JP7195847B2 - Measurement of glycated protein - Google Patents

Description

TECHNICAL FIELD The present disclosure relates to measurement of glycated proteins, and more specifically to measurement of glycated proteins, including stabilization of leuco-type dyes.

The concentration of glycated proteins, which are reaction products of blood glucose and various proteins, is an indicator of blood sugar levels over the medium and long term. In particular, hemoglobin A1c (HbA1c), glycoalbumin, fructosamine, and the like are substances frequently measured in clinical examinations, and are widely used for diabetes diagnosis and treatment monitoring. HbA1c %, which is the ratio of HbA1c, which is hemoglobin with a β-chain N-terminal glycated, to total hemoglobin, is used for monitoring blood sugar level and is an important test item related to diagnosis of diabetes.

HbA1c% is measured by high performance liquid chromatography (HPLC) method, boronic acid affinity method, immunoturbidimetric method, enzymatic method and the like. Among them, the enzymatic reagent is attracting social attention as a methodology capable of measuring HbA1c% at the lowest cost.

An example of measuring HbA1c% using an enzymatic reagent is a method including the following steps (1) to (4).
(1) a step of denaturing hemoglobin and quantifying it by an absorbance method;
(2) hydrolyzing the vicinity of the glycated N-terminus of HbA1c with a protease;
(3) oxidizing the hydrolyzed oligopeptide having a saccharified N-terminus with an enzyme to produce hydrogen peroxide;
(4) A step of quantifying HbA1c by a spectrophotometric method by developing a leuco-type dye as a color-developing substrate by reacting hydrogen peroxide and peroxidase.

Leuco-type dyes DA-64 and DA-67, which are highly sensitive color formers, are used for the measurement of trace components such as HbA1c. While these color formers have excellent sensitivity, they also have the disadvantage of being colored upon exposure to oxygen, light, water, and the like. In particular, such deterioration is significant when the measurement reagent is a solution reagent rather than a dry reagent. In order to solve this problem, Patent Literatures 1 to 3 disclose techniques for stabilizing the coloring agent by adding a reducing agent to the coloring agent solution for the purpose of preventing the oxidation of the coloring agent. Further, Patent Document 4 discloses a technique for stabilizing a coloring agent by adding a surfactant.

Japanese Patent No. 5274590 Japanese Patent No. 3604198 JP 2013-104007 A Japanese Patent No. 5616498

In one aspect, the present disclosure provides a reagent suitable for enzymatic measurement of glycated proteins and containing a compound capable of stabilizing a leuco dye, and a method for measuring glycated proteins using the same.

In one aspect, the present disclosure relates to a reagent used for measuring a glycated protein by an enzymatic method, the reagent containing a leuco dye and further containing a compound of the following formula (I) (hereinafter referred to as "reagent according to the present disclosure (Also referred to as “reagent”).

式（Ｉ）において、Ｒは、炭素数８－１７の炭化水素鎖を表す。

In formula (I), R represents a hydrocarbon chain of 8-17 carbon atoms.

In another aspect, the present disclosure relates to a kit for measuring glycated protein by an enzymatic method, comprising a reagent containing peroxidase and a reagent according to the present disclosure as a reagent separate from the reagent.

In another aspect, the present disclosure relates to a method for measuring HbA1c including (1) to (5) below, which method includes mixing a sample with a reagent according to the present disclosure.
(1) Denaturing the hemoglobin in the sample.
(2) generating an N-terminal glycated peptide by reacting glycated hemoglobin in the sample with protease;
(3) to generate hydrogen peroxide by the reaction of N-terminal glycosylated peptide and fructosyl peptide oxidase;
(4) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(5) Calculating the amount of HbA1c and the amount of hemoglobin by measuring the coloring signal.

In another aspect, the present disclosure relates to a method for measuring hemoglobin A1c including (1) to (4) below, which method includes mixing a sample with a reagent according to the present disclosure.
(1) Denaturing the hemoglobin in the sample.
(2) Producing hydrogen peroxide through the reaction between glycated hemoglobin in the sample and glycated protein direct fructosyl peptide oxidase.
(3) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(4) Calculating the amount of HbA1c and the amount of hemoglobin by measuring the coloring signal.

According to the present disclosure, since the stability of the leuco-type dye is improved, it is possible to suppress a decrease in accuracy in measurement of glycated protein, for example, measurement of HbA1c.

FIG. 1 is a graph showing the evaluation of the effect of a test compound on suppressing spontaneous coloring of a leuco dye due to heat treatment. FIG. 2 is a graph evaluating the concentration dependence of the effect of heat treatment of a test compound to suppress the spontaneous coloring of a leuco dye. FIG. 3 is a graph evaluating the hemoglobin-modifying ability of test compounds. FIG. 4 is a graph evaluating the ability of test compounds to promote protease reactions.

The present disclosure is based on the finding that the stability of leuco-type dyes used for enzymatic measurement of glycated proteins is improved in the presence of sulfobetaine having an alkyl group with a predetermined number of carbon atoms. According to the present disclosure, the stability of leuco-type dyes in reagent solutions can be improved.

In the present disclosure, measurement of glycated protein includes measuring the amount of glycated protein in one embodiment, includes measuring the amount of glycated protein and the amount of protein in another embodiment, and further includes measuring the amount of glycated protein and the amount of protein. Embodiments may include measuring the amount of glycated protein and the amount of protein to determine their ratio.

The glycated protein to be measured in the measurement method according to the present disclosure includes fructosamine, glycated albumin, glycated hemoglobin, etc. In one embodiment, it is glycated albumin or glycated hemoglobin.
Glycated hemoglobin includes the following.
HbA1c: glycated Hb β-chain N-terminus GHbLys: glycated Hb Lys amino group GHbα: glycated Hb α-chain N-terminus

[Compound capable of stabilizing leuco-type dye]
A reagent according to the present disclosure contains a leuco-type dye and a compound of formula (I) below. A compound of the following formula (I) can stabilize a leuco dye. Accordingly, in one or more embodiments, the reagents of the present disclosure contain a compound of formula (I) below in an amount effective to stabilize the leuco-type dye.

式（Ｉ）において、Ｒは、炭素数８－１７の炭化水素基を表す。

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

In formula (I), the number of carbon atoms in R is preferably 12 to 16, more preferably 14, from the viewpoint of not inhibiting enzymatic measurement of glycated proteins and improving the stability of the leuco dye. From the same viewpoint, the hydrocarbon group for R is preferably an alkyl group, preferably a linear alkyl group. From the same viewpoint, R is preferably a dodecyl group or a tetradecyl group, more preferably a tetradecyl group.

In one or more embodiments, the content of the compound of formula (I) in the reagent according to the present disclosure is 0.1 g, from the viewpoint of not inhibiting the enzymatic measurement of glycated proteins and from the viewpoint of improving the stability of the leuco dye. /L or more is preferred, 1 g/L or more is more preferred, 5 g/L or more is even more preferred, 10 g/L or more is even more preferred, and 20 g/L or more is even more preferred. Also, the content of the compound of formula (I) is, for example, 50 g/L or less, or 40 g/L or less.

[Leuco dye]
As the leuco-type dye in the present disclosure, a highly sensitive one that can be used for minute amount detection is preferred. In one or more embodiments, phenothiazine-based dyes, triphenylmethane-based dyes, diphenylamine-based dyes, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, tetramethylbenzidine, and the like are included, and are used to improve the stability of leuco-type dyes. From this point of view, phenothiazine dyes are preferred.
Phenothiazine dyes include 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine or salts thereof, and 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) Phenothiazine sodium (DA-67) can be mentioned.
Triphenylmethane-based dyes include N,N,N',N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triaminotriphenylmethane or Salts thereof include N,N,N′,N′,N″,N″-hexa(3-sulfopropyl)-4,4′,4″-triaminotriphenylmethane hexasodium ( TMPPS).
Diphenylamine dyes include N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine (DA-64).

In one or more embodiments, the content of the leuco dye in the reagent according to the present disclosure is preferably 0.005 mmol/L or more, more preferably 0.01 mmol/L or more, from the viewpoint of ensuring sufficient measurement sensitivity. , more preferably 0.03 mmol/L or more. In addition, the content of the leuco dye is preferably 2 mmol/L or less, more preferably 0.5 mmol/L or less, from the viewpoint of improving the stability of the leuco dye.

The content ratio of the compound of formula (I) to the leuco dye in the reagent according to the present disclosure (molar ratio, substance amount of compound of formula (I)/substance amount of leuco dye) obtains a sufficient stabilizing effect. From a viewpoint, in one or more embodiments, 5 or more is preferable, 50 or more is more preferable, and 1000 or more is even more preferable. Moreover, the content ratio of the leuco dye is preferably 10,000 or less, more preferably 5,000 or less, from the viewpoint of ensuring sufficient measurement sensitivity.

Reagents according to the present disclosure may be in the form of solution reagents or in the form of dried reagents. In the case of solution reagents, the reagent pH according to the present disclosure is preferably 5 or higher, more preferably 6 or higher, in one or more embodiments. In one or more embodiments, the reagent pH according to the present disclosure is preferably 9 or less, more preferably 8 or less. In addition, pH is the value measured with the pH meter at the temperature of 25 degreeC.

According to the reagent according to the present disclosure, in one or more embodiments, the storage period of the reagent can be extended. Also, in one or more other embodiments, the number of calibrations can be reduced by reducing the change in sensitivity. Furthermore, in one or more embodiments, reagents can be stored at elevated temperatures (eg, 2-50° C.).

In one or more embodiments, the amount of glycated protein such as HbA1c or the amount of protein such as hemoglobin is calculated from the coloring signal of the leuco dye in the measurement of glycated protein using the reagent according to the present disclosure by a known method. can be done.

[Embodiment A]
In a non-limiting embodiment A, the reagent according to the present disclosure can be used as a reagent to be mixed with a sample in a method for measuring HbA1c including the following (1) to (5).
(1) Denaturing the hemoglobin in the sample.
(2) generating an N-terminal glycated peptide by reacting glycated hemoglobin in the sample with protease;
(3) to generate hydrogen peroxide by the reaction of N-terminal glycosylated peptide and fructosyl peptide oxidase (FPOX);
(4) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase (POD).
(5) Calculating the amount of HbA1c and the amount of hemoglobin by measuring the coloring signal.

In the present disclosure, "mixing the reagent with the sample" includes adding the reagent to the sample and adding the sample to the reagent.
In one or more embodiments of Embodiment A, the reagent according to the present disclosure is mixed with the sample to supply at least the leuco dye of (4) above. That is, the leuco-type dye used in the method for measuring HbA1c including (1) to (5) above is contained in the reagent according to the present disclosure. In one or more other embodiments of Embodiment A, the reagent according to the present disclosure is mixed with the sample at least in order to cause the leuco dye to develop color in (4) above.

When embodiment A is performed in a two-reagent system, generally two reagents, a first reagent and a second reagent, are added to the sample in that order, or the sample and these reagents are mixed. . In one or more non-limiting embodiments, the denaturation of hemoglobin in (1) above is performed by adding or mixing the first reagent, and hemoglobin is denatured after the addition or mixing of the first reagent and before the addition or mixing of the second reagent. Absorbance for quantity determination is measured. Depending on the composition of the first reagent, the above (1) and (2) are performed by adding the first reagent, and (1) to (4) are performed by adding or mixing with the second reagent. ) everything goes on. Therefore, the absorbance for measuring the HbA1c amount is measured immediately before or after the addition or mixing of the second reagent and after a certain period of time has passed since the addition or mixing of the second reagent. The HbA1c amount is calculated based on the absorbance change value.

In one or more non-limiting embodiments, reagents according to the present disclosure can be used as second reagents in configurations A1 and A2 below and as first reagents in configurations A3 and A4 below. However, reagents according to the present disclosure are not limited to these embodiments. The first and second reagents may further contain other components (eg, additional buffers, denaturants, leuco-type dye stabilizers, etc.).
Configuration A1
1st reagent: FPOX, POD, denaturant, buffer 2nd reagent: protease, compound of formula (I), leuco-type dye, buffer Composition A2
First reagent: protease, POD, denaturant, buffer Second reagent: FPOX, compound of formula (I), leuco-type dye, buffer Composition A3
First reagent: FPOX, compound of formula (I), leuco-type dye, buffer Second reagent: protease, POD, buffer Configuration A4
First reagent: protease, compound of formula (I), leuco-type dye, buffer Second reagent: FPOX, POD, buffer

Accordingly, reagents according to the present disclosure may further comprise at least one of buffer, protease, and fructosyl peptide oxidase (FPOX).

As the buffering agent in the embodiment A, one that can be adjusted to near neutrality and that does not impair the reaction system can be used. Non-limiting examples of buffering agents 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-piperazinyl]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, glutamic acid, glutamine, valine, cysteine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, tryptophan, trishydroxymethylaminomethane, dimethylaminoethanol, triethanolamine, diethanolamine, mono ethanolamine, N-methylaminoethanol, creatinine, imidazole, barbital, ammonia, ethylamine, diethylamine, triethylamine and the like.

A protease in Embodiment A can be one that has near-neutral activity and can react with glycated hemoglobin to produce an N-terminal glycated peptide (including glycated amino acids in the present disclosure). The producing 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. Exoproteases are preferred over endoproteases.

The fructosyl peptide oxidase (FPOX) in Embodiment A can use an N-terminal glycosylated peptide (including glycosylated amino acids) as a substrate and produce hydrogen peroxide as a detectable intermediate. There are no particular restrictions on the producing species. Amadoriase, termed fructosyl amino acid oxidase, can also be used and included in FPOX in this disclosure.

Peroxidase (POD) in Embodiment A can be one that reacts with hydrogen peroxide to develop a leuco-type dye, which is a chromogenic substrate. There are no particular restrictions on the producing species. Non-limiting examples include horseradish peroxidase.

As the denaturing agent in Embodiment A, one that can denature hemoglobin and does not significantly impair the activity of the enzyme contained in the first reagent can be used. Non-limiting examples of modifiers include (1) to (8) below. Incidentally, (1) to (7) may be used in combination with nitrite.
(1) 3-lauryldimethylaminobutyric acid (2) 3-myristyldimethylaminobutyric acid (3) lauryldimethylaminopropanesulfonic acid (4) myristyldimethylaminopropanesulfonic acid (5) laurylamidopropyldimethylaminobutyric acid (6) myristamide Propyl betaine (7) n-dodecyl-βD-maltoside (8) WST-3
Alternatively, the modifier in Embodiment A may be a compound of formula (I) instead of (1) to (8) above. The compounds of formula (I) are capable of denaturing hemoglobin and promoting protease reactions.

A sample to be measured in Embodiment A includes a sample containing hemoglobin and glycated hemoglobin. In one or a plurality of non-limiting embodiments, the sample to be measured includes 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 erythrocytes, it may be hemolyzed with the first reagent, or may be hemolyzed by means other than the first reagent to obtain a hemolyzed sample. 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 using freezing, a method using ultrasonic waves, and the like.

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

Calculation of the HbA1c amount includes, in one or more embodiments, converting the chromogenic signal obtained by the measurement into the HbA1c amount based on a predetermined conversion factor. Conversion of the amount of HbA1c based on a predetermined conversion factor is performed in one or more embodiments by converting color signals such as absorbance obtained by measurement into HbA1c based on any of the following conversion rules (i) to (iv): It can be done by converting to quantity. (iv) may be performed in combination with (i) to (iii).
(i) obtaining a calibration curve based on known calibration substances in the sample, and converting the absorbance derived from HbA1c into the amount of HbA1c based on the calibration curve; (iii) taking absorbance ratios determined at different wavelengths, and calculating a calibration curve for the absorbance ratio; Converting to HbA1c amount (iv) Obtaining the amount of change in absorbance by taking the difference in absorbance measured immediately after mixing the sample and the reagent and after a predetermined time has elapsed after mixing, and converting to the amount of HbA1c based on the calibration curve for the amount of absorbance change. The known calibrator in (i) includes, in one or more embodiments, a calibration standard containing a known amount of HbA1c. As a calibration standard containing a known amount of HbA1c, in one or more embodiments, whole blood or frozen blood cells, Hb solutions containing HbA1c purified from them, or buffer compositions and Hb stabilization Substances including agents and the like are included. Known calibrators, in one or more embodiments, include primary or working standards provided by public institutions, calibrators provided with kits, and the like.
In one or more embodiments, the time immediately after mixing in (iv) is about 5 to 30 seconds after the sample and reagent are mixed. In one or a plurality of embodiments, the elapse of a predetermined time is about 1 to 3 minutes after the sample and the reagent are mixed.
In the above embodiment, the coloring signal is an example of absorbance, and reflectance and transmittance other than absorbance can be used in the same manner.

[Embodiment B]
In a non-limiting embodiment B, the reagent according to the present disclosure can be used as a reagent mixed with a sample in a method for measuring HbA1c including the following (1) to (4).
(1) Denaturing the hemoglobin in the sample.
(2) Producing hydrogen peroxide through the reaction between glycated hemoglobin in the sample and glycated protein direct fructosyl peptide oxidase (direct FPOX).
(3) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(4) Calculating the amount of HbA1c and the amount of hemoglobin by measuring the coloring signal.

In one or more embodiments of Embodiment B, the reagent according to the present disclosure is mixed with the sample to supply at least the leuco dye of (3) above. That is, the leuco-type dye used in the method for measuring HbA1c including (1) to (4) above is contained in the reagent according to the present disclosure. In one or more other embodiments of Embodiment B, the reagent according to the present disclosure is mixed with the sample at least in order to cause the leuco dye to develop color in (3) above.

Embodiment B is a form in which the fructosyl peptide oxidase (FPOX) in Embodiment A is changed to glycated protein direct fructosyl peptide oxidase (direct FPOX), and protease is no longer required in the reaction system.

In one or more non-limiting embodiments, reagents according to the present disclosure can be used as second reagents in configurations B1 and B2 below, and as first reagents in configurations B3 and B4 below. However, reagents according to the present disclosure are not limited to these embodiments. The first reagent and the second reagent may further contain other components.
Configuration B1
First reagent: POD, denaturant, buffer Second reagent: Direct FPOX, compound of formula (I), leuco dye, buffer Composition B2
First reagent: direct FPOX, denaturant, buffer Second reagent: POD, compound of formula (I), leuco dye, buffer Configuration B3
First reagent: POD, compound of formula (I), leuco dye, denaturant, buffer Second reagent: direct FPOX, buffer Configuration B4
First reagent: direct FPOX, compound of formula (I), leuco dye, denaturant, buffer Second reagent: POD, buffer

In the case of configuration B1, in one or a plurality of non-limiting embodiments, the denaturation of hemoglobin in (1) above is performed by adding the first reagent, and the amount of hemoglobin is changed after addition of the first reagent and before addition of the second reagent. Absorbance for measurement is measured. (2) and (3) proceed as the second reagent is added. Therefore, the absorbance for measuring the HbA1c amount is measured immediately before or after the addition of the second reagent and after a certain period of time from the addition of the second reagent. The HbA1c amount is calculated based on the absorbance change value.

Accordingly, reagents according to the present disclosure may further include glycated protein direct fructosyl peptide oxidase (direct FPOX).

The buffer, protease, peroxidase (POD), denaturant, sample to be measured, chromogenic signal, and HbA1c calculation method in Embodiment B are the same as in Embodiment A.

The glycated protein direct fructosyl peptide oxidase (direct FPOX) in Embodiment B is an improved FPOX that has substrate specificity for glycated hemoglobin (acts directly on glycated hemoglobin) (WO2015-005257 and WO2015-060429).

[kit]
In another aspect, the present disclosure relates to a kit for measuring glycated protein by an enzymatic method, comprising a reagent containing peroxidase and a reagent according to the present disclosure as a reagent separate from the reagent.
The kit according to the present disclosure may be a two-reagent kit or a three-reagent kit for enzymatic measurement of glycated proteins. In one or more non-limiting embodiments, configurations of the two-reagent system kit include configurations A1 to A4 and B1 described above.

[Measuring method]
In another aspect, the present disclosure provides a method for measuring HbA1c including the following (1) to (5), wherein reagents according to the present disclosure are mixed to supply at least the following leuco-type dye of (4) It relates to a measurement method including
(1) Denaturing the hemoglobin in the sample.
(2) generating an N-terminal glycated peptide by reacting glycated hemoglobin in the sample with protease;
(3) to generate hydrogen peroxide by the reaction of N-terminal glycosylated peptide and fructosyl peptide oxidase (FPOX);
(4) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase (POD).
(5) Calculating the amount of HbA1c and the amount of hemoglobin by measuring the coloring signal.

In another aspect, the present disclosure provides a method for measuring HbA1c including the following (1) to (4), wherein reagents according to the present disclosure are mixed to supply at least the following leuco-type dye (3) It relates to a measurement method including
(1) Denaturing the hemoglobin in the sample.
(2) Producing hydrogen peroxide through the reaction between glycated hemoglobin in the sample and glycated protein direct fructosyl peptide oxidase (direct FPOX).
(3) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(4) Calculating the amount of HbA1c and the amount of hemoglobin by measuring the coloring signal.

The measurement method according to the present disclosure is as described in Embodiments A and B above.

式（Ｉ）において、Ｒは、炭素数８－１７の炭化水素鎖を表す。
〔２〕 糖化蛋白質が、糖化ヘモグロビン又は糖化アルブミンである、〔１〕に記載の試薬。
〔３〕 糖化蛋白質が、ヘモグロビンＡ１ｃである、〔１〕又は〔２〕に記載の試薬。
〔４〕 前記試薬は、式（Ｉ）の化合物を、少なくとも、前記ロイコ型色素の安定化剤として含む、〔１〕から〔３〕のいずれかに記載の試薬。
〔５〕 前記ロイコ型色素が、１０－（カルボキシメチルアミノカルボニル）－３，７－ビス（ジメチルアミノ）フェノチアジン、Ｎ，Ｎ，Ｎ'，Ｎ'，Ｎ''，Ｎ''－ヘキサ（３－スルフォプロピル）－４，４'，４''－トリアミノトリフェニルメタン、Ｎ－（カルボキシメチルアミノカルボニル）－４，４'－ビス（ジメチルアミノ）ジフェニルアミン、又はこれらの塩である、〔１〕から〔４〕のいずれかに記載の試薬。
〔６〕 ペルオキシダーゼを含む試薬と、該試薬とは別の試薬として〔１〕から〔５〕のいずれかに記載の試薬とを備える、糖化蛋白質を酵素法により測定するためのキット。
〔７〕 下記（１）から（５）を含むヘモグロビンＡ１ｃの測定方法であって、
試料と〔１〕から〔５〕のいずれかに記載の試薬とを混合することを含む、測定方法。
（１）試料中のヘモグロビンを変性させること。
（２）試料中の糖化ヘモグロビンとプロテアーゼと反応によりＮ末端糖化ペプチドを生成させること。
（３）Ｎ末端糖化ペプチドとフルクトシルペプチドオキシダーゼとの反応により過酸化水素を生成させること。
（４）生成した過酸化水素とペルオキシダーゼとの反応によりロイコ型色素を発色させること。
（５）吸光度を測定してヘモグロビンＡ１ｃ量及びヘモグロビン量を算出すること。
〔８〕 下記（１）から（４）を含むヘモグロビンＡ１ｃの測定方法であって、
試料と〔１〕から〔５〕のいずれかに記載の試薬とを混合することを含む、測定方法。
（１）試料中のヘモグロビンを変性させること。
（２）試料中の糖化ヘモグロビンと糖化蛋白直接型フルクトシルペプチドオキシダーゼとの反応により過酸化水素を生成させること。
（３）生成した過酸化水素とペルオキシダーゼとの反応によりロイコ型色素を発色させること。
（４）吸光度を測定してヘモグロビンＡ１ｃ量及びヘモグロビン量を算出すること。 The present disclosure further relates to one or more of the following non-limiting embodiments.
[1] A reagent used for measurement of glycated protein by an enzymatic method, comprising:
contains a leuco-type dye,
A reagent further comprising a compound of formula (I) below.

In formula (I), R represents a hydrocarbon chain of 8-17 carbon atoms.
[2] The reagent of [1], wherein the glycated protein is glycated hemoglobin or glycated albumin.
[3] The reagent of [1] or [2], wherein the glycated protein is hemoglobin A1c.
[4] The reagent according to any one of [1] to [3], wherein the reagent contains at least the compound of formula (I) as a stabilizer for the leuco dye.
[5] The leuco dye is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, N,N,N',N',N'',N''-hexa(3 -sulfopropyl)-4,4′,4″-triaminotriphenylmethane, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine, or salts thereof [ The reagent according to any one of 1] to [4].
[6] A kit for measuring glycated protein by an enzymatic method, comprising a reagent containing peroxidase and the reagent according to any one of [1] to [5] as a separate reagent.
[7] A method for measuring hemoglobin A1c including the following (1) to (5),
A measuring method comprising mixing a sample with the reagent according to any one of [1] to [5].
(1) Denaturing the hemoglobin in the sample.
(2) generating an N-terminal glycated peptide by reacting glycated hemoglobin in the sample with protease;
(3) to generate hydrogen peroxide by the reaction of N-terminal glycosylated peptide and fructosyl peptide oxidase;
(4) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(5) Measuring the absorbance and calculating the amount of hemoglobin A1c and the amount of hemoglobin.
[8] A method for measuring hemoglobin A1c including the following (1) to (4),
A measuring method comprising mixing a sample with the reagent according to any one of [1] to [5].
(1) Denaturing the hemoglobin in the sample.
(2) Producing hydrogen peroxide through the reaction between glycated hemoglobin in the sample and glycated protein direct fructosyl peptide oxidase.
(3) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(4) Measuring the absorbance and calculating the amount of hemoglobin A1c and the amount of hemoglobin.

The following compounds were used in the examples below.
Octylsulfobetaine (sulfobetaine C8): N-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
Decylsulfobetaine (sulfobetaine C10): N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
Laurylsulfobetaine (sulfobetaine C12): N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
Tetradecylsulfobetaine (sulfobetaine C14): N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
Hexadecylsulfobetaine (sulfobetaine C16): N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
Octadecylsulfobetaine (sulfobetaine C18): N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
DA-67 (leuco dye): 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium (manufactured by Tokyo Chemical Industry Co., Ltd.)

[Evaluation of Stabilization of Leuco Dye 1]
This experiment was conducted to confirm that sulfobetaine contributes to the stabilization of leuco-type dyes. A leuco dye (DA-67) was dissolved in a 100 mmol/L Tris-PIPES buffer (pH 6.8) to a concentration of 0.045 mmol/L, and a stabilizer shown in Table 1 was added to 20 mL of this solution. A reagent was prepared and heat-treated at 25° C. for 24 hours. Thereafter, using a biochemical automatic analyzer (JCA-BM-6010, manufactured by JEOL Ltd.), purified water was measured as a sample according to the following measurement parameters to measure the coloring amount of the reagent. The same amount of coloring was measured for the reagent before heat treatment at 25 ° C. for 24 hours. The stabilizing ability was evaluated. The results are shown in Table 1 and FIG.
<First reagent>
PIPES buffer (pH 6.4)
<Second reagent>
Protease (trade name NEP-209, manufactured by Toyobo): 2U/mL
DA-67: 45 µmol/L
Sulfobetaine shown in Table 1: 1 g / L
PIPES buffer (pH 6.8)
The measurement parameters used for evaluating the coloring amount of the reagent are as follows.
Measurement dominant wavelength: 658 nm
Measurement sub-wavelength: 694 nm
Specimen (purified water) dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
1st metering point: 17-19
Second metering point: 21

As shown in FIG. 1 and Table 1, in Examples 1-5, compared with Comparative Example 1, the coloring rate was reduced. In particular, the reduction in coloring rate was remarkable in Examples 3-5.

[Evaluation of Stabilization of Leuco Dye 2]
This experiment was conducted to confirm that the stability of the leuco dye is improved depending on the concentration of sulfobetaine. A leuco dye (DA-67) was dissolved in a 100 mmol/L Tris-PIPES buffer (pH 6.8) to a concentration of 0.045 mmol/L, and a stabilizer shown in Table 2 was added to 5 mL of this solution. A reagent was prepared and heat-treated at 25° C. for 24 hours. Thereafter, using a biochemical automatic analyzer (JCA-BM-6010, manufactured by JEOL Ltd.), purified water was measured as a sample according to the following measurement parameters to measure the coloring amount of the reagent. The same amount of coloring was measured for the reagent before heat treatment at 25 ° C. for 24 hours. The stabilizing ability was evaluated. The results are shown in Table 2 and FIG.

The measurement parameters used for evaluating the coloring amount of the reagent are as follows.
Measurement dominant wavelength: 658 nm
Measurement sub-wavelength: 694 nm
Specimen (purified water) dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Second reagent dispensing volume: 24 μL
1st metering point: 17-19
Second metering point: 21
The results are shown in Table 2 and FIG.

As shown in FIG. 2 and Table 2, the greater the amount of sulfobetaine C12 and sulfobetaine C14 added, the more inhibited the leuco dye. That is, the leuco-type dye was stabilized.

[Measurement 1 of hemoglobin A1c (HbA1c)]
This experiment was conducted to demonstrate that HbA1c% can be measured with high accuracy using an enzymatic reagent containing sulfobetaine. Diabetic patient-derived blood cell specimens (37 specimens) were measured with a commercially available measurement reagent and a reagent containing the stabilizer of Examples 13-20 (first and second reagents below), and the measured values were correlated. Analysis evaluated the practical applicability of these examples to clinical testing. The results are shown in Table 3 below.
The hemoglobin concentration and the hemoglobin A1c concentration were obtained by taking absorbance ratios obtained at different wavelengths (measurement dominant wavelength and measurement sub-wavelength) and converting them based on a calibration curve for the absorbance ratios.
<Sample>
Blood cell samples from diabetic patients (37 samples)
<Sample for calibration>
Commercially available HbA1c calibrator (manufactured by ARKRAY)
<First reagent>
Horseradish-derived peroxidase: 15 U/mL
Fructosyl peptide oxidase (trade name FPO-302, manufactured by Toyobo): 3 U/mL
Sulfobetaine same as the second reagent (various): 3 g / L
PIPES buffer (pH 6.4)
<Second reagent>
Protease (trade name NEP-209, manufactured by Toyobo): 2U/mL
DA-67: 45 µmol/L
Sulfobetaine C12/C14 (concentrations in Table 3 below)
PIPES buffer (pH 6.8)
<Measurement method 1: Hemoglobin concentration>
Measurement dominant wavelength: 596 nm
Measurement sub-wavelength: 694 nm
Specimen dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
1st metering point: 17-19
<Measurement method 2: Hemoglobin A1c concentration>
Measurement dominant wavelength: 658 nm
Measurement sub-wavelength: 694 nm
Specimen dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
1st metering point: 21
Second metering point: 40-42
<Measurement method 3: Calculation of HbA1c value (NGSP)>
When the hemoglobin concentration measured by measurement methods 1 and 2 is C _Hb and the hemoglobin A1c concentration is C _HbA1c ,
HbA1c=C _HbA1c /C _Hb ×0.915×100+2.15
Calculated by

As shown in Table 3, for the same specimen (37 specimens), the results of correlation analysis between the HbA1c value measured by the commercially available measurement kit and the HbA1c value measured under the stabilizer addition conditions of Examples 13-20. showed good correlation.

[Measurement 2 of hemoglobin A1c (HbA1c)]
This experiment was conducted to show that the same measurement performance as described above can be obtained even with a reagent having a composition different from that described above. As a result, it was confirmed that the reagent having the following composition has the same performance as the reagent having the above composition.
<Sample for calibration>
Commercially available HbA1c calibrator (manufactured by ARKRAY)
<First reagent>
DA-67: 10 µmol/L
Protease (trade name NEP-209, manufactured by Toyobo): 8 U/mL
Sulfobetaine (C12 or C14): 30 g/L
PIPES buffer (pH 6.8)
<Second reagent>
Horseradish-derived peroxidase: 60 U/mL
Fructosyl peptide oxidase (trade name FPO-302, manufactured by Toyobo): 12 U/mL
PIPES buffer (pH 6.4)
<Measurement method 1: Hemoglobin concentration>
Measurement dominant wavelength: 596 nm
Measurement sub-wavelength: 694 nm
Specimen dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
1st metering point: 17-19
<Measurement method 2: Hemoglobin A1c concentration>
Measurement dominant wavelength: 658 nm
Measurement sub-wavelength: 694 nm
Specimen dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
1st metering point: 21
Second metering point: 40-42
<Measurement method 3: Calculation of HbA1c value (NGSP)>
When the hemoglobin concentration measured by measurement methods 1 and 2 is C _Hb and the hemoglobin A1c concentration is C _HbA1c ,
HbA1c=C _HbA1c /C _Hb ×0.915×100+2.15
Calculated by

[Evaluation of hemoglobin modification ability and protease reaction promotion ability of sulfobetaine]
In order to measure HbA1c% with an enzymatic reagent, hemoglobin is preferably denatured appropriately for accurate quantification of hemoglobin and for promoting hydrolysis by protease. Therefore, this experiment was conducted to evaluate the Hb denaturing ability and protease reaction promoting ability of sulfobetaines C12 and C14 under the following conditions.
<Evaluation method for hemoglobin alteration ability>
Using a biochemical automatic analyzer (JCA-BM-6010, manufactured by JEOL Ltd.), changes in absorbance of hemoglobin were observed for 5 minutes after the sample was dispensed until the second reagent was dispensed. The measurement results are shown in FIG.
<Measurement parameters>
Measurement dominant wavelength: 596 nm
Measurement sub-wavelength: 694 nm
Specimen dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
<Sample>
Specimen obtained by diluting healthy human whole blood 23-fold with purified water <First reagent>
Horseradish-derived peroxidase: 15 U/mL
Fructosyl peptide oxidase (trade name FPO-302, manufactured by Toyobo): 3 U/mL
Sulfobetaine (C12 or 14): 3 g/L
PIPES buffer: 50 mmol/L (pH 6.4)
<Second reagent>
Protease (trade name NEP-209, manufactured by Toyobo): 2U/mL
DA-67: 45 µmol/L
Sulfobetaine same as the first reagent: 3 g/L
PIPES buffer: 100 mmol/L (pH 6.8)
<Method for evaluating ability to promote protease reaction>
Using a biochemical automatic analyzer (JCA-BM-6010, manufactured by JEOL Ltd.) and a measurement reagent that is protease rate-limiting, after the sample is dispensed, the coloring reagent derived from the second reagent is dispensed. Absorbance changes were observed. The measurement results are shown in FIG.
<Measurement parameters>
Measurement dominant wavelength: 658 nm
Measurement sub-wavelength: 694 nm
Specimen dispensing volume: 8 μL
Amount of first reagent dispensed: 96 μL
Amount of second reagent dispensed: 24 μL
<Sample>
Specimen obtained by diluting healthy human whole blood 23-fold with purified water <First reagent>
Horseradish-derived peroxidase: 15 U/mL
Fructosyl peptide oxidase (trade name FPO-302, manufactured by Toyobo): 3 U/mL
Sulfobetaine (C12/C14): 3 g/L
PIPES buffer: 50 mmol/L (pH 6.4)
<Second reagent>
Protease (trade name NEP-209, manufactured by Toyobo): 2U/mL
DA-67: 45 µmol/L
Sulfobetaine same as the first reagent: 3 g/L
PIPES buffer: 100 mmol/L (pH 6.8)

As shown in FIGS. 3 and 4, sulfobetaines C12 and C14 were shown to have Hb denaturing ability and protease reaction promoting ability.

Claims (7)

A reagent used for measuring glycated protein by an enzymatic method,
contains a leuco-type dye,
further comprising an effective amount of a compound of formula (I) below for stabilizing the leuco dye ;
The leuco dye is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, N,N,N',N',N'',N''-hexa(3-sulfo propyl)-4,4′,4″-triaminotriphenylmethane, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine, or salts thereof;
The reagent , wherein the glycated protein is glycated hemoglobin or glycated albumin .

In formula (I), R represents a hydrocarbon chain of 8-17 carbon atoms. The reagent according to claim 1 , wherein the glycated protein is glycated hemoglobin. The reagent according to claim 1 or 2, wherein the glycated protein is hemoglobin A1c. The content of the compound of formula (I) is 0.1 to 50 g/L,
4. The reagent according to any one of claims 1 to 3, wherein the content ratio of the compound of formula (I) to the leuco dye is 50 or more and 5000 or less . A kit for measuring glycated protein by an enzymatic method, comprising a reagent containing peroxidase and the reagent according to any one of claims 1 to 4 as a reagent separate from said reagent. A method for measuring hemoglobin A1c including the following (1) to (5),
A measuring method comprising mixing a sample with the reagent according to any one of claims 1 to 4 .
(1) Denaturing the hemoglobin in the sample.
(2) generating an N-terminal glycated peptide by reacting glycated hemoglobin in the sample with protease;
(3) to generate hydrogen peroxide by the reaction of N-terminal glycosylated peptide and fructosyl peptide oxidase;
(4) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(5) Calculating the amount of hemoglobin A1c and the amount of hemoglobin by measuring the coloring signal. A method for measuring hemoglobin A1c including the following (1) to (4),
A measuring method comprising mixing a sample with the reagent according to any one of claims 1 to 4 .
(1) Denaturing the hemoglobin in the sample.
(2) Producing hydrogen peroxide through the reaction between glycated hemoglobin in the sample and glycated protein direct fructosyl peptide oxidase.
(3) Color development of the leuco-type dye by the reaction of the generated hydrogen peroxide and peroxidase.
(4) Calculating the amount of hemoglobin A1c and the amount of hemoglobin by measuring the coloring signal.

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