JP2009072136A - Stable measurement method using reducing pigment, and reagent thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining and measuring specific ingredients in a biological specimen with a liquid reagent that is constituted of two or more reagents, produced by making an enzyme having dehydrogenation-oxidizing ability that acts on the biological ingredients, in the presence of an electron receptor, treating the produced reduced electron acceptor with an electron carrier and a reducing pigment to make formazan formed, and then determining the formazan, and makes both the "high degree of accuracy" and "stability" of the reagent compatible. <P>SOLUTION: This method for determining and measuring the specific ingredient in the biological specimen uses tetrazolium or its compound as the reducing pigment, controlling the pH of the reagent that contains the reducing pigment to be 8.0 or lower, and controlling the pH of the reagent to be 7.5 or higher, when reacting. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention provides a stable measurement method that achieves the following (1) to (4) and a reagent therefor in a liquid clinical test reagent that uses a reducing dye for color determination.
(1) Suppresses nonspecific color development of a reducing dye during storage in a liquid reagent.
(2) High accuracy measurement is possible.
(3) To avoid the influence of ascorbic acid and lactate dehydrogenase (hereinafter referred to as LDH) in the biological sample.
(4) Avoid the influence of reducing dyes and proteins in biological samples.

Conventionally, an enzymatic analysis method for measuring absorbance has been developed as a method for detecting various biological components contained in biological samples such as blood and urine, and is widely used in daily clinical tests.
As one of the methods to quantify various specific components contained in biological samples using enzymatic analysis,
For example, a method using a redox reaction is known. That is, in the presence of an electron acceptor, an enzyme capable of oxidizing by dehydrogenation is applied to a specific sample or a substance derived from the specific component on a biological sample, and a reduced form of the generated electron acceptor is measured. This is a method for indirectly quantifying a specific component in a sample.

In particular, when the specific component to be quantified is a trace component, the reduced form of the electron acceptor generated above is detected using a color former with higher color development sensitivity in order to measure with higher sensitivity. Quantification is also performed. For example, when NAD or NADP is used as an electron acceptor, formazan is formed by reacting an electron carrier such as diaphorase with tetrazolium or a reducing dye, or a compound thereof, on NADH or NADPH which is a reduced form thereof. Detection and quantification are performed.

In the clinical test reagents using the reducing dyes that have been sold so far, the formazan produced is poorly soluble and adsorbs on the cell of the analyzer, affecting the measurement. Therefore, recently, tetrazolium salts that produce formazan with high water solubility have been developed, and products that are suitable for clinical laboratory reagents have been sold.

For example, Patent Documents 1 to 9 listed below are known as prior art relating to a measurement method using tetrazolium or a compound thereof, which is a reducing dye.
Patent Document 1 discloses a compound contained in the general formula (1) of claim 3 of the present application.
Patent Document 2 discloses a method for measuring active oxygen using a compound contained in the general formula (1) of claim 3 of the present application.
In Patent Document 3, a tetrazolium compound is added to a pretreatment solution in advance to avoid the influence of a reducing substance in a biological sample, and the tetrazolium compound is used for the purpose of avoiding the influence of a reducing substance in a biological sample. Is disclosed.
Patent Document 4 discloses that formazan generated from tetrazolium is added to a pretreatment liquid in advance to avoid the influence of a reducing substance in a biological sample.
These Patent Documents 3 and 4 use a tetrazolium salt for the purpose of pretreatment of a biological sample, and are clearly different from the present invention used for color development quantification of a specific component in a biological sample.
Patent Document 5 discloses that a tetrazolium salt is used in contact with a test sample in the range of pH 8.5 to 11.0 (preferably 9.5 to 10.5).
Patent Document 6 discloses pH values in Examples, and discloses that the pH of a reagent containing a tetrazolium salt is set to pH 10.0 as in Examples.
In Patent Documents 5 and 6, when the pH of a reagent containing a tetrazolium salt is 8.5 or higher, and the tetrazolium salt is stored in the pH solution, the non-specific color development of the tetrazolium salt over time during storage The present inventors have confirmed that the above becomes remarkable.
Patent Documents 7 and 8 disclose dry reagents using tetrazolium salts, but do not mention the stability of tetrazolium salts in liquid reagents. The embodiment is also a dry reagent and is clearly different from the liquid reagent of the present invention.
Patent Document 9 discloses a method for avoiding the influence of a biological sample containing hemoglobin by adding albumin to a reagent in a measurement system using a tetrazolium salt.
In any of these documents, non-specific color development during storage in a liquid reagent of a tetrazolium salt is not described.
Japanese Patent No. 2592436 Japanese Patent No. 3606026 JP 2000-210100 A Republished WO2003 / 033730 Japanese Patent No. 2796150 JP 2000-3000293 A JP 2000-93198 A JP 2000-93199 A JP 2004-61263 A

In the liquid clinical test reagent using the tetrazolium salt which is a reducing dye, the following two points are serious problems.
(1) Reagent blank rises day by day during storage.
(2) It is easily affected by components in the biological sample.
In (1), problems such as a decrease in sensitivity and a decrease in the upper limit of the measurement range occur in the absorbance measurement. Therefore, in the measurement, a standard substance is measured every time and a calibration curve is drawn (hereinafter referred to as calibration). It becomes necessary to subtract the light absorption amount of the colored dye. Moreover, it becomes difficult to measure a measurement object that exists only in a minute amount in a biological sample due to a decrease in measurement sensitivity.
In (2), the redox reaction measurement system using a tetrazolium salt is particularly affected by the influence of a reducing substance such as ascorbic acid, glutathione, protein, or a redox enzyme such as lactate dehydrogenase (LDH). It has become.

In order to use tetrazolium salt with high sensitivity, it is known that the pH is set to the alkaline side. However, when the pH of the storage solution is set to the alkaline side for use with high sensitivity, The inventors have found that a typical blank rise is more pronounced.

Furthermore, when the reaction pH is set to the alkaline side for measurement with high sensitivity, and the biological sample to be measured contains a large amount of lactic acid, the reaction activity of LDH from lactic acid to pyruvic acid is promoted, so the tetrazolium salt A problem arises in that it affects the oxidation-reduction reaction using.

That is, the problem to be solved by the present invention is to achieve both “high accuracy” and “stability” of a reagent in a liquid clinical test reagent using a tetrazolium salt.

［一般式（１）］
（式中、Ｒ１は水素、ニトロ基、または、メトキシ基、Ｒ２は水素またはニトロ基、Ｒ３はハロゲン、ニトロ基または、以下の式（２）で表されるアゾ化合物、Ｒ４は水素またはスルホ基、Ｒ５は水素または、スルホ基、Ｒ６は水素、ニトロ基、またはスルホ基、Ｒ７は水素または、スルホ基を示す。）

［式（２）］
［項４］
還元系色素を含む試薬に、アスコルビン酸オキシダーゼを含有する項１〜３のいずれかに記載の方法。
［項５］
還元系色素を含む試薬に、電子受容体を含有する項１〜４のいずれかに記載の方法。
［項６］
請求項５記載の電子受容体が、ニコチンアミドアデニンジヌクレオチドニコチンアミドアデニンジヌクレオチドホスフェイト、アセチルピリジンアデニンジヌクレオチド、アセチルピリジンアデニンジヌクレオチドホスフェイト、ニコチンアミドヒポキサンチンジヌクレオチド、あるいはニコチンアミドヒポキサンチンジヌクレオチドホスフェイト、ニコチンアミド１,Ｎ６-エテノアデニンジヌクレオチド、ニコチンアミド１,Ｎ６-エテノアデニンジヌクレオチドホスフェイト、ピロロキノリンキノンである項１〜５のいずれかに記載の方法。
［項７］
還元系色素を含む試薬に、電子受容体およびアスコルビン酸オキシダーゼを含有する項１〜６のいずれかに記載の方法。
［項８］
電子受容体の存在下で、該特定成分に対して脱水素による酸化能を有する酵素を生体試料に作用させ、
生成された電子受容体の還元体を、さらに、電子キャリアーと還元系色素を作用させてホルマザンを形成させることにより生体試料中の特定成分を定量する方法に用いる液状試薬であって、
以下の（ａ）〜（ｄ）の構成を全て有する、液状試薬。
（ａ）２試薬以上で構成される。
（ｂ）第一試薬に還元系色素としてテトラゾリウム、またはその化合物を含む。
（ｃ）第一試薬のｐＨがｐＨ８．０以下である。
（ｄ）反応時における試薬のｐＨがｐＨ７．５以上である。
［項９］
電子受容体の存在下で、該特定成分から別の反応により派生した物質に対して脱水素による酸化能を有する酵素を生体試料に作用させ、
生成された電子受容体の還元体を、さらに、電子キャリアーと還元系色素を作用させてホルマザンを形成させることにより生体試料中の特定成分を定量する方法に用いる液状試薬であって、
以下の（ａ）〜（ｄ）の構成を全て有する、液状試薬。
（ａ）２試薬以上で構成される。
（ｂ）第一試薬に還元系色素としてテトラゾリウム、またはその化合物を含む。
（ｃ）第一試薬のｐＨがｐＨ８．０以下である。
（ｄ）反応時における試薬のｐＨがｐＨ７．５以上である。
［項１０］
還元系色素を含む試薬に、アスコルビン酸オキシダーゼを含有する項８または９に記載の試薬。
［項１１］
還元系色素を含む試薬に、電子受容体を含有する項８または９に記載の試薬。
［項１２］
請求項１１記載の電子受容体が、ニコチンアミドアデニンジヌクレオチド、ニコチンアミドアデニンジヌクレオチドホスフェイト、アセチルピリジンアデニンジヌクレオチド、アセチルピリジンアデニンジヌクレオチドホスフェイト、ニコチンアミドヒポキサンチンジヌクレオチド、あるいはニコチンアミドヒポキサンチンジヌクレオチドホスフェイト、ニコチンアミド１,Ｎ６-エテノアデニンジヌクレオチド、ニコチンアミド１,Ｎ６-エテノアデニンジヌクレオチドホスフェイト、ピロロキノリンキノンである請求項８〜１１のいずれかに記載の試薬。
［項１３］
還元系色素を含む試薬に、電子受容体およびアスコルビン酸オキシダーゼを含有する請求項８〜１２のいずれかに記載の試薬。 As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention.
That is, this invention consists of the following structures.
[Claim 1]
A method for quantifying a specific component in a biological sample using a liquid reagent composed of two or more reagents,
In the presence of an electron acceptor, an enzyme having the ability to oxidize by dehydrogenating the specific component is allowed to act on a biological sample,
In the method of detecting and quantifying the reduced form of the generated electron acceptor by further forming an formazan by acting an electron carrier and a reducing dye,
A method for quantifying a component in a biological sample having all of the following configurations (a) to (c):
(A) Tetrazolium or a compound thereof is used as the reducing dye.
(B) The reagent containing the reducing dye has a pH of 8.0 or less.
(C) The pH of the reagent during the reaction is pH 7.5 or higher.
[Section 2]
A method for quantifying a specific component in a biological sample using a liquid reagent composed of two or more reagents,
In the presence of an electron acceptor, an enzyme having an ability to oxidize by dehydrogenating a substance derived from the specific component by another reaction is allowed to act on a biological sample,
In the method of detecting and quantifying the reduced form of the generated electron acceptor by further forming an formazan by acting an electron carrier and a reducing dye,
A method for quantifying a component in a biological sample having all of the following configurations (a) to (c):
(A) Tetrazolium or a compound thereof is used as the reducing dye.
(B) The reagent containing the reducing dye has a pH of 8.0 or less.
(C) The pH of the reagent during the reaction is pH 7.5 or higher.
[Section 3]
Item 3. The method for quantifying a specific component in a biological sample according to Item 1 or 2, wherein the reducing dye is a reducing dye represented by the general formula (1).

[General formula (1)]
Wherein R1 is hydrogen, nitro group, or methoxy group, R2 is hydrogen or nitro group, R3 is halogen, nitro group or an azo compound represented by the following formula (2), R4 is hydrogen or sulfo group R5 represents hydrogen or a sulfo group, R6 represents hydrogen, a nitro group, or a sulfo group, and R7 represents hydrogen or a sulfo group.)

[Formula (2)]
[Claim 4]
Item 4. The method according to any one of Items 1 to 3, wherein the reagent containing a reducing dye contains ascorbate oxidase.
[Section 5]
Item 5. The method according to any one of Items 1 to 4, wherein the reagent containing a reducing dye contains an electron acceptor.
[Claim 6]
6. The electron acceptor according to claim 5, wherein nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, acetylpyridine adenine dinucleotide, acetylpyridine adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, or nicotinamide hypoxanthine dinucleotide. Item 6. The method according to any one of Items 1 to 5, which is nucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate, or pyrroloquinoline quinone.
[Claim 7]
Item 7. The method according to any one of Items 1 to 6, wherein the reagent containing a reducing dye contains an electron acceptor and ascorbate oxidase.
[Section 8]
In the presence of an electron acceptor, an enzyme having the ability to oxidize by dehydrogenating the specific component is allowed to act on a biological sample,
A liquid reagent used in a method for quantifying a specific component in a biological sample by forming a formazan by further reacting an electron carrier and a reducing dye to form a reduced form of an electron acceptor,
The liquid reagent which has all the structures of the following (a)-(d).
(A) Consists of two or more reagents.
(B) The first reagent contains tetrazolium or a compound thereof as a reducing dye.
(C) The pH of the first reagent is pH 8.0 or less.
(D) The reagent has a pH of 7.5 or higher during the reaction.
[Claim 9]
In the presence of an electron acceptor, an enzyme having an ability to oxidize by dehydrogenating a substance derived from the specific component by another reaction is allowed to act on a biological sample,
A liquid reagent used in a method for quantifying a specific component in a biological sample by forming a formazan by further reacting an electron carrier and a reducing dye to form a reduced form of an electron acceptor,
The liquid reagent which has all the structures of the following (a)-(d).
(A) Consists of two or more reagents.
(B) The first reagent contains tetrazolium or a compound thereof as a reducing dye.
(C) The pH of the first reagent is pH 8.0 or less.
(D) The reagent has a pH of 7.5 or higher during the reaction.
[Section 10]
Item 10. The reagent according to Item 8 or 9, wherein the reagent containing a reducing dye contains ascorbate oxidase.
[Section 11]
Item 10. The reagent according to Item 8 or 9, wherein the reagent containing a reducing dye contains an electron acceptor.
[Claim 12]
The electron acceptor according to claim 11, wherein nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, acetylpyridine adenine dinucleotide, acetylpyridine adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, or nicotinamide hypoxanthine The reagent according to any one of claims 8 to 11, which is dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate, or pyrroloquinoline quinone.
[Claim 13]
The reagent according to any one of claims 8 to 12, wherein the reagent containing a reducing dye contains an electron acceptor and ascorbate oxidase.

In the present invention, “high accuracy” means that the measurement object contained in a biological sample has a low concentration, for example, a measurement object having a concentration of 2 mM or less, and the fluctuation range of the measured value of the known concentration is ± 20%. Hereinafter, it means that measurement of preferably 10% or less, more preferably 5% or less is possible.

In the present invention, “stability” means that the calibration curve is drawn (calibration) by the method described below for the first time of measurement, and if the calibration is not performed in the subsequent measurement, It means that the measured value of a specific component in a sample is stabilized over a long period of time. More strictly, it means that the measured value during 2 weeks and after 2 weeks does not fall below 90%, preferably less than 95% of the theoretical value in an opened storage condition of 10 ° C. or less.

Calibration method with calibration curve (calibration method)
[1] Purified water or physiological saline,
[2] It is preferable to use a standard substance or standard solution having a concentration of one level or more.

The calibration method is not limited to a straight line method or a spline method, but the straight line method is particularly preferable.

An object of the present invention is to achieve both “high accuracy” and “stability” of a reagent in a liquid clinical test reagent using a tetrazolium salt. Specifically, in a liquid clinical test reagent using a tetrazolium salt, in the measurement of a measurement object having a concentration of 2 mM or less contained in a biological sample, the fluctuation range of the measurement value of the known concentration is ± Measurements of 20% or less, preferably 10% or less, and more preferably 5% or less are possible. Furthermore, in the measurement of specific components in biological samples by suppressing non-specific color development of reducing dyes during liquid storage, and reagents with suppressed non-specific color development, proteins, ascorbic acid, glutathione, etc. in biological samples Provides a measurement method that stabilizes the measured value of a specific component in a biological sample over a long period of time by avoiding the effects of reducing substances in the sample and the effects of oxidoreductases such as LDH in the biological sample, and its reagents It is to be.

Further, when the method of the present invention is used, if only the first measurement is calibrated, the measured value of the specific component in the biological sample is stabilized over a long period of time without performing calibration in the subsequent measurement.

By suppressing the non-specific color development of the reducing dye and avoiding the influence of the components in the biological sample, if only the first measurement is calibrated, it will not be calibrated in the biological sample for a long time. It becomes possible to stabilize the measured value of the specific component.

In the present invention, in order to stabilize the measurement value in the non-calibration measurement, the relationship between the degree of increase of the reagent blank and the sensitivity becomes important.
In the present invention, it is preferable to have sensitivity that can tolerate an error due to an increase in absorbance of the reagent blank due to storage. More specifically, it is preferable that the blank increase degree by storage is within 10% of the sensitivity.

The present invention is a method for quantifying a specific component in a biological sample using a liquid reagent composed of two or more reagents, and has the ability to oxidize the specific component by dehydrogenation in the presence of an electron acceptor. In a method of detecting and quantifying a reduced form of an electron acceptor produced by reacting an enzyme with a biological sample, and further forming a formazan by acting an electron carrier and a reducing dye, (a) as a reducing dye Using tetrazolium or a compound thereof, (b) The pH of the reagent containing the reducing dye is pH 8.0 or lower, and (c) The pH of the reagent during the reaction is pH 7.5 or higher. It is characterized by that.

In addition, “at the time of reaction” described in this patent means “a reaction when a reducing dye is led to a color-forming reaction in an enzyme-specific manner”.

（式１）
式１では、電子受容体であるＮＡＤ＋の存在下で特定成分に対して脱水素酵素が作用することにより、特定成分が酸化されるとともに電子受容体の還元体が得られ、次いで、電子受容体の還元体が電子キャリアーと還元系色素であるテトラゾリウム化合物を作用させてホルマザン色素を形成させることにより、比色による検出・定量ができることが例示される。 The method for quantifying a specific component in a biological sample of the present invention can be typically applied to the reaction represented by the following formula 1.

(Formula 1)
In Formula 1, a dehydrogenase acts on a specific component in the presence of the electron acceptor NAD + to oxidize the specific component and obtain a reduced form of the electron acceptor. It is exemplified that colorimetric detection and quantification can be performed by forming a formazan dye by allowing a tetrazolium compound, which is an electron carrier and a reducing dye, to act as a reduced form.

Diaphorase is included in the electron carrier in the present invention.

（式２） Alternatively, in another exemplary embodiment, as shown in Formula 2, another substance is obtained by the action of an enzyme on a specific component, and the substance is an electron acceptor. A dehydrogenase acts in the presence of a certain NAD + to obtain a reduced form of the body. Next, the reduced form of the electron acceptor acts by reacting a tetrazolium compound, which is an electron carrier and a reducing dye, to form a formazan dye. By forming it, a method of detecting and quantifying by colorimetry can also be exemplified.

(Formula 2)

（式３）

（式４）
As an example, reaction formulas in total branched chain amino acid (hereinafter referred to as BCAA) measurement and tyrosine measurement are shown in Formula 3 and Formula 4, respectively.

(Formula 3)

(Formula 4)

In Formula 3, as a measuring method of the BCAA measuring reagent, a method in which leucine dehydrogenase is allowed to act on BCAA in the presence of NAD, and the reduced NAD reagent is reduced in the presence of diaphorase by the generated reduced NAD to lead to color development is exemplified. (An electron carrier may be used instead of diaphorase).

In Equation 4, as a measuring method of tyrosine measuring reagent, pyruvate obtained by allowing β-tyrosinase to act on tyrosine is reduced by reducing chromogenic reagent by the action of lactate dehydrogenase in the presence of reduced NAD and diaphorase. (The electron carrier may be used instead of diaphorase).

The liquid reagent used in the method for quantifying a specific component in the biological sample of the present invention is not particularly limited as long as it is a reagent configuration that can be handled by a general-purpose automatic analyzer, but it is 2 or more, preferably 2 to 4 parts. It is desirable to be configured. A two-reagent system composed of two parts is preferred.
The reagent state may be a freeze-dried reagent or a liquid reagent, but a liquid reagent is particularly preferable.
That is, the liquid reagent is preferably composed of 2 to 4 parts. A liquid reagent composed of two parts is preferable.

The biological sample that is the measurement target is not particularly limited, but is preferably serum, plasma, whole blood, or urine.

The specific component to be measured contained in the biological sample includes (I) an enzyme that can be used in the presence of an electron acceptor and an enzyme capable of oxidizing by dehydrogenation to the specific component, or (II) There is no particular limitation as long as an enzyme capable of being oxidized by dehydrogenation can be used in the presence of an electron acceptor for a substance derived from a specific component by another reaction.

Specific examples of specific components include, for example, total branched chain amino acids such as valine, leucine, and isoleucine, various amino acids such as tyrosine, glucose, cholesterol, HDL-C, LDL-C, urea nitrogen, uric acid, and neutral fat. , Creatinine, inulin, inorganic phosphorus, calcium, lactic acid, pyruvate, mannose, 1,5-anhydroglucitol, arabinitol, inositol, sorbitol, fructose, galactose, bile acid, 3-deoxyglucosone, alcohol, glucoalbumin , HbA1c and the like.
In particular, various amino acids such as total branched chain amino acids, tyrosine, mannose, 1,5-anhydroglucitol, arabinitol, inositol, sorbitol, fructose, galactose, bile acids, 3-deoxyglucosone, alcohol, glucoalbumin, HbA1c It is suitably applied when quantifying trace components that require high-sensitivity detection. Preferably, it is more suitably applied when quantifying a trace component present in a biological sample at a concentration of 2 mM or less, more strictly, a trace component present at about 1 to 1000 μM.

The enzyme used in the present invention is an enzyme having an ability to oxidize by dehydrogenation in the presence of an electron acceptor (hereinafter referred to as dehydration) for a specific component to be measured or a substance derived from the specific component by another reaction. It is sufficient if it is called elementary enzyme.) That is, the enzyme is not limited to an enzyme that directly acts on a specific component, and a dehydrogenase for a substance that is quantitatively derived from the specific component by an enzyme conjugate system or the like can also be used. For example, when the specific component is a total branched chain amino acid (valine, leucine, isoleucine), a leucine dehydrogenase that acts directly on the total branched chain amino acid is used. Lactate dehydrogenase that converts to acid and acts on pyruvate derived from tyrosine, which is a specific component, can be used. In addition, when the specific component is alcohol, alcohol dehydrogenase, when it is 1,5-anhydroglucitol, 1,5-anhydroglucitol dehydrogenase, when glucose is glucose dehydrogenase, when cholesterol is cholesterol dehydrogenase What is it? Other dehydrogenases used in the present invention include homoserine dehydrogenase, glycerol dehydrogenase, mannitol-1-phosphate-5-dehydrogenase, various amino acid dehydrogenases, xanthine dehydrogenase, sorbitol dehydrogenase, carnitine dehydrogenase, aldohexose dehydrogenase, aldehyde dehydrogenase, histamine Examples include dehydrogenase and 3-hydroxybutyrate dehydrogenase.
For the other specific components exemplified above, not only an enzyme that directly acts on the specific component, but also a dehydrogenase for a substance quantitatively derived from the specific component by an enzyme conjugate system or the like can be used. Well-known enzymes can be used for these enzymes.
The enzyme concentration used in the present invention is preferably 0.01 to 200 U / L, more preferably 0.05 to 100 U / L in the reaction solution.

The electron acceptor can be used without particular limitation as long as the reduced form of the generated electron acceptor can be directly quantified or the reduced form can be colorimetrically quantified using a color former. . It is preferable to perform colorimetric determination of formazan produced by the action of electron carriers such as tetrazolium and its compounds, phenazine methosulfates, dichlorophenolindophenol, and ferricyan compounds on coenzymes such as NAD and NADP which are electron acceptors. .
Specifically, as nicotinamide coenzyme, nicotinamide adenine dinucleotide (NADH: reduced type, NAD: oxidized type, the same applies hereinafter), nicotinamide adenine dinucleotide phosphate (NADPH, NADP), acetylpyridine adenine dinucleotide Specific examples include acetylpyridine adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, and nicotinamide hypoxanthine dinucleotide phosphate. As thionicotinamide coenzyme, thionicotinamide adenine dinucleotide (thioNADH, thioNAD), thionicotinamide adenine dinucleotide phosphate (thioNADPH, thioNADP), thionicotinamide hypoxanthine dinucleotide, or thio Examples thereof include nicotinamide hypoxanthine dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate, pyrroloquinoline quinone, and flavin compounds such as FAD and FMN.
The electron acceptor used in the present invention is preferably used at a concentration of 0.01 to 50 mmol / L, more preferably 0.05 to 10 mmol / L in the reaction solution.

The reducing dye used in the present invention is a dye that develops color when reduced. In the present invention, tetrazolium and its compound are used as the reducing dye.
Tetrazolium and its compounds are led to formazan by cleavage of the π-electron bond that forms a double bond between nitrogen atoms in the pentacycle, which is the main skeleton, and develop color by the excitation wavelength generated at that time. .

The color development mechanism in non-specific color development is the same, but according to the present invention, the pH of the reagent containing the reducing dye is adjusted to pH 8.0 or less, preferably less than pH 8.0, and more preferably pH 6.0 to 7.5. By suppressing the non-specific cleavage of π electrons that form a double bond between nitrogen in the pentacycle, which is the main skeleton of tetrazolium and its compounds, it is possible to suppress non-specific color development over time. It becomes possible to strengthen the degree.

Examples of tetrazolium and compounds used in the present invention include 2- (2′benzothiazolyl) -5-styryl-3- (4′-phthalhydrazyl) tetrazolium (BSPT), 2-benzothiazolyl- (2) -3. , 5-diphenyltetrazolium (BTDP), 2,3-di (4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3- (4-styrylphenyl) tetrazolium (DPSP), distyrylnitroblue tetrazolium ( DSNBT), 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4,4'-diyl] -bis [2- (4-nitrophenyl) -5-phenyl (-2H Tetrazolium (NBT), 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium (M T), 2-phenyl-3- (4-carboxyphenyl) -5-methyltetrazolium (PCPM), tetrazolium blue (TB), thiocarbamylnitroblue tetrazolium (TCNBT), tetranitroblue tetrazolium (TNBT), tetrazolium violet (TV), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (INT), 3,3 ′-[3,3′-dimethoxy- (1, 1'-biphenyl) -4,4'-diyl] -bis (2,5-diphenyl) -2H-tetrazolium chloride, 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl)- 4,4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride], 2,3 Diphenyl-5- (4-chlorophenyl) tetrazolium chloride, 2,5-diphenyl-3- (p-diphenyl) tetrazolium chloride, 2,3-diphenyl-5- (p-diphenyl) tetrazolium chloride, 2,5-diphenyl- 3- (m-Tolyl) tetrazolium chloride, 2,5-diphenyl-3- (p-tolyl) tetrazolium chloride, 2,3-diphenyl-5- (2-thienyl) tetrazolium chloride, 2-benzothiazoyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H-tetrazolium, 2,2′-dibenzothiazoyl-5,5′-bis [4-di (2 -Sulfoethyl) carbamoylphenyl] -3,3 '-(3,3'-dimethoxy -4,4'-biphenylene) ditetrazolium salt, 2,3-diphenyl-5-cyanotetrazolium chloride, 2,3-diphenyl-5-carboxytetrazolium chloride, 2,3-diphenyl-5-methyltetrazolium chloride, 2, 3-diphenyl-5-ethyltetrazolium chloride, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium salt, 2,3,5-triphenyltetrazolium bromide, 2,3 5-triphenyltetrazolium chloride, 2,3,5-triphenyltetrazolium oxide, 2,3,5-tris (p-tolyl) tetrazolium chloride, 2,3-bis (3-chlorophenyl) -5-phenyltetrazolium chloride, 2,3-bis (3-full Orophenyl) -5-phenyltetrazolium chloride, 2,3-bis (3-methylphenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-chlorophenyl) -5-phenyltetrazolium chloride, 2,3-bis ( 4-ethylphenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-fluorophenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-methoxyphenyl) -5- (4-cyanophenyl) Tetrazolium chloride, 2,3-bis (4-methoxyphenyl) -5-phenyltetrazolium chloride, 2,3-bis (4-methylphenyl) -5- (4-cyanophenyl) tetrazolium chloride, 2,3-bis ( 4-Nitrophenyl) -5-phenyltetra Rium chloride, 2,3-di (p-tolyl) -5-phenyltetrazolium chloride, 2,3-bis (3-methylphenyl) -5-phenyltetrazolium chloride, 2,3-diphenyl-5- (p-tolyl) ) Tetrazolium chloride, 2,3-diphenyl-5-aminotetrazolium chloride, 2,3-diphenyl-5-carboxytetrazolium chloride, 2,5-di (p-tolyl) -3-phenyltetrazolium chloride, 2- (2 -Benzothiazolyl) -3,5-diphenyltetrazolium bromide, 2- (2-benzothiazolyl) -3- (4-nitrophenyl) -5-phenyltetrazolium bromide, 2- (4-ididophenyl) -3- (4-nitro Phenyl) -5-phenyltetrazolium chlora 2-phenyl-3- (4-biphenylyl) -5-methyltetrazolium chloride, 3,3 ′-(3,3′-dimethoxy-4,4-diphenylene) bis (2-phenyl-5-veratritetrazolium) Chloride), 3- (3-nitrophenyl) -5-methyl-2-phenyltetrazolium chloride, 3- (4-nitrophenyl) -5-methyl-2-phenyltetrazolium chloride, m-tritetrazolium red, neo-tetri Tetrazolium chloride, nitroblue tetrazolium, O-tritetrazolium red, p-tritetrazolium red, and the like can be mentioned. In particular, the following tetrazolium that produces water-soluble formazan and its compounds are preferred.
That is, tetrazolium and its compound used in the present invention are 2-benzothiazothiazolyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl]. -2H-tetrazolium, 2,2'-dibenzothiazolyl-5,5'-bis [4-di (2-sulfoethyl) carbamoylphenyl] -3,3 '-(3,3'-dimethoxy-4,4 '-Biphenylene) ditetrazolium, disodium salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt, 2- (4 -Iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt, 2- (4-nitro, 2-methoxypheny ) -3- (4-Nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H- Tetrazolium salt, 2- (4-nitrophenyl) -5-phenyl-3- [4- (4-sulfophenylazo) -2-sulfophenyl] -2H-tetrazolium salt, 2,5-di (4-nitrophenyl) ) -3- [4- (4-sulfophenylazo) -2-sulfophenyl] -2H-tetrazolium salt, 2- (4-nitrophenyl) -5- (2-sulfophenyl) -3- [4- ( 4-sulfophenylazo) -2-sulfophenyl] -2H-tetrazolium salt is preferred.
These are preferably used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

In the present invention, tetrazolium and its compound are used for color determination, and are not added to a pretreatment solution or the like in order to avoid the influence of a reducing substance in a biological sample.

In the liquid clinical laboratory reagent, the arrangement of tetrazolium and its compound in the present invention is not particularly limited. For example, in the case of a clinical laboratory reagent composed of two reagents, it may be added to tetrazolium and its compound first reagent. preferable.

Examples of the diaphorase used in the present invention include Bacillus megaterium and Clostridium SP. The reaction solution is preferably used at a concentration of 0.01 to 100 U / L, preferably 0.05 to 50 U / L.

The reduced NAD oxidase used in the present invention is preferably used in the reaction solution at a concentration of 0.5 to 100 U / mL, preferably 1 to 50 U / mL.

The buffer used in the present invention is not particularly limited as long as it does not contain amines or the like that can cause non-specific color development as an impurity. Tris buffer, phosphate buffer, citrate buffer, phthalic acid Buffering agents, maleic acid buffering agents, glycine buffering agents, boric acid buffering agents, carbonate buffering agents, dimethylglycine buffering agents, Good buffering agents and the like can be mentioned. Good buffering agents include N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N-cyclohexyl-2 -Aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-morpholinoethane Sulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-amino Propanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropa Sulfonic acid (CAPSO), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propane Sulfonic acid (EPPS), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholino Propanesulfonic acid (MOPSO), piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N- Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TA S), N- (2-acetamido) iminoniacetic acid (ADA), N, N-bis (2-hydroxyethyl) glycine (Bicine), N- [tris (hydroxymethyl) methyl] glycine (Tricine), etc. Is done. Among them, N, N-bis (2-hydroxyethyl) glycine (Bicine), N- [tris (hydroxymethyl) methyl] glycine (Tricine), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl- 3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), Tris buffer Carbonic acid buffer, boric acid buffer, dimethylglycine buffer, and diethanolamine buffer are preferred because they reduce the non-specific coloration of the reducing dye. The pH of the buffer is adjusted in the range of 5 to 10, but the pH of the reagent containing the reducing dye should be less than pH 8.0, more preferably pH 6 to 7.5. It is preferable in suppressing nonspecific color development.
The principle of coloring of reducing dyes is led to formazan by the cleavage of the π-electron bond that forms a double bond between the nitrogen in the pentacycle, which is the main skeleton of tetrazolium and its compounds. This is the principle that color is generated by the excitation wavelength generated in the substrate.
The color development mechanism in non-specific color development is the same, but according to the present invention, the pH of the reagent containing the reducing dye is pH 8.0 or less, preferably pH 6.0 to 8.0, more preferably pH 6.0 to 7. .5 prevents non-specific cleavage of π-electrons that form double bonds between nitrogen in the pentacycle, which is the main skeleton of tetrazolium and its compounds, thereby preventing non-specific color development over time. Can be suppressed.

Examples of the surfactant used in the present invention include fatty acid salts, alpha sulfo fatty acid ester salts, alkylbenzene sulfonates, alkyl sulfates, alkyl ether sulfate esters, alkyl sulfate triethanolamines, fatty acid diethanolamides, polyoxyethylene alkyl ethers. Polyoxyethylene alkylphenyl ether and alkylbetaine are preferable.

Examples of polyoxyethylene lauryl ethers include Emulgen (registered trademark) 104P, Emulgen (registered trademark) 105, Emulgen (registered trademark) 106, Emulgen (registered trademark) 108, Emulgen (registered trademark) 109P, Emulgen (registered trademark) 120, Emulgen (registered trademark) 123P, Emulgen (registered trademark) 147, Emargen (registered trademark) 130K, Nonion (registered trademark) K-204, Nonion (registered trademark) K-215, Nonion (registered trademark) K-220, Nonion ( Registered trademark) K-230, NIKKOL (registered trademark) BL-2, NIKKOL (registered trademark) BL-4.2, NIKKOL (registered trademark) BL-9EX, NIKKOL (registered trademark) BL-21, NIKKOL (registered trademark) BL-25, polyoxyethylene cetyl As ethers, Emulgen (registered trademark) 210, Emulgen (registered trademark) 220, NIKKOL (registered trademark) BC-2, NIKKOL (registered trademark) BC-5.5, NIKKOL (registered trademark) BC-7, NIKKOL (registered) Trademarks) BC-10TX, NIKKOL (registered trademark) BC-15TX, NIKKOL (registered trademark) BC-20TX, NIKKOL (registered trademark) BC-23, NIKKOL (registered trademark) BC-25TX, NIKOL (registered trademark) BC-30X NIKKOL (registered trademark) BC-40TX, nonion (registered trademark) P-208, nonion (registered trademark) P-210, nonion (registered trademark) P-213, and polyoxyethylene stearyl ethers as Emulgen (registered trademark) 306P, Emargen (registered merchant ) 320P, NIKKOL (registered trademark) BS-2, NIKKOL (registered trademark) BS-4, NIKKOL (registered trademark) BS-20, nonion (registered trademark) S-206, nonion (registered trademark) S-207, nonion ( (Registered Trademark) S-215, Nonion (Registered Trademark) S-220, Polyoxyethylene Oleyl Ethers include Emulgen (Registered Trademark) 404, Emulgen (Registered Trademark) 408, Emulgen (Registered Trademark) 409P, and Emulgen (Registered Trademark) ) 420, Emulgen (registered trademark) 430, NIKKOL (registered trademark) BO-2, NIKKOL (registered trademark) BO-7, NIKKOL (registered trademark) BO-10TX, NIKKOL (registered trademark) BO-20, NIKKOL (registered trademark) ) BO-50, Nonion (registered trademark) E-206, NION (registered trademark) E-215, NONION (registered trademark) E-230, and polyoxyethylene behenyl ethers include NIKKOL (registered trademark) BB-5, NIKKOL (registered trademark) BB-10, NIKKOL (registered trademark). BB-20, NIKKOL (registered trademark) BB-30, and the like.

In addition, as polyoxyethylene secondary alkyl ethers, Emulgen (registered trademark) 707, NIKKOL (registered trademark) BT-5, NIKKOL (registered trademark) BT-7, NIKKOL (registered trademark) BT-9, Adecatol (registered) Trademark) SO-80, Adecatol (registered trademark) SO-105, Adecatol (registered trademark) SO-120, Adecatol (registered trademark) SO-135, Adecatol (registered trademark) SO-145, Adecatol (registered trademark) SO-160 , Emulgen (registered trademark) 705, Emulgen (registered trademark) 707, Emargen (registered trademark) 709, and the like. Examples of polyoxyethylene alkylphenyl ethers include Emulgen (registered trademark) 810, Emulgen (registered trademark) 840S, Emulgen (registered trademark) 909, Emulgen (registered trademark) 910, Emulgen (registered trademark) 930, and Emulgen (registered trademark). 950, Triton X-100, Triton X-114, NIKKOL (registered trademark) NP-5, NIKKOL (registered trademark) NP-7.5, NIKKOL (registered trademark) NP-10, NIKKOL (registered trademark) NP-15, NIKKOL (registered trademark) NP-20, NIKKOL (registered trademark) OP-10, NIKKOL (registered trademark) OP-30, and the like. Examples of polyoxyethylene styryl phenyl ethers include Emulgen (registered trademark) A60, Emulgen (registered trademark) A90, and Emulgen (registered trademark) B66. As oxyethylene / oxypropylene block copolymers, Emulgen (registered trademark) PP-150, Emulgen (registered trademark) PP-230, Emargen (registered trademark) PP-250, Emargen (registered trademark) PP-290, NIKKOL (registered) Trademark) PBC-34, NIKKOL (registered trademark) PBC-44, and the like. Examples of polyoxypropylene (2) polyoxyethylene decyl ethers include EMALEX (registered trademark) DAPE0207, EMALEX (registered trademark) DAPE0210, EMALEX (registered trademark) DAPE0212, EMALEX (registered trademark) DAPE0215, Emalex ( (Registered trademark) DAPE0220, Emarex (registered trademark) DAPE0220, and the like. Examples of fatty acid esters include Rheodor (registered trademark) TW-L120, Rhedol (registered trademark) TW-L106, Rhedol (registered trademark) TW-P120, Rheodor (registered trademark) TW-S120, Rhedol (registered trademark). ) TW-O120, Rheodor (registered trademark) 460, Emanon (registered trademark) 1112, Emanon (registered trademark) 3115, Emanon (registered trademark) 3170, Emanon (registered trademark) 3299, Emanon (registered trademark) 3130

Examples of the polyoxyethylene sterols include NIKKOL (registered trademark) BPS-10, NIKKOL (registered trademark) BPS-20, NIKKOL (registered trademark) BPS-30, NIKKOL (registered trademark) PSH-25, NIKKOL (registered trademark) DHC. -30 etc. are mentioned. Others include n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside, N, N-bis (3-D-gluconoamidopropyl) colamide, N, N-bis (3-D -Gluconoamidopropyl) deoxycolamide, n-octanoyl-N-methylglucoamide, n-nonanoyl-N-methylglucoamide, n-decanoyl-N-methylglucoamide, sucrose monocaprate, sucrose monolaurate , Sucrose monocholate, digitonin and the like.

Furthermore, alkylbetaines, alkylimidazolium betaines, alkylamidobetaines, alkylalanines, alkylamine oxides, derivatives thereof and the like can be mentioned. These Amphitol (registered trademark) 20BS, Amphitol (registered trademark) 24B, Amphitol (registered trademark) 86B, Amphitol (registered trademark) 20Z, 3-[(3-Colamidopropyl) dimethylammonio] propanesulfone Acid, 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid, and the like.

The concentration of the surfactant is not particularly specified, but in a reagent containing a reducing dye in order to suppress a decrease in absorbance due to the action of specific or non-specific formazan and the protein in the sample. It is preferable to add a surfactant in an amount of 0.01% by weight or more, preferably 0.05% by weight or more, and more preferably 0.1% by weight or more.

Further, among the surfactants, the HLB value is particularly preferably 8.8 to 19.1, more preferably 11.0 to 19.1, and the molecular weight is 167 to 1200. And those containing 12 or more hydrogen atoms and 23 or more hydrogen atoms are preferred.

Examples of the preservative used in the present invention include azides, chelating agents, antibiotics, and antibacterial agents. Among them, sodium azide is suitable, and is 0.001 to 1% by weight, preferably 0.01 to 0.1% by weight in the reaction solution.

Examples of the stabilizer used in the present invention include cyclodextrin and modified products thereof, cluster dextrin, calixarene derivative, ethylenediaminetetraacetate, aminosulfonic acid compound, smectite and other swellable layered clay minerals, borate, catalase, etc. There is. Among them, ethylenediaminetetraacetate is suitable, and 0.01 to 10 mmol / L, preferably 0.05 to 2 mmol / L in the reaction solution.
In addition, when a calixarene derivative such as calixarene sulfate (6) and calixarene sulfate (8) and dextrins such as G1αCD, G2αCD, G1βCD, and G2βCD are used as a dye stabilizer, in order to suppress nonspecific color development It is preferable to add the reagent separately from the reagent containing the reducing dye.

In order to avoid the influence of ascorbic acid in the biological sample, examples of the source of ascorbate oxidase used in the present invention include Cucumis sp., Cucurbita sp. And Acremonium sp. The reaction solution is preferably used at a concentration of 0.01 to 100 U / L, preferably 0.05 to 50 U / L. The pH of the reagent containing ascorbate oxidase is preferably less than 8.0 in consideration of the stability and activity of the enzyme. That is, in this invention, it is preferable to make it contain in the reagent containing a tetrazolium salt.

Examples of agents for reducing and avoiding the effects of LDH in biological samples include oxalic acid, oxamic acid and their salts, substances generated from iodine, silver, mercury and their respective ions, p-chloromercuribenzoate, NAD + or NADH, etc. Can be mentioned. In the reaction solution, it is preferably used at a concentration of 0.01 g / L or more, preferably 0.05 g / L or more, more preferably 0.1 g / L or more.

Examples of the reagent kit according to the present invention include the following, but are not limited thereto.

Kit 1 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier, LDH inhibitor Second reagent Reagent containing dehydrogenase acting on specific components in biological sample

Kit 2 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier Second reagent Reagent that acts on specific components in biological sample, reagent containing LDH inhibitor

Kit 3 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, LDH inhibitor Second reagent Reagent that acts on specific components in biological sample, reagent containing electron carrier

Kit 4 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, ascorbate oxidase, electron carrier, LDH inhibitor Second reagent Reagent that acts on specific components in biological sample, reagent containing electron acceptor

Kit 5 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase Second reagent Reagent containing dehydrogenase, electron carrier, LDH inhibitor acting on specific components in biological sample

Kit 6 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron carrier, ascorbate oxidase, reagent containing dehydrogenase, electron acceptor and LDH inhibitor acting on specific components in biological sample

Kit 7 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, ascorbate oxidase, LDH inhibitor Second reagent Reagent containing dehydrogenase, electron acceptor and electron carrier acting on specific components in biological sample

Kit 8 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt and ascorbate oxidase Second reagent Reagent containing dehydrogenase, electron carrier, LDH inhibitor and electron acceptor acting on specific components in biological sample

Kit 9 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
A reagent containing a tetrazolium salt, an electron acceptor, ascorbate oxidase, an electron carrier, an LDH inhibitor, a dehydrogenase that acts on a derivative of a specific component of a biological sample, a second reagent, an enzyme that acts on a specific component in a biological sample Reagents contained

Kit 10 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, electron acceptors, ascorbate oxidase, electron carriers, dehydrogenases that act on derivatives of specific components of biological samples Second reagents Enzymes that act on specific components in biological samples, LDH inhibitors Reagents contained

Kit 11 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, LDH inhibitor, dehydrogenase that acts on derivatives of specific components of biological samples Second reagent Enzymes that act on specific components in biological samples, electron carriers Reagents contained

Kit 12 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, ascorbate oxidase, electron carriers, LDH inhibitors, dehydrogenases that act on derivatives of specific components of biological samples Second reagents Enzymes that act on specific components in biological samples, electron acceptors Reagents contained

Kit 13 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, electron acceptors, ascorbate oxidase, dehydrogenases that act on derivatives of specific components of biological samples Second reagents Enzymes that act on specific components in biological samples, electron carriers, LDH inhibitors Reagents contained

Kit 14 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, electron carriers, ascorbate oxidase, dehydrogenases that act on derivatives of specific components in biological samples Second reagents Enzymes that act on specific components in biological samples, electron receptors, LDH inhibitors Containing reagents

Kit 15 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, ascorbate oxidase, LDH inhibitors, reagents containing dehydrogenases that act on derivatives of specific components of biological samples Second reagents Enzymes that act on specific components in biological samples, electron acceptors , A reagent containing an electron carrier

Kit 16 (reaction pH 7.5 or higher)
1st reagent (pH 8.0 or less) tetrazolium salt, ascorbate oxidase, a reagent containing a reagent containing dehydrogenase that acts on a derivative of a specific component of a biological sample 2nd reagent acts on a specific component in a biological sample Reagent containing enzyme, electron carrier, LDH inhibitor, electron acceptor

Kit 17 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier, LDH inhibitor, enzyme acting on specific component in biological sample Second reagent Dehydrogenase acting on derivative of specific component of biological sample Reagents contained

Kit 18 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier, reagent containing an enzyme that acts on a specific component in a biological sample Second reagent Dehydrogenase that acts on a derivative of a specific component in a biological sample, LDH inhibitor Reagents contained

Kit 19 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salts, electron acceptors, ascorbate oxidase, LDH inhibitors, reagents containing enzymes that act on specific components in biological samples, dehydrogenases that act on derivatives of specific components in biological samples, electron carriers Reagents contained

Kit 20 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, ascorbate oxidase, electron carrier, LDH inhibitor, reagent containing an enzyme that acts on a specific component in a biological sample Second reagent Dehydrogenase that acts on a derivative of a specific component in a biological sample, an electron acceptor Reagents contained

Kit 21 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, electron acceptors, ascorbate oxidase, enzymes that act on specific components in biological samples Second reagents Dehydrogenases, electron carriers, and LDH inhibitors that act on derivatives of specific components in biological samples Reagents containing

Kit 22 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron carrier, ascorbate oxidase, enzyme acting on specific component in biological sample Second reagent Contains dehydrogenase electron acceptor acting on derivative of biological component, LDH inhibitor Reagent


Kit 23 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, ascorbate oxidase, LDH inhibitors, reagents containing enzymes that act on specific components in biological samples Second reagents Dehydrogenases that act on derivatives of specific components in biological samples, electron acceptors , A reagent containing an electron carrier

Kit 24 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagents containing tetrazolium salts, ascorbate oxidase, enzymes that act on specific components in biological samples Second reagents Dehydrogenases, electron carriers, LDH inhibitors, electron acceptors that act on derivatives of specific components in biological samples Reagents containing reagents

Kit 25 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent second reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier, LDH inhibitor, enzyme acting on specific component in biological sample, dehydrogenase acting on derivative of specific component of biological sample A reagent containing a buffer adjusted so that the pH during the reaction is 7.5 or more

Kit 26 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent second reagent LDH inhibitor containing tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier, enzyme acting on specific component in biological sample, dehydrogenase acting on derivative of specific component of biological sample Reagents contained

Kit 27 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Contains reagent second reagent electron carrier containing tetrazolium salt, electron acceptor, ascorbate oxidase, LDH inhibitor, dehydrating enzyme acting on specific component in biological sample, acting on derivative of specific component of biological sample reagent

Kit 28 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent second reagent electron acceptor containing tetrazolium salt, ascorbate oxidase, electron carrier, LDH inhibitor, dehydrogenase acting on derivative of specific component of biological sample, enzyme acting on specific component in biological sample Reagents containing

Kit 29 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, electron acceptor, ascorbate oxidase, dehydrogenase that acts on derivatives of specific components in biological samples, reagent second reagent containing an enzyme that acts on specific components in biological samples, electron carrier, LDH inhibitor Reagents containing

Kit 30 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent second reagent electron acceptor containing tetrazolium salt, electron carrier, ascorbate oxidase, enzyme acting on specific component in biological sample, reagent containing dehydrogenase acting on derivative of specific component of biological sample, Reagent containing LDH inhibitor

Kit 31 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, ascorbate oxidase, LDH inhibitor, reagent containing an enzyme that acts on a specific component in a biological sample, reagent second reagent that contains a dehydrogenase acting on a derivative of a specific component of a biological sample, electron acceptor , A reagent containing an electron carrier

Kit 32 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, ascorbate oxidase, an enzyme that acts on a specific component in a biological sample, a reagent containing a dehydrogenase that acts on a derivative of a specific component of a biological sample, an electron carrier, an LDH inhibitor, and an electron acceptor Reagents containing reagents

Kit 33 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, electron acceptor, ascorbate oxidase, electron carrier, reagent containing LDH inhibitor Second reagent Dehydrogenase that acts on specific components in biological samples, Dehydrogenase that acts on derivatives of specific components of biological samples Reagents containing enzymes

Kit 34 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, electron acceptor, ascorbate oxidase, reagent containing electron carrier Second reagent Dehydrogenase that acts on specific components in biological samples, dehydrogenase that acts on derivatives of specific components of biological samples, LDH inhibition Reagents containing agents

Kit 35 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase, LDH inhibitor Second reagent Dehydrogenase acting on specific components in biological samples, Dehydrating enzyme acting on derivatives of specific components of biological samples, carriers Reagents contained

Kit 36 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, ascorbate oxidase, electron carrier, LDH inhibitor Second reagent Dehydrogenase acting on specific components in biological sample, Dehydrating enzyme acting on derivatives of specific components of biological sample, electron acceptor Reagents containing

Kit 37 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron acceptor, ascorbate oxidase Second reagent Dehydrogenase acting on specific components in biological samples, Dehydrating enzyme acting on derivatives of specific components of biological samples, electron carriers, LDH inhibitors Reagents containing reagents containing

Kit 38 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, electron carrier, reagent containing ascorbate oxidase Second reagent Dehydrogenase acting on specific components in biological samples, Dehydrating enzyme acting on derivatives of specific components of biological samples, electron acceptors , A reagent containing an LDH inhibitor

Kit 39 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Reagent containing tetrazolium salt, ascorbate oxidase, LDH inhibitor Second reagent Dehydrogenase acting on specific components in biological samples, Dehydrating enzyme acting on derivatives of specific components of biological samples, electron acceptors, electron carriers Reagents containing

Kit 40 (reaction pH 7.5 or higher)
First reagent (pH 8.0 or less)
Tetrazolium salt, reagent containing ascorbate oxidase Second reagent Dehydrogenase acting on specific components in biological samples, Dehydrating enzyme acting on derivatives of specific components of biological samples, electron carriers, LDH inhibitors, electron acceptors Reagents containing reagents containing

The solution containing the reducing pigment of the present invention can be provided in the form of filling into a glass bottle, a plastic container or the like, but it is more desirable to shield these containers from light.

Although the calibration method (calibration method) with the calibration curve in the present invention is not particularly limited, [1] purified water or physiological saline,
[2] It is preferable to use a standard substance or standard solution having a concentration of one level or more.

The calibration method is not limited to a straight line method or a spline method, but the straight line method is particularly preferable.

The analysis method is not limited to the following method, but is preferably any of the following [1], [2], and [3].
[1] One-point method; end-point method in which a reagent is added and the absorbance is measured after a certain time [2] two-point method; end-point method in which the difference is measured twice and the difference absorbance is obtained [3 ] Rate method; a method for determining the change in absorbance per unit time between specified photometric points

Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.
Examples of the enzyme used in this example include, but are not limited to, the following.
As leucine dehydrogenase, those derived from Bacillus SP and Bacillus stearothermophilus can be obtained from Toyobo and Unitika. Examples of diaphorase include Clostridium SP and Bacillus stearothermophilus, which are available from Toyobo and Unitika. Further, in the present invention, for the purpose of avoiding the influence of ascorbic acid in the biological sample, ascorbate oxidase may be contained, and those derived from Cucumis SP or Cucurita SP are available from Toyobo, Roche and Asahi Kasei.

[Examples 1 to 6 and Comparative Examples 1 to 6] Relationship between the arrangement of the reducing dye and the degree of increase in the reagent blank due to nonspecific color development (Table 1, FIG. 1)
A total branched chain amino acid (hereinafter referred to as BCAA) measurement reagent having the following composition was prepared (1,5-anhydro-D-glucitol (1,5-AG) measurement reagent only in Comparative Example 6).

Example 1 Reaction pH 7.5
First reagent (pH 6.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 2 Reaction pH 7.75
First reagent (pH 6.5)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Roche) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 3) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 15g / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 4) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene octyl phenyl ether (Nippon Yushi Co., Ltd.) 25g / L
Diaphorase (Unitika) 10U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene octyl phenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 5) Reaction pH 8.3
First reagent (pH 7.5)
PIPES buffer 50 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 5g / L
1-M-PMS 0.1 μmol / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 6) Reaction pH 8.5
First reagent (pH 8.0)
PIPES buffer 50 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 5g / L
1-M-PMS 0.1 μmol / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Comparative Example 1) Reaction pH 8.2
First reagent (pH 8.2)
Bicine buffer 50 mmol / L
Polyoxyethylene pt-octylphenyl ether (Nacalai Tesque) 20g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 8.2)
Bicine buffer 150 mmol / L
Polyoxyethylene pt-octylphenyl ether (Nacalai Tesque) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 100 U / mL

(Comparative Example 2) Reaction pH 7.6
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene pt-octylphenyl ether (Nacalai Tesque) 20g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Second reagent (pH 8.2)
Bicine buffer 150 mmol / L
Polyoxyethylene pt-octylphenyl ether (Nacalai Tesque) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Comparative Example 3) Reaction pH 7.8
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent (pH 8.5)
Bicine buffer 150 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Comparative Example 4) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
N, N-Bis (3-D-gluconamidopropyl) cholamide (Dojin Chemical Co., Ltd.) 20g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
N, N-Bis (3-D-glucanamidopropyl) cholamide (Dojin Chemical Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Comparative Example 5) Reaction pH 8.3
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
n-Octanoyl-N-methyl-D-glucamine (Dojin Chemical Co., Ltd.) 20 g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent (pH 9.5)
CHES buffer 150 mmol / L
n-Octanoyl-N-methyl-D-glucamine (Dojin Chemical Co., Ltd.) 1 g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Comparative Example 6) Reaction pH 8.1
First reagent (pH 6.8)
Buffer
ADP-dependent hexokinase 3U / mL
Diaphorase 9U / mL
Adenosine-5′-diphosphate (ADP) 0.4 mmol / L
Oxidized NADP + 9.2mmol / L
Second reagent (pH 8.7)
Buffer
AG-6-phosphate dehydrogenase 80U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

The BCAA measurement reagents of Examples 1 to 6 and Comparative Examples 1 to 5 and the 1,5AG measurement reagent of Comparative Example 6 were stored at 35 ° C. for 7 days, and the difference in reagent blank from each reagent immediately after preparation was examined. .

Examples 1-6, BCAA measurement reagents of Comparative Examples 1-5, and 1,5AG reagent of Comparative Example 6 immediately after preparation and reagents stored for 7 days at 35 ° C., respectively, purified water as a sample by the following measurement method Was repeatedly measured at n = 3, and a reagent blank was determined from the average value.

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample (2.0 μL only for Comparative Example 6), and incubated at 37 ° C. for 5 minutes to form the first reaction. Thereafter, 40 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The measurement wavelength was measured by the two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume, and the absorbance at the main wavelength of 450 nm was measured for all the reagents in Examples 1 to 6 and Comparative Examples 1 to 6. .

Table 1 and FIG. 1 show the relationship between the arrangement of the reducing dye and the degree of increase in the reagent blank due to non-specific color development.
In Examples 1 to 6 in which the reducing dye was contained in the first reagent, the increase in the reagent blank was 18 mABS or less, whereas in Comparative Examples 1 to 6 in which the reducing dye was contained in the second reagent, it was 40 mABS or more. It was. (In particular, in Example 3 and Comparative Example 4, the first test solution and the second test solution are compared under the same pH conditions.)

[Examples 7 to 14 and Comparative Examples 7 and 8] Relationship between pH and sensitivity during mixing of the first reagent and the second reagent (Table 2, FIG. 2)
A reagent for measuring total branched chain amino acid (hereinafter referred to as BCAA) having the following composition was prepared.

Example 7 Reaction pH 7.5
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 8.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 8 Reaction pH 7.6
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Roche) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 8.5)
TAPS buffer 160 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 9 Reaction pH 7.75
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 15g / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 160 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 10 Reaction pH 7.9
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene octyl phenyl ether (Nippon Yushi Co., Ltd.) 25g / L
Diaphorase (Unitika) 10U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 154 mmol / L
Polyoxyethylene octyl phenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 11 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 5g / L
1-M-PMS 0.1 μmol / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 12) Reaction pH 8.5
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 5g / L
1-M-PMS 0.1 μmol / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.5)
CHES buffer 141 mmol / L
Polyoxyethylene sorbitan fatty acid ester (Nacalai Tesque Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 13) Reaction pH 9.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Roche) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 10.0)
CAPSO buffer 133 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 14 Reaction pH 9.5
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Roche) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 10.0)
CAPSO buffer 118 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Comparative Example 7) Reaction pH 7.25
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Roche) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 7.5)
TES buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Comparative Example 8) Reaction pH 7.4
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Roche) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 8.0)
TAPS buffer 154 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

For the BCAA measurement reagents of Examples 7 to 14 and Comparative Examples 7 and 8, a standard solution and purified water were repeatedly measured as samples by the following measurement method at n = 2, and the standard solution of each reagent was determined from the average value. The sensitivity of was calculated from the following equation. Note that an L-isoleucine solution (500 μmol / L) was used as the standard solution.

Sensitivity of standard solution (mABS) = absorbance of standard solution (mABS) −absorbance of reagent blank (mABS)

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample, and incubated at 37 ° C. for 5 minutes to prepare the first reaction. Thereafter, 40 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The measurement wavelength is measured by the two-point end method in which the absorbances of the first reaction and the second reaction are corrected for the liquid volume, and the absorbance is measured at the main wavelength of 450 nm in Examples 7 to 14 and Comparative Examples 6 and 7. did.

The relationship between the pH and sensitivity during mixing of the first reagent and the second reagent is shown in Table 2 and FIG.
In Examples 7 to 14 where the pH at the time of mixing the first reagent and the second reagent was 7.5 or more, the sensitivity was 150 mABS or more, whereas the pH at the time of mixing the first reagent and the second reagent was In Comparative Examples 7 and 8, which were less than 7.5, the sensitivity was about 140 mABS or less.
Moreover, the sensitivity became higher as the pH at the time of mixing the first reagent and the second reagent was higher than 7.5.

Next, what is the value of the increase and sensitivity of the reagent blank due to non-specific color development for the combination of “reducing dye arrangement” and “pH when mixing the first and second reagents”? Are summarized in Table 3.
Examples 1 to 6 and Comparative Examples 1 to 6 in Table 3 are the same as those in Table 1.
Table 3 further shows the numerical value of the degree of increase in the reagent blank with respect to the sensitivity obtained from the above results.

[About the arrangement of reducing dyes]
In the present invention, in order to realize high-accuracy and stable measurement in non-calibration measurement, it is preferable that the blank increase degree due to storage is within 10% of the sensitivity.
As can be seen from Table 3, in Examples 1 to 6 in which the reducing dye is arranged in the first test solution, the blank increasing degree due to storage is within 10% of the sensitivity, whereas the reducing dye is the first. In Comparative Examples 1 to 6 arranged in the second reagent, the degree of increase of the storage blank with respect to sensitivity is as high as 18 to 72%. As represented by Comparative Examples 1 to 6, in the reagent composition in which the degree of increase of the storage blank exceeds 10% with respect to the sensitivity during storage at 35 ° C. for 7 days, even in non-calibration measurement, it is 35 ° C. As with the day storage, the blank rises with time, and the rise of the storage blank becomes unacceptable.

[About pH when mixing first reagent and second reagent]
Regarding the pH at the time of mixing the first reagent and the second reagent, as described in Table 2 and FIG. 2, the sensitivity became higher as the pH increased beyond 7.5.
On the other hand, although no data is described, when the reaction pH is 7.5 or less, particularly 7.0 or less, the sensitivity of the reducing dye is lowered and the enzyme activity of the leucine dehydrogenase used in the present invention is low. And the reaction represented by (Formula 1) in the present invention is delayed. Therefore, measurement stability and high accuracy are impaired.

On the other hand, in the biological sample, lactic acid or pyruvic acid is contained, but when lactic acid dehydrogenase is further contained, the reaction represented by Formula 5 occurs. In particular, the reaction from lactic acid to pyruvic acid (hereinafter referred to as L → P reaction) is promoted at pH 8.5 to 9.0, and the reaction from pyruvic acid to lactic acid (hereinafter referred to as P → L reaction) is promoted at pH 7 to 8. In particular, the L → P reaction competes with the oxidation-reduction reaction represented by (Formula 1) of the present invention. Therefore, it is important to increase the reactivity of leucine dehydrogenase and suppress the reactivity of lactate dehydrogenase. In the present invention, it has been confirmed that when the reaction pH is 7.5 or higher, preferably 8.0 or higher, the competitive reaction of lactate dehydrogenase can be suppressed and the redox reaction of the present invention can be enhanced.

（式５）

(Formula 5)

Furthermore, when the activity of diaphorase preferably used as an electron carrier in the present invention is taken into account, the diaphorase activity is remarkably reduced at a reaction pH of 10 or more.
Therefore, the pH at the time of mixing the first reagent solution and the second reagent solution, that is, the pH at the time of reaction, is pH 7.5 or more, more preferably pH 7.5 to pH 10.0, and further preferably pH 7.5 to pH 9.5. More preferably, the pH is preferably 8.0 to 9.5.

Furthermore, with respect to the combination of “pH of the first reagent in which the reducing dye is arranged” and “pH at the time of mixing the first reagent and the second reagent”, the degree of increase in the reagent blank due to non-specific color development, measurement sensitivity Table 4 summarizes whether high-accuracy and stable measurement in non-calibration measurement is possible.

From Table 4, the pH of the first test solution containing the reducing dye is pH 6.0 to 8.0, and the pH at the time of mixing the first test solution and the second test solution containing leucine dehydrogenase is pH 7.5 or more. By doing so, it can be seen that it is possible to achieve both long-term stability in liquid form and high-accuracy measurement.

In order to realize this highly accurate and stable measurement, it is necessary to set the pH during the reaction to pH 7.5 or higher for the purpose of increasing sensitivity, but the pH of the first reagent solution containing the reducing dye is pH 6.0. If it is less than the above, the blank rise due to storage is good, but in order to make the sensitivity at the time of reaction 7.5 or more, the pH of the test solution not containing the reducing dye is set high as pH 10-11, or When the pH is set to 8 to 10, it is necessary to increase the buffer concentration. The former may reduce the long-term storage stability of the contained enzyme in the liquid state, and the latter may similarly reduce the enzyme stability and reactivity because the ionic strength increases.
Moreover, when the pH is less than 5.5, there is no good buffer having a good buffering capacity, so that the stability of enzymes and dyes during long-term storage becomes a problem.
Moreover, although diaphorase is suitably used as an electron carrier in the present invention, when the enzyme is stored in a test solution containing a reducing dye, the storage stability is significantly lowered at a pH of less than 6.0. In the present invention, ascorbic acid oxidase which is preferably used for the purpose of avoiding the influence of ascorbic acid in a biological sample is preferably added to the first reagent containing a reducing dye, but has a pH of 6.0. If the ratio is less than 1, the storage stability in the liquid state is significantly lowered.
Therefore, in the present invention, in order to suppress the rise of the reagent blank due to non-specific color development of the reducing dye, the pH of the first reagent containing the reducing dye is pH 5.5 to 8.0, preferably pH 6 It is preferable that it is 0.0-8.0.

  Further, regarding the addition position of the reducing dye, when it is arranged in the second reagent, as in the case of adding to the first reagent, if the pH of the second reagent is adjusted to pH 8.0 or less, the non-specification of the reducing dye is not performed. Although the blank rise due to color development is suppressed, it is necessary to set the pH of the first test solution to be neutral or higher in order to set the reaction pH to 7.5 or higher. In this case, long-term storage stability of ascorbate oxidase and diaphorase contained in the first test solution is lowered. Further, since the long-term storage stability and activity of the leucine dehydrogenase in the second test solution are reduced by reducing the pH of the second test solution to pH 8.0 or less, a reducing dye is included in the second test solution. That is not preferable.

  From the above, in order to achieve the present invention, the reducing dye is contained in the first test solution, and the pH of the first test solution is pH 5.5 to 8.0, preferably pH 6.0 to 8.0. It is essential.

[Example 15 and Comparative Examples 9 and 10] Regarding the rise of the reagent blank when the reagent was stored for a long time (Table 5, FIG. 3) (same as Example 16 and Comparative Examples 11 and 12 described later) Study using composition reagents)
A reagent for measuring total branched chain amino acid (hereinafter referred to as BCAA) having the following composition was prepared.
(Example 15) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Comparative Example 9) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

Comparative Example 10 Diamond Color / BTR Total Branched Chain Amino Acid Measuring Reagent (Freeze-dried)
[1] Enzyme reagent BR1
1-methoxy-5-methylphenadium methyl sulfate nicotinamide adenine dinucleotide [2] enzyme solution BR1
Tris-HCl buffer (pH 7.5)
[3] Enzyme reagent BR2
Leucine dehydrogenase [4] enzyme solution BR2
3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide first reagent: a test solution (pH 7.5) in which [1] is dissolved in [2]
Second reagent: Reagent obtained by dissolving [3] in [4]

The BCAA measurement reagents of Example 15 and Comparative Examples 9 and 10 were opened and stored for 10 weeks (71 days) in the reagent table (10 ° C. or lower) of the H7180 automatic analyzer, and the first day, the first week, At 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks, purified water was repeatedly measured as a sample by the following measurement method, and n = 3, and the average value of n = 3 measurements More reagent blanks were obtained.

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample (1.7 μL in Comparative Example 10), and incubated at 37 ° C. for 5 minutes to form the first reaction. Thereafter, 40 μL of the second reagent (30 μL in Comparative Example 10) was added and incubated for 5 minutes to form the second reaction. A two-point end method is used in which the absorbances of the first reaction and the second reaction are corrected for the liquid volume, and the measurement wavelength is the absorbance at the main wavelength of 450 nm for both the reagents of Example 15 and Comparative Example 9. Comparative Example 10 measured the absorbance at a dominant wavelength of 570 nm.

The results of the increase in the reagent blank when the reagent is stored for a long period of time are shown in Table 5 and FIG.
In Comparative Examples 9 and 10, the increase in the reagent blank due to nonspecific color development was observed over time, and at the stage of the 10th week, the degree of increase in the reagent blank due to nonspecific color development was 98 in Comparative Example 9 compared with that at the start. .9 mABS, which is 20.1 mABS in Comparative Example 10, whereas Example 15 shows no non-specific color development over time, and the increase in reagent blanks due to non-specific color development at the 10th week is also started. Compared to the time, it was as good as about 3.6 mABS, indicating the usefulness of this method.

[Example 16 and Comparative Examples 11 and 12] Regarding the precision and stability of measured values in non-calibration measurement when the reagent was stored for a long period of time (Table 6, FIGS. 4 to 6) (Example 15 described above, And studies using reagents of the same composition as Comparative Examples 9 and 10)
A reagent for measuring total branched chain amino acid (hereinafter referred to as BCAA) having the following composition was prepared.
Examples 15 and 16, Comparative Examples 9 and 11, and Comparative Examples 10 and 12 are the same reagent.
(Example 16)
First reagent (pH 7.0) Reaction pH 8.0
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Comparative Example 11) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

Comparative Example 12 Diamond Color / BTR Total Branched Chain Amino Acid Measuring Reagent (Freeze-dried)
[1] Enzyme reagent BR1
1-methoxy-5-methylphenadium methyl sulfate nicotinamide adenine dinucleotide [2] enzyme solution BR1
Tris-HCl buffer (pH 7.5)
[3] Enzyme reagent BR2
Leucine dehydrogenase [4] enzyme solution BR2
3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide first reagent: a test solution (pH 7.5) in which [1] is dissolved in [2]
Second reagent: Reagent obtained by dissolving [3] in [4]

The reagent for BCAA measurement of Example 16 and Comparative Examples 11 and 12 was opened and stored for 10 weeks (71 days) in the reagent table (10 ° C. or lower) of the H7180 automatic analyzer, and the first day, the first week, In the second week, the third week, the fourth week, the sixth week, the eighth week, and the tenth week, liquid nescoal N, liquid nescoal A (Alfresa Pharma), QAP Troll IIX (as a sample) by the following measurement method were used. Sysmex) was measured repeatedly at n = 5.

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample (1.7 μL in Comparative Example 12), and incubated at 37 ° C. for 5 minutes for the first reaction. Thereafter, 40 μL of the second reagent (30 μL in Comparative Example 12) was added and incubated for 5 minutes to form a second reaction. A two-point end method is used in which the absorbances of the first reaction and the second reaction are corrected for the liquid volume, and the measurement wavelength is the absorbance at the main wavelength of 450 nm for both the reagents of Example 16 and Comparative Example 11. Comparative Example 12 measured the absorbance at a dominant wavelength of 570 nm.

Also, the measurement is calibrated by the following method only for the first day measurement, and the subsequent measurement is a so-called non-calibration measurement method without calibration, and liquid nescoal N and liquid nescoal A (Alfresa Pharma Co., Ltd.) , QAP Troll IIX (Sysmex Corporation) was repeatedly measured at n = 5. The results show the average value of n = 5 measurements.

(Calibration method)
The purified water and 500 μmol / L L-isoleucine aqueous solution were measured twice, and the absorbance (sensitivity) obtained by subtracting the purified water measured absorbance from the 500 μmol / L L-isoleucine aqueous solution measured absorbance was calculated and corrected.

The results are shown in Table 6 and FIGS. 4, 5, and 6 for the precision of the measured values when the reagents are stored for a long period of time.
In Comparative Example 11, the serum measurement value increases due to the influence of the non-specific color development of the reducing dye over time. On the other hand, in Comparative Example 12, which is a lyophilized product, the serum measurement value increased due to the influence of non-specific color development of the reducing dye over time until the 4th week, and thereafter the storage stability of the enzyme decreased. Therefore, the serum measurement value decreases.
About Example 16, the fluctuation | variation of the serum value with time is not seen, It has shown that the highly accurate and long-term stable measurement by non-calibration is possible.

In addition, although data is not described as examples and comparative examples, the pH of the first reagent solution containing the reducing dye is pH 6.0 to 8.0, and the pH when the first reagent solution and the second reagent solution are mixed is pH 7. Under the condition of .5 or more, the same result as in Example 16 was obtained, and high-precision and stable measurement with non-calibration in the present invention was possible.

[Examples 17 to 34 and Comparative Example 13] Avoidance of influence of protein in biological sample on reducing dye by addition of surfactant (Tables 7 to 9, FIGS. 7 to 9)
(Example 17) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 18) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 5g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 19) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 20) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 15g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 21) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 22 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 30g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 23) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 24) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 5g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 25) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 10g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 26 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 15g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 27) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 20g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 28 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 30g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene nonylphenyl ether (Nippon Yushi Co., Ltd.) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 29 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 30) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 5g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 31 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 10g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 32) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 15g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

Example 33 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 20g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

(Example 34) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 30g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene lauryl ether (Kao Corporation) 1 g / L Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 1 U / mL

(Comparative Example 13) Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

QAP Troll IIX (Sysmex Corporation) was repeatedly measured at n = 5 using the BCAA measurement reagents of Examples 17 to 34 and Comparative Example 13 as samples by the following measurement method.

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample, and incubated at 37 ° C. for 5 minutes to prepare the first reaction. Thereafter, 40 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The absorbance at the main wavelength of 450 nm is measured for each of the reagents in Examples 17 to 34 and Comparative Example 13 by the two-point end method in which the absorbance of the first reaction and the second reaction is corrected for each liquid volume. did.

The results are shown in Tables 7 to 9 and FIGS. A value obtained by measuring the same sample with a BCAA measuring reagent (total branched chain amino acid measuring reagent) of a commercially available diamond color BTR was regarded as a true value. In addition, the diamond color BTR reagent has been approved for production, and the measurement is good in correlation with the HPLC method.
From the results, in the case where the surfactant is not added to the reagent containing the tetrazolium salt (Comparative Example 13), the measured value is lowered with respect to the true value, while the interface with the reagent containing the tetrazolium salt is reduced. The reagent containing the activator (Examples 17 to 34) was a good measured value with respect to the true value. The decrease in the measured value is due to the decrease in the color absorbance of the reducing dye due to the action of the reducing dye and a protein such as albumin in the sample. As shown in Examples 17 to 34, in order to suppress a decrease in absorbance due to the action of a protein such as formazan dye and albumin in the sample, a surfactant is added to the reagent containing a reducing dye in the present invention. .1% by weight or more, preferably 0.5% by weight or more.

The type of the surfactant is not particularly limited, but the concentration is preferably 5.0% by weight, more preferably 3.0%, in consideration of the stability of the enzyme in the same arrangement. preferable.
Therefore, in order to suppress a decrease in absorbance due to the action of the protein in the biological sample to the reducing dye, the additive concentration of the surfactant to the test solution containing the reducing dye is 0.1 to 5.0% by weight. The content is preferably 0.1 to 3.0% by weight, more preferably 0.5 to 3.0% by weight, and still more preferably 1.0 to 3.0% by weight.

[Example 35] Avoidance of influence of ascorbic acid (Table 10, Fig. 10)
A reagent for measuring total branched chain amino acid (hereinafter referred to as BCAA) having the following composition was prepared.
Example 35 Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

The BCAA measurement reagent of Example 35 was measured with the following measurement method, and the following samples were repeatedly measured at n = 2.

(sample)
QAP Trol IIX (Sysmex Corporation): L-isoleucine solution (5000 μmol / L) = 9: 1 mixed with ascorbic acid in the sample at final concentrations of 0 mg / dL, 20 mg / dL, 40 mg / dL, 60 mg / dL, Samples added to 80 mg / dL and 100 mg / dL.

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample, and incubated at 37 ° C. for 5 minutes to prepare the first reaction. Thereafter, 40 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The absorbance at the main wavelength of 450 nm was measured by a two-point end method in which the absorbance of the first reaction and the second reaction was corrected for the liquid volume and the difference between each absorbance was taken.

(Concentration conversion)
The L-isoleucine concentration in the sample was calculated from the results of the following calibration method.
Isoleucine concentration in sample (μmol / L) = (measured absorbance of sample) / (500 μmol / L measured absorbance of L-isoleucine aqueous solution) × (500 μmol / L)

(Calibration method)
The purified water and 500 μmol / L L-isoleucine aqueous solution were measured twice, and the absorbance (sensitivity) obtained by subtracting the purified water measured absorbance from the 500 μmol / L L-isoleucine aqueous solution measured absorbance was calculated and corrected.

The results are shown in Table 10 and FIG. In Example 35, it is shown that the influence of ascorbic acid in the sample can be avoided. Although not shown in the data, the same is true for the reagent in which the pH of the first reagent solution containing ascorbate oxidase and the reducing dye is pH 6.0 to 8.0 and the pH at the time of reaction is 7.5 or higher. Results were obtained.

In the present invention, ascorbate oxidase is used by being arranged in the first test solution in the same manner as the reducing dye. Furthermore, by adjusting the pH of the first reagent solution to pH 5.5 to 8.0, preferably pH 6.0 to 8.0, not only the nonspecific color development of the reducing reagent is suppressed, but also the ascorbic acid reagent solution It is also possible to maintain the storage stability in the inside.

[Example 36] Avoidance of effects of lactate dehydrogenase (Table 11, Fig. 11)
A reagent for measuring total branched chain amino acid (hereinafter referred to as BCAA) having the following composition was prepared.
(Example 36)
Reaction pH 8.0
First reagent (pH 7.0)
PIPES buffer 50 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 20g / L
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 5 U / mL
Oxidized NAD 10mmol / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Sodium oxamate 10mM
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L
Second reagent (pH 9.0)
TAPS buffer 150 mmol / L
Polyoxyethylene oleyl ether (Kao Corporation) 1g / L
Leucine dehydrogenase (LED-201 manufactured by Toyobo Co., Ltd.) 70 U / mL

For the BCAA measurement reagent of Example 36, the following samples were repeatedly measured at n = 2 by the following measurement method.

(sample)
In a mixed solution containing L-isoleucine (500 μmol / L) and lactic acid (20 mM), lactate dehydrogenase is added in a final concentration of 0 u / L, 2000 u / L, 4000 u / L, 6000 u / L, 8000 u / L, 10000 u / L in the sample. A sample added so that

(Measurement method)
A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 1.8 μL of the sample, and incubated at 37 ° C. for 5 minutes to prepare the first reaction. Thereafter, 40 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The absorbance at the main wavelength of 450 nm was measured by a two-point end method in which the absorbance of the first reaction and the second reaction was corrected for the liquid volume and the difference between each absorbance was taken.

(Concentration conversion)
The L-isoleucine concentration in the sample was calculated from the results of the following calibration method.
Isoleucine concentration in sample (μmol / L) = (measured absorbance of sample) / (500 μmol / L measured absorbance of L-isoleucine aqueous solution) × (500 μmol / L)

(Calibration method)
The purified water and 500 μmol / L L-isoleucine aqueous solution were measured twice, and the absorbance (sensitivity) obtained by subtracting the purified water measured absorbance from the 500 μmol / L L-isoleucine aqueous solution measured absorbance was calculated and corrected.

The results are shown in Table 11 and FIG. In the measurement system using the oxidation-reduction reaction, there is a problem that the catalytic reaction of lactic acid dehydrogenase in the sample from lactic acid to pyruvic acid or the catalytic reaction from pyruvic acid to lactic acid affects the measurement. In Example 36, it is shown that the influence of lactate dehydrogenase in the sample can be avoided by adding sodium oxamate, which is an inhibitor of lactate dehydrogenase.

Further, although not shown in the data, the same result was obtained with a reagent in which the pH of the first reagent solution containing the reducing dye was pH 6.0 to 8.0 and the pH at the time of reaction was 7.5 or higher. Obtained.

The liquid reagent for measuring total branched-chain amino acids of the present invention can be used in fields of application such as in vitro diagnostic drugs and contributes greatly to the industry.

The relationship between the reagent pH containing a reducing pigment and the reagent blank is shown (Examples 1-6, Comparative Examples 1-5). The vertical axis shows the degree of increase in reagent blank (mABS; milliabsorbance) compared with the reagent immediately after preparation after storage at 35 ° C. for 7 days. The horizontal axis indicates the pH of the reagent containing the reducing dye in each example and comparative example. The relationship between the reaction pH of the reagent and the sensitivity is shown (Examples 7 to 14, Comparative Examples 6 and 7). The vertical axis shows the measurement sensitivity (mABS; milli-absorbance) of the standard solution. The reagent blank raise degree with time of Example 15 and Comparative Examples 9 and 10 is shown. The horizontal axis is the number of days elapsed, and W in the figure indicates week (week). The vertical axis indicates the reagent blank, and mABS indicates milli absorbance. The fluctuation | variation with time of each serum measurement value in the non-calibration measurement of Example 16 is shown. The vertical axis indicates the total branched chain amino acid (BCAA) concentration (μmol / L) in serum. The horizontal axis is the number of days elapsed, and W in the figure indicates week (week). The time-dependent fluctuation | variation of each serum measured value in the non-calibration measurement of the comparative example 11 is shown. The vertical axis indicates the total branched chain amino acid (BCAA) concentration (μmol / L) in serum. The horizontal axis is the number of days elapsed, and W in the figure indicates week (week). The fluctuation | variation with time of each serum measurement value in the non-calibration measurement of Comparative Example 12 is shown. The vertical axis indicates the total branched chain amino acid (BCAA) concentration (μmol / L) in serum. The horizontal axis is the number of days elapsed, and W in the figure indicates week (week). The relationship between the surfactant addition concentration and the measured value with respect to the true value is shown (Examples 17 to 22, Comparative Example 13). A vertical axis | shaft shows the measured value (micromol / L) with respect to the BCAA density | concentration (micromol / L) in a biological sample (%). The horizontal axis indicates the surfactant addition concentration. The relationship between the surfactant addition concentration and the measured value with respect to the true value is shown (Examples 23 to 28, Comparative Example 13). A vertical axis | shaft shows the measured value (micromol / L) with respect to the BCAA density | concentration (micromol / L) in a biological sample (%). The horizontal axis indicates the surfactant addition concentration. The relationship between the surfactant addition concentration and the measured value with respect to the true value is shown (Examples 29 to 34, Comparative Example 13). A vertical axis | shaft shows the measured value (micromol / L) with respect to the BCAA density | concentration (micromol / L) in a biological sample (%). The horizontal axis indicates the surfactant addition concentration. The influence of ascorbic acid in the sample of Example 35 is shown. The vertical axis represents the concentration of L-isoleucine in the sample, and the horizontal axis represents the concentration of ascorbic acid added to the sample. The influence of lactate dehydrogenase in the sample of Example 36 is shown. The vertical axis represents the concentration of L-isoleucine in the sample, and the horizontal axis represents the concentration of lactate dehydrogenase added to the sample.

Claims (13)

A method for quantifying a specific component in a biological sample using a liquid reagent composed of two or more reagents,
In the presence of an electron acceptor, an enzyme having the ability to oxidize by dehydrogenating the specific component is allowed to act on a biological sample,
In the method of detecting and quantifying the reduced form of the generated electron acceptor by further forming an formazan by acting an electron carrier and a reducing dye,
A method for quantifying a component in a biological sample having all of the following configurations (a) to (c):
(A) Tetrazolium or a compound thereof is used as the reducing dye.
(B) The reagent containing the reducing dye has a pH of 8.0 or less.
(C) The pH of the reagent during the reaction is pH 7.5 or higher. A method for quantifying a specific component in a biological sample using a liquid reagent composed of two or more reagents,
In the presence of an electron acceptor, an enzyme having an ability to oxidize by dehydrogenating a substance derived from the specific component by another reaction is allowed to act on a biological sample,
In the method of detecting and quantifying the reduced form of the generated electron acceptor by further forming an formazan by acting an electron carrier and a reducing dye,
A method for quantifying a component in a biological sample having all of the following configurations (a) to (c):
(A) Tetrazolium or a compound thereof is used as the reducing dye.
(B) The reagent containing the reducing dye has a pH of 8.0 or less.
(C) The pH of the reagent during the reaction is pH 7.5 or higher. The method for quantifying a specific component in a biological sample according to claim 1 or 2, wherein the reducing dye is a reducing dye represented by the general formula (1).

[General formula (1)]
Wherein R1 is hydrogen, nitro group, or methoxy group, R2 is hydrogen or nitro group, R3 is halogen, nitro group or an azo compound represented by the following formula (2), R4 is hydrogen or sulfo group R5 represents hydrogen or a sulfo group, R6 represents hydrogen, a nitro group, or a sulfo group, and R7 represents hydrogen or a sulfo group.)

[Formula (2)] The method according to claim 1, wherein the reagent containing a reducing dye contains ascorbate oxidase. The method according to claim 1, wherein the reagent containing a reducing dye contains an electron acceptor. 6. The electron acceptor according to claim 5, wherein nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, acetylpyridine adenine dinucleotide, acetylpyridine adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, or nicotinamide hypoxanthine dinucleotide. 6. The method according to claim 1, which is nucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate, or pyrroloquinoline quinone. The method according to claim 1, wherein the reagent containing a reducing dye contains an electron acceptor and ascorbate oxidase. In the presence of an electron acceptor, an enzyme having the ability to oxidize by dehydrogenating the specific component is allowed to act on a biological sample,
A liquid reagent used in a method for quantifying a specific component in a biological sample by forming a formazan by further reacting an electron carrier and a reducing dye to form a reduced form of an electron acceptor,
The liquid reagent which has all the structures of the following (a)-(d).
(A) Consists of two or more reagents.
(B) The first reagent contains tetrazolium or a compound thereof as a reducing dye.
(C) The pH of the first reagent is pH 8.0 or less.
(D) The reagent has a pH of 7.5 or higher during the reaction. In the presence of an electron acceptor, an enzyme having an ability to oxidize by dehydrogenating a substance derived from the specific component by another reaction is allowed to act on a biological sample,
A liquid reagent used in a method for quantifying a specific component in a biological sample by forming a formazan by further reacting an electron carrier and a reducing dye to form a reduced form of an electron acceptor,
The liquid reagent which has all the structures of the following (a)-(d).
(A) Consists of two or more reagents.
(B) The first reagent contains tetrazolium or a compound thereof as a reducing dye.
(C) The pH of the first reagent is pH 8.0 or less.
(D) The reagent has a pH of 7.5 or higher during the reaction. The reagent according to claim 8 or 9, wherein the reagent containing a reducing dye contains ascorbate oxidase. The reagent according to claim 8 or 9, wherein the reagent containing a reducing dye contains an electron acceptor. The electron acceptor according to claim 11, wherein nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, acetylpyridine adenine dinucleotide, acetylpyridine adenine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide, or nicotinamide hypoxanthine The reagent according to any one of claims 8 to 11, which is dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate, pyrroloquinoline quinone. The reagent according to claims 8 to 12, wherein the reagent containing a reducing dye contains an electron acceptor and ascorbate oxidase.

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