JP2000232898A - Quantitative analysis of substance and reagent therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quantitatively analyzing substance to be analyzed in a converted state into ammonia after eliminating ammonia previously existing in a sample and to provide a reagent therefor. SOLUTION: After eliminating ammonia by making glutamine synthetase act in the presence of glutamic acid, adenosine triphosphate and a metal salt on ammonia in a sample containing a substance (or an enzyme) to be quantitatively analyzed and ammonia, a reaction terminating reagent containing a glutamine synthetase inhibitor selected from methionine sulfoximine or methionine sulfone is added to terminate glutamine synthetase reaction, and then a reagent containing an enzyme acting on the substance to be quantitatively analyzed or a substrate for an enzyme to be quantitatively analyzed is made to act to quantitatively analyzing the formed substance directly or in an enzymatically converted state into other substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for quantifying a substance producing ammonia by an enzyme such as urea, creatine, creatinine or an enzyme involved in the reaction and a reagent therefor in the field of clinical diagnosis or food ingredient analysis. About.

[0002]

2. Description of the Related Art Conventionally, when quantifying substances such as urea, creatine, and creatinine that produce ammonia by an enzyme or enzymes involved in the reaction, it is necessary to eliminate ammonia present in a sample in advance, and the substance to be quantified It is necessary to stop the reaction at the stage where the ammonia to be quantified is generated by the reaction. Also, when quantifying the enzymatic activity of an enzyme whose activity changes in the presence of ammonia, it is necessary to eliminate ammonia in the sample in advance.

[0003] As a method for eliminating ammonia, for example, a method is known in which glutamate dehydrogenase and isocitrate dehydrogenase are allowed to act in the presence of reduced nicotinamide adenine dinucleotide phosphate, α-ketoglutaric acid and isocitrate. The method of stopping the present elimination system is performed by treating a magnesium ion required by isocitrate dehydrogenase with a chelating agent [clinical test-instrument / reagent, 14 , 183 (1)
99)]. However, in this scavenging enzyme system, the supply of a liquid reagent is limited because reduced nicotinamide adenine dinucleotide phosphate which is unstable in a solution is used as a substrate. In some cases, the activity of the target enzyme is reduced or suppressed in the presence of a chelating agent.

[0004] A method is known in which ammonia is eliminated using glutamine synthetase, and after the ammonia is eliminated, the reaction by glutamine synthetase is stopped with a chelating agent, and then the target biological substance is measured (Japanese Patent Laid-Open No. Hei 7 (1999)).
-51094). Glutamine synthetase is known to be inactivated by glutamine analog methionine sulfoximine in the presence of adenosine triphosphate and magnesium ion (Biochemical Dictionary 1st edition, Tokyo Chemical Dojin, 1
984).

On the other hand, a method for quantifying ammonia using deamide nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide synthetase, adenosine triphosphate or the like (JP-A-63-185378) is known.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for quantifying a substance or an enzyme to be quantified by removing ammonia existing in a sample and then producing ammonia from the substance or an enzyme to be quantified. To provide reagents. It is another object of the present invention to provide a method for quantifying an enzyme whose activity is affected by ammonia after eliminating ammonia present in a sample in advance, and a reagent used therefor.

In particular, it is an object of the present invention to provide a reagent for quantifying a substance or an enzyme which has a high storage stability and incorporates an ammonia eliminating reagent which has little influence on the quantification of the substance or the enzyme to be quantified.

[0008]

The present invention provides the following (1).
To (24). (1) Glutamic acid, ammonia in the sample containing the substance or enzyme to be quantified and containing ammonia
Glutamine synthetase is allowed to act in the presence of adenosine triphosphate and a metal salt to eliminate ammonia, and then a reaction terminating reagent containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone is added, and glutamine synthase is added. The enzyme reaction on the substance to be quantified in the sample or the reagent containing the substrate of the enzyme to be quantified is allowed to act simultaneously or after the addition of the quenching reagent with the reaction stop reagent, and the substance to be produced is directly or A method for quantifying a substance or an enzyme, characterized in that the substance or enzyme is enzymatically converted into a substance and quantified.

(2) The method according to (1), wherein the substance to be quantified by enzymatic conversion into a produced substance or another substance is ammonia. (3) The method for quantifying ammonia involves quantifying nicotinamide adenine dinucleotide produced by allowing nicotinamide adenine dinucleotide synthase to act in the presence of adenosine triphosphate and deamidonicotinamide adenine dinucleotide ( The quantification method according to 2).

(4) The quantification of nicotinamide adenine dinucleotide is carried out by the action of an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme and the resulting reduced nicotinamide adenine dinucleotide being colorimetrically determined. (3) The method of quantification according to (3). (5) The quantification of nicotinamide adenine dinucleotide is carried out by reacting glucose dehydrogenase in the presence of glucose and colorimetrically determining the reduced nicotinamide adenine dinucleotide produced. Quantitation method.

(6) The method according to any one of (1) to (5), wherein the substance to be quantified is urea, and the reagent containing an enzyme acting on the substance to be quantified is a reagent containing urease. . (7) The substance to be quantified is creatine, and the reagent containing an enzyme acting on the substance to be quantified is a reagent containing creatinase and urease (1)-
The quantification method according to any one of (5).

(8) The substance to be quantified is creatinine, and the reagent containing an enzyme acting on the substance to be quantified is a reagent containing creatininase, creatinase and urease. The quantification method according to any of the above. (9) An enzyme in which the enzyme activity of the enzyme acting on the substance to be quantified, the enzyme to be quantified or the enzyme used to convert the produced substance into another substance is promoted or suppressed by ammonia (1). The method described in.

(10) Glutamine synthetase is allowed to act on ammonia in a sample containing the substance or enzyme to be quantified and ammonia in the presence of glutamic acid, adenosine triphosphate and a metal salt to eliminate the ammonia, and then to remove methionine sulfo. A method for eliminating ammonia, comprising adding a reaction terminating reagent containing a glutamine synthetase inhibitor selected from xymin and methionine sulfone to terminate the glutamine synthetase reaction, and then quantifying the substance or enzyme to be quantified.

(11) a first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; and a second reagent: a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. An ammonia elimination reagent kit containing a reaction stopping reagent.

(12) a first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; and a second reagent: a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. A reagent kit for quantifying a substance or enzyme, comprising a reaction stopping reagent to be contained and an enzyme acting on a substance to be quantified in a sample or a reagent containing a substrate of the enzyme to be quantified.

(13) a first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; a second reagent: a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. A reagent for quantifying a substance or enzyme comprising a reaction-stopping reagent and a third reagent: a reagent containing an enzyme acting on a substance to be quantified in a sample or a substrate of the enzyme to be quantified.

(14) a first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; a second reagent: a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. Reaction stop reagents contained, Third reagent: A reagent containing an enzyme that acts on the substance to be quantified in the sample or a substrate of the enzyme to be quantified, and a fourth reagent: Contains a reagent necessary for quantifying the generated substance Quantitative reagent kit for a substance or enzyme containing a reagent to be used.

(15) (12) or (13) containing a reagent necessary for quantifying a substance produced in the second reagent
(16) The quantitative reagent kit according to (13), which contains a reagent necessary for quantifying a substance generated in the third reagent. (17) The reagent according to any one of (12) to (16), wherein the enzyme that acts on the substance to be quantified in the sample or the reagent containing the substrate of the enzyme to be quantified is a reagent necessary for producing ammonia. Quantitative reagent kit.

(18) A reagent containing an enzyme acting on a substance to be quantified in a sample or a substrate of the enzyme to be quantified is a reagent necessary for producing ammonia, and a reagent for quantifying the produced substance. (14) to (16), wherein the reagent necessary for (1) is a reagent necessary for quantifying ammonia. (19) Reagents necessary for quantifying ammonia include deamide nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide synthase (1)
The quantitative reagent kit according to 8).

(20) The quantitative reagent kit according to (19), which comprises a substrate of an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme or an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme. (21) An oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme is glucose dehydrogenase, and a substrate of an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme is glucose (2)
The quantitative reagent kit according to 0).

(22) The reagent kit according to (17) or (18), wherein the reagent necessary for producing ammonia is urease. (23) The reagent necessary for producing ammonia is a reagent containing creatinase (17) or (1).
The quantitative reagent kit according to 8). (24) The quantitative reagent kit according to (17) or (18), wherein the reagent necessary for producing ammonia is a creatininase-containing reagent.

Hereinafter, the present invention will be described in detail.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION As a sample, any sample may be used as long as it contains ammonia and contains substances or enzymes involved in the production of ammonia. In addition, any sample may be used as long as it contains ammonia and an enzyme whose activity is affected by ammonia. Examples of the sample include biological samples such as blood, plasma, serum, and urine.

The elimination of ammonia in a sample is carried out by reacting ammonia with glutamine synthetase in the presence of glutamic acid, a metal salt and adenosine triphosphate. The method for eliminating ammonia in a sample of the present invention comprises:
The sample contains glutamic acid, a metal salt, adenosine triphosphate and glutamine synthetase, and if necessary, in an aqueous medium containing an aqueous medium, an enzyme activity regulator, a stabilizer, a surfactant, a preservative, and the like. Addition is performed. The reaction conditions are not particularly limited. The reaction temperature is preferably from 0 to 60 ° C,
10-50 degreeC is more preferable, and 20-40 degreeC is especially preferable. The reaction time is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 2 to 10 minutes. The pH of the reaction solution is preferably from 4 to 12, more preferably from 5 to 10.

The concentrations of glutamic acid and adenosine triphosphate are not particularly limited as long as they are equal to or higher than the equimolar concentration of the ammonia to be eliminated, but are preferably 2 to 100 times the concentration of the ammonia to be eliminated. Adenosine triphosphate is preferably 0.01 to 100 mM, and 0.1 to 5 mM.
0 mM is more preferred, and 1-20 mM is particularly preferred.

Glutamic acid is preferably 0.01 to 100 mM, more preferably 0.1 to 50 mM, and 1 to 20 mM.
mM is particularly preferred. Examples of the metal salt include salts of divalent metals such as magnesium sulfate, calcium chloride, and manganese chloride.
50 mM is more preferred.

Glutamine synthetase is not particularly limited as long as it is an enzyme that catalyzes a reaction for converting glutamic acid to glutamine in the presence of ammonia and adenosine triphosphate. Enzymes can be used, and the concentration in the reaction solution is 0.1 to 1
00U / ml is preferred, 0.5-50U / ml is more preferred, and 5-50U / ml is particularly preferred. This enzyme is easily available as a commercial product. For example, glutamine synthetase manufactured by Kikkoman Corporation can be used.

After eliminating ammonia by the reaction, a glutamine synthetase inhibitor is added to stop the enzyme activity. Examples of the glutamine synthetase inhibitor include methionine sulfoximine and methionine sulfone, and a mixture of D and L forms may be used, but L form is preferred. The concentration in the reaction solution is preferably 0.1 to 400 mM, more preferably 1 to 200 mM, and 10 to 100 mM.
mM is particularly preferred. The pH of the reaction solution is preferably 5 to 10. Thereby, the glutamine synthetase activity is rapidly suppressed, and the glutamine synthetase activity completely disappears within one minute.

Next, or simultaneously with the addition of the inhibitor,
A reagent containing an enzyme acting on the substance to be quantified or a substrate of the enzyme to be quantified in the sample is added, and the substance or enzyme to be quantified is quantified. The substance or enzyme to be quantified is not particularly limited as long as it uses an enzymatic reaction, but the substance or enzyme to be quantified is preferably a substance which produces ammonia when the enzyme or the substrate is allowed to act. As the enzyme to be quantified, an enzyme whose activity is influenced by promotion or suppression by ammonia is preferable. As an enzyme acting on a substance to be quantified or an enzyme used for converting a produced substance into another substance, an enzyme whose activity is influenced by promotion or suppression by ammonia is preferable.

The reaction for producing ammonia is carried out by adding a reagent containing a substrate and an enzyme for producing ammonia from a substance or an enzyme to be quantified in a sample, and optionally containing a surfactant, a metal salt and the like.

Examples of the substance to be quantified and the enzyme acting on the substance to be quantified include the following. Urease when the substance to be quantified is urea, nicotinamide when the substance to be quantified is nicotinamide, glutaminyl peptide-glutaminase when the substance to be quantified is glutaminyl peptide, guanine when the substance to be quantified is guanine Deaminase, adenosine deaminase when the substance to be quantified is adenosine, threonine dehydrolase when the substance to be quantified is threonine, aspartate ammonia lyase when the substance to be quantified is aspartic acid, and when the substance to be quantified is methionine Is methionine lyase, methylamine glutamate methyltransferase and methylamine if the substance to be quantified is guanine, guanine deaminase if the substance to be quantified is guanine, and quinine if the substance to be quantified is creatinine. Achi Nin deiminase peptidase and the like.
When the substance to be quantified is creatine, creatinase and urease are included, and when the substance to be quantified is creatinine, creatininase, creatinase and urease are exemplified.

The concentration of these enzymes in the reaction solution is as follows:
0.1-100 U / ml is preferred, and 0.5-50 U /
ml is more preferable, and 5 to 50 U / ml is particularly preferable. In addition, the concentration of these substrates in the reaction solution is set to 0.1.
It is preferably from 01 to 100 mM, more preferably from 0.1 to 50 mM, particularly preferably from 1 to 20 mM. When urease is used, it is preferable to add an amine such as triethanolamine borate.

The reaction conditions are not particularly limited. The reaction temperature is preferably from 0 to 60 ° C, more preferably from 10 to 50 ° C,
20-40 ° C is particularly preferred. The reaction time is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 2 to 10 minutes. The pH of the reaction is not particularly limited as long as the ammonia generation reaction proceeds, but it is 4 to 12
Is preferable, and 5 to 10 are more preferable.

The produced ammonia can be quantified by a reaction using an enzyme other than glutamine synthetase. The quantitative reaction is not particularly limited, and includes, for example, a reaction for converting ammonia to nicotinamide adenine dinucleotide. The reaction to convert the produced ammonia to nicotinamide adenine dinucleotide is adenosine triphosphate,
The reaction is carried out by adding a reagent containing deamide nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide synthetase and, if necessary, a surfactant and a metal salt to the aqueous medium in which the ammonia generation reaction has been performed. This reaction may be carried out simultaneously with the reaction for producing ammonia from the substance to be quantified, during the reaction, or after the reaction.

The reaction conditions are not particularly limited. The reaction temperature is preferably from 0 to 60 ° C, more preferably from 10 to 50 ° C,
20-40 ° C is particularly preferred. The reaction time is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 2 to 10 minutes. The pH of the reaction solution is not particularly limited as long as the nicotinamide adenine dinucleotide synthesis reaction proceeds, but is preferably 4 to 12, and more preferably 5 to 10.

As nicotinamide adenine dinucleotide synthase, the reaction of producing adenosine monophosphate, pyrophosphate and nicotinamide adenine dinucleotide by acting on ammonia, adenosine triphosphate and deamide nicotinamide adenine dinucleotide. The enzyme is not particularly limited as long as it is an enzyme that catalyzes and does not react with glutamine. Examples of the enzyme include, for example, JP-A-63-185.
The enzyme described in Japanese Patent No. 378 is used.

The substance to be quantified is determined, for example, by quantifying the nicotinamide adenine dinucleotide formed. The quantification of nicotinamide adenine dinucleotide can be performed by colorimetrically determining the reaction solution after the reaction using light having a wavelength of, for example, 340 nm, and quantifying the decrease in absorbance. After enzymatic conversion to a compound, the conversion may be performed by quantifying the converted substance.

The conversion reaction and the method of quantifying the converted substance include, for example, a redox reaction using nicotinamide adenine dinucleotide as a coenzyme to generate reduced nicotinamide adenine dinucleotide phosphate, It can be performed by measuring the absorbance at 340 nm of type nicotinamide adenine dinucleotide phosphate.

In the oxidation-reduction reaction for converting nicotinamide adenine dinucleotide into reduced nicotinamide adenine dinucleotide, the above-described reaction for converting the substance to be quantified to ammonia and the reaction for converting ammonia to nicotinamide adenine dinucleotide are completed. Although the reaction can be performed after the reaction, the reaction may be started at the same time. Examples of the redox reaction include nicotine, such as a combination of glucose and glucose dehydrogenase, a combination of glucose-6-phosphate and glucose-6-phosphate dehydrogenase, and a combination of cholic acid and 3-α-hydroxysteroid dehydrogenase. An enzyme reaction system that can convert amide adenine dinucleotide into a reduced nicotinamide adenine dinucleotide using amide adenine dinucleotide as a substrate is exemplified. Among the reaction systems, those in which the reaction equilibrium is inclined toward the production of reduced nicotinamide adenine dinucleotide are preferable.

The reaction conditions are not particularly limited. The reaction temperature is preferably from 0 to 60 ° C, more preferably from 10 to 50 ° C,
20-40 ° C is particularly preferred. The reaction time is preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and particularly preferably 2 to 10 minutes. The pH of the reaction solution is not particularly limited as long as the reduced nicotinamide adenine dinucleotide synthesis reaction proceeds, but is preferably 4 to 12, and is preferably 5 to 12.
10 is more preferred.

Examples of the aqueous medium include physiological saline and an aqueous solution containing a buffer. As a buffer,
In addition to phosphates, triethanolamine borate and organic acid salts, Good's buffer may be used. Concentration is 1-500m
M. Examples of the metal salt include metal salts such as magnesium sulfate, potassium chloride, calcium chloride, and manganese chloride. Examples of the stabilizer include bovine serum albumin, amino acids, saccharides, and the like, and triethanolamine borate is an example of the urease stabilizer. As the surfactant, a nonionic, anionic or cationic surfactant can be used in a concentration that does not extremely inhibit the enzyme, usually in a range of 0.01% to 5%. For example, a nonionic HS210, a nonionic NS270, and the like can be given. Preservatives include sodium azide and the like.

The kit of the present invention comprises: a first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; and a second reagent: a glutamine synthetase selected from methionine sulfoximine and methionine sulfone. A reaction termination reagent containing an inhibitor;

Also, the kit of the present invention comprises: a first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; and a second reagent: glutamine selected from methionine sulfoximine and methionine sulfone. It comprises a substance or enzyme quantification reagent containing a reaction stop reagent containing a synthetase inhibitor and a reagent containing an enzyme acting on a substance to be quantified or a substrate of the enzyme to be quantified in a sample.

Further, the kit of the present invention comprises: a first reagent: an ammonia eliminating reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; and a second reagent: glutamine selected from methionine sulfoximine and methionine sulfone. A reaction stopping reagent containing a synthetase inhibitor and a third reagent: a reagent containing an enzyme acting on a substance to be quantified in a sample or a reagent containing a substrate of the enzyme to be quantified, or a reagent for quantifying an enzyme.

The kit of the present invention comprises: a first reagent: an ammonia eliminating reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase; a second reagent: glutamine selected from methionine sulfoximine and methionine sulfone. Reaction stop reagent containing a synthetase inhibitor, Third reagent: An enzyme that acts on the substance to be quantified in the sample or a reagent containing the substrate of the enzyme to be quantified, and Fourth reagent: For quantifying the generated substance A reagent containing a necessary reagent, a reagent containing a substance containing the necessary reagent, or a reagent for quantifying an enzyme.

Examples of the ammonia determination reagent include adenosine triphosphate, deamide nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide synthetase. If necessary, nicotinamide adenine dinucleotide can be converted to reduced nicotinamide adenine dinucleotide. And a reagent that converts ammonia containing nicotinamide adenine dinucleotide, including a reagent, a buffer, an enzyme activity regulator, a stabilizer, a surfactant, a preservative, and the like. Preferably, the ammonia determination reagent contains glucose and glucose dehydrogenase.

The reaction stopping reagent containing the glutamine synthetase inhibitor, the reagent containing the reagent necessary for producing ammonia, and the ammonia quantitative reagent may be contained in the same kit. Further, the ammonia-eliminating reagent may contain some components of a reagent necessary for producing ammonia and some components of an ammonia quantification reagent, unless ammonia is generated in the presence of the substance or enzyme to be quantified. .

Each reagent of the present invention may contain other enzymes, a substrate of the enzyme, a metal salt, a buffer, a stabilizer, a surfactant, a preservative, and the like, if necessary. Metal salts, buffers,
The stabilizer, surfactant and preservative are the same as described above. These reagents may be prepared as a dry product, or may be dissolved in water. The pH of the reagent dissolved in water is not particularly limited, but the pH of the reagent containing a glutamine synthetase inhibitor is preferably 4 to 12, and particularly preferably 5 to 10.

Hereinafter, examples, comparative examples and test examples of the present invention will be described. The sources of the enzymes and compounds used are as shown below. Trishydroxyaminomethane: Wako Pure Chemical Industries, magnesium sulfate: Kanto Chemical, potassium chloride: Wako Pure Chemical Industries, Nonion NS270: Nippon Yushi, sodium glutamate: Wako Pure Chemical Industries, adenosine triphosphate: Oriental yeast, glutamine synthetase ( Bacterial origin): prosperity, deamide nicotinamide adenine dinucleotide: Asahi Kasei Kogyo, glucose: Kanto Chemical, glucose dehydrogenase (microbial origin): Asahi Kasei Kogyo, triethanolamine trishydroxyaminomethane borate: Nacalai Tesque, methionine sulfoxy Min: Sigma, nicotinamide adenine dinucleotide synthase (from Bacillus): Asahi Kasei Kogyo, urease (from microorganism): Kyowa Hakko Kogyo.

Example 1 A urea quantitative reagent having the composition shown in Reagents 1A and 2A in Table 1 was prepared. Even if this quantitative reagent kit was stored at 30 ° C. for one month, no influence on the quantitative property was observed.

[0051]

[Table 1]

Comparative Example 1 Reagents having the compositions shown in Reagents 1B and 2B in Table 1 were prepared.

Comparative Example 2 Reagents having the compositions shown in Tables 1 and 2C were prepared.

Example 2 Reagent 1 prepared in Example 1, Comparative Example 1 and Comparative Example 2
A, 1B, and 1C, 3 ml each, were placed in a test tube, 30 μl of an aqueous solution containing 40 mg / dl of ammonia and 500 mg / dl of urea were added, and the mixture was incubated at 37 ° C. for 5 minutes.

Next, 1 ml of each of the reagents 2A, 2B and 2C was
The mixture was added to each corresponding test tube, and the change in absorbance was observed while heating at 37 ° C. The results are shown in FIG. Since ammonia cannot be eliminated from the reagent prepared in Comparative Example 1, the absorbance sharply increases after the addition of reagent 2B, and
After a minute, it remains slightly and is added to the ammonia generated from urea itself.

In the reagent prepared in Comparative Example 2, the ammonia elimination reaction is dominant, and the absorbance does not increase even if ammonia is generated. This makes measurement itself impossible.
In the reagent prepared in Example 1, a sudden increase in the absorbance due to ammonia did not occur, and the change in the absorbance corresponding to the urea content was observed because the reaction for eliminating ammonia was completely inhibited.

Example 3 A sample was prepared by adding ammonia at a concentration of 10 mg / dl to a serum sample containing 26 mg / dl of urea.

Reagent 1 prepared in Example 1 and Comparative Example 1
A and 1B were placed in 3 ml test tubes, and the above sample 1 was placed in each tube.
00 µl was added, mixed and heated at 37 ° C for 5 minutes. Subsequently, after adding 1 ml of each of the reagents 2A and 2B heated to 37 ° C. to the corresponding test tube, the change in absorbance from 2 minutes to 5 minutes was measured. Separately purified water and urea 50m
g / dl of standard solution was operated in exactly the same way. The result of the purified water was subtracted from the obtained result, and the result was compared with that obtained by subtracting the result of the purified water from the standard solution. The urea content in the serum was calculated. In Example 1, 26.2 mg / dl was obtained. The result of Example 1 was 29.8 mg / dl, indicating that the system of the present invention was excellent without the influence of ammonia.

Example 4 As a conventional method, ammonia was eliminated using glutamate dehydrogenase and isocitrate dehydrogenase.
Determinator U sold as a reagent for measuring urea using urease and glutamate dehydrogenase
Using NI (manufactured by Kyowa Medex) and the reagent of Example 1, a low-concentration sample having a urea content of 15.7 mg / dl was measured five times. Coefficient of variation (C
V) was 4.3% in the conventional method and 1.2% in the present method, and it was found that stable measurement values were obtained by the present method.

[0060]

Industrial Applicability According to the present invention, there is provided a method for quantifying a substance to be quantified by converting the substance to be quantified into ammonia, after eliminating ammonia present in a sample in advance, and a reagent used for the method. The reagent has high storage stability,
Also provided are a method and a reagent for quantifying a substance or an enzyme, which incorporates an ammonia elimination reagent that has little effect on the quantification of the substance or the enzyme to be quantified.

[Brief description of the drawings]

FIG. 1 shows the change over time in the absorbance of a reaction solution when reacted using the reagents prepared in Example 1, Comparative Examples 1 and 2.

[Explanation of symbols]

 ● The results of Example 1 are shown. (2) The results of Comparative Example 1 are shown. The results of Comparative Example 2 are shown.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Akira Miike 600-1 Kamiyamachi, Minami-Ishiki, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture Inside Kyowa Medex Laboratory, Kyowa Medex Co., Ltd. Kamiyamachi 600-1 Kyowa Medex Co., Ltd. Kyowa Medex Research Laboratory F-term (reference) 2G042 AA01 BB06 CA10 CB03 DA03 FB02 GA01 GA05 4B063 QA01 QA19 QQ03 QQ16 QQ21 QQ64 QQ84 QQ87 QQ90 QR02 QR40 QR40 QR40 QR41 QR42 QS20 QS28 QX01

Claims (24)

[Claims] The present invention relates to a method comprising the steps of: (a) adding a substance or an enzyme to be quantified to a sample containing ammonia and subjecting the ammonia to glutamine synthetase in the presence of glutamic acid, adenosine triphosphate and a metal salt to eliminate the ammonia; A glutamine synthetase inhibitor containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone to stop the glutamine synthetase reaction, and the substance to be quantified in the sample simultaneously with or after the addition of the reaction stop reagent A method for quantifying a substance or an enzyme, which comprises reacting an enzyme acting on a substance or a reagent containing a substrate of the enzyme to be quantified, and directly or enzymatically converting the generated substance into another substance for quantification. 2. The method according to claim 1, wherein the substance to be quantified by enzymatically converting it into a substance to be produced or another substance is ammonia. 3. A method for quantifying ammonia comprising quantifying nicotinamide adenine dinucleotide produced by allowing nicotinamide adenine dinucleotide synthase to act in the presence of adenosine triphosphate and deamide nicotinamide adenine dinucleotide. The method for quantification according to claim 2. 4. The method for quantifying nicotinamide adenine dinucleotide by reacting an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme and colorimetrically determining the reduced nicotinamide adenine dinucleotide produced. The quantification method according to claim 3, wherein 5. The method according to claim 4, wherein the quantification of nicotinamide adenine dinucleotide is carried out by reacting glucose dehydrogenase in the presence of glucose and colorimetrically determining the reduced nicotinamide adenine dinucleotide produced. Quantitation method as described. 6. The method according to claim 1, wherein the substance to be quantified is urea, and the reagent containing an enzyme acting on the substance to be quantified is a reagent containing urease. 7. The method according to claim 1, wherein the substance to be quantified is creatine, and the reagent containing an enzyme acting on the substance to be quantified is a reagent containing creatinase and urease. . 8. The substance to be quantified is creatinine,
The method according to any one of claims 1 to 5, wherein the reagent containing an enzyme acting on the substance to be quantified is a reagent containing creatininase, creatinase, and urease. 9. An enzyme in which the enzyme activity of an enzyme acting on a substance to be quantified, an enzyme to be quantified or an enzyme used to convert a produced substance into another substance is promoted or suppressed by ammonia. Item 1. The method according to Item 1. 10. Glutamate, ammonia in a sample containing a substance or enzyme to be quantified and ammonia.
Glutamine synthetase is allowed to act in the presence of adenosine triphosphate and a metal salt to eliminate ammonia, and then a reaction terminating reagent containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone is added, and glutamine synthase is added. A method for elimination of ammonia, which comprises stopping the enzyme reaction and then quantifying the substance or enzyme to be quantified. 11. A first reagent: an ammonia scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase and a second reagent: containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. Ammonia elimination reagent kit, comprising: 12. A first reagent: an ammonia scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt, and glutamine synthetase; and a second reagent: containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. A reagent kit for determining a substance or an enzyme, comprising: a reaction-stopping reagent that performs the reaction; 13. A first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase, a second reagent: containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. And a third reagent: a reagent for quantifying a substance or an enzyme, the reagent comprising an enzyme acting on the substance to be quantified in the sample or a reagent containing a substrate of the enzyme to be quantified. 14. A first reagent: an ammonia-scavenging reagent containing glutamic acid, adenosine triphosphate, a metal salt and glutamine synthetase, and a second reagent: containing a glutamine synthetase inhibitor selected from methionine sulfoximine and methionine sulfone. Reaction stopping reagent, third reagent: a reagent containing an enzyme acting on a substance to be determined in a sample or a substrate of the enzyme to be determined, and fourth reagent: containing a reagent necessary for quantifying a generated substance. A reagent kit containing a reagent or a substance containing an enzyme. 15. The kit according to claim 12, further comprising a reagent necessary for quantifying a substance produced in the second reagent. 16. The kit according to claim 13, further comprising a reagent necessary for quantifying a substance produced in the third reagent. 17. The reagent according to claim 12, wherein the reagent containing the enzyme acting on the substance to be quantified or the substrate of the enzyme to be quantified in the sample is a reagent necessary for producing ammonia.
7. The quantitative reagent kit according to any one of 6. 18. A reagent containing an enzyme acting on a substance to be quantified or a substrate of the enzyme to be quantified in a sample is a reagent necessary for producing ammonia, and a reagent for quantifying the produced substance. The quantitative reagent kit according to any one of claims 14 to 16, wherein the necessary reagent is a reagent necessary for quantitatively determining ammonia. 19. The quantification reagent kit according to claim 18, wherein the reagent necessary for quantifying ammonia includes deamidiconicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide synthase. 20. The quantitative reagent kit according to claim 19, further comprising a substrate of an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme or an oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme. 21. The method according to claim 20, wherein the oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme is glucose dehydrogenase, and the substrate of the oxidoreductase having nicotinamide adenine dinucleotide as a coenzyme is glucose. Reagent kit. 22. The kit according to claim 17, wherein the reagent necessary for producing ammonia is urease. 23. The quantitative reagent kit according to claim 17, wherein the reagent necessary for producing ammonia is a creatinase-containing reagent. 24. The reagent necessary for producing ammonia is a reagent containing creatininase.
Or the quantitative reagent kit according to 18.