To calculate the amount of hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).

[Embodiment using glycated protein direct type amadoriase]
In one embodiment, the measurement method according to the present disclosure uses, as an amadoriase, an amadoriase (which directly acts on a glycated protein) having substrate specificity for a glycated protein (glycated protein direct type amadoriase).
In the present disclosure, "glycated protein direct type amadoriase" refers to an amadoriase that can be reacted using a glycated portion of a glycated protein as a substrate without degrading the glycated protein into a peptide by a protease or the like.

  As glycated protein direct type amadoriases, glycated protein direct type fructosyl peptide oxidase (direct type FPOX) and glycated protein direct type fructosyl peptide dehydrogenase (direct type FPDH) can be mentioned (Patent Documents 8 and 9).

[Embodiment A using direct type FPOX]
The measurement method according to the present disclosure includes the following (1) to (4) in non-limiting embodiment A, and to perform the following (1) to (4), or the following (2) to (4) It is a measuring method of a glycated protein including mixing one reagent (one reagent system reagent) and a sample in order to carry out.
(1) Denature the protein in the sample.
(2) Generating hydrogen peroxide by the reaction of glycated protein in the sample with direct FPOX.
(3) Coloring the oxidized color former by the reaction between the generated hydrogen peroxide and peroxidase (POD).
(4) To calculate the amount of glycated protein by measuring the color development signal of the oxidized color developing agent that has developed color.

式（Ｉ）において、Ｒは、炭素数８−１７の炭化水素基を表す。 In one or more non-limiting embodiments, the following configurations A1 to A3 may be mentioned as one reagent-based reagent in embodiment A. However, the reagent according to the present disclosure is not limited to these embodiments. One reagent-based reagent may further contain other components.
Configuration A1
1 Reagent system reagent: Buffer, denaturant, direct type FPOX, POD, oxidation color former Composition A2
1 Reagent system reagent: Buffer, direct type FPOX, POD, oxidation color former Composition A3
1 Reagent system reagent: Buffer, direct type FPOX, POD, leuco type dye, compound of the following formula (I)

In formula (I), R represents a hydrocarbon group having 8 to 17 carbon atoms.

  As the buffer in the embodiment A, one which can be adjusted to near neutrality and which does not impair the reaction system can be used. Nonlimiting examples of buffers include 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-piperidinyl] ethanesulfonic acid (HEPES), N- [Tris (hydroxymethyl) methyl] glycine (TRICINE), Piperazine-1,4-bis (2-ethanesulfonic acid) ( PIPES), Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) dehydrate (POPSO), carbonic acid, phosphoric acid, boric acid, glycine, alanine, leucine, arginine, lysine, histidine, taurine, aspartic acid, Asparagine, hydroxyproline, proline, threonine, serine, glutamate, glutamine, valine, cysteine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, tryptophan Trishydroxymethylaminomethane, dimethylaminoethanol, triethanolamine, diethanolamine, monoethanolamine, N- methylaminoethanol, creatinine, imidazole, barbital, ammonia, ethylamine, diethylamine, triethylamine, and the like.

  As the peroxidase (POD) in the embodiment A, one which can react with hydrogen peroxide to cause an oxidative color agent such as leuco type dye which is a color forming substrate to emit light can be used. The species of organism from which the POD is derived is not particularly limited. A non-limiting example includes horseradish peroxidase.

As the denaturing agent in the constitution A1 of Embodiment A, a denaturing agent which can denature proteins and which does not significantly impair the activity of the enzyme can be used. The following (1) to (8) may be mentioned as non-limiting examples of the modifier. In addition, (1) to (7) may be used in combination with nitrous acid.
(1) 3-lauryl dimethylaminobutyric acid (2) 3-myristyl dimethylaminobutyric acid (3) lauryl dimethylaminopropanesulfonic acid (4) myrityl dimethylaminopropanesulfonic acid (5) laurylamidopropyl dimethylaminobutyric acid (6) myristal Mydopropyl betaine (7) n-dodecyl-βD-maltoside (8) WST-3 (2- (4-iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfophenyl) ) -2H-tetrazolium)

The oxidized color former in the configurations A1 and A2 of the embodiment A is preferably a leuco-type dye when the amount of glycated hemoglobin and the amount of hemoglobin are measured.
As the leuco-type dye, for example, N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA-64), 10- (carboxymethylaminocarbonyl) -3, which is easily available 7-bis (dimethylamino) phenothiazine (DA-67), 2,2'-aminobis (3-ethylbenzothiazolinone-6-sulfonic acid (ABTS), bis- (4-diethylaminophenyl) -2-sulfo Phenylmethane (BSPM), bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA), 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP), o-tolidine, 3,3'-diaminobenzidine 4HCl (DAB), 3- (4- (4-) Droxyphenyl) propionic acid (HPPA), 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), N- (3-sulfopropyl) -3,3 ′, 5,5′-tetramethylbenzidine. Na (TMBZ-PS), N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 ', 4''-triaminotriphenylmethane · 6Na (TPM- PS etc.

In the case of measurement of fructosamine and glycated albumin, the oxidative color former in configurations A1 and A2 of Embodiment A is, for example, 4-aminoantipyrine (4-AA) or 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the case of measurement of glycated albumin. A Trinder reagent or a leuco-type dye can be used which produces a dye by oxidative condensation of a coupler and a chromogen such as phenol.
A phenol derivative, an aniline derivative, a toluidine derivative etc. can be used as a hydrogen donor of Trinder's reagent, A N- (3-sulfopropyl) aniline sodium 1 water (HALPS), N-ethyl-N- can be used as a specific example. (3-sulfopropyl) -3-methylaniline sodium 1 water (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium water 1 (MAOS), N- (3-sulfopropyl) -3,5-dimethoxyaniline sodium 1 water (HDAPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (HDAOS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline sodium 1 water (DAPS), N-ethyl- -(2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (DAOS), N-ethyl-N- (3-sulfopropyl) aniline sodium (ALPS), N-ethyl-N- (3-) Sulfopropyl) -3-methoxyaniline sodium 1 water (ADPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline sodium water 2 (ADOS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N, N-bis (4-sulfobutyl) -3-methylaniline disodium (TODB), and the like.

  Configurations A1 and A2 of Embodiment A may further include a dye stabilizer, such as a reducing agent or surfactant.

  Configuration A3 of Embodiment A includes compounds of Formula (I) that can perform the dual functions of modifier and dye stabilizer. In the formula (I), the number of carbon atoms of R is preferably 8 to 17, more preferably 12 to 16, and still more preferably 14 from the viewpoint of not inhibiting the measurement by the enzyme method and the stability improvement of the leuco dye. The hydrocarbon group of R is preferably an alkyl group from the same viewpoint, and a linear alkyl group is preferable. From the same viewpoint, R is preferably a dodecyl group and a tetradecyl group, and more preferably a tetradecyl group.

  The sample to be measured in Embodiment A includes a sample containing hemoglobin and glycated hemoglobin. The sample to be measured includes, in one or more non-limiting embodiments, a sample containing red blood cells such as whole blood and blood cells, and a sample obtained by hemolyzing the sample. When the sample to be measured is a sample containing red blood cells, hemolysis may be performed with one reagent-based reagent, or hemolysis sample may be used as hemolysis by means other than the one reagent-based reagent. Hemolysis of red blood cells can be performed by existing methods. For example, there are a method using osmotic pressure (eg, water), a method using a surfactant, a method of freezing, a method using ultrasonic waves, and the like.

  In the present disclosure, the chromogenic signal includes, in one or more embodiments, absorbance, reflectance, transmittance, and the like.

The calculation of the amount of glycated protein includes, in one or more embodiments, converting the chromogenic signal obtained by the measurement into the amount of glycated protein based on a predetermined conversion factor. In one or more embodiments, conversion of the glycated protein mass based on a predetermined conversion factor is based on one of the following conversion rules (i) to (iv) of a chromogenic signal such as absorbance obtained by measurement: It can carry out by converting into the amount of glycated protein. (Iv) may be performed in combination with (i) to (iii).
(I) Obtain a calibration curve based on a known calibration substance in the sample, and convert the absorbance derived from HbA1c to the amount of HbA1c based on the calibration curve (ii) Using the chromogenic dye as a calibration substance (substance for calibration) And calculate the absorbance of the color forming dye based on the calibration curve of the color forming dye (iii) taking the absorbance ratios determined at different wavelengths, the weight of the glycated protein based on the calibration curve relative to the absorbance ratio Convert to (iv) Calculate the difference in absorbance after taking the difference between the absorbance measured immediately after mixing the sample and the reagent and after a predetermined time from mixing, and convert it to the amount of glycated protein based on the calibration curve for the amount of absorbance change. Known calibrators in (i) include, in one or more embodiments, calibration standards containing known amounts of HbA1c. The calibration standard containing known amounts of HbA1c includes, in one or more embodiments, frozen whole blood or blood cells, Hb solution containing HbA1c purified from them, or buffer composition and Hb stability thereof And the like. The known calibration substance includes, in one or more embodiments, a primary standard substance or routine standard substance provided by a public organization, a calibration substance attached to a kit, and the like.
Immediately after mixing in (iv), in one or more embodiments, about 5 seconds to 30 seconds may be mentioned from the mixing of the sample and the reagent. After a predetermined time has elapsed, in one or more embodiments, about 1 minute to 3 minutes may be mentioned from the mixing of the sample and the reagent.
In addition, in the said form, it is an example in case a color development signal is a light absorbency, Comprising: It can carry out similarly, even if it is reflectance and the transmittance | permeability other than a light absorbency.

An object to be measured in Embodiment A is glycated hemoglobin, and Embodiment Aa in which the ratio of glycated hemoglobin (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) is measured includes the following (1) to (5). The method of measuring the glycated hemoglobin ratio includes mixing one sample (one reagent-based reagent) with the sample to perform 5).
Embodiment Aa
(1) Denature the hemoglobin in the sample.
(2) Generating hydrogen peroxide by the reaction of glycated hemoglobin in a sample with direct FPOX.
(3) Coloring a leuco-type dye by the reaction of generated hydrogen peroxide and peroxidase (POD).
(4) Calculate the amount of glycated hemoglobin by measuring the color development signal of the colored leuco dye.
(5) To calculate the amount of hemoglobin by measuring the color development signal of denatured hemoglobin.

An object to be measured in embodiment A is glycated hemoglobin, and an embodiment Ab for measuring a glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) includes the following (0) to (5). The method of measuring the glycated hemoglobin ratio includes mixing one sample (one reagent-based reagent) with the sample to perform 5).
Embodiment Ab
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) Generating hydrogen peroxide by the reaction of glycated hemoglobin in a sample with direct FPOX.
(3) Coloring a leuco-type dye by the reaction of generated hydrogen peroxide and peroxidase (POD).
(4) Calculate the amount of glycated hemoglobin by measuring the color development signal of the colored leuco dye.
(5) To calculate the amount of hemoglobin by measuring the color development signal of denatured hemoglobin.

Embodiments Aa and Ab may further include calculating a glycated hemoglobin ratio from the amount of glycated hemoglobin and the amount of hemoglobin.
In embodiments Aa and Ab, glycated hemoglobin is hemoglobin A1c in one or more embodiments.

In one or more embodiments, when the measurement target in embodiment A is a glycated protein other than glycated hemoglobin (for example, glycated albumin etc.) and the glycated protein ratio (ratio of glycated protein mass to protein mass) is measured. The ratio of glycated protein can be determined from the amount of glycated protein obtained by the measurement according to the disclosure and the amount of protein obtained by another method. The glycated hemoglobin ratio may also be determined by this method.
Examples of methods for determining the amount of protein include the Biuret reaction method, Lowry method using Folin phenol reagent, Bradford (Coomassie) method using Coomassie G-250, etc. In the case of albumin, BCG method and BCP method Can be mentioned.

[Form of Administration Preparation of Embodiment A]
One reagent-based reagent in Embodiment A may not be a single reagent in terms of the stability of the reagent in the distribution stage, and the like. The preparation may be made at the stage of performing the measurement method according to the present disclosure. The following configurations A4 to A9 can be mentioned as configurations of two non-limiting reagents for preparing one reagent-based reagent in such an embodiment. However, the reagent according to the present disclosure is not limited to these embodiments. The reagent may further contain other components.
Configuration A4
Reagent 1: Buffer, denaturant, direct FPOX, POD
Reagent 2: Buffer, oxidative color former Composition A5
Reagent 1: Buffer, direct type FPOX, POD
Reagent 2: Buffer, denaturing agent, oxidation color former Composition A6
Reagent 1: Buffer, POD
Reagent 2: Buffering agent, denaturing agent, direct type FPOX, oxidation color former Composition A7
Reagent 1: Buffer, denaturant, POD
Reagent 2: Buffer, Direct FPOX, Oxidative Coloring Agent Composition A8
Reagent 1: Buffer, direct type FPOX, POD
Reagent 2: Buffer, Oxidative Colorant Composition A9
Reagent 1: Buffer, POD
Reagent 2: Buffer, direct type FPOX, oxidation color former

  Configurations A4 to A9 of Embodiment A may further comprise dye stabilizers, such as reducing agents, surfactants, and the like. Further, as the buffer, the modifier, and the oxidative color former in the configurations A4 to A9, those described above can be used. Also, in configurations A4 to A9, the modifier and / or the dye stabilizer may be a compound of formula (I).

  In the present disclosure, the “form of business preparation” includes preparation of one reagent-based reagent from a plurality of reagents for one or a plurality of samples when the measurement method according to the present disclosure is performed on one or a plurality of samples. It says a form.

[Embodiment B using direct type FPDH]
The measurement method according to the present disclosure includes the following (1) to (3) in non-limiting embodiment B, and to perform the following (1) to (3), or the following (2) to (3) It is a measuring method of a glycated protein including mixing one reagent (one reagent system reagent) and a sample in order to carry out.
(1) Denature the protein in the sample.
(2) To develop a reduced color former by the reaction between the glycated protein in the sample and the direct type FPDH.
(3) To calculate the amount of glycated protein by measuring the color development signal of the reduced color former that has developed color.

In one or more non-limiting embodiments, one reagent-based reagent in embodiment B includes the following configurations B1 and B2. However, the reagent according to the present disclosure is not limited to these embodiments. One reagent-based reagent may further contain other components.
Configuration B1
1 Reagent-based reagent: Buffer, denaturant, direct type FPDH, reduced color former Composition B2
1 Reagent-based reagent: Buffer, direct type FPDH, reduced color former

  The buffer, the denaturing agent, and the sample to be measured in Embodiment B are the same as in Embodiment A.

As the reduced color former in Embodiment B, a tetrazolium salt can be used.
As a tetrazolium salt, 2,5-diphenyl-3- (1-naphthyl) -2H-tetrazolium chloride <abbreviated tetrazolium violet>, 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene)- Bis [2- (p-nitrophenyl) -5-phenyltetrazolium chloride] <abbreviated as nitro blue tetrazolium>, 3,3 ′-(3,3′-dimethoxy-4,4′-biphenylene) -bis (2,5) -Diphenyltetrazolium chloride) <abbreviated blue tetrazolium>, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazolium bromide <abbreviated MTT>, 3- (p-iodophenyl) -2- (p -Nitrophenyl) -5-phenyl-tetrazolium chloride, 2,2 ', 5,5'-tetra- (p-nit) Phenyl) -3,3 '-(3-dimethoxy-4-diphenylene) -ditetrazolium chloride (abbreviated as nitro blue tetrazolium), 2,3,5-triphenyl tetrazolium chloride, 3,3'-(3,3 '-(3,3'-) Dimethoxy-4,4'-biphenylene) -bis- [2,5-bis (p-nitrophenyl) tetrazolium chloride], 3,3 '-(4,4'-biphenylene) -bis (2,5-diphenyltetrazolium) Chloride), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride <abbreviation INT>, 3,3 '-[3,3'-dimethoxy- (1 1,1'-biphenyl) -4,4'-diyl] -bis [2- (4-nitrophenyl) -5-phenyl] -2H tetrazolium chloride <abbr. B>, 2-benzothiazoyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H-tetrazolium <abbreviation WST-4>, 2, 2 '-Dibenzothiazoyl-5,5'-bis [4-di (2-sulfoethyl) carbamoylphenyl] -3,3'-(3,3'-dimethoxy-4,4'-diphenylene) ditetrazolium disodium salt <Abbreviated WST-5> and the like can be mentioned.
Among these, WST-4 and WST-5 are high in water solubility, easy to be adjusted as a reagent, and have characteristics of having absorption in a long wavelength range and being less susceptible to absorption of proteins such as hemoglobin.

  Configurations B1 and B2 of Embodiment B may further include a dye stabilizer. Examples of dye stabilizers include sodium azide (WO 2003/029229), pH adjusters (JP 2009-072136) and the like.

  The configurations B1 and B2 of Embodiment B may include an electron transfer agent. Examples of electron transfer agents include diaphorase, N-methylphenazine methosulfates (eg N-methylphenazine methosulfate, 1-methoxy-5-methylphenazine methosulfate (1-methoxy PMS), etc.), meldola blue, Methylene blue etc. are mentioned.

  The measurement of the chromogenic signal and the amount of glycated protein in Embodiment B are the same as in Embodiment A.

An object to be measured in Embodiment B is glycated hemoglobin, and an embodiment Ba for measuring a glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) includes the following (1) to (4). The method of measuring the glycated hemoglobin ratio includes mixing one sample (one reagent-based reagent) and the sample to perform 4).
Embodiment Ba
(1) Denature the hemoglobin in the sample.
(2) To develop a reduced color former by the reaction of glycated hemoglobin in the sample with direct FPDH.
(3) To calculate the amount of glycated protein by measuring the color development signal of the reduced color former that has developed color.
(4) To calculate the amount of hemoglobin by measuring the chromogenic signal of denatured hemoglobin.

The measurement target in the embodiment B is glycated hemoglobin, and the embodiment Bb for measuring the glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) includes the following (0) to (4), and The method of measuring the glycated hemoglobin ratio includes mixing one sample (one reagent-based reagent) and the sample to perform 4).
Embodiment Bb
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) To develop a reduced color former by the reaction of glycated hemoglobin in the sample with direct FPDH.
(3) To calculate the amount of glycated protein by measuring the color development signal of the reduced color former that has developed color.
(4) To calculate the amount of hemoglobin by measuring the chromogenic signal of denatured hemoglobin.

Embodiments Ba and Bb may further include calculating a glycated hemoglobin ratio from the amount of glycated hemoglobin and the amount of hemoglobin.
In the present embodiments Ba and Bb, glycated hemoglobin is hemoglobin A1c in one or more embodiments.

  In one or more embodiments, when the measurement target in embodiment B is a glycated protein other than glycated hemoglobin (for example, glycated albumin etc.) and the glycated protein ratio (ratio of glycated protein mass to protein mass) is measured. The ratio of glycated protein can be determined from the amount of glycated protein obtained by the measurement according to the disclosure and the amount of protein obtained by another method. The glycated hemoglobin ratio may also be determined by this method. Examples of methods for determining the amount of protein include those described above.

[Form of Administration Preparation of Embodiment B]
One reagent-based reagent in the embodiment B may not be one reagent in terms of the stability of the reagent or the like at the distribution stage. The preparation may be made at the stage of performing the measurement method according to the present disclosure. The following configurations B3 and B4 can be mentioned as configurations of two non-limiting reagents for preparing one reagent-based reagent in such an embodiment.
Configuration B3
Reagent 1: Buffer, denaturant, direct type FPDH
Reagent 2: Buffer, reducing color former Composition B4
Reagent 1: Buffer, direct type FPDH
Reagent 2: Buffer, reducing color former

Configurations B3 and B4 of Embodiment B may further include the above-mentioned dye stabilizer and / or electron transfer agent.
As the buffer, the modifier and the reducing color former in the configurations B3 and B4, those described above can be used.

[Embodiment Using Amadoriase and Protease]
In another embodiment, the measurement method according to the present disclosure uses, as an amadoriase, an N-terminal glycated peptide and an amadoriase having substrate specificity for a glycated amino acid (hereinafter, also simply referred to as "glycated peptide"). The amadoriase includes fructosyl peptide oxidase (FPOX) and fructosyl peptide dehydrogenase (FPDH).
As an amadoriase used for the measurement of fructosamine and glycated albumin, an amino acid in which the ε amino group of lysine is glycated and / or an enzyme which acts on a glycated amino acid and / or a glycated peptide which functions well on a peptide in which the ε amino group is glycosylated It can be used. For example, an oxidase derived from Gibberella, Aspergillus, Candida, Penicillium, Fusarium, Acremonium or Debaryomyces, etc. Can be mentioned.

[Embodiment C using FPOX]
The measurement method according to the present disclosure includes the following (1) to (5) in non-limiting embodiment C, and to perform the following (1) to (5), or the following (2) to (5) It is a measuring method of a glycated protein including mixing one reagent (one reagent system reagent) and a sample in order to carry out.
(1) Denature the protein in the sample.
(2) To form a glycated peptide by the reaction of a glycated protein in a sample with a protease.
(3) Generating hydrogen peroxide by the reaction of a glycated peptide with FPOX.
(4) Coloring the oxidized color former by the reaction between the generated hydrogen peroxide and the peroxidase.
(5) To calculate the amount of glycated protein by measuring the color development signal of the oxidized color developing agent that has developed color.

In one or more non-limiting embodiments, one reagent-based reagent in embodiment C includes the following configurations C1 to C3. However, the reagent according to the present disclosure is not limited to these embodiments. One reagent-based reagent may further contain other components.
Configuration C1
1 Reagent-based reagent: Buffer, denaturant, protease, FPOX, POD, oxidative color former Composition C2
1 Reagent-based reagent: Buffer, Protease, FPOX, POD, Oxidative coloring agent Composition C3
1 Reagent system reagent: buffer, protease, FPOX, POD, leuco type dye, compound of formula (I)

Configurations C1 to C3 of Embodiment C may further comprise the dye stabilizers described above.
The buffer, the modifier, the POD, the oxidative color developing agent, the leuco-type dye, the compound of the formula (I), and the sample to be measured in the configurations C1 to C3 are the same as in the embodiment A.

As the protease in embodiment C, one having an activity near neutral and capable of reacting with glycated hemoglobin to produce an N-terminal glycated peptide (including a glycated amino acid in the present disclosure) can be used. The production species and enzyme family are not particularly limited. Non-limiting examples of proteases include serine proteases, threonine proteases, glutamic proteases, asparatic proteases, metalloproteases and the like.
Examples of the protease in Embodiment C include proteases derived from microorganisms such as Bacillus, Aspergillus, Streptomyces and the like. In addition, serine proteases are also preferred.
When the measurement target is glycated albumin, a microorganism-derived protease of Bacillus and Streptomyces is more preferable because it has a greater effect on human albumin, and a Bacillus-derived protease such as subtilisin, Nagase, protease-type- VIII, -IX, -X, -XV, -XXIV, -XXVII, -XXXI (all manufactured by Sigma), thermolysin (Thermolysin), neutrase, esperase, savinase, durazyme, biofeed pro, alcalase, Disc bio-industry) and the like are particularly preferable, and subtilisin, nagase, protease-type-XXVII and the like are more preferable.

  The measurement of the chromogenic signal and the amount of glycated protein in Embodiment C are the same as in Embodiment A.

An object to be measured in Embodiment C is glycated hemoglobin, and as a embodiment Ca for measuring a glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin), the following (1) to (6) are included, The method of measuring the ratio of glycated hemoglobin may be mentioned, which comprises mixing one reagent (one reagent type reagent) and the sample to perform 6).
Embodiment Ca
(1) Denature the hemoglobin in the sample.
(2) Generating a glycated peptide by the reaction of glycated hemoglobin in a sample with a protease.
(3) Generating hydrogen peroxide by the reaction of a glycated peptide with FPOX.
(4) Coloring the oxidized color former by the reaction between the generated hydrogen peroxide and the peroxidase.
(5) To calculate the amount of glycated protein by measuring the color development signal of the oxidized color developing agent that has developed color.
(6) To calculate the amount of hemoglobin by measuring the color development signal of denatured hemoglobin.

An object to be measured in Embodiment C is glycated hemoglobin, and Embodiment Cb for measuring a glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) includes the following (0) to (6), and The method of measuring the ratio of glycated hemoglobin may be mentioned, which comprises mixing one reagent (one reagent type reagent) and the sample to perform 6).
Embodiment Cb
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) Generating a glycated peptide by the reaction of glycated hemoglobin in a sample with a protease.
(3) Generating hydrogen peroxide by the reaction of a glycated peptide with FPOX.
(4) Coloring the oxidized color former by the reaction between the generated hydrogen peroxide and the peroxidase.
(5) To calculate the amount of glycated protein by measuring the color development signal of the oxidized color developing agent that has developed color.
(6) To calculate the amount of hemoglobin by measuring the color development signal of denatured hemoglobin.

Embodiments Ca and Cb may further include calculating the glycated hemoglobin ratio from the amount of glycated hemoglobin and the amount of hemoglobin.
In embodiments Ca and Cb, glycated hemoglobin is hemoglobin A1c in one or more embodiments.

  In one or more embodiments, when the measurement target in embodiment C is a glycated protein other than glycated hemoglobin (for example, glycated albumin etc.) and the glycated protein ratio (ratio of glycated protein mass to protein mass) is measured, The ratio of glycated protein can be determined from the amount of glycated protein obtained by the measurement according to the disclosure and the amount of protein obtained by another method. The glycated hemoglobin ratio may also be determined by this method. Examples of methods for determining the amount of protein include those described above.

[Form of preparation of embodiment C]
One reagent-based reagent in the embodiment C may not be one reagent in terms of the stability of the reagent in the distribution stage, and the like. The preparation may be made at the stage of performing the measurement method according to the present disclosure. The following configurations C4 to C11 can be mentioned as configurations of two non-limiting reagents for preparing one reagent-based reagent in such an embodiment.
Configuration C4
First reagent: buffer, denaturant, FPOX, POD
Second Reagent: Buffer, Protease, Oxidative Coloring Agent Composition C5
First reagent: buffer, denaturant, protease, POD
Second reagent: Buffer, FPOX, oxidative color former Composition C6
First Reagent: Buffer, Denaturant, FPOX, Oxidized Coloring Agent Second Reagent: Buffer, Protease, POD
Configuration C7
First Reagent: Buffer, Denaturant, Protease, Oxidative Coloring Agent Second Reagent: Buffer, FPOX, POD
Configuration C8
First reagent: Buffer, FPOX, POD
Second Reagent: Buffer, Protease, Oxidative Coloring Agent Composition C9
First reagent: buffer, protease, POD
Second reagent: buffer, FPOX, oxidation color former composition C10
First Reagent: Buffer, FPOX, Oxidative Coloring Agent Second Reagent: Buffer, Protease, POD
Configuration C11
First Reagent: Buffer, Protease, Oxidative Coloring Agent Second Reagent: Buffer, FPOX, POD

  Configurations C4 to C11 of Embodiment C may further contain a dye stabilizer, such as a reducing agent or surfactant. Further, the buffer, the modifier, the oxidative color developing agent, and the sample to be measured in configurations C4 to C11 are the same as in the embodiment A. Also, in configurations C4 to C11, the modifier and / or the dye stabilizer may be a compound of formula (I).

[Embodiment D using FPDH]
The measurement method according to the present disclosure includes the following (1) to (4) in non-limiting embodiment D, and to perform the following (1) to (4), or the following (2) to (4) It is a measuring method of a glycated protein including mixing one reagent (one reagent system reagent) and a sample in order to carry out.
(1) Denature the protein in the sample.
(2) To form a glycated peptide by the reaction of a glycated protein in a sample with a protease.
(3) To develop a reduced color former by the reaction of a glycated peptide with FPDH.
(4) To calculate the amount of glycated protein by measuring the color development signal of the reduced color former that has developed color.

In one or more non-limiting embodiments, one reagent-based reagent in embodiment D includes the following configurations D1 and D2. However, the reagent according to the present disclosure is not limited to these embodiments. One reagent-based reagent may further contain other components.
Configuration D1
1 Reagent-based reagent: Buffer, denaturant, protease, FPDH, reduced color former Composition D2
1 Reagent-based reagent: Buffer, FPDH, reduced color former

  Configuration D1 and D2 of Embodiment D may further include the above-described dye stabilizer and / or electron transfer agent.

  The buffer, the denaturant, the reduced color former, and the measurement target sample in configurations D1 and D2 are the same as in Embodiment B, and the protease is the same as in Embodiment C.

  The measurement of the chromogenic signal and the amount of glycated protein in Embodiment D is the same as in Embodiment A.

An object to be measured in embodiment D is glycated hemoglobin, and an embodiment Da for measuring a glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) includes the following (1) to (5). The method of measuring the glycated hemoglobin ratio includes mixing one sample (one reagent-based reagent) with the sample to perform 5).
Embodiment Da
(1) Denature the hemoglobin in the sample.
(2) Generating a glycated peptide by the reaction of glycated hemoglobin in a sample with a protease.
(3) To develop a reduced color former by the reaction of a glycated peptide with FPDH.
(4) To calculate the amount of glycated protein by measuring the color development signal of the reduced color former that has developed color.
(5) To calculate the amount of hemoglobin by measuring the color development signal of denatured hemoglobin.

An object to be measured in embodiment D is glycated hemoglobin, and the embodiment Db for measuring the glycated hemoglobin ratio (ratio of the amount of glycated hemoglobin to the amount of hemoglobin) includes the following (0) to (5), and The method of measuring the glycated hemoglobin ratio includes mixing one sample (one reagent-based reagent) with the sample to perform 5).
Embodiment Db
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) Generating a glycated peptide by the reaction of glycated hemoglobin in a sample with a protease.
(3) To develop a reduced color former by the reaction of a glycated peptide with FPDH.
(4) To calculate the amount of glycated protein by measuring the color development signal of the reduced color former that has developed color.
(5) To calculate the amount of hemoglobin by measuring the color development signal of denatured hemoglobin.

Embodiments Da and Db may further include calculating the glycated hemoglobin ratio from the amount of glycated hemoglobin and the amount of hemoglobin.
In embodiments Da and Db, glycated hemoglobin is hemoglobin A1c in one or more embodiments.

  In one or more embodiments, when the measurement target in Embodiment D is a glycated protein other than glycated hemoglobin (for example, glycated albumin etc.) and the glycated protein ratio (ratio of glycated protein mass to protein mass) is measured, The ratio of glycated protein can be determined from the amount of glycated protein obtained by the measurement according to the disclosure and the amount of protein obtained by another method. The glycated hemoglobin ratio may also be determined by this method. Examples of methods for determining the amount of protein include those described above.

[Form of Administration Preparation of Embodiment D]
One reagent-based reagent in the embodiment D may not be one reagent in the distribution stage, from the viewpoint of the stability of the reagent. The preparation may be made at the stage of performing the measurement method according to the present disclosure. The following configurations D3 to D10 can be mentioned as configurations of two non-limiting reagents for preparing one reagent-based reagent in such an embodiment.
Configuration D3
First reagent: buffer, denaturant, FPDH
Second reagent: buffer, protease, reduced color former Composition D4
First reagent: buffer, denaturant, protease second reagent: buffer, FPDH, reduced color former Composition D5
First reagent: buffer, denaturant, FPDH, reduced color former Second reagent: buffer, protease Composition D6
First reagent: buffer, denaturing agent, protease, reduced color former Second reagent: buffer, FPDH
Configuration D7
First reagent: buffer, FPDH
Second reagent: buffer, protease, reduced color former Composition D8
First reagent: buffer, protease second reagent: buffer, FPDH, reduced color former Composition D9
First Reagent: Buffer, FPDH, Reduced Colorant Second Reagent: Buffer, Protease Constituent D10
First reagent: buffer, protease, reduced color former Second reagent: buffer, FPDH

  Configuration D3 to D10 of Embodiment D may further comprise the above-mentioned dye stabilizer and / or electron transfer agent.

  The buffer, the denaturing agent, the reduced color developing agent, and the measurement target sample in configurations D3 to D10 are the same as in embodiment B, and the protease is the same as in embodiment C.

[Measurement device / measurement system]
In another aspect, the present disclosure relates to a glycated protein measurement device or measurement system that implements the measurement method according to the present disclosure.
In one or more embodiments (FIG. 1), the measuring device or system according to the present disclosure supplies a sampling unit 1 that supplies a sample to a reaction container (photometric cell), and supplies one reagent-based reagent to the reaction container (photometric cell) Reagent supply unit 2, photometry unit 3 for measuring a color forming signal such as absorbance of a photometry cell, control unit 4 for controlling sampling unit 1, reagent supply unit 2 and photometry unit 3, recording unit 5 for recording photometric data etc. The calculation unit 6 that calculates the amount of glycated protein and the like, and the data output unit 7 are provided. These units may be included in one device or may be included in separate devices to configure the system.

As a method of measuring the amount of glycated protein, there is an HPLC method in addition to the measurement method according to the present disclosure. The HPLC method has become faster in recent years, and measurement with one sample (only) can be performed in about 1 minute, and multiple samples can be measured at a rate of about 5.5 minutes for every 10 samples (about 30 seconds with one sample) became. On the other hand, in the case of the conventional two-reagent system, the enzyme method required 8 to 10 minutes of measurement time for one sample. However, since the reaction time can be advanced in parallel and photometry (measurement) at sampling intervals is possible, the measurement time per sample can be made closer to the sampling interval time as the number of samples increases, and it is faster than HPLC. Processing becomes possible.
With the measurement method according to the present disclosure, in one or more embodiments, measurement can be performed in 10 seconds to 180 seconds from the mixing of the sample and the one reagent-based reagent, and the speed can be further increased than in the two reagent-based system.
Thus, in one or more embodiments, the devices of the present disclosure are suitable for large automated analyzers that process a large number of samples at one time.
Alternatively, in one or more embodiments, the measurement method according to the present disclosure can reduce the time required for the conventional enzyme method by using one reagent-based reagent, so a compact automatic analyzer with a small number of measurement target samples Is also suitable.

One or more non-limiting embodiments of an apparatus or system according to the present disclosure will be described.
The sample is supplied to the sampling unit 1 by setting the blood collection tube used for blood collection as it is in the sampling unit 1 or transferring the blood etc. to a sample cup or setting a sampling tool to the blood collection tube. Be done. When the blood collection tube is set as it is, it is also possible to use a piercing nozzle which can pierce the cap.
In the sampling unit 1, the sample is moved from the supplied sample to the reaction container (photometric cell). The sampling amount is, for example, 0.1 to 10 μL. In the case of direct transfer to the photometric cell, 0.1-2 μL is preferable from the viewpoint of dilution rate. On the other hand, the sample may be moved from the sample to the photometric cell through the dilution tank, and the sampling amount in that case is, for example, 0.5 to 10 μL.
When hemolyzing erythrocytes in a sample, when using a dilution tank, the sample is mixed with purified water and / or a solution containing a surfactant or the like (hereinafter, these are also referred to as “hemolytic solution”) in the dilution tank Hemolysis can be done by After hemolysis, it moves to a photometric cell as a sample to be measured. Thus, an apparatus or system according to the present disclosure may include a hemolytic solution supply that supplies a hemolytic solution.
On the other hand, the sample may be moved directly to the photometric cell. In this case, a hemolytic solution may be supplied to the photometric cell for hemolysis, or hemolysis may be performed by supplying one reagent system reagent.
In any case, for hemolysis, physical stirring with a stirrer or ultrasonic stirring can be used.

  One reagent-based reagent is supplied from the reagent supply unit 2 to the photometric cell. In the reagent supply unit 2, a solution of one reagent type reagent supplied as one solution may be set, and it is prepared by mixing the reagents supplied as the second solution (as a preparation form) by a necessary specimen. One reagent type reagent may be set. For example, one day or several days may be produced, and for example, several samples to several thousand samples may be produced.

  Either of the supply of the sample and the one reagent-based reagent to the photometric cell may be first or simultaneous. In the case of a whole blood sample, the mixing ratio of the sample and the reagent is, for example, a ratio of 1:30 to 2,000, preferably 1: 100 to 1000, and more preferably 1: 150 to 500. In the case of a blood cell sample, the mixing ratio of the sample and the reagent is, for example, a ratio of 1:50 to 4000, preferably 1: 100 to 2000, and more preferably 1: 250 to 1000.

  In the present specification, one reagent-based reagent may be prepared. In one or more embodiments, one reagent-based reagent may be prepared within 5 minutes prior to mixing with the sample. This preparation, in one or more embodiments, refers to the final steps required to make contact with all the reagent components. In one or more non-limiting embodiments, a first reagent comprising an enzyme may be combined with a second reagent comprising a color forming agent to form a one-reagent based reagent. After mixing the sample and the one reagent-based reagent, the time to measurement can be set, for example, between 10 seconds and 180 seconds. The time after mixing and before measurement is, for example, 30 seconds or more, 45 seconds or more, or 60 seconds or more. The time after mixing and before measurement is, for example, 30 seconds or less, 45 seconds or less, 60 seconds or less, 90 seconds or less, 120 seconds or less, or 180 seconds or less.

The measurement is performed by measuring a color forming signal such as the absorbance of the photometric cell by the photometry unit 3.
The measurement of the glycated protein is carried out immediately after the addition of the measurement reagent, and the second measurement is made at the end of the measurement, and the difference between the second measurement and the first photometry is taken as the measurement value.
Hemoglobin is measured at a wavelength at which Hb is absorbed, but a wavelength which is not affected by the color reaction of glycated hemoglobin is preferable. For example, when the color former is DA-67, for example, 550 nm or less, preferably 500 nm or less, more preferably Preferably, it measures at about 450-480 nm.
The measuring cell or the hemolyzing cell may be in the form of being washed and used many times but may be in the form of being disposable at one time.

  The control unit 4 controls, for example, the sampling unit 1, the reagent supply unit 2, the photometry unit 3, and selectively the hemolytic solution supply unit to operate as described above.

The data measured by the photometry unit is recorded by the recording unit 5. Further, based on the data, the amount of glycated protein, the amount of protein, and / or the glycated protein ratio is calculated by the calculation unit 6 and recorded in the recording unit 5. The recorded data is output from the data output unit.
The calculation unit 6 calculates the amount of glycated protein by converting the measurement value (for example, the amount of change in absorbance) obtained by the photometry unit into the amount of glycated protein based on the calibration curve for the measurement value.

The present disclosure may relate to one or more of the following non-limiting embodiments.
[1] A method for measuring glycated protein in a sample, comprising the following (1) to (3),
A measurement method in which one reagent is mixed with the sample to perform the following (2) to (3).
(1) Denature the protein in the sample.
(2) To develop a color former by the reaction of glycated protein in the sample with amadoriase. (3) To calculate the amount of glycated protein by measuring the absorbance of the sample after (1) and (2). [2] The measurement method according to [1], wherein the glycated protein is glycated hemoglobin or glycated albumin, and the protein is hemoglobin or albumin.
[3] A method for measuring glycated hemoglobin in a sample, which comprises the following (1) to (4), wherein one reagent is mixed with the sample to perform the following (1) to (4): Method.
(1) Denature the hemoglobin in the sample.
(2) To develop a color former by the reaction of glycated hemoglobin in the sample with amadoriase.
(3) Calculate the amount of glycated hemoglobin by measuring the absorbance of the sample after (1) and (2).
(4) Calculate the amount of hemoglobin by measuring the absorbance of the sample after (1) and (2).
[4] A method for measuring glycated hemoglobin in a sample, comprising the following (0) to (4), wherein one reagent is mixed with the sample to perform the following (2) to (4): Method.
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) To develop a color former by the reaction of glycated hemoglobin in the sample with amadoriase.
(3) Calculate the amount of glycated hemoglobin by measuring the absorbance of the sample after (1) and (2).
(4) Calculate the amount of hemoglobin by measuring the absorbance of the sample after (1) and (2).
[5] The method according to any one of [2] to [4], wherein the glycated hemoglobin is hemoglobin A1c.
[6] The measurement method according to any one of [1] to [5], wherein the amadoriase is a direct glycated protein amadoriase.
[7] The measuring method according to any one of [1] to [6], wherein the reagent to be added contains a denaturing agent, glycated protein direct type fructosyl peptide oxidase, peroxidase, and an oxidative coloring agent.
[8] The measurement method according to any one of [1] to [6], wherein the reagent to be added contains a denaturing agent, glycated protein direct type fructosyl peptide dehydrogenase, and a reducing color former.
[9] The above (2) is the formation of an N-terminal glycated peptide by the reaction of glycated hemoglobin in the sample and the protease, and the coloration of the color former by the reaction of the N-terminal glycated peptide with the amadoriase. The measuring method in any one of [2] to [8].
[10] The measuring method according to any one of [1] to [5] and [9], wherein the reagent to be added contains a denaturing agent, a protease, fructosyl peptide oxidase, a peroxidase, and a color former.
[11] The measuring method according to any one of [1] to [5] and [9], wherein the reagent to be added contains a denaturing agent, a protease, fructosyl peptide dehydrogenase, and a color former.
[12] A measuring device or measuring system for glycated hemoglobin, which executes the measuring method according to any one of [1] to [11].

  Hereinafter, the present disclosure will be described in more detail by way of examples, but these are illustrative and the present disclosure is not limited to these examples.

Example 1
1 Reagent system reagent glycated protein direct type fructosyl peptide oxidase 3000 U / L
Myristyl dimethylamino propane sulfonic acid (Tokyo Chemical Industry) 3 g / L
POD 10KU / L
DA-67 (Wako Pure Chemical Industries) 0.1 mmol / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
Measuring device BM-6010 (manufactured by Nippon Denshi)
Operation 2 μL of human whole blood and 148 μL of one reagent-based reagent are mixed, and incubated at 37 ° C. for 2 minutes.
Absorbance measurement: Measured after about 20 seconds from mixing of reagent-based reagents and about 2 minutes after mixing of reagents based on absorbance at about 654 nm and 694 nm, and after about 20 seconds from about 2 minutes The amount of change in absorbance is determined by subtracting the absorbance of.
Hemoglobin measurement: The absorbance after about 2 minutes from the mixing of the reagent based reagents is measured at a main wavelength of 478 nm and a sub wavelength of 694 nm.
Calculation of the amount of glycated hemoglobin The amount of glycated hemoglobin is calculated by converting it into the amount of glycated hemoglobin based on the calibration curve for the amount of change in absorbance.

Example 2
1 Reagents First reagent for preparation of reagents (enzyme reagent)
Glycated protein direct type fructosyl peptide oxidase 400 U / L
POD 20KU / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
1 Reagents Second reagent for preparation of reagents (chromogen reagent)
Myristyl dimethylamino propane sulfonic acid (Tokyo Chemical Industry) 30 g / L
DA-67 (Wako Pure Chemical Industries) 1.0 mmol / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
Measuring device BM-6010 (manufactured by Nippon Denshi)
Operation 9 mL of the first reagent and 1 mL of the second reagent are mixed to prepare 1 reagent system reagent.
2 μL of human whole blood and 148 μL of one reagent-based reagent are mixed, and incubated at 37 ° C. for 2 minutes.
Absorbance measurement: Measured after about 20 seconds from mixing of reagent-based reagents and about 2 minutes after mixing of reagents based on absorbance at about 654 nm and 694 nm, and after about 20 seconds from about 2 minutes The amount of change in absorbance is determined by subtracting the absorbance of.
Hemoglobin measurement: The absorbance after about 2 minutes from the mixing of the reagent based reagents is measured at a main wavelength of 478 nm and a sub wavelength of 694 nm.
Calculation of the amount of glycated hemoglobin The amount of glycated hemoglobin is calculated by converting it into the amount of glycated hemoglobin based on the calibration curve for the amount of change in absorbance.

[Example 3]
1 Reagents First reagent for preparation of reagents (enzyme reagent)
Fructosyl peptide dehydrogenase 500 U / L
1-methoxy PMS (Dojin Chemical Research Institute) 1 mmol / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
1 Reagents Second reagent for preparation of reagents (chromogen reagent)
Metalloprotease 20000 KU / L
Myristyl dimethylamino propane sulfonic acid (Tokyo Chemical Industry) 30 g / L
WST-4 (Dojin Chemical Research Institute) 1 mmol / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
Measuring device BM-6010 (manufactured by Nippon Denshi)
Operation 9 mL of the first reagent and 1 mL of the second reagent are mixed to prepare 1 reagent system reagent.
2 μL of human whole blood and 148 μL of one reagent-based reagent are mixed, and incubated at 37 ° C. for 2 minutes.
Absorbance measurement: Measurement of glycated hemoglobin: The absorbance at about 20 seconds after mixing of the reagent based reagents and at about 2 minutes is measured at a main wavelength of 694 nm and a sub wavelength of 805 nm, and after about 20 seconds from the absorbance after about 2 minutes The amount of change in absorbance is determined by subtracting the absorbance of.
Hemoglobin measurement: The absorbance after about 2 minutes from the mixing of the reagent based reagents is measured at a main wavelength of 478 nm and a sub wavelength of 694 nm.
Calculation of the amount of glycated hemoglobin The amount of glycated hemoglobin is calculated by converting it into the amount of glycated hemoglobin based on the calibration curve for the amount of change in absorbance.

Example 4
1 Reagents First reagent for preparation of reagents (enzyme reagent)
Fructosyl peptide oxidase 500 U / L
POD 20KU / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
1 Reagents Second reagent for preparation of reagents (chromogen reagent)
Metalloprotease 30000KU / L
Myristyl dimethylamino propane sulfonic acid (Tokyo Chemical Industry) 30 g / L
DA-67 (Wako Pure Chemical Industries) 1.0 mmol / L
MOPS (Dojin Chemical Research Institute) 50 mmol / L
NaOH pH 6.5
Measuring device BM-6010 (manufactured by Nippon Denshi)
Operation 9 mL of the first reagent and 1 mL of the second reagent were mixed to prepare one reagent system reagent.
2 μL of human whole blood and 148 μL of one reagent-based reagent were mixed and incubated at 37 ° C.
Absorbance measurement: Measurement of glycated hemoglobin: About 1 minute after about 20 seconds, about 1 minute, about 2 minutes and about 5 minutes of the mixing of the reagent based reagents, the absorbance is measured at the main wavelength of 658 nm and the sub wavelength of 694 nm. After that, each absorbance change was determined by subtracting the absorbance after about 20 seconds from each absorbance after about 2 minutes and about 5 minutes.
Hemoglobin measurement: The absorbance after about 2 minutes from the mixing of 1 reagent-based reagent was measured at a main wavelength of 478 nm and a sub wavelength of 694 nm.
Calculation of the amount of glycated hemoglobin The amount of glycated hemoglobin was calculated by converting it into the amount of glycated hemoglobin based on the calibration curve for the amount of change in absorbance.
Results The same measurement results were obtained after about 1 minute, about 2 minutes and about 5 minutes.

Claims (14)

A method for measuring glycated protein in a sample, comprising the following (1) to (3),
A measuring method in which one reagent is mixed with the sample to perform the following (2) and (3).
(1) Denature the protein in the sample.
(2) To develop a color former by the reaction of glycated protein in the sample with amadoriase. (3) To calculate the amount of glycated protein by measuring the chromogenic signal of the sample after (1) and (2).   The measurement method according to claim 1, wherein the calculation of the amount of glycated protein includes converting the measured chromogenic signal into the amount of glycated protein based on a predetermined conversion factor.   The method according to claim 1 or 2, wherein the glycated protein is glycated hemoglobin or glycated albumin, and the protein is hemoglobin or albumin. A method for measuring glycated hemoglobin in a sample, comprising the following (1) to (4),
A measuring method in which one reagent is mixed with the sample to perform the following (1) to (4).
(1) Denature the hemoglobin in the sample.
(2) To develop a color former by the reaction of glycated hemoglobin in the sample with amadoriase.
(3) To calculate the amount of glycated hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).
(4) To calculate the amount of hemoglobin by measuring the chromogenic signal of the sample after (1) and (2). A method of measuring glycated hemoglobin in a sample, comprising the following (0) to (4),
A measuring method in which one reagent is mixed with the sample to perform the following (2) to (4).
(0) Hemolysis of erythrocytes in a sample and releasing hemoglobin from the erythrocytes.
(1) Denature the hemoglobin in the sample.
(2) To develop a color former by the reaction of glycated hemoglobin in the sample with amadoriase.
(3) To calculate the amount of glycated hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).
(4) To calculate the amount of hemoglobin by measuring the chromogenic signal of the sample after (1) and (2).   The method according to claim 4 or 5, wherein the calculation of the amount of glycated protein includes converting the measured chromogenic signal into the amount of glycated protein based on a predetermined conversion factor.   The measurement method according to any one of claims 2 to 6, wherein the glycated hemoglobin is hemoglobin A1c.   The measurement method according to any one of claims 1 to 7, wherein the amadoriase is a glycated protein direct type amadoriase.   The measuring method according to any one of claims 1 to 8, wherein the reagent to be added contains a denaturing agent, glycated protein direct type fructosyl peptide oxidase, peroxidase, and an oxidative coloring agent.   The measuring method according to any one of claims 1 to 8, wherein the reagent to be added contains a denaturing agent, glycated protein direct type fructosyl peptide dehydrogenase, and a reducing color former.   The method according to claim 2, wherein (2) is generating an N-terminal glycated peptide by reacting glycated hemoglobin in a sample with a protease, and causing a color former to react with the reaction of the N-terminal glycated peptide with amadoriase. The measuring method in any one of to 10.   The measuring method according to any one of claims 1 to 7 and 11, wherein the reagent to be added contains a denaturing agent, a protease, fructosyl peptide oxidase, a peroxidase, and a coloring agent.   The measuring method according to any one of claims 1 to 7 and 11, wherein the reagent to be added contains a denaturing agent, a protease, fructosyl peptide dehydrogenase, and a color former.   The measuring apparatus or measuring system of glycated hemoglobin which implements the measuring method in any one of Claims 1-13.

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