JP2007289097A - Liquid reagent for determination of molar ratio of total branched-chain amino acid/tyrosine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a BCAA determination reagent, a TYR determination reagent or a BTR determination reagent stable for a long period in the state of solution. <P>SOLUTION: The reagent for the determination of total branched-chain amino acid with an enzyme comprises a liquid reagent for the determination of total branched-chain amino acid containing a total branched-chain amino acid decomposition enzyme in combination with a chromogen having reduced non-specific color development, or a liquid reagent for BCAA determination containing a BCAA decomposition enzyme and a chromogen in separate state. The reagent for the determination of tyrosine with an enzyme comprises a liquid reagent containing a tyrosine decomposition enzyme in combination with a metal salt. The liquid reagent for the determination of total branched-chain amino acid/tyrosine molar ratio calculates the total branched-chain amino acid/tyrosine molar ratio by using the total branched-chain amino acid determination liquid reagent and/or the tyrosine determination liquid reagent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a reagent composition for measuring total branched chain amino acid (abbreviated as BCAA) or tyrosine (abbreviated as TYR) or total branched chain amino acid / tyrosine molar ratio (abbreviated as BTR). More specifically, the present invention relates to a liquid reagent for BCAA, TYR or BTR measurement that is stable for a long time in a solution state.

Among the clinical laboratory drugs used in hospital laboratories and laboratories, reagents that use unstable enzymes are distributed in the form of lyophilized products for stabilization, and are dissolved in the solution when used. It has become the mainstream. Reagents for measuring BTR are also unstable in enzymes, dyes, etc., and are distributed in the form of lyophilized products. On the other hand, in clinical laboratories, since many items are measured and the number of specimens is large, it is often a burden to dissolve lyophilized products. Development is desired. The same applies to the BTR measurement reagent, and the development of a measurement reagent that is stable for a long time in a solution state is desired.

Currently, the BTR measurement reagent used in clinical laboratories is a set of two measurement reagents, BCAA measurement reagent and TYR measurement reagent, each measured by the following reaction formula, and the obtained BCAA value, TYR The BTR value is obtained by taking the ratio of the values. For example, the methods as shown in FIGS.

The reagent for BTR measurement used in the BTR measurement method is a BCAA measurement reagent because the blood BCAA concentration decreases during the transition from chronic hepatitis to cirrhosis. High sensitivity measurement is necessary. Therefore, a system that leads to high sensitivity color development is required, and MTT is used as a high sensitivity color former. However, there is a problem that MTT has natural non-specific color development during storage and the reagent blank rises. There is also a problem of reduced activity of leucine dehydrogenase. In addition, tyrosine decarboxylase was unstable in the TYR measurement reagent, and the sensitivity decreased after long-term storage even at low temperatures, and the stability was not sufficient. Accordingly, an object of the present invention is to provide a reagent for BCAA measurement, a reagent for TYR measurement, or a reagent for BTR measurement that is stable for a long time in a solution state.

In view of the above problems, the present invention provides a liquid reagent for BCAA measurement by allowing a BCAA-degrading enzyme and a color source with reduced nonspecific coloration to coexist as a method for stabilizing a BCAA measurement reagent, or BCAA It is a liquid reagent for BCAA measurement by using a degrading enzyme and a color source as separate formulations. Further, as a method for stabilizing the reagent for TYR measurement, a liquid reagent for TYR measurement by allowing a TYR degrading enzyme and a metal compound to coexist. The present invention also relates to a liquid reagent for BTR measurement using the liquid reagent for BCAA measurement or the liquid reagent for TYR measurement.

That is, the present invention has the following configuration.
[Item 1] A tyrosine-decomposing liquid reagent for measuring tyrosine, wherein a tyrosine-degrading enzyme and a buffer containing a metal compound and / or an amine coexist in a reagent for measuring tyrosine [Item 2] Item 1. The liquid reagent for tyrosine measurement according to Item 1, which is tyrosine decarboxylase or β-tyrosinase [Item 3] The metal compound is iron (III) ethylenediaminetetraacetate, ferrous (III) chloride, potassium ferrocyanide, sodium ferrocyanide The liquid reagent for tyrosine measurement according to Item 1 which is any one of [4] A buffer containing an amine is N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis ( 2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 2- [4- (2-hydroxyethyl) -1-pi Razinyl] ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris (hydroxymethyl) methyl-2-aminomethane Sulfonic acid (TES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholino Item 2. The tyrosine according to Item 1, which is any one of propanesulfonic acid (MOPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), and N- (2-acetamido) iminoniacetic acid (ADA). Liquid reagent for measurement [Claim 5] Liquid reagent for tyrosine measurement according to Items 1 to 4, Beauty, total branched-chain amino acids / Chiroshinmoru ratio measurement liquid reagent for calculating the total branched-chain amino acids / Chiroshinmoru ratio using the total branched-chain amino acids for measuring liquid reagent

Furthermore, the present invention is a tyrosine measuring method characterized in that a tyrosine degrading enzyme and a buffer containing a metal compound and / or an amine coexist in a reagent for measuring tyrosine using an enzyme. This is a total branched chain amino acid / tyrosine molar ratio measurement method for calculating a total branched chain amino acid / tyrosine molar ratio using a liquid reagent and a total branched chain amino acid measurement liquid reagent.

Alternatively, the present invention is a method for stabilizing a tyrosine measuring reagent, comprising coexisting a tyrosine-degrading enzyme and a buffer containing metal compound and / or amine in a reagent for measuring tyrosine using an enzyme. This is a method for producing a tyrosine measuring reagent.

According to the present invention, the BCAA-degrading enzyme is improved in stability by improving the stability of BCAA-degrading enzyme by coexisting BCAA-degrading enzyme and non-specific color-developing color source in the BCAA-measuring reagent. Therefore, stability and precision can be improved. Further, by allowing the TYR-degrading enzyme and the metal compound to coexist in the TYR-measuring reagent, the stability of the TYR-degrading enzyme is improved and the stability and precision of the TYR-measuring reagent can be improved. From this, it is possible to provide a BTR measurement liquid reagent having good stability and precision by making a BCAA measurement reagent or a TYR measurement reagent into a liquid reagent.

As an embodiment of the present invention, as a measuring method for BCAA measuring reagents, (1) leucine dehydrogenase is allowed to act on BCAA in the presence of oxidized nicotinamide adenine dinucleotide (NAD), in the presence of an electron carrier. (2) Reduce chromogenic reagent by reducing chromogenic reagent with the generated reduced NAD, and (2) let BCAA react with leucine dehydrogenase in the presence of NAD and reduce the chromogenic reagent in the presence of diaphorase with the generated reduced NAD. (3) A leucine dehydrogenase is allowed to act on BCAA in the presence of NAD, and the resulting reduced NAD is allowed to act on lactate dehydrogenase in the presence of pyruvate to produce lactic acid. A method of oxidizing hydrogen peroxide and oxidizing the generated hydrogen peroxide in the presence of peroxidase to lead to color development, (4) By reacting BCAA with leucine dehydrogenase in the presence of NAD, in the presence of an electron carrier, the generated reduced NAD is converted into hydrogen peroxide by the action of superoxide dismutase, and the generated hydrogen peroxide is oxidized in the presence of peroxidase. (5) BCAA is reacted with leucine dehydrogenase in the presence of NAD, and the resulting reduced NAD is converted into hydrogen peroxide by the action of reduced NAD oxidase. A method of oxidizing an oxidative coloring reagent in the presence of peroxidase to lead to color development, (6) BCAA reacts with valine decarboxylase to form a monoamine, the produced minoamine is converted to hydrogen peroxide by monoamine oxidase, and hydrogen peroxide is present to peroxidase (7) Oxidation type thionicochi with BCAA. There is a method in which leucine dehydrogenase is allowed to act in the presence of amide adenine dinucleotide (abbreviated as thio NAD) to lead to reduced thio NAD.

In these measurement systems, factors that impair long-term storage stability in the solution state include the stability of BCAA-degrading enzymes and non-specific color development of the color source. It was found that the stability of BCAA-degrading enzyme was improved and the reagent blank of the measuring reagent was reduced by coexisting with BCAA-degrading enzyme in a composition that coexists or reduces non-specific color development of the color source.

BCAA-degrading enzymes are not particularly limited, and include leucine dehydrogenase and valine decarboxylase. Leucine dehydrogenase is obtained from Bacillus sphaericus and the like, but its origin is not particularly limited. Moreover, although valine decarboxylase is obtained from Proteus vulgaris, its origin is not particularly limited. In the reaction solution, it is used at a concentration of 0.01 to 200 U / L, preferably 0.05 to 100 U / L.

The color source body with reduced non-specific color development of the present invention is not particularly limited as long as it is a color source body with little increase in reagent blank after long-term storage, and specifically, 35 ° C., before and after storage for 1 week. The difference in absorbance at the maximum absorption of the dye is 0.2 Abs or less, preferably 0.1 Abs or less, more preferably 0.05 Abs or less.

Examples of such a color source include N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3, as a hydrogen donor. 5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl -N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3 5-dimethylaniline, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-sulfopropylaniline, N- (2-hydroxy-3-sulfopropyl) -2,5-dimethylaniline and the like can be mentioned. Examples of these couplers include 4-aminoantipyrine, MBTH, and NCP. The hydrogen donor and coupler are used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

As leuco dyes, 4,4′-benzylidenebis (N, N-dimethylaniline), 4,4′-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl] Methane, 1- (ethylaminothiocarbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole, bis [3-bis (4-chlorophenyl)- Methyl-4-dimethylaminophenyl] amine, 4,4′-bis (dimethylamino) diphenylamine, N-carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt, 10- (carboxymethylaminocarbonyl) ) -3,7-bis (dimethylamino) phenothiazine salt, 10-N-carboxymethylcarbamoyl-3,7-dimethylamino-10H -Phenothiazine salt etc. are mentioned. These are used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

Further, as tetrazolium 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- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl)- 5- (2,4-disulfophenyl) -2H-tetrazolium salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride, 3,3 '-( 1,1'-biphenyl-4,4'-diyl) -bis (2,5-diphenyl) -2H-tetrazolium chloride, 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4,4'-zyl] -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-di Enyl) tetrazolium chloride, 2,5-diphenyl-3- (4-styrylphenyl) 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 chlora 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium salt, 2- (4-nitrophenyl) -5-phenyl-3- [4- (4-sulfophenyl) Azo) -2-sulfophenyl] -2H-tetrazolium salt, 2,5-di (4-nitrophenyl) -3- [4- (4-sulfophenylazo) -2-sulfophenyl] -2H-tetrazolium salt, Examples include 2- (4-nitrophenyl) -5- (2-sulfophenyl) -3- [4- (4-sulfophenylazo) -2-sulfophenyl] -2H-tetrazolium salt. These are used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

Examples of coenzymes that can be measured in the visible region include thio NAD and thio NADP. These are used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

Examples of the electron mediator used in the present invention include phenazine methosulfate, 1-methoxy-5-methylphenazinium methylsulfate, and Meldola Blue. The initial concentration of the electron carrier in the reaction solution is 0.001 to 50 μmol / L, preferably 0.01 to 10 μmol / L.

Examples of the diaphorase used in the present invention include Bacillus megaterium and Clostridium SP. In the reaction solution, it is 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 used in the reaction solution at a concentration of 0.5 to 100 U / mL, preferably 1 to 50 U / mL.

Further, the non-specific color development of the color source is also affected by the solution composition. Factors affecting the non-specific color development of the color source include buffers, pH, surfactants, preservatives, stabilizers, and the like. In applying the above-described color source, non-specific color development can be reduced by a combination of these solution compositions.

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 (TAPS) ), N- (2-acetamido) iminoniacetic acid (ADA), N, N-bis (2-hydroxyethyl) glycine (Bicine), N- [tris (hydroxymethyl) methyl] glycine (Tricine), etc. The 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 Carbonate buffer, borate buffer, dimethylglycine buffer, and diethanolamine buffer are preferred because they reduce the non-specific color development of the color source. The pH of the buffer is adjusted in the range of 5 to 10, preferably a pH containing a reagent containing oxidized NAD or oxidized thio NAD is set to 5 to 7.5 and the oxidized thio NAD is excluded. The reagent containing is preferably adjusted to pH 7.5-10.

Although it does not specifically limit as surfactant used by this invention, A nonionic surfactant and zwitterionic surfactant are suitable.

Nonionic surfactants include polyoxyethylene lauryl ethers such as Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 147, Emulgen 130K, Nonion K-204, Nonion K -215, Nonion K-220, Nonion K-230, NIKKOL BL-2, NIKKOL BL-4.2, NIKKOL BL-9EX, NIKKOL BL-21, NIKKOL BL-25, polyoxyethylene cetyl ethers, Emulgen 210 , Emulgen 220, NIKKOL BC-2, NIKKOL BC-5.5, NIKKOL BC-7, NIKKOL BC-10TX, NIKKOL BC-15TX, IKKOL BC-20TX, NIKKOL BC-23, NIKKOL BC-25TX, NIKKOL BC-30TX, NIKKOL BC-40TX, nonionic P-208, nonionic P-210, nonionic P-213, polyoxyethylene stearylgen P 306 , Emulgen 320P, NIKKOL BS-2, NIKKOL BS-4, NIKKOL BS-20, Nonion S-206, Nonion S-207, Nonion S-215, Nonion S-220, and polyoxyethylene oleyl ethers include Emulgen 404 , Emulgen 408, Emulgen 409P, Emulgen 420, Emulgen 430, NIKKOL BO-2, NIKKOL BO-7, NIKKOL BO-10TX, NIKKOL BO-20, NIKKOL BO-50, Nonion E-206, Nonion E-215, Nonion E-230, and polyoxyethylene behenyl ethers include NIKKOL BB-5, NIKKOL BB-10, NIKKOL BB-20, NIKKOL BB. -30 etc. are mentioned.

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

Examples of the polyoxyethylene sterols include NIKKOL BPS-10, NIKKOL BPS-20, NIKKOL BPS-30, NIKKOL BPSH-25, and NIKKOL DHC-30. 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.

Examples of the zwitterionic surfactant include alkylimidazolium betaines, alkylbetaines, alkylamidobetaines, alkylalanines, alkylamine oxides, and derivatives thereof. These Amphithr 20BS, Amhitoal 24B, Amhitoal 86B, Amhitoal 20Z, 3-[(3-Colamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-Colamidopropyl) dimethylammonio] -2-hydroxy And propane sulfonic acid.

The concentration of the surfactant is not particularly limited, but is 0.01 to 10% by weight, preferably 0.1 to 5% by weight in the reaction solution.

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 is 0.01 to 10 mmol / L, preferably 0.05 to 2 mmol / L in the reaction solution.

In the present invention, ascorbate oxidase may be contained for the purpose of avoiding the influence of ascorbic acid in the sample, and it is 0.1 to 20 U / mL, preferably 0.5 to 10 U / mL in the reaction solution.

Reagents for TYR measurement are as follows: (1) Tyrosine decarboxylase is allowed to act on TYR, the generated tyramine is converted into hydrogen peroxide using tyramine oxidase, and the generated hydrogen peroxide is used in the presence of peroxidase. (2) Pyruvate is produced by the action of β-tyrosinase on TYR, and hydrogen peroxide is produced by the action of pyruvate oxidase in the presence of thiamine pyrophosphate and flavin adenine dinucleotide. , Oxidation of the generated hydrogen peroxide in the presence of peroxidase to lead to color development, (3) β-tyrosinase is allowed to act on TYR to produce pyruvic acid, and lactate dehydrogenase in the presence of reduced NAD Acts to produce lactic acid, and the produced lactic acid is peroxidized with lactate oxidase And iodine, in the presence of hydrogen peroxide produced peroxidase method leading to oxidation coloring the oxidative color-developing reagent is. Among these, a preferred embodiment is (1), and tyrosine decarboxylase is exemplified as the TYR degrading enzyme.

In these measurement systems, the stability of TYR-degrading enzyme can be cited as a factor that impairs long-term storage stability in solution. By containing TYR-degrading enzyme and metal compound, the stability of TYR-degrading enzyme is improved and measured. It was found that the long-term storage stability of the reagent for use was improved.

The TYR degrading enzyme is not particularly limited, and examples include tyrosine decarboxylase and β-tyrosinase. Tyrosine decarboxylase can be obtained from Streptococcus faecaris etc., but its origin is not particularly limited. Β-tyrosinase is obtained from Escherichia intermedia, but its origin is not particularly limited. In the reaction solution, it is used at a concentration of 0.01 to 200 U / L, preferably 0.05 to 100 U / L.

The metal compound of the present invention includes alkali metal ions, alkaline earth metal ions, manganese ions, iron ions, cobalt ions, nickel ions and salts with inorganic acids such as sulfuric acid, nitric acid, carbonic acid, iodine, bromine, chlorine, etc. A salt with a halogen atom, or a salt with an organic acid such as acetic acid, gluconic acid, propionic acid, pantentic acid, or ethylenediaminetetraacetic acid. Among these, iron ions are preferable, and examples thereof include iron (III) ethylenediaminetetraacetate, ferrous chloride (III), potassium ferrocyanide, sodium ferrocyanide, and the like. The concentration of the metal compound is 0.05 to 5 mmol / L, preferably 0.1 to 0.5 mmol / L in the reaction solution. A plurality of these metal compounds can be used in combination.

Examples of the buffer containing the amine of the present invention include Tris buffer and Good buffer. 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), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2- Hydroxy-3-aminopropanesulfonic acid (CAPSO), 3- [N, N-bis (2-hydroxy Ethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), 2-hydroxy-3- [4- (2- Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis (2-hydroxy) -3-propanesulfonic acid) (POPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N- (2-acetamido) iminoniacetic acid (ADA), N, N-bis (2 -Hydroxyethyl) glycine (Bicine), N- [Tris (hydroxy) Methyl) methyl] glycine (Tricine), etc. are exemplified. Preferably, N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 2- [4- (2 -Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris (hydroxymethyl) ) Methyl-2-aminomethanesulfonic acid (TES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N-tris (hydroxymethyl) methyl -3-amino-propanesulfonic acid (TAPSO), N- (2- acetamido) Iminoni acetate (ADA), a.

The concentration of the buffer is preferably 0.01 to 0.5 mol / L, more preferably 0.05 to 0.3 mol / L. The pH of the buffer is adjusted in the range of 5-9. Furthermore, 5.5-8 are preferable. Among these, 6 to 7 is preferable. In addition, it can be used in combination with a buffer containing no amine such as a phosphate buffer, a citrate buffer, a phthalate buffer, a maleate buffer, a glycine buffer, and a borate buffer.

Examples of the color source used in the TYR measuring reagent include hydrogen donors such as N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, and N-ethyl-N-sulfopropyl. -3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy -3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylanily N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-sulfopropylaniline, N- (2-hydroxy-3-sulfopropyl) -2,5-dimethylaniline, etc. Can be mentioned. Examples of these couplers include 4-aminoantipyrine, MBTH, and NCP. The hydrogen donor and coupler are used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

As leuco dyes, 4,4′-benzylidenebis (N, N-dimethylaniline), 4,4′-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl] Methane, 1- (ethylaminothiocarbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole, bis [3-bis (4-chlorophenyl)- Methyl-4-dimethylaminophenyl] amine, 4,4′-bis (dimethylamino) diphenylamine, N-carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt, 10- (carboxymethylaminocarbonyl) ) -3,7-bis (dimethylamino) phenothiazine salt, 10-N-carboxymethylcarbamoyl-3,7-dimethylamino-10H -Phenothiazine salt etc. are mentioned. These are used in the reaction solution at a concentration of 0.01 to 50 mmol / L, preferably 0.05 to 10 mmol / L.

Although it does not specifically limit as surfactant used by this invention, A nonionic surfactant and zwitterionic surfactant are suitable.

Nonionic surfactants include polyoxyethylene lauryl ethers such as Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 147, Emulgen 130K, Nonion K-204, Nonion K -215, Nonion K-220, Nonion K-230, NIKKOL BL-2, NIKKOL BL-4.2, NIKKOL BL-9EX, NIKKOL BL-21, NIKKOL BL-25, polyoxyethylene cetyl ethers, Emulgen 210 , Emulgen 220, NIKKOL BC-2, NIKKOL BC-5.5, NIKKOL BC-7, NIKKOL BC-10TX, NIKKOL BC-15TX, IKKOL BC-20TX, NIKKOL BC-23, NIKKOL BC-25TX, NIKKOL BC-30TX, NIKKOL BC-40TX, nonionic P-208, nonionic P-210, nonionic P-213, polyoxyethylene stearylgen P 306 , Emulgen 320P, NIKKOL BS-2, NIKKOL BS-4, NIKKOL BS-20, Nonion S-206, Nonion S-207, Nonion S-215, Nonion S-220, and polyoxyethylene oleyl ethers include Emulgen 404 , Emulgen 408, Emulgen 409P, Emulgen 420, Emulgen 430, NIKKOL BO-2, NIKKOL BO-7, NIKKOL BO-10TX, NIKKOL BO-20, NIKKOL BO-50, Nonion E-206, Nonion E-215, Nonion E-230, and polyoxyethylene behenyl ethers include NIKKOL BB-5, NIKKOL BB-10, NIKKOL BB-20, NIKKOL BB. -30 etc. are mentioned.

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

Examples of the polyoxyethylene sterols include NIKKOL BPS-10, NIKKOL BPS-20, NIKKOL BPS-30, NIKKOL BPSH-25, and NIKKOL DHC-30. 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.

Examples of the zwitterionic surfactant include alkylimidazolium betaines, alkylbetaines, alkylamidobetaines, alkylalanines, alkylamine oxides, and derivatives thereof. These Amphithr 20BS, Amhitoal 24B, Amhitoal 86B, Amhitoal 20Z, 3-[(3-Colamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-Colamidopropyl) dimethylammonio] -2-hydroxy And propane sulfonic acid.

The concentration of the surfactant is not particularly limited, but is 0.01 to 10% by weight, preferably 0.1 to 5% by weight in the reaction solution.

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.

In the present invention, a stabilizer, ascorbic acid oxidase, catalase and the like may be contained in addition. Examples of the stabilizer include pyridoxal 5'-phosphate, albumin and the like.

Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.

(Preparation of reagents)
A BCAA measuring reagent having the following composition was prepared.

Example 1
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
1-M-PMS 0.1 μmol / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
PIPES buffer 200 mmol / L pH 7.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 2)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
PIPES buffer 200 mmol / L pH 7.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 3)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
Bicine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

Example 4
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
Bicine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Ethylenediaminetetraacetic acid dihydrogen dipotassium 10mmol / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 5)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
Bicine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2,2'-Dibenzothiazoyl-5,5'-bis [4-di (2-sulfoethyl) carbamoylphenyl] -3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) ditetrazolium Salt (λmax = 580nm) 0.5mmol / L

(Example 6)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
Bicine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 433 nm) 0.5 mmol / L

(Example 7)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
Bicine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 460 nm) 0.5 mmol / L

(Example 8)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized thio NAD 10mmol / L
Second reagent
PIPES buffer 200 mmol / L pH 7.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL

Example 9
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
PIPES buffer 200 mmol / L pH 7.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 10)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
Bicine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 11)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
TAPS buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 12)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent borate buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt (λmax = 438 nm) 0.5 mmol / L

(Example 13)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
Diaphorase (Toyobo DAD-301) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent Dimethylglycine buffer 200 mmol / L pH 8.5
Triton X-100 1g / L
Leucine dehydrogenase 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 1)
First reagent
PIPES buffer 50 mmol / L pH 7.0
Triton X-100 1g / L
1-M-PMS 0.1 μmol / L
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Oxidized NAD 10mmol / L
Second reagent
PIPES buffer 200 mmol / L pH 7.5
Triton X-100 1g / L
Leucine dehydrogenase 100 U / mL
3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium salt (λmax = 565 nm) 0.5 mmol / L

The reagents for BCAA measurement of Examples 1 to 13 and Comparative Example 1 were stored at 35 ° C. for 7 days, and the residual activity of leucine dehydrogenase (the ratio of the activity value after storage to the activity value immediately after preparation) was examined.

Results are shown in Table 1. The residual rate of leucine dehydrogenase of Comparative Example 1 was 56%, while the residual rate of Examples 1 to 13 was as good as 72 to 98%.

Next, for each of the reagents immediately after preparation of the BCAA measurement reagents of Examples 1 to 13 and Comparative Example 1 and stored for 7 days at 35 ° C., purified water and 500 μmol / L isoleucine aqueous solution were measured as samples by the following measurement method. Absorbance (sensitivity) obtained by subtracting the purified water measurement absorbance from the 500 μmol / L isoleucine measurement absorbance was calculated and compared with the absorbance immediately after preparation to obtain the relative percentage.

(Measurement method)
A Hitachi 7180 automatic analyzer was used. The first reaction was performed by adding 120 μL of the first reagent to 1.7 μL of the sample and incubating at 37 ° C. for 5 minutes. Thereafter, 30 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The measurement wavelength is 405 nm for Examples 8 and 450 nm for Examples 1-4, 6 and 7, respectively. Absorbance at 9 to 13 was measured at 505 nm, Example 5 and Comparative Example 1 at 570 nm.
The result was calculated from the measured absorbance of purified water and 500 μmol / L isoleucine aqueous solution, and obtained as the BCAA concentration.

Results are shown in Table 2. The purified water measurement absorbance of Comparative Example 1 showed an increase in absorbance of 0.2 Abs or more after 1 week at 35 ° C., and the standard solution measurement sensitivity decreased by 28.9% relative to immediately after preparation at 35 ° C. for 1 week. On the other hand, the absorbance of the purified water measured in Examples 1 to 13 was 35 ° C. and the absorbance increase after 1 week was 0.0019 to 0.1553 Abs and not more than 0.2 Abs, and the standard after 35 ° C. and 1 week The liquid measurement sensitivity was as good as 77.5-102.2% immediately after preparation.

Next, the simultaneous reproducibility of each of the reagents immediately after preparation of the BCAA measurement reagents of Examples 1 to 13 and Comparative Example 1 and stored at 35 ° C. for 7 days was examined. Simultaneous reproducibility was measured with n = 5 repeatedly using commercially available control serum QAP trawls IX and IIX (Sysmex) as samples, and the coefficient of variation CV (%) was calculated.

Results are shown in Table 3. The CV immediately after production of Comparative Example 1 was 1.9% with QAP Troll IX, 1.2% with QAP Troll IIX, 35 ° C., and the CV after 7 days storage was 4.7% with QAP Troll IX and QAP Troll IIX. In contrast to 2.1%, in Examples 1 to 13, the CV immediately after production was 0.7 to 1.8% for QAP Troll IX, 0.3 to 1.1% for QAP Troll IIX, and stored at 35 ° C. for 7 days. The later CV was 2.0-3.2% for QAP Troll IX and 0.5-1.8% for QAP Troll IIX, which was good. In addition, the relative percentage of the measured value of the reagent after storage for 7 days at 35 ° C. with respect to the reagent immediately after production was 144.0% for QAP Troll IX and 110.3% for QAP Troll IIX, whereas in Examples 1 to 13 QAP Troll IX was 96.7 to 105.3%, and QAP Troll IIX was 98.6 to 104.5%.

(Preparation of reagents)
A TYR measuring reagent having the following composition was prepared.

(Example 14)
First reagent Mcylvine buffer 50 mM pH 6.0
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent Mcylvine buffer 50 mM pH 6.0
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL
Potassium ferrocyanide 0.15mmol / L

(Example 15)
First reagent
PIPES buffer 50 mM pH 6.0
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent
PIPES buffer 50 mM pH 6.0
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL

(Example 16)
First reagent
PIPES buffer 100 mM pH 6.0
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent
PIPES buffer 100 mM pH 6.0
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL

(Example 17)
First reagent
PIPES buffer 50 mM pH 6.5
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent
PIPES buffer 50 mM pH 6.5
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL

(Example 18)
First reagent
PIPES buffer 50 mM pH 7.0
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent
PIPES buffer 50 mM pH 7.0
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL

Example 19
First reagent
PIPES buffer 50 mM pH 6.5
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent
PIPES buffer 50 mM pH 6.5
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL
Potassium ferrocyanide 0.15mmol / L

(Comparative Example 2)
First reagent Mcylvine buffer 50 mM pH 6.0
Triton X-100 1g / L
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent Mcylvine buffer 50 mM pH 6.0
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL

The TYR measurement reagents of Examples 14 to 19 and Comparative Example 2 were stored at 35 ° C. for 7 days, and the residual activity of tyrosine decarboxylase (the ratio of the activity value after storage to the activity value immediately after preparation) was examined.

Results are shown in Table 4. The residual rate of h sirocin decarboxylase of Comparative Example 2 was 28%, while the residual rate of Examples 14 to 19 was 46 to 91%, which was good.

Next, for each of the reagents stored in Examples 14 to 19 and Comparative Example 2 immediately after preparation of the TYR measurement reagent and stored at 35 ° C. for 7 days, purified water and 200 μmol / L tyrosine aqueous solution were measured as samples by the following measurement method. Absorbance (sensitivity) obtained by subtracting the purified water measurement absorbance from the 200 μmol / L tyrosine aqueous solution absorbance was calculated and compared with the absorbance immediately after preparation to obtain the relative percentage.

(Measurement method)
A Hitachi 7170 automatic analyzer was used. The first reaction was performed by adding 120 μL of the first reagent to 3.5 μL of the sample and incubating at 37 ° C. for 5 minutes. Thereafter, 30 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The absorbance at 570 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume and the difference between the absorbances was taken.
The result was calculated from the measured absorbance of purified water and 200 μmol / LL-tyrosine aqueous solution, and obtained as the tyrosine concentration.

Results are shown in Table 5. The standard solution measurement sensitivity of Comparative Example 2 was 35.3% after 1 week at 35 ° C., whereas a decrease of 38.3% was observed with respect to immediately after the preparation, whereas the standard solution measurement sensitivity after 1 week at 35 ° C. in Examples 14-19. Was as good as 83.7 to 100.5% immediately after preparation.

Next, the simultaneous reproducibility of each of the reagents immediately after preparation of the TYR measurement reagents of Examples 14 to 19 and Comparative Example 2 and stored at 35 ° C. for 7 days was examined. Simultaneous reproducibility was measured with n = 5 repeatedly using commercially available control serum QAP trawls IX and IIX (Sysmex) as samples, and the coefficient of variation CV (%) was calculated.

Results are shown in Table 6. The CV immediately after production of Comparative Example 2 was 5.9% with QAP Troll IX, 1.5% with QAP Troll IIX, 35 ° C., after storage for 7 days, CV was 6.7% with QAP Troll IX and QAP Troll IIX. Compared to 2.1%, in Examples 14 to 19, the CV immediately after production was 3.8 to 5.6% for QAP Troll IX, 0.8 to 1.2% for QAP Troll IIX, stored at 35 ° C. for 7 days. The later CV was 5.2-6.6% for QAP Troll IX and 1.2-1.8% for QAP Troll IIX, which was good. The relative percentage of the measured value of the reagent after storage for 7 days at 35 ° C. with respect to the reagent immediately after production was 86.7% for QAP Troll IX in Comparative Example 2 and 93.3 to 100 for QAP Troll IX in Examples 14-19. It was as good as 0.0%.

Next, the BTR value (BCAA measurement value ÷ TYR measurement value) was calculated by changing the combination from the commercially available serum QAP Troll IIX (Sysmex) measurement values of the BCAA measurement reagent of Examples 1 to 13 and the TYR measurement reagent of Example 17 The relative% of the BTR value of the reagent after storage for 7 days at 35 ° C. with respect to the BTR value immediately after production was calculated. Similarly, the BTR value (BCAA measurement value / TYR measurement value) is calculated from the measurement values of the BCAA measurement reagent of Comparative Example 1 and the TYR measurement reagent of Comparative Example 2, and stored at 35 ° C. for 7 days immediately after the production. The relative% of reagent BTR value was calculated.

Results are shown in Table 7. The relative% of the measured value of the reagent after storage for 7 days at 35 ° C. with respect to the reagent immediately after production is 106.8% in Comparative Example 1, whereas the BTR value calculated from the BCAA value in Examples 1 to 13 and the TYR value in Example 17 is It was as good as 98.5 to 104.3%.

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

Example of BCAA measurement method. Illustration of tyrosine measurement method.

Claims (5)

A reagent for measuring tyrosine using an enzyme, wherein a tyrosine-degrading enzyme and a metal compound and / or a buffer containing an amine coexist in a liquid reagent for tyrosine measurement The liquid reagent for tyrosine measurement according to claim 1, wherein the tyrosine degrading enzyme is tyrosine decarboxylase or β-tyrosinase. The liquid reagent for tyrosine measurement according to claim 1, wherein the metal compound is any one of iron (III) ethylenediaminetetraacetate, ferrous (III) chloride, potassium ferrocyanide, and sodium ferrocyanide. Buffers containing amines include N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 2- [ 4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N- Tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (TES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N-tris (hydroxymes ) Methyl-3-amino-propanesulfonic acid (TAPSO), N- (2- acetamido) Iminoni acetate (ADA), a liquid reagent tyrosine measurement according to claim 1 is either A liquid reagent for measuring the total branched chain amino acid / tyrosine molar ratio, wherein the liquid reagent for measuring tyrosine according to claim 1 and the liquid reagent for measuring total branched chain amino acid are calculated.

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