JP6049795B2 - L-glutamine measurement kit - Google Patents

Description

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

  L-glutamine is known to affect protein production efficiency and sugar chain structure in the production process of biopharmaceuticals such as antibody drugs and recombinant protein preparations. In addition, it is a very useful component as a functional component of various supplements and a nutrient source in the culture medium of microorganisms and cultured cells, and its concentration control is very important.

  Conventionally, as a method for measuring L-glutamine, a method using high performance liquid chromatography and a method using an enzyme have been used in many cases. As a method using an enzyme, in particular, glutaminase is allowed to act on L-glutamine in a sample to give L-glutamic acid, and then L-glutamate oxidase is allowed to act on liberated L-glutamic acid to thereby react oxygen with the reaction. A method of detecting consumption or detecting the amount of hydrogen peroxide, ammonia or α-ketoglutarate produced during the reaction (Patent Documents 1 to 3), and acting on L-glutamate dehydrogenase on liberated L-glutamate Then, a method (Non-patent Document 1) is known in which the change in absorbance at 340 nm accompanying the production of coenzyme NAD (P) H is measured.

  In the method for measuring L-glutamine using the above L-glutaminase and L-glutamate oxidase, if the sample contains endogenous L-glutamate, an error may occur in the measured value. In order to correct from the measurement value of endogenous L-glutamic acid, for example, measurement is performed in two measurement cells, a measurement cell holding both glutaminase and L-glutamate oxidase, and a measurement cell holding only L-glutamate oxidase, Measure L-glutamine from the difference between the two measured values (publicly known document 2), L-glutaminase alone, L-glutamate oxidase alone, or an immobilized body in which both enzymes are immobilized. A method of calculating the amount of L-glutamine based on a calibration curve obtained from the detected value (known document 3) is known.

More specifically, a method is known in which L-glutamine is quantified after removing endogenous L-glutamic acid in advance by a three-step reaction such as the following (1) to (3).
(1) Step of removing endogenous glutamate L-glutamate oxidase and catalase are allowed to act on the sample. Endogenous glutamic acid is decomposed by the action of L-glutamic acid oxidase, and hydrogen peroxide generated at this time is also decomposed by catalase and thus removed from the sample. On the other hand, L-glutamine remains unreacted in the sample.
(2) Step of reacting L-glutamine with L-glutamic acid L-glutaminase and sodium azide are allowed to act on a sample in which the reaction of (1) has been sufficiently performed. At this time, the catalase contained in the reaction solution I is immediately deactivated by the action of sodium azide.

On the other hand, L-glutamine in the sample is converted into L-glutamic acid by the action of L-glutaminase, and then decomposed by the action of L-glutamate oxidase added in the step (1) to produce hydrogen peroxide.
(3) Step of quantifying generated hydrogen peroxide L-glutamine is quantified by measuring the amount of hydrogen peroxide produced.

Here, as a method for measuring the production amount of the various substances at the time of L-glutamine measurement, a color former and peroxidase are added to the enzyme reaction solution in addition to the above enzyme, and the reaction between the color former, peroxidase and hydrogen peroxide is added. the color detection amount caused by, there is a method of measuring by spectrophotometer. As the color former at this time, for example, a combination of a new Trinder reagent and a coupler compound is used.

  Conventionally, a glutamine measurement kit using L-glutamate oxidase provides a measurement reagent as a lyophilized product. Moreover, the kit using the conventional freeze-dried product contains a coupler compound in the freeze-dried reagent of the reaction solution I containing L-glutamate oxidase and catalase, and contains the reaction solution I, L-glutaminase and catalase inactivating agent. The solution for dissolving the reaction solution II contained a new Trinder reagent. In the conventional method using a freeze-dried measurement reagent, the operation is complicated such as the need for dissolution before use. After the lyophilized product that is the measurement reagent is made into a solution, the absorbance of the reagent blank increases due to foaming of the solution, destabilization of the measurement reagent itself due to the inactivation of the enzyme and the color developing agent over time, or natural coloring of the color developing agent As a result, the absorbance of the sample and the standard sample fluctuates, and it is impossible to calculate an accurate measurement value of the sample. Therefore, it is necessary to limit the expiration date after dissolution to about one week. .

  If it is a kit which provides a measuring reagent in the form of a solution from the beginning, it can be easily applied to an automatic colorimetric analyzer, and L-glutamine can be measured more easily. In order to stably store the enzyme, it is essential to thoroughly examine the composition of the reaction solution. However, such examination has not been made in the past and no knowledge has been obtained.

Japanese Patent Publication No. 4-24997 Japanese Patent Laid-Open No. 2-501009 JP 2003-322635 A

Methods of Enzymatic Analysis Vol.8, 357-363 (Weinheim, 1986)

  The problem to be solved by the present invention is to accurately measure L-glutamine by a simple method. Another subject of the present invention is a problem in a method using an enzyme of a lyophilized product, specifically, destabilization of a measurement reagent itself due to foaming of a solution, deactivation of an enzyme and a color former with the passage of time, Alternatively, it is to provide a method using a liquid reaction reagent in order to suppress an increase in absorbance of the reagent blank due to natural coloring of the color former. Furthermore, the further subject of this invention is providing the measurement kit which can store the said liquid reagent in a stable state for a long period of time.

  The inventors of the present invention have made an appropriate combination of a new Trinder reagent and 4-aminoantipyrine in the L-glutamine measurement kit according to the above steps (1) to (3) in which an enzyme is stored in a solution state during intensive studies. I found a new issue that is important for improving stability.

Therefore, by conducting a more detailed study, the inventors of the L-glutamine measurement kit containing the following (A) and (B), a combination different from the conventional method using a lyophilized reagent, specifically, By adding a new Trinder reagent to reaction solution I and peroxidase and a coupler compound to reaction solution II, unexpectedly, even when measuring reagents containing enzymes are stored in a liquid state, the storage time has passed and the temperature load during storage It has been found that an L-glutamine measurement kit using an enzyme in the form of a solution can be produced and used so that the increase in the reagent blank absorbance and the decrease in the absorbance of the sample associated with can be suppressed and a stable measurement value can be provided over a long period of time. The present invention has been completed:
(A) Reaction solution I (including L-glutamate oxidase, catalase, and new Trinder reagent),
(B) Reaction solution II (including glutaminase, peroxidase, catalase inactivator, coupler compound).

  The kit of the present invention can accurately measure L-glutamine by a simple method. In addition, since the enzyme is supplied as a liquid reagent, the reagent blank is caused by foaming of the solution, destabilization of the measuring reagent itself due to the deactivation of the enzyme and the coloring agent over time, or natural coloring of the coloring agent. In addition to solving the problems associated with conventional freeze-dried products such as an increase in the absorbance of the liquid, the liquid reagent can be stored in a stable state for a long period of time, and is extremely useful as a measurement kit.

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

  The analysis sample used in the present invention is not particularly limited as long as it is expected to contain L-glutamine. 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.

  Known L-glutamate oxidase, catalase, glutaminase and peroxidase used in the present invention can be used. 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. Catalase includes catalase derived from microorganisms such as Aspergillus niger and Corynebacterium glutamicum, in addition to catalase derived from bovine liver. Examples of the glutaminase include glutaminase derived from Bacillus amyloriquefaciens. Examples of peroxidase include horseradish peroxidase.

  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-sulfopropyl-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), and the like can be used. 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 It is preferred to use either sulfopropylaniline (ALPS) or N-ethyl-N-sulfopropyl-3-methylaniline (TOPS).

  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. Of these, 4-aminoantipyrine (4-AA) is preferably used.

  The reaction solution I used in the present invention contains L-glutamate oxidase, catalase, 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.

  The contents of each enzyme and new Trinder reagent in the buffer solution in the reaction solution I are L-glutamate oxidase 0.05-2 U / mL, catalase 100-3000 U / mL, new Trinder reagent 0.01-2 μmol / mL In particular, L-glutamate oxidase 0.2 to 0.8 U / mL, catalase 500 to 2000 U / mL, and new Trinder reagent 0.4 to 0.8 μmol / mL are more preferable. .

  Next, the reaction solution II used in the present invention contains L-glutaminase, peroxidase, a catalase inactivator and a coupler compound. Various buffers can be used as the solution containing the enzyme, catalase quencher and coupler compound. 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 each enzyme, catalase inactivator and coupler compound in the buffer solution, etc. is as follows: glutaminase 0.01-2 U / mL, peroxidase 1-30 U / mL, catalase inactivator 0.01-0.1%, coupler Preferably the compound is in the range of 0.01 to 2 μmol / mL, in particular glutaminase 0.1 to 0.5 U / mL, peroxidase 5 to 20 U / mL, catalase deactivator 0.05 to 0.1%, coupler compound More preferably, it is in the range of 0.2 to 0.8 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 materials such as Procrine 300, Procrine 950, and chloramphenicol can be used.

  In addition to the above reaction solutions I and II, the kit of the present invention can be attached with an L-glutamine standard, a 96-well plate or cell for absorbance measurement, a sample diluent, and the like.

  The L-glutamine 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 comprises the absorbance when a reaction solution I and a reaction solution II stored at 37 ° C. for 10 weeks are added in this order to a solution containing L-glutamine at a predetermined concentration, and storage. The difference in the measured value of the absorbance obtained by adding the reaction solution I and the reaction solution II on the 0th day to the same L-glutamine solution in this order in this order is as high as ± 10%. In this embodiment, as the “solution containing L-glutamine at a predetermined concentration”, for example, the L-glutamine 20 mg / L aqueous solution, the L-glutamine 100 mg / L aqueous solution, and the L-glutamine 1000 mg / L used in Example 2 of the present application are used. An aqueous solution may be mentioned. In the present invention, when one of these three types of L-glutamine 20 mg / L aqueous solution is measured, the reaction solution I and the reaction solution II stored at 37 ° C. for 10 weeks and the reaction solution I on the 0th storage day and It is preferable that the difference in absorbance measured with the reaction solution II is within ± 10%, and when all these three types of L-glutamine 20 mg / L aqueous solutions were measured, all were stored at 37 ° C. for 10 weeks. It is more preferable that the difference in the measured value of absorbance between the reaction solution I and the reaction solution II and the reaction solution I and the reaction solution II on storage day 0 is within ± 10%.

  In the present invention, in order to measure L-glutamine, reaction solution I is first added to the sample. If endogenous L-glutamic 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 with the reaction solution I has sufficiently progressed, the reaction solution II is further added to convert L-glutamine into L-glutamic acid, which is further decomposed to produce hydrogen peroxide. Color development occurs by reacting hydrogen peroxide with a color former and peroxidase. 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 development that occurs is measured by absorbance, and the L-glutamine 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-glutamine solution having a known concentration (for example, a 100 mg / L L-glutamine solution) as a standard solution instead of the sample. In this embodiment, after measuring the absorbance of the sample and the L-glutamine standard solution, the amount of L-glutamine in the sample is subtracted by subtracting the absorbance of the blank using water instead of the sample and using Equation 1 below. Can be calculated.

  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.

(Reference example) Examination of coupler compound and new Trinder reagent (1)
In order to examine the stability of the enzyme reagent in which the coupler compound and the new Trinder reagent were added to the reaction solution, a stability test was performed. 4-Aminoantipyrine was used as the coupler compound.

  N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (abbreviation: TOOS) and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5 -Prepared a solution-like enzyme reagent having a composition containing both 4-aminoantipyrine and the new Trinder reagent in the reaction solution I as shown in Table 1 below using two new Trinder reagents of dimethoxyaniline (abbreviation: DAOS) did. In the table, “L-GLOX” indicates L-glutamate oxidase, “4-AA” indicates 4-aminoantipyrine, and the abbreviations in the following are also the same. A HEPES buffer (pH 7.1) was used as a buffer for dissolving each component.

  Dispensing the prepared reagents in equal amounts into polypropylene centrifuge tubes, storing them at 30 ° C as an accelerated test, and performing sample measurement using each enzyme reagent on the 0th and 4th week after the start of storage, Stability was evaluated.

<Measurement method, reaction solution volume, reaction time>
Sample measurement was performed according to the following procedure.
First, 450 μL of the reaction solution I and a sample were placed in a glass test tube and reacted at room temperature for 10 minutes. Thereafter, 450 μL of the reaction solution II was further added and reacted in the same manner for 20 minutes. After completion of the reaction, 0.9 mL of the solution was transferred to a disposable semi-micro cuvette (Bio-Rad), and absorbance was measured at 555 nm (measured using pure water as a control) with a spectrophotometer. The liquid volume of the sample was determined so that the calibration curve was a linear expression at an L-glutamine concentration of 10 to 500 mg / L. The amount of sample added was 60 μL in Test 1 and 30 μL in Test 2.

<Measurement results and discussion>
The measurement results (absorbance of each specimen) are shown in Table 2. In Test 2 using DAOS as the new Trinder reagent, the absorbance of the reagent blank when stored at 30 ° C. for 4 weeks increased 38 times compared to that at 0 days of storage, and the absorbance of each sample was stored accordingly. It was higher than that of the day. It was thought that the presence of 4-aminoantipyrine and a new Trinder reagent naturally caused an oxidative condensation reaction to cause coloration, resulting in an increase in absorbance.

  Moreover, about the light absorbency of the L-glutamine solution of Test 2, what was stored for 4 weeks at 30 ° C., the measured value of L-glutamine 500 mg / L decreased to about 40% compared to the case of 0 days. The cause was considered to be a decrease in the activity of the enzyme in the reaction solution. At the same time, the measurement of L-glutamic acid, which should have been removed, was also detected, suggesting inactivation of the enzyme in the reaction solution as well.

  On the other hand, even in Test 1 using TOOS, after storage at 30 ° C. for 4 weeks, the absorbance of the reagent blank increased 15 times compared to the storage day 0, and accordingly, the absorbance of each sample increased compared to Day 0. The natural coloring of the coloring reagent was suggested.

  As shown in Table 2, since the measurement of L-glutamic acid was detected at 30 ° C. for 4 weeks, it was considered that the enzyme in the reaction solution was inactivated when stored at 30 ° C. for 4 weeks.

  From the above results, it is considered that the coexistence of the coupler compound and the new Trinder reagent in the reaction solution I causes natural coloring of the coloring reagent, and also decreases the activities of the enzyme contained in the reaction solution and the new Trinder reagent itself. It was.

(Example 1) Examination of combination of coupler compound and new Trinder reagent (2)
From the results of the reference example, adding both a new Trinder reagent such as DAOS and TOOS and 4-aminoantipyrine to the reaction solution I may cause an increase in reagent blank absorbance with time and inactivation of enzymes such as catalase. Sex was considered.

  Therefore, in order to further examine and optimize the combination of the coloring reagents in the reaction liquids I and II, six types of enzyme reagents shown in Table 3 below were prepared ((1) to (6)), and an accelerated test was performed. Carried out. The concentrations of the enzyme and coloring reagent were the same as in the reference example, and TOOS was used as the new Trinder reagent.

  After preparation, the solution enzyme reagent was dispensed in equal amounts into polypropylene centrifuge tubes and stored at 37 ° C. as an accelerated test. Sample measurement was performed on the 0th, 1st and 4th weeks after the start of storage. Samples and standard solutions were dispensed, stored at −80 ° C., and used as needed.

  In addition, in order to ensure linearity in the vicinity of L-glutamine 500 mg / L, measurement was performed with the sample solution amount being 20 μL. The amount of enzyme reagent, reaction time, and measured absorbance were the same as in the reference example.

<Measurement results and discussion>
Table 4 shows the measurement results of absorbance in each test. As for the absorbance of the L-glutamine solution, “(1) 4-AA + (2) TOOS” showed a decrease in the value when stored at 37 ° C. for 4 weeks, whereas “(3) TOOS + (4) 4-AA” Even at 37 ° C. for 4 weeks, the same absorbance as Day 0 was exhibited. In the case of “(5) 4-AA, TOOS + (6) None”, the absorbance of L-glutamine 500 mg / L was significantly reduced by storage for 1 week at 37 ° C. compared to Day 0 of the L-glutamine solution, and 4 weeks at 37 ° C. Then, all the samples no longer developed color.

  Therefore, the combination of “(3) TOOS + (4) 4-AA” showed the best result.

  Summing up the above results, only the combination of “(3) TOOS + (4) 4-AA” in which the new Trinder reagent is added to the reaction solution I and the coupler compound is added to the reaction solution II, the results show very high stability. The results show that this combination is optimal.

(Example 2) Stability test 1
A solution enzyme reagent having the composition shown in Table 5 below was prepared, and an accelerated test at a temperature load of 37 ° C. was performed in order to confirm the stability of the enzyme reagent. Dispense equal amounts of the prepared enzyme reagents into polypropylene tubes, store separately for refrigeration and 37 ° C storage, and use each enzyme reagent on the 0th, 4th, and 10th weeks after the start of storage. Specimen measurement was performed.

  The standard solution used was an L-glutamine 100 mg / L aqueous solution, the L-glutamine sample used was a 20 mg / L, 1000 mg / L aqueous solution, and the L-glutamic acid 1000 mg / L aqueous solution was used as a sample for confirming the measurement of L-glutamic acid. . Samples and standard solutions were dispensed, stored at −80 ° C., and used as needed.

<Measurement method, reaction solution volume, reaction time>
In each well of a 96-well round bottom assay plate (IWAKI), 4 μL of the sample and 90 μL of the reaction solution I were placed and reacted for 10 minutes. Thereafter, 90 μL of the reaction solution II was further added and reacted for 20 minutes, and then absorbance at 555 nm was measured with a plate reader (TECAN Infinite). In this test, the sample amount was set to 4 μL as the liquid amount of the sample having an L-glutamine concentration of 10 to 1500 mg / L and a calibration curve having a linear expression.

<Measurement results and discussion>
The measurement results are shown in Table 6 below. In the measurement with the enzyme reagent stored at 37 ° C. for 10 weeks, the absorbance at the time of glutamine measurement and the measured value of L-glutamine were within the Day 0 value ± 10%, and the values were judged to be stable. Moreover, measurement of L-glutamic acid was not detected. Further, the absorbance of L-glutamine 0 mg / L (reagent blank) was less than 0.01. That is, it was considered that the enzyme reagent maintained stability even after storage at 37 ° C. for 10 weeks.

(Example 3) Examination of various new Trinder reagents In order to examine the difference in stability among various new Trinder reagents, solution enzyme reagents having the compositions shown in Table 7 below were prepared. In addition, various reagents shown in Table 8 were added to the reaction solutions I (1) to (6) as final Trinder reagents to a final concentration of 0.8 (μmol / mL). New Trinder reagents include 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) and N-ethyl-N-sulfopropyl-3-methylaniline (TOPS) were used.

  In order to confirm the stability of the enzyme reagent, an accelerated test was performed at a temperature load of 37 ° C. Aliquots of the prepared enzyme reagent were dispensed into tubes and stored at 37 ° C., and sample measurement was performed using each enzyme reagent on the 0th and 6th weeks after the start of storage.

<Measurement method, reaction solution volume, reaction time>
In the measurement, 700 μL of the reaction solution I was added to 20 μL of the sample and allowed to react for 10 minutes. Thereafter, 700 μL of the reaction solution II was added and reacted for 10 minutes, and then the absorbance was measured. The measurement OD was a value as shown in Table 8 according to the type of each new Trinder reagent.

  As a measurement sample, L-glutamine and / or L-glutamic acid was added and used so that the concentration at the time of measurement would be the value shown in Table 9.

<Measurement results and discussion>
Table 10 below shows the absorbance values when the samples (a) to (d) were measured using each new Trinder reagent. As new Trinder reagents, TOOS, ADOS, HDAOS, ADPS, ALPS, and TOPS are suitable for use in an L-glutamine measurement kit for storing an enzyme in solution, and it was found that TOOS and ALPS can be measured particularly with high sensitivity.

(Example 4) Stability test 2
A solution enzyme reagent having the composition shown in Table 11 was prepared, and a long-term stability test under refrigerated conditions was performed in order to confirm the long-term storage stability of the enzyme reagent. Enzyme reagents were dispensed in equal amounts into polypropylene tubes, and sample measurement was performed on the 0th day (Day 0), 6 months, 9 months, and 12 months after the start of storage.

  The sample used was purified water as an L-glutamine 0 mg / L aqueous solution, L-glutamine 100 mg / L aqueous solution as a standard solution, and the L-glutamine sample used 15 mg / L aqueous solution, 150 mg / L aqueous solution, and 1500 mg / L aqueous solution. -An L-glutamic acid 1500 mg / L aqueous solution was used as a sample for confirming the measurement of glutamic acid. The standard solution was lyophilized and stored refrigerated, and dissolved in purified water before use. The L-glutamine sample was dispensed and stored at −80 ° C. and used as needed.

<Measurement method, reaction solution volume, reaction time>
In each well of a 96-well round bottom assay plate (IWAKI), 4 μL of the sample and 90 μL of the reaction solution I were placed and reacted for 10 minutes. Thereafter, 90 μL of the reaction solution II was further added and reacted for 20 minutes, and then the absorbance at 555 nm was measured with a plate reader (TECAN Infinite). In this test, the sample amount was set to 4 μL as the liquid amount of the sample having an L-glutamine concentration of 10 to 1500 mg / L and a calibration curve having a linear expression.

<Measurement results and discussion>
Table 13 shows the absorbance and measured values at each time point after the storage day 0 (Day 0), 6 months, 9 months, and 12 months. In the measurement with the enzyme reagent stored for up to 12 months, the absorbances of the standard solution and the sample showed almost the same value as Day 0, the L-glutamine measurement value was within Day 0 value ± 10%, and the value was stable. It was judged. Moreover, the measured value of the L-glutamic acid 1500 mg / L aqueous solution showed around 0 mg / L, and the measurement of L-glutamic acid was completely suppressed. Furthermore, abnormalities such as a rise over time were not observed in the absorbance of the L-glutamine 0 mg / L aqueous solution, and the reagent blank absorbance was kept low. From the above results, it was considered that the enzyme reagent maintained stability even after refrigerated storage for 12 months.

Claims (6)

A kit for measuring L-glutamine in a sample, comprising the following (A) and (B).
(A) Reaction solution I containing L-glutamate oxidase, catalase, and a new Trinder reagent,
(B) Reaction liquid II containing L-glutaminase, peroxidase, catalase inactivator, and coupler compound. 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- The measurement kit according to claim 1, which is selected from the group consisting of ethyl-N-sulfopropylaniline (ALPS) and N-ethyl-N-sulfopropyl-3-methylaniline (TOPS). 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. The absorbance when reaction solution I and reaction solution II stored at 37 ° C. for 10 weeks were added to a solution containing glutamine in a predetermined concentration in this order was the same as that of solution 0 and reaction solution I on storage day 0 in this order. The measurement kit according to any one of claims 1 to 4, which is within ± 10% of the absorbance value added to the glutamine solution. A method for measuring L-glutamine in a sample, comprising the following steps (1) to (3), wherein the reaction solution I comprises L-glutamate oxidase, catalase, a new Trinder reagent, and the reaction solution II is L- A method for measuring L-glutamine, comprising glutaminase, peroxidase, a catalase quencher, and a coupler compound.
(1) adding reaction solution I to the sample;
(2) By further adding reaction solution II to the sample to which reaction solution I has been added in the above step (1), L-glutamine is converted to L-glutamic acid, which is further decomposed to produce hydrogen peroxide. A step of causing color development by reaction of the hydrogen peroxide with a color former and peroxidase,
(3) A step of measuring the degree of color development produced by absorbance.