JP6218894B2 - L-glutamic acid measurement kit - Google Patents

Description

  The present invention relates to a reagent for measuring L-glutamic acid and a kit containing the reagent.

  L-glutamic acid is known as one of the “umami” components, and sodium L-glutamate, which is a sodium salt, is used as the main component of “umami seasoning”. It also functions as an excitatory neurotransmitter that performs neurotransmission via glutamate receptors in the animal body. Therefore, detecting and quantifying L-glutamic acid is very important in the fields of food and biochemistry. There are amino acid analyzers, glutamate dehydrogenases, and methods using decarboxylase for the measurement of L-glutamic acid, but all of them are inaccurate and reproducible, complicated and expensive. Problems such as were pointed out. The applicant of the present application found that a kind of Streptomyces produced an enzyme L-glutamate oxidase that acts specifically on L-glutamate in the process of soy sauce research in 1983, and has succeeded in commercializing the product. Furthermore, a method capable of easily measuring L-glutamic acid using this enzyme has been developed and marketed as an L-glutamic acid measurement kit (Non-patent Documents 1 and 2).

  Conventionally, L-glutamic acid measurement kits using L-glutamate oxidase provide the measurement reagent as a lyophilized product. Specifically, the kit includes a lyophilized product of L-glutamate oxidase, a color former and peroxidase, and a buffer solution for dissolving the lyophilized product. When measuring L-glutamic acid, a freeze-dried product of glutamate oxidase, color former and peroxidase is dissolved in a buffer solution, and a sample is added to the obtained measurement reagent. Through this step, L-glutamic acid in the sample is decomposed by the action of L-glutamic acid oxidase to produce hydrogen peroxide. Then, the color developed by the reaction of the hydrogen peroxide, the color former and peroxidase is measured with an absorptiometer. As the color former at this time, for example, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS) which is a new Trinder reagent and 4-aminoantipyrine (DAOS) which is a coupler compound. 4-AA) combination is used. Traditionally, DAOS and 4-AA have been mixed into glutamate oxidase and peroxidase lyophilized products. Here, a kit that provides the measurement reagent in the form of a solution from the beginning has been desired because the operation is simpler than that of a lyophilized product that requires dissolution before use in a buffer solution. In addition, in the kit using the above lyophilized product, after the lyophilized product, which is the measurement reagent, is made into a solution, the coloring agent is naturally colored with time and the absorbance of the reagent blank increases. The expiration date after dissolution is set to about one month, and it has been demanded to extend the expiration date.

  In the above method, if ascorbic acid is present in the sample, the color development reaction is inhibited. Therefore, a kit capable of measuring L-glutamic acid while eliminating the influence of ascorbic acid in the sample has been desired.

Japan Soy Sauce Research Institute Journal, 13 (1); 8,1987 Agric. Biol. Chem., 48 (1); 181, 1984

  The problem to be solved by the present invention is to accurately measure L-glutamic acid by a simple method. Another object of the present invention is to use a liquid reaction reagent that can measure the L-glutamic acid concentration while removing ascorbic acid, and is stable over a long period, even for a sample containing ascorbic acid. Is to provide a kit.

In order to solve the above problems, the inventors have decomposed L-glutamic acid to produce hydrogen peroxide, L-glutamic acid oxidase; a new color former for quantitatively measuring the obtained hydrogen peroxide. A kit with two types of reaction liquids, reaction liquid I and reaction liquid II, is studied by examining a kit that includes a liquid that includes a Trinder reagent, a coupler compound, and a peroxidase; and a catalase quencher. Tried to prevent coloring. And as a result of further examining the composition of the reaction solution,
When divided into two reaction solutions, reaction solution I containing ascorbate oxidase, peroxidase and new Trinder reagent, and reaction solution II containing L-glutamate oxidase, coupler compound and catalase quencher, it is not a lyophilized product but a liquid It was found that a kit using only a reagent in the form of a reagent and can provide a stable measurement value over a long period of time. The present invention is based on such novel findings. Accordingly, the present invention provides the following sections:
Item 1. A kit for measuring L-glutamic acid in a sample, comprising the following (A) and (B).
(A) Reaction solution I containing ascorbate oxidase, peroxidase and a new Trinder reagent,
(B) Reaction liquid II containing L-glutamate oxidase, a coupler compound, and a catalase quencher.

  Item 2. New Trinder reagent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl)- 3-methoxyaniline (ADOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N- Ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyl Item 2. The measurement kit according to Item 1, which is selected from the group consisting of aniline (MAOS).

  Item 3. Item 3. The measurement kit according to Item 1 or 2, wherein the coupler compound is 4-aminoantipyrine.

  Item 4. Item 4. The measurement kit according to any one of Items 1 to 3, wherein a reagent is added to the sample in this order to carry out the reaction.

  Item 5. Reaction solution I and reaction solution II stored at 37 ° C. for 12 weeks are added to a solution containing L-glutamic acid and ascorbic acid in this order in this order. Item 5. The measurement kit according to any one of Items 1 to 4, wherein II is within ± 10% with respect to the absorbance value added in this order to the same L-glutamic acid and ascorbic acid solution as the solution.

Item 6. A method for measuring L-glutamic acid in a sample comprising the following steps (1) to (3), wherein reaction solution I contains ascorbate oxidase, peroxidase and a new Trinder reagent, and reaction solution II contains L-glutamic acid A method for measuring L-glutamic acid, comprising an oxidase, a coupler compound, and a catalase quencher.
(1) adding reaction solution I to the sample;
(2) The reaction solution II is further added to the sample to which the reaction solution I has been added in the above step (1) to decompose L-glutamic acid to produce hydrogen peroxide. , A step of causing color development by the reaction of the coupler compound and peroxidase, and (3) a step of measuring the degree of color development caused by absorbance.

  The kit of the present invention can accurately measure L-glutamic acid by a simple method. In addition, since the enzyme is supplied as a liquid reagent, the problems associated with freeze-dried products such as foaming of solutions have been solved, and the reagent for measuring coloration due to natural coloration of the colorant over time or enzyme deactivation The liquid reagent can be stored in a stable state for a long period of time without destabilizing itself, and is extremely useful as a measurement kit. Furthermore, according to the kit of the present invention, even if the sample contains ascorbic acid and / or catalase, the amount of L-glutamic acid can be measured with high accuracy while reducing these effects.

L-glutamic acid measurement kit The L-glutamic acid measurement kit of the present invention contains at least the following constituent reagents (A) and (B).
(A) Reaction solution I (including ascorbate oxidase, peroxidase and a new Trinder reagent),
(B) Reaction solution II (including L-glutamate oxidase, coupler compound and catalase quencher).

  The analysis sample used in the present invention is not particularly limited as long as it is expected to contain L-glutamic acid. Specific examples include biological samples such as serum, plasma, urine, amniotic fluid, and tissue extract, culture fluids such as tissues, cells, and microorganisms, foods, food materials, and the like.

  As the L-glutamate oxidase, peroxidase and ascorbate oxidase used in the present invention, known ones can be used, and they may be derived from living organisms or produced by recombination. Specific examples of L-glutamate oxidase include L-glutamate oxidase derived from microorganisms such as Streptomyces sp. X-119-6, Streptomyces violascens, and Streptomyces endus. Examples of peroxidase include horseradish peroxidase. Examples of ascorbate oxidase include ascorbate oxidase derived from pumpkin and cucumber.

  In addition, as the new Trinder reagent used in the present invention, a known color developing reagent can be used, for example, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium Salt (TOOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline (ADOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline ( HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N-sulfate Hopropyl-3,5-dimethoxyaniline (DAPS), N- (2-carboxyethyl) -N-ethyl-3,5-dimethoxyaniline (CEDB), or N- (2-carboxyethyl) -N-ethyl-3 -Methoxyaniline (CEMO), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-sulfopropyl-3,5 -Dimethoxy-4-fluoroaniline (FDAPS) or the like can be used. These new Trinder reagents can be used alone or in combination of two or more. Among them, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxy Aniline (ADOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N -Sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS) or N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS) N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline. It is more preferable to use sodium salt (TOOS).

  The coupler compound used in the present invention may be any compound that generates color when combined with a new Trinder reagent. For example, 4-aminoantipyrine (4-AA), vanillin diamine sulfonic acid, methylbenzthiazoli Nonhydrazone (MBTH), sulfonated methylbenzthiazolinone hydrazone (SMBTH), aminodiphenylamine, 1- (4-sulfophenyl) -2,3-dimethyl-4-amino-5-pyrazolone (CP2-4), N -Ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine or a derivative thereof can be used. These coupler compounds can be used singly or in combination of two or more. Of these, 4-aminoantipyrine (4-AA) is preferably used.

  The reaction solution I used in the present invention contains ascorbate oxidase, peroxidase, and a new Trinder reagent. Various buffers can be used as the liquid containing the enzyme and the new Trinder reagent. Buffers include acetic acid, phosphoric acid, citric acid, boric acid, trisaminomethane, HEPES, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, MOPS, BES, TES, DIPSO, TAPSO, TAPS, CHES, CAPSO, CAPS and their salts can be used. In the present invention, the reaction liquid I preferably does not contain a coupler compound. In the present invention, the reaction solution I “contains no coupler compound” means that the reaction solution I does not substantially contain a coupler compound. More specifically, in the present invention, the phrase “reaction liquid I containing no coupler compound” includes not only the reaction liquid I containing no coupler compound but also a very small amount of coupler compound as long as the effects of the present invention are not impaired. The reaction liquid I containing is also included.

  As a compounding quantity of ascorbic acid oxidase to the buffer solution etc. in the reaction liquid I, it is preferable to exist in the range of 1-30 U / mL, for example, and it is more preferable to exist in the range of 2-20 U / mL especially. The content of peroxidase in the buffer solution or the like in the reaction solution I is, for example, preferably in the range of 1 to 30 U / mL, and more preferably in the range of 5 to 20 U / mL. The content of the new Trinder reagent in the buffer solution or the like in the reaction solution I is, for example, preferably in the range of 0.1 to 4 μmol / mL, and more preferably in the range of 0.4 to 1 μmol / mL. preferable.

  Next, the reaction solution II used in the present invention contains L-glutamate oxidase, a coupler compound, and a catalase quencher. Various buffers can be used as the liquid containing the enzyme, coupler compound and catalase quencher. Examples of buffers include acetic acid, phosphoric acid, citric acid, boric acid, trisaminomethane, HEPES, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, MOPS, BES, TES, DIPSO, TAPSO, TAPS, CHES, CAPSO, CAPS, and salts thereof can be used. In the present invention, the reaction solution II preferably does not contain a new Trinder reagent. In the present invention, “the reaction liquid II does not contain a new Trinder reagent” means that the reaction liquid II does not substantially contain the new Trinder reagent. More specifically, in the present invention, the phrase “reaction liquid II that does not contain a new Trinder reagent” includes not only the reaction liquid II that does not contain any new Trinder reagent, but also a new Trinder reagent as long as the effects of the present invention are not impaired. The reaction solution II containing is also included.

  As the catalase deactivator, any substance may be used as long as it has a function of quickly deactivating catalase. For example, sodium azide, 3-amino-1,2,4-triazole, or the like may be used. it can.

  The content of L-glutamate oxidase in the buffer or the like is preferably, for example, in the range of 0.05 to 2 U / mL, and more preferably in the range of 0.2 to 0.8 U / mL. For example, the content of the coupler compound in the buffer solution is preferably in the range of 0.1 to 4 μmol / mL, and more preferably in the range of 0.4 to 1 μmol / mL.

  The reaction liquids I and II used in the present invention may contain preservatives and the like in addition to the above reagents. As the preservative, known ones such as procrine and chloramphenicol can be used. Procrine is widely used as a preservative for clinical diagnosis, and four types of procrine 150, 200, 300 and 950 are commercially available. In any case, isothiazolone (5-chloro-2-methyl-4-isothiazolin-3-one and / or 2-methyl-4-isothiazolin-3-one) is used as an active ingredient, and the concentration used as a preservative is 6 to 15 ppm (procrine). 150, 200, 300) or 48-95 ppm (Procrine 950) is recommended, and the recommended upper limit concentration is 150 ppm (Procrine 150, 200, 300) or 200 ppm (Procrine 950). In this invention, the compounding quantity to the reaction liquid I or the reaction liquid II can be suitably adjusted within the range of 6-200 ppm, More preferably, 50-150 ppm etc. in conversion of an active ingredient.

  In addition to the above reaction solutions I and II, the kit of the present invention includes an L-glutamic acid standard, a 96-well plate or cell for absorbance measurement, a sample diluent, a container for filling the reaction solutions I and II, and the like. It can also be attached. The material of the container for filling the reaction liquids I and II is not particularly limited. For example, polypropylene, polyethylene, polyethylene terephthalate and the like are preferable from the viewpoint of enzyme stability.

  The L-glutamic acid measurement kit of the present invention can stably store an enzyme in a liquid state for a long period of time. Specifically, in a preferred embodiment, the kit of the present invention has an absorbance when reaction solution I and reaction solution II stored at 37 ° C. for 12 weeks are added to a solution containing L-glutamic acid and ascorbic acid in this order in this order. And the difference of the measured value of the light absorbency which added the reaction liquid I and the reaction liquid II of the storage 0th day in this order to the same L-glutamic acid solution as the said solution has high stability which is less than +/- 10%. In this embodiment, the “solution containing L-glutamic acid at a predetermined concentration” is, for example, a standard solution (L-Glu250 + ASA1000 containing L-glutamic acid 250 mg / L and ascorbic acid 1000 mg / L used in Example 2 of the present application). ). In the present invention, when the standard solution is measured, there is a difference in measured values of absorbance between the reaction solution I and the reaction solution II stored at 37 ° C. for 10 weeks and the reaction solution I and the reaction solution II on the 0th storage day. It is preferably within ± 10%.

  In the present invention, in order to measure L-glutamic acid, first, the reaction solution I is added to the sample. If endogenous ascorbic acid is present in the sample, it is removed by this step. The reaction conditions may be set according to the optimum pH and temperature of the enzyme to be used, but are generally carried out for about 5 to 20 minutes under conditions of pH 6.0 to 8.0 and temperature 15 to 30 ° C. can do.

  Next, after the reaction by the reaction solution I has sufficiently progressed, the reaction solution II is further added to decompose L-glutamic acid to produce hydrogen peroxide, and the hydrogen peroxide, the color former and peroxidase are removed. Color development is caused by the reaction. The reaction conditions may be set according to the optimum pH and temperature of the enzyme to be used, but are generally carried out for about 5 to 30 minutes under conditions of pH 6.0 to 8.0 and temperature 15 to 30 ° C. can do.

  Finally, the degree of color developed is measured by absorbance, and the L-glutamic acid concentration in the sample is calculated. In the absorbance measurement, the wavelength to be measured may be selected according to the type of color reagent used.

In the present invention, in addition to the above method, a similar method may be further performed using an L-glutamic acid solution having a known concentration (for example, a 100 mg / L L-glutamic acid solution) as a standard solution instead of the sample. In this embodiment, after measuring the absorbance of the sample and the L-glutamic acid standard solution, the amount of L-glutamic acid in the sample is obtained by subtracting the absorbance of the blank using water instead of the sample, and using the following formula 1. Can be calculated.
L-glutamic acid concentration (mg / L) = absorbance of sample ÷ absorbance of standard solution × standard solution concentration (mg / L)

  Hereinafter, the present invention will be described in detail with reference to reference examples and examples, but it is clear that the present invention is not limited thereto.

1. Preparation of enzyme reagent solution
1-1. Preparation of Enzyme Reagent Solution under Condition 1 As reaction solutions I and II and L-glutamic acid standard solutions under condition 1 in the examples described later, solutions having the compositions described in Table 1 below were prepared.

1-2. Preparation of Enzyme Reagent Solution Under Conditions 2-6 Ascorbate oxidase (abbreviation: abbreviation: ASOX, Wako Pure Chemical Industries, 2000 U / vial), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (abbreviation: TOOS), peroxidase (oriental yeast), proclin 950 (Preservative: 2-methyl-4-isothiazolin-3-one concentration 9.5 to 9.9%), glutamate oxidase (abbreviation: GLOX, manufactured by Enzyme Sensor Co., Ltd.), 4-aminoantipyrine (abbreviation: 4) -AA, coupler compound) and sodium azide (NaN3, catalase quencher) were used to prepare enzyme reagent solutions under conditions 2-6.

2. Measurement of glutamic acid in sample The measuring method of glutamic acid in this example was as follows:
[1] To each well of a 96-well round bottom assay plate (IWAKI), 4 μL of a sample and 90 μL of the reaction solution were added, stirred, and allowed to stand for several minutes.
[2] Add 90 μL of Reaction Solution II, stir, and let stand at room temperature for 20 minutes.
[3] Absorbance at 555 nm is measured 20 minutes after the addition of Reaction Solution II. (Purified water is used as a control.) L-glutamic acid measurement value is calculated by the following formula.
([Sample Absorbance] − [0 mg / L Sample Absorbance]) ÷ ([Standard Solution Absorbance] − [0 mg / L Sample Absorbance]) × Standard Solution Concentration (250 mg / L)
2-1. Reagent component combination acceleration test (up to 12 weeks of storage)
The following samples were used for measurement:
STD L-Glu:
Standard solution prepared by dissolving L-glutamic acid in water to 250 mg / L
L-Glu250 + ASA1000:
L-Glu L solution containing L-glutamic acid 250mg / L and ascorbic acid 1000mg / L:
Solution with L-glutamic acid concentration of 15mg / L
L-Glu M:
Solution with L-glutamic acid concentration of 150mg / L
L-Glu H:
A solution having an L-glutamic acid concentration of 1500 mg / L.

  About the enzyme reagent liquid of the conditions 1-6 mentioned above, the measurement of glutamic acid in the said sample was measured according to the said "measurement method of glutamic acid in a sample" immediately after adjustment and after storage for 12 weeks at 37 degreeC.

  For conditions 1 to 6, the measurement results immediately after preparation are shown in Table 3 below, and the measurement results after storage at 37 ° C. for 12 weeks are shown in Table 4 below.

  As shown in Table 4, when stored at 37 ° C. for 12 weeks, the absorbance and measured value in “L-Glu250 + ASA1000” were almost zero under conditions other than Condition 1, and ascorbate oxidase in the sample was inactivated. It was thought that. However, a sample containing only L-glutamic acid could be measured to some extent even under conditions other than condition 1.

Conditions 1 and 2 differ only in the combination of TOOS and 4-AA, and the other compositions are exactly the same. Since L-glutamic acid alone could be measured under any condition, 4-AA could lose its activity by coexistence with ascorbate oxidase. Under other conditions 3 to 6, the absorbance and measured value of “L-Glu250 + ASA1000” are almost 0 after storage at 37 ° C. for 12 weeks, and condition 1 is the most stable condition for storing the reaction solution. It turned out.
Condition 1 was also considered to be a composition in which the absorbance increase of the reagent blank was the lowest compared to the other conditions, and the composition did not easily raise the reagent blank.

  Next, the ratio (%) of the absorbance value after storage at 37 ° C. for 12 weeks with respect to the absorbance value immediately after preparation is shown in Table 5 below. As a result, in only condition 1, in all the L-glutamic acid-containing samples, the absorbance value measured after storage at 37 ° C. for 12 weeks was within ± 10% of the absorbance value measured immediately after preparation.

2-2. Reagent component combination storage test (up to 12 months of storage)
The following samples were used for measurement:
STD L-Glu:
Standard solution prepared by dissolving L-glutamic acid in water to 250 mg / L
L-Glu250 + ASA1000:
A solution containing 1000 mg / L of ascorbic acid in addition to 250 mg / L of L-glutamic acid
L-Glu L:
Solution with L-glutamic acid concentration of 15mg / L
L-Glu M:
Solution with L-glutamic acid concentration of 150mg / L
L-Glu H:
A solution having an L-glutamic acid concentration of 1500 mg / L.

  For the enzyme reagent solution of Condition 1 described above in “1. Preparation of enzyme reagent solution”, after refrigerated storage at 7-8 ° C. for 12 months immediately after adjustment and after adjustment, according to the above “Method for measuring glutamic acid in sample” The measurement of L-glutamic acid in the sample was measured.

  For Condition 1, the measurement results immediately after preparation are shown in Table 6 below, and the measurement results after refrigerated storage at 7-8 ° C. for 12 months are shown in Table 7 below. For the samples immediately after preparation and after storage for 12 months, the measurement was repeated 5 times, and the average value was calculated. And the operation of the said 5 measurement and average value calculation was performed 3 times (Therefore, it measured 15 times in total about the sample immediately after preparation and after storage for 12 months, respectively). Tables 6 and 7 show the average of three times, the standard deviation (SD), and the coefficient of variation (CV). Furthermore, Table 8 shows% of the average value of the measurement results after storage for 12 months with respect to the average value of the measurement results immediately after preparation.

  As is clear from Table 6 above, the absorbance (subtracted value of the water blank) of the L-glutamic acid 0 mg / mL sample (purified water (test blank)) was less than 0.015. The average value of three measurements on Day 0 was used as the reference value for stability determination.

The results after storage for 12 months are summarized in Tables 7 and 8.
L-glutamic acid control samples (L, M, H) after storage for 12 months and L-glutamic acid 250 mg / L solution containing 1000 mg / L of ascorbic acid, measured values when each sample was measured on Day 0 Within ± 20% of the value. Although not clearly shown in the above table, the CV of the measured value when an L-glutamic acid control sample (L, M, H) and an ascorbic acid 1000 mg / L-containing L-glutamic acid 250 mg / L solution were measured 5 times simultaneously Within 15%. When each sample was measured on Day 0, the absorbance of each sample of the standard solution after storage for 12 months, L-glutamic acid control sample H, and ascorbic acid 1000 mg / L containing L-glutamic acid 250 mg / L solution Was within ± 20% of the mean value. Furthermore, the absorbance (subtraction value of water blank) of the L-glutamic acid 0 mg / mL sample (purified water) was less than 0.015.
From the above results, the kit was judged to be stable even after storage for 12 months.

Claims (6)

A kit for measuring L-glutamic acid in a sample containing the following (A) and (B):
(A) Reaction solution I containing ascorbate oxidase, peroxidase and a new Trinder reagent,
(B) Reaction liquid II containing L-glutamate oxidase, a coupler compound, and a catalase quencher. New Trinder reagent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl)- 3-methoxyaniline (ADOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N- Ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyl The measurement kit according to claim 1, which is selected from the group consisting of aniline (MAOS). The measurement kit according to claim 1 or 2, wherein the coupler compound is 4-aminoantipyrine. The measurement kit according to any one of claims 1 to 3, wherein a reagent is added in this order to the sample to carry out the reaction. When the reaction solution I and the reaction solution II stored at 37 ° C. for 12 weeks were added in this order to a solution containing L-glutamic acid and ascorbic acid at a predetermined concentration, the absorbance of the reaction solution I and the reaction solution II on storage day 0 The measurement kit according to any one of claims 1 to 4, which is within ± 10% with respect to the absorbance value added to the same L-glutamic acid and ascorbic acid solution in the same order as the solution. A method for measuring L-glutamic acid in a sample comprising the following steps (1) to (3), wherein reaction solution I contains ascorbate oxidase, peroxidase and a new Trinder reagent, and reaction solution II contains L-glutamic acid A method for measuring L-glutamic acid, comprising an oxidase, a coupler compound, and a catalase quencher.
(1) adding reaction solution I to the sample;
(2) The reaction solution II is further added to the sample to which the reaction solution I has been added in the above step (1) to decompose L-glutamic acid to produce hydrogen peroxide. , A step of producing color by the reaction of the coupler compound and peroxidase, and (3) a step of measuring the degree of color produced by the absorbance.