Measurement reagent containing coenzyme, and method for stabilizing the same

Abstract

[Problem] To provide a measurement reagent which is composed of at least two reagents and contains a coenzyme and an amino acid, wherein the stability of the coenzyme is improved in order to enable the measurement reagent to be used for a long period of time; and to provide a method for impoving the stability of the coenzyme. [Solution] A measurement reagent which is composed of at least two reagents and contains a coenzyme and an amino acid, wherein the amino acid concentration of the reagent containing the most of the coenzyme is made lower than the amino acid concentration of the other reagent.

Description

Coenzyme-containing assay reagent and method for stabilizing the same

[0001] The present invention relates to a measurement reagent in which the stability of a coenzyme in a solution is improved. The present invention also relates to a method for improving the stability of a coenzyme contained in a measurement reagent. The present invention is particularly useful in the fields of chemistry, life science, analytical science, clinical testing, etc.

[0002] Measuring enzyme activity in biological samples such as blood and observing its fluctuations is essential for the diagnosis, treatment, early detection, and prevention of diseases, and is widely practiced. For example, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are enzymes found in large amounts in the liver, and their levels fluctuate due to liver disease, etc., making their measurement extremely important in clinical practice. In addition, AST is also found in large amounts in cardiac muscle and skeletal muscle, etc., and its levels fluctuate due to myocardial damage, muscular damage, etc., making its measurement extremely important in clinical practice.

[0003] The method for measuring AST and ALT is known as the JSCC-recommended method (Japan Society of Clinical Chemistry (JSCC)). Specifically, when measuring AST, L-aspartic acid and α-ketoglutaric acid are reacted with AST derived from a measurement sample to generate oxaloacetic acid and glutamic acid. The resulting oxaloacetic acid is then reacted with malate dehydrogenase (MDH) in the presence of reduced nicotinamide adenine dinucleotide (NADH), a coenzyme, to convert it to malic acid and oxidized nicotinamide adenine dinucleotide (NAD). When NADH is converted to NAD, the absorbance at 340 nm decreases, and AST is measured by measuring the rate of this decrease (Non-Patent Document 1). Furthermore, when measuring ALT, L-alanine and α-ketoglutaric acid are reacted with ALT derived from the measurement sample to produce pyruvate and glutamate, and the resulting pyruvate is then reacted with lactate dehydrogenase (LDH) in the presence of the coenzyme NADH to convert it to lactate and NAD. When NADH is converted to NAD, the absorbance at 340 nm decreases, and ALT is measured by measuring the rate of this decrease (Non-Patent Document 2). Another known measurement method is the IFCC-recommended method. This method involves adding pyridoxal phosphate to the measurement reagent to fully activate AST and ALT derived from the measurement sample before measurement (Non-Patent Document 3). Many manufacturers sell measurement reagents compatible with automated analyzers that are compatible with this measurement method and are used in hospital laboratories.

[0004] However, measurement reagents are not necessarily used immediately after purchase by users, but may be used after long-term storage in a refrigerator, etc., and it is known that making measurement reagents usable for long periods of time is a problem. To address this problem, it is required that the content of coenzymes such as NADH does not decrease even after long-term storage, that is, that the reagents have good stability, and there are conventional techniques such as a method for stabilizing coenzymes using secondary amines or tertiary amines (see Patent Document 1).

[0005] Japanese Patent Application Laid-Open No. 2020-048473

[0006] Japanese Society of Clinical Chemistry, "Recommended Method for Measurement of Enzyme Activity in Human Serum - Aspartate Aminotransferase -", Clinical Chemistry, Japanese Society of Clinical Chemistry, December 30, 1989, Vol. 18, No. 4, pp. 226-230; Japanese Society of Clinical Chemistry, "Recommended Method for Measurement of Enzyme Activity in Human Serum - Alanine Aminotransferase -", Clinical Chemistry, Japanese Society of Clinical Chemistry, December 30, 1989, Vol. 18, No. 4, pp. 250-254; Masaomi Kono and Midori Ishibashi, "Additional Comment: AST / ALT Holoenzyme Measurement - Comparison of the IFCC Method and the JSCC Method -", Clinical Pathology, Japanese Society of Clinical Laboratory Medicine, April 25, 2020, Vol. 68, No. 4, pp. 318-324

[0007] The addition of secondary amines or tertiary amines to measurement reagents has been considered for reasons such as enabling the use of measurement reagents over long periods of time. However, due to their basicity, amines can inhibit the reaction originally intended by the measurement reagent to measure the analyte contained in the sample, or can denature other components contained in the measurement reagent. In such cases, problems arise, such as the inability to obtain accurate measurements or the possibility of measurement being impossible. The inability to obtain accurate measurements or the inability to perform measurements is a serious problem that can lead to incorrect or delayed diagnosis of disease. There is a demand for measurement reagents that can obtain accurate measurements. Therefore, there is a need for methods to improve the stability of coenzymes other than by adding secondary amines or tertiary amines.

[0008] As a result of intensive research to solve the above problems, the present inventors have found that amino acids are related to the stability of coenzymes, and have thus completed the present invention.

[0009] That is, the present invention is as follows: (1) A measurement reagent comprising two or more reagents and containing a coenzyme and an amino acid, characterized in that the amino acid concentration of the reagent containing the most coenzyme is lower than the amino acid concentrations of the other reagents. (2) The measurement reagent according to (1) above, in which the coenzyme is NADH. (3) The measurement reagent according to (2) above, in which the amino acid is at least one of alanine, phenylalanine, asparagine, aspartic acid, and glycine. (4) The measurement reagent according to (3) above, in which the amino acid concentration of the reagent containing the most coenzyme is 50 mM or less. (5) The measurement reagent according to (3) above, in which the amino acid concentration of the reagent containing the most coenzyme is 20 mM or less. (6) The measurement reagent according to (3) above, in which the amino acid concentration of the reagent containing the most coenzyme is 1 mM or less. (7) The measurement reagent according to (3) above, in which the reagent containing the most coenzyme does not contain an amino acid. (8) The measuring reagent according to any one of (3) to (7) above, wherein the coenzyme is contained in only one reagent. (9) The measuring reagent according to any one of (3) to (7) above, wherein the measuring reagent is an AST measuring reagent. (10) The measuring reagent according to any one of (3) to (7) above, wherein the measuring reagent is an ALT measuring reagent. (11) The measuring reagent according to (8) above, wherein the measuring reagent is an AST measuring reagent. (12) The measuring reagent according to (8) above, wherein the measuring reagent is an ALT measuring reagent. (13) The measuring reagent according to (9) above, wherein the measuring reagent contains pyridoxal phosphate. (14) The measuring reagent according to (10) above, wherein the measuring reagent contains pyridoxal phosphate. (15) The measuring reagent according to (11) above, wherein the measuring reagent contains pyridoxal phosphate. (16) The measuring reagent according to (12) above, wherein the measuring reagent contains pyridoxal phosphate. (17) A method for improving the stability of a coenzyme contained in a measurement reagent, the method comprising: (a) making the concentration of the amino acid in the reagent containing the coenzyme in a large amount lower than that in the other reagents;(19) The method according to (18) above, wherein the amino acid is at least one of alanine, phenylalanine, asparagine, aspartic acid, and glycine. (20) The method according to (19) above, wherein the coenzyme is contained in only one reagent. (21) The method according to (19) or (20) above, wherein the measurement reagent is an AST or ALT measurement reagent. (22) The method according to (21) above, wherein the measurement reagent contains pyridoxal phosphate.

[0010] According to the present invention, the stability of the coenzyme can be improved, and therefore the measurement reagent can be used for a long period of time, and accurate measurement values ​​can be obtained even when used after long-term storage.

[0011] The present invention will be described in detail below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention.

[0012] 1. Overview Measurement reagents are not always used immediately after purchase; they may be stored in a refrigerator or other storage facility for extended periods before use. Therefore, ensuring long-term usability of measurement reagents is a known challenge. To address this challenge, it is necessary to maintain the coenzyme content even after long-term storage. Conventional techniques, such as a method for stabilizing coenzymes using secondary or tertiary amines (see Patent Document 1), exist. However, due to their basicity, amines can inhibit the intended reaction of the measurement reagent to measure the analyte contained in the sample or denature other components contained in the measurement reagent. In such cases, accurate measurements may not be obtained or the measurement itself may not be possible. The inability to obtain accurate measurements or the inability to perform the measurement itself is a serious problem that can lead to incorrect or delayed diagnosis of disease. A measurement reagent that can provide accurate measurements is desired. Therefore, there is a need for methods to improve coenzyme stability other than by adding secondary or tertiary amines. The present inventors conducted extensive research to solve the above problem and discovered that amino acids are related to coenzyme stability. The present invention was made based on these findings. In this specification, "stabilization" has the same meaning as "improving stability."

[0013] 2. Coenzyme The coenzyme that can be contained in the measuring reagent of the present invention is not particularly limited, and can be NADH or NADPH.The purpose of containing coenzyme is not particularly limited, and for example, in the AST measuring reagent, the oxaloacetic acid that is produced as a result of the reaction between the measuring sample and the measuring reagent is reacted with malate dehydrogenase in the presence of NADH to be converted into malic acid and NAD, and in the ALT measuring reagent, the pyruvic acid that is produced as a result of the reaction between the measuring sample and the measuring reagent is reacted with lactate dehydrogenase in the presence of NADH to be converted into lactic acid and NAD.When containing NADH for the above-mentioned purpose, it is not particularly limited as long as it has this function.

[0014] The measuring reagent may contain at least one type of coenzyme, but may also contain two or more types.

[0015] The concentration of coenzyme contained in the measurement reagent is not particularly limited.For example, in the AST measurement reagent, the oxaloacetic acid resulting from the reaction between the measurement sample and the measurement reagent is reacted with malate dehydrogenase in the presence of NADH to convert it into malic acid and NAD, and in the ALT measurement reagent, the pyruvic acid resulting from the reaction between the measurement sample and the measurement reagent is reacted with lactate dehydrogenase in the presence of NADH to convert it into lactate and NAD.When NADH is contained, the concentration of NADH is not particularly limited as long as it can achieve the above-mentioned intended effect.When the measurement reagent and the measurement sample are all mixed, the lower limit of the concentration of NADH is preferably 0.01 mM, and particularly preferably 0.05 mM.In addition, the upper limit is preferably 0.6 mM, and particularly preferably 0.3 mM. Regarding the concentration of NADH, for example, when the lower limit is 0.01 mM, the concentration can be 0.01 mM to 0.3 mM or 0.01 mM to 0.6 mM, and when the lower limit is 0.05 mM, the concentration can be 0.05 mM to 0.3 mM or 0.05 mM to 0.6 mM.

[0016] When the measurement reagent is composed of multiple reagents, the coenzyme may be contained in one reagent or in two or more reagents. The coenzyme may be contained in the reagent that is first mixed with the measurement sample, or in the reagent that is mixed second or later. The coenzyme may be contained only in the reagent that is first mixed with the measurement sample, or in the reagent that is mixed second or later. If the coenzyme is contained in the reagent that is first mixed with the measurement sample, the coenzyme may also be contained in the reagent that is mixed second or later.

[0017] 3. Amino Acids The purpose of the amino acids contained in the measurement reagent of the present invention is not particularly limited, but examples include alanine, phenylalanine, asparagine, aspartic acid, and glycine. For example, in the case of an AST measurement reagent, an amino acid and α-ketoglutaric acid can be reacted with AST derived from the measurement sample to produce oxaloacetic acid and glutamic acid, and in the case of an ALT measurement reagent, an amino acid and α-ketoglutaric acid can be reacted with ALT derived from the measurement sample to produce pyruvate and glutamic acid. When an amino acid is contained for the above purposes, it is not particularly limited as long as it has the desired effect, but alanine and aspartic acid are preferred. Furthermore, when an amino acid is contained for the above purposes, it is not particularly limited as long as it has the desired effect, but examples include L-amino acids, D-amino acids, and mixtures of L- and D-amino acids. Furthermore, when an amino acid is contained for the above purposes, it is not particularly limited as long as it has the desired effect, but examples include sodium salts, potassium salts, calcium salts, and the like.

[0018] The measuring reagent may contain at least one type of amino acid, but may contain two or more types.

[0019] The concentration of amino acids contained in the measurement reagent is not particularly limited. For example, when an amino acid is contained in an AST measurement reagent for the purpose of generating oxaloacetic acid and glutamic acid by reacting an amino acid and α-ketoglutaric acid with AST derived from the measurement sample, or when an amino acid is contained in an ALT measurement reagent for the purpose of generating pyruvate and glutamic acid by reacting an amino acid and α-ketoglutaric acid with ALT derived from the measurement sample, the concentration of the amino acid is not particularly limited as long as it exerts the above-mentioned intended effect. In a measurement reagent composed of two or more reagents, it is desirable that the concentration of amino acids in the reagent containing the highest amount of coenzyme is lower than the amino acid concentrations of the other reagents. In a measurement reagent composed of two or more reagents, the lower limit of the concentration of amino acids in the reagent containing the highest amount of coenzyme is preferably 0.5 mM, and particularly preferably 0 mM. Furthermore, the upper limit is preferably 50 mM, particularly preferably 20 mM, more preferably 10 mM, and even more preferably 1 mM. Regarding the concentration of this amino acid, for example, when the lower limit is 0 mM, the concentration can be 0 mM to 1 mM, 0 mM to 10 mM, 0 mM to 20 mM, or 0 mM to 50 mM; when the lower limit is 0.5 mM, the concentration can be 0.5 mM to 1 mM, 0.5 mM to 10 mM, 0.5 mM to 20 mM, or 0.5 mM to 50 mM.

[0020] When the measurement reagent is composed of multiple reagents, the amino acid may be contained in one reagent or in two or more reagents. The amino acid may be contained in the reagent that is first mixed with the measurement sample, or in the reagent that is mixed second or later. The amino acid may be contained only in the reagent that is first mixed with the measurement sample, or in the reagent that is mixed second or later. If the amino acid is contained in the reagent that is first mixed with the measurement sample, the amino acid may also be contained in the reagent that is mixed second or later.

[0021] 4. Measurement Reagents In the present invention, a measurement reagent refers to a reagent capable of measuring enzyme activity, substance concentration, etc. in a measurement sample, and is not particularly limited as long as it is capable of measuring such a property. Measurement reagents include biochemical measurement reagents that utilize biochemical reactions, immunological measurement reagents that utilize antigen-antibody reactions, and genetic measurement reagents that utilize gene analysis techniques, but in the present invention, biochemical measurement reagents are preferred. Examples of biochemical measurement reagents include AST measurement reagents and ALT measurement reagents.

[0022] The measuring reagent of the present invention may be one that performs measurement by an end-point method or a reaction rate method, and may be selected appropriately.

[0023] The measurement reagent of the present invention may be a two-reagent method in which a measurement sample and two reagents are mixed simultaneously or in an appropriate order to perform measurement, or a multi-reagent method in which a measurement sample and three or more reagents are mixed simultaneously or in an appropriate order to perform measurement, and may be selected as appropriate.

[0024] In the measurement reagent of the present invention, the measurement may be performed manually or using an apparatus such as an automatic analyzer.

[0025] The measuring reagent of the present invention may be a liquid reagent, all or part of which is a constituent reagent.

[0026] The measuring reagent of the present invention can be sold by itself or used for measuring the enzyme activity in a measurement sample.

[0027] The measurement reagent of the present invention can be sold or used in combination with other reagents other than the above-mentioned measurement reagent for measuring a substance to be measured in a sample. Examples of other reagents other than the above-mentioned measurement reagent include buffer solutions, sample dilutions, reagent dilutions, reagents containing substances for calibration, and reagents containing substances for quality control.

[0028] The measurement reagent of the present invention may be a measurement reagent kit consisting of a plurality of constituent reagents such as a first reagent and a second reagent, or other reagents.

[0029] The pH of the measurement reagent of the present invention can be appropriately selected taking into consideration the stability of the components during storage, the reaction rate during measurement of the measurement sample, etc. Furthermore, when the measurement reagent is composed of multiple reagents, the pH of each reagent constituting the measurement reagent can be appropriately selected taking into consideration the stability of the components during storage, the reaction rate during measurement of the measurement sample, etc.

[0030] For example, in the case of an AST measurement reagent, when the measurement reagent and the measurement sample are completely mixed, the lower limit of the pH is preferably 6.0 (37°C), with 7.0 (37°C) being particularly preferred. The upper limit is preferably 9.0 (37°C), with 8.0 (37°C) being particularly preferred. For example, when the lower limit of the pH of this AST measurement reagent is 6.0 (37°C), the range may be 6.0 to 8.0 (37°C) or 6.0 to 9.0 (37°C). When the lower limit is 7.0 (37°C), the range may be 7.0 to 8.0 (37°C) or 7.0 to 9.0 (37°C). In addition, when the ALT measurement reagent is completely mixed with the measurement reagent and the measurement sample, the lower limit of the pH is preferably 5.5 (37°C), with 6.5 (37°C) being particularly preferred. The upper limit is preferably 8.5 (37°C), and particularly preferably 7.5 (37°C). For example, when the lower limit is 5.5 (37°C), the pH of the ALT measurement reagent may be 5.5 to 7.5 (37°C) or 5.5 to 8.5 (37°C). When the lower limit is 6.5 (37°C), the pH may be 6.5 to 7.5 (37°C) or 6.5 to 8.5 (37°C).

[0031] In addition to the coenzyme and amino acid, the measurement reagent of the present invention may contain known surfactants, buffers, enzymes, coenzymes, reaction substrates, pH adjusters, preservatives, etc. as needed. The type of stereoisomer of each of these components can be appropriately selected, taking into account the stability of the components during storage, the reaction rate during measurement of the measurement sample, etc. Furthermore, the concentration of each of these components can be appropriately selected, taking into account the stability of the components during storage, the reaction rate during measurement of the measurement sample, etc. When the measurement reagent is composed of multiple reagents, the concentration of each of these components in each reagent constituting the measurement reagent can be appropriately selected, taking into account the stability of the components during storage, the reaction rate during measurement of the measurement sample, etc. In addition, the AST measurement reagent and the ALT measurement reagent may or may not contain pyridoxal phosphate.

[0032] 5. Biological Samples In the present invention, measurement samples include artificially prepared solutions and biological samples. Biological samples are those for which enzyme activity, substance concentration, etc. contained in a living organism are to be measured, and are not particularly limited as long as they are of this nature. Examples of such biological samples include human or animal blood, serum, plasma, urine, feces, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, and extracts of tissues and cells, such as organs (e.g., brain), hair, skin, nails, muscles, or nerves.

[0033] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0034] In the following examples, rLDH(PH) is L-type lactate dehydrogenase and D-LDH is D-type lactate dehydrogenase.

[0035] Example 1 Confirmation of the Effect of Amino Acids on NADH Stability In a solution containing amino acids and NADH, the residual rate of NADH was measured to confirm the effect of amino acids on NADH stability.

[0036] 1. Preparation of solutions

[0037] (1) Preparation of Solution A-1 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-1. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) (2) Preparation of Solution A-2 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 10 mM (Fujifilm Wako Pure Chemical Industries) (3) Preparation of Solution A-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 20 mM (Fujifilm Wako Pure Chemical Industries) (4) Preparation of Solution A-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-4.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 50 mM (Fujifilm Wako Pure Chemical Industries) (5) Preparation of Solution A-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-5. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 100 mM (Fujifilm Wako Pure Chemical Industries) (6) Preparation of Solution A-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 200 mM (Fujifilm Wako Pure Chemical Industries) (7) Preparation of Solution A-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution A-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), rLDH (PH) 3458 U / L (Oriental Yeast), pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), L-aspartic acid 400 mM (Fujifilm Wako Pure Chemical).

[0038] (8) Preparation of Solution B-1 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-1. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) (9) Preparation of Solution B-2 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 10 mM (Actec) (10) Preparation of Solution B-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 20 mM (Actec) (11) Preparation of Solution B-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-4.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 50 mM (Actec) (12) Preparation of Solution B-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-5. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 100 mM (Actec) (13) Preparation of Solution B-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 200 mM (Actec) (14) Preparation of Solution B-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution B-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), rLDH (PH) 3458 U / L (Oriental Yeast), pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), sodium L-aspartate 400 mM (Actec).

[0039] (15) Preparation of Solution C-1 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-1. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), Glycerol 5% (v / v) (Kanto Chemical), D-LDH 2155 U / L (Toyobo), Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries, Ltd.). (16) Preparation of Solution C-2 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 10 mM (Fujifilm Wako Pure Chemical Industries) (17) Preparation of Solution C-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 20 mM (Fujifilm Wako Pure Chemical Industries) (18) Preparation of Solution C-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-4.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 50 mM (Fujifilm Wako Pure Chemical Industries) (19) Preparation of Solution C-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-5. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 100 mM (Fujifilm Wako Pure Chemical Industries) (20) Preparation of Solution C-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-aspartic acid 200 mM (Fujifilm Wako Pure Chemical Industries) (21) Preparation of Solution C-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution C-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), D-LDH 2155 U / L (Toyobo), pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), L-aspartic acid 400 mM (Fujifilm Wako Pure Chemical).

[0040] (22) Preparation of Solution D-1 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-1. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), Glycerol 5% (v / v) (Kanto Chemical), D-LDH 2155 U / L (Toyobo), Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries, Ltd.). (23) Preparation of Solution D-2 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 10 mM (Actec) (24) Preparation of Solution D-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 20 mM (Actec) (25) Preparation of Solution D-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-4.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 50 mM (Actec) (26) Preparation of Solution D-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-5. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 100 mM (Actec) (27) Preparation of Solution D-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium L-aspartate 200 mM (Actec) (28) Preparation of Solution D-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution D-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), D-LDH 2155 U / L (Toyobo), pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), L-sodium aspartate 400 mM (Actec).

[0041] 2. Storage of the solution

[0042] Each of the solutions prepared in (1) to (28) above was stored in an incubator at 30° C. for 7 days.

[0043] 3. Measurement of residual NADH rate

[0044] The NADH content of each solution was measured before and after performing step 2. The NADH content after performing step 2 was divided by the NADH content before performing step 2, and the resulting value was multiplied by 100 to determine the NADH residual rate (percentage). The higher the NADH residual rate, the better the stability of NADH. The values ​​are shown in Table 1. The NADH content was measured as follows: a. Preparation of NADH Content Measurement Reagent: Potassium dihydrogen phosphate (Kanto Chemical) was dissolved in pure water to a concentration of 0.1 M, and dipotassium phosphate (Fujifilm Wako Pure Chemical Industries) was dissolved in pure water to a concentration of 0.1 M. These were mixed to a pH of 7.0 (25°C), and Triton® X-100 (Nacalai Tesque) was added to this to a concentration of 1 g / L to prepare reagent α. Furthermore, sodium pyruvate (Kanto Chemical) was added to reagent α to a concentration of 15.5 mM, and rLDH (PH) (Oriental Yeast) was added to a concentration of 46.5 kU / L to prepare reagent β. b. Method for measuring NADH content Measurements were performed using a Canon Medical Systems Corporation TBA-120FR Sora Edition automatic analyzer. 4 μL of reagent α and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was measured after this time. 4 μL of reagent β and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was measured after this time. The difference between the absorbance measured using reagent α and the absorbance measured using reagent β was taken as the NADH content.

[0045]

[0046] 4. Summary

[0047] The NADH residual rates of solutions A-1 to A-3 were higher than 90%, but the NADH residual rates of solutions A-4 to A-7 were lower than 90%. The NADH residual rates of solutions B-1 to B-3 were higher than 90%, but the NADH residual rates of solutions B-4 to B-7 were lower than 90%. The NADH residual rates of solutions C-1 to C-3 were higher than 90%, but the NADH residual rates of solutions C-4 to C-7 were lower than 90%. The NADH residual rates of solutions D-1 to D-3 were higher than 90%, but the NADH residual rates of solutions D-4 to D-7 were lower than 90%.

[0048] The NADH residual rate in solutions A-1 to A-3, in which the aspartic acid concentration was 20 mM or less, was higher than 90%, whereas the NADH residual rate in solutions A-4 to A-7, in which the aspartic acid concentration was 50 mM or more, was lower than 90%, indicating that the stability of NADH was reduced by adding a certain amount of aspartic acid. Furthermore, the NADH residual rate was lower in solution A-5 than in solution A-4, solution A-6 than in solution A-5, and solution A-7 than in solution A-6, indicating that the stability of NADH decreased depending on the concentration of added aspartic acid.

[0049] The NADH residual rate was higher than 90% in solutions B-1 to B-3, which contained 20 mM or less sodium aspartate, whereas the NADH residual rate was lower than 90% in solutions B-4 to B-7, which contained 50 mM or more sodium aspartate. This indicates that the addition of a certain amount of sodium aspartate reduces NADH stability. Furthermore, the NADH residual rate was lower in solution B-5 than in solution B-4, solution B-6 than in solution B-5, and solution B-7 than in solution B-6. This indicates that the reduction in NADH stability depends on the concentration of added sodium aspartate.

[0050] The NADH residual rate in solutions C-1 to C-3, which contained 20 mM or less aspartic acid, was higher than 90%, whereas the NADH residual rate in solutions C-4 to C-7, which contained 50 mM or more aspartic acid, was lower than 90%, indicating that the stability of NADH was reduced by the addition of a certain amount of aspartic acid. Furthermore, the NADH residual rate was lower in solution C-5 than in solution C-4, solution C-6 than in solution C-5, and solution C-7 than in solution C-6, indicating that the stability of NADH decreased depending on the concentration of added aspartic acid.

[0051] The NADH residual rate was higher than 90% in solutions D-1 to D-3, which contained 20 mM or less sodium aspartate, whereas the NADH residual rate was lower than 90% in solutions D-4 to D-7, which contained 50 mM or more sodium aspartate, indicating that the addition of a certain amount of sodium aspartate reduced the stability of NADH. Furthermore, the NADH residual rate was lower in solution D-5 than in solution D-4, solution D-6 than in solution D-5, and solution D-7 than in solution D-6, indicating that the stability of NADH decreased depending on the concentration of added sodium asparagine.

[0052] This confirms that amino acids decrease the stability of NADH in solutions containing pyridoxal phosphate, and this phenomenon was observed whether L- or D-lactate dehydrogenase was added.

[0053] Example 2 (Confirmation of the Influence of Amino Acids on NADH Stability) The influence of amino acids on NADH stability was confirmed by measuring the residual rate of NADH in a solution containing amino acids and NADH.

[0054] 1. Preparation of solutions

[0055] (29) Preparation of Solution E-1 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-1. NADH disodium 0.2 mM (Oriental Yeast Co., Ltd.), Tris-HCl 100 mM (MP Biomedicals), Glycerol 5% (v / v) (Kanto Chemical Co., Ltd.), rLDH (PH) 3458 U / L (Oriental Yeast Co., Ltd.), Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries, Ltd.). (30) Preparation of Solution E-2 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-alanine 0.5 mM (Actec) (31) Preparation of Solution E-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-alanine 1 mM (Actec) (32) Preparation of Solution E-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-4.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-alanine 2 mM (Actec) (33) Preparation of Solution E-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-5. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-alanine 3 mM (Actec) (34) Preparation of Solution E-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L-alanine 5 mM (Actec) (35) Preparation of Solution E-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution E-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), rLDH (PH) 3458 U / L (Oriental Yeast), pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), L-alanine 10 mM (Actec).

[0056] 2. Storage of the solution

[0057] Each of the solutions prepared in (29) to (35) of 1 above was stored in an incubator at 30° C. for 7 days.

[0058] 3. Measurement of residual NADH rate

[0059] The NADH content of each solution was measured before and after performing step 2. The NADH content after performing step 2 was divided by the NADH content before performing step 2, and the resulting value was multiplied by 100 to determine the NADH residual rate (percent). The higher the NADH residual rate, the better the stability of NADH. The values ​​are shown in Table 2. The NADH content was measured as follows: a. Preparation of NADH content measurement reagent: Potassium dihydrogen phosphate (Kanto Chemical) was dissolved in pure water to a concentration of 0.1 M, and dipotassium phosphate (Fujifilm Wako Pure Chemical Industries) was dissolved in pure water to a concentration of 0.1 M. These were mixed to a pH of 7.0 (25°C), and Triton (registered trademark) X-100 (Nacalai Tesque) was added to this to a concentration of 1 g / L to prepare reagent α. Furthermore, reagent β was prepared by adding sodium pyruvate (Kanto Chemical) to reagent α to a concentration of 15.5 mM and rLDH (PH) (Oriental Yeast) to a concentration of 46.5 kU / L. b. Method for Measuring NADH Content Measurements were performed using a Canon Medical Systems Corporation TBA-120FR Sora Edition automated analyzer. 4 μL of reagent α and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was then measured. 4 μL of reagent β and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was then measured. The difference between the absorbance measured using reagent α and that measured using reagent β was taken as the NADH content.

[0060]

[0061] 4. Summary

[0062] The residual NADH rates of solutions E-1 to E-3 were higher than 90%, while the residual NADH rates of solutions E-4 to E-7 were lower than 90%.

[0063] The NADH residual rate in solutions E-1 to E-3, which contained 1 mM or less alanine, was higher than 90%, whereas the NADH residual rate in solutions E-4 to E-7, which contained 2 mM or more alanine, was lower than 90%, indicating that the addition of a certain amount of alanine reduced the stability of NADH. Furthermore, the NADH residual rate was lower in solution E-5 than in solution E-4, solution E-6 than in solution E-5, and solution E-7 than in solution E-6, indicating that the stability of NADH decreased depending on the concentration of alanine added.

[0064] This confirmed that the stability of NADH was reduced by the presence of amino acids in a solution containing pyridoxal phosphate.

[0065] Example 3 Confirmation of the Influence of Amino Acids on NADH Stability In a solution containing amino acids and NADH, the residual rate of NADH was measured to confirm the influence of amino acids on NADH stability.

[0066] 1. Preparation of solutions

[0067] (36) Preparation of Solution F-1 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-1. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) (37) Preparation of Solution F-2 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-aspartic acid 10 mM (Fujifilm Wako Pure Chemical Industries) (38) Preparation of Solution F-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-aspartic acid 20 mM (Fujifilm Wako Pure Chemical Industries) (39) Preparation of Solution F-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-4. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-aspartic acid 50 mM (Fujifilm Wako Pure Chemical Industries) (40) Preparation of Solution F-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-5.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-aspartic acid 100 mM (Fujifilm Wako Pure Chemical Industries) (41) Preparation of Solution F-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-aspartic acid 200 mM (Fujifilm Wako Pure Chemical Industries) (42) Preparation of Solution F-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution F-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), rLDH (PH) 3458 U / L (Oriental Yeast), L-aspartic acid 400 mM (Fujifilm Wako Pure Chemical).

[0068] (43) Preparation of Solution G-1 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-1. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) (44) Preparation of Solution G-2 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium L-aspartate 10 mM (Fujifilm Wako Pure Chemical Industries) (45) Preparation of Solution G-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium L-aspartate 20 mM (Fujifilm Wako Pure Chemical Industries) (46) Preparation of Solution G-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-4. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium L-aspartate 50 mM (Fujifilm Wako Pure Chemical Industries) (47) Preparation of Solution G-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-5.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium L-aspartate 100 mM (Fujifilm Wako Pure Chemical Industries) (48) Preparation of Solution G-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium L-aspartate 200 mM (Fujifilm Wako Pure Chemical Industries) (49) Preparation of Solution G-7 The following components were dissolved in pure water to the indicated concentrations, and the pH was adjusted to 9.0 (30°C) to prepare Solution G-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), rLDH (PH) 3458 U / L (Oriental Yeast), L-aspartic acid sodium 400 mM (Fujifilm Wako Pure Chemical).

[0069] (50) Preparation of Solution H-1 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-1. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) (51) Preparation of Solution H-2 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-alanine 10 mM (Fujifilm Wako Pure Chemical Industries) (52) Preparation of Solution H-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-alanine 20 mM (Fujifilm Wako Pure Chemical Industries) (53) Preparation of Solution H-4 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-4. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-alanine 50 mM (Fujifilm Wako Pure Chemical Industries) (54) Preparation of Solution H-5 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-5.NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-alanine 100 mM (Fujifilm Wako Pure Chemical Industries) (55) Preparation of Solution H-6 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-6. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) L-alanine 200 mM (Fujifilm Wako Pure Chemical Industries) (56) Preparation of Solution H-7 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution H-7. NADH disodium 0.2 mM (Oriental Yeast), Tris-HCl 100 mM (MP Biomedicals), glycerol 5% (v / v) (Kanto Chemical), rLDH (PH) 3458 U / L (Oriental Yeast), L-alanine 400 mM (Fujifilm Wako Pure Chemical).

[0070] 2. Storage of the solution

[0071] Each of the solutions prepared in (36) to (56) of 1 above was stored in an incubator at 37° C. for 21 days.

[0072] 3. Measurement of residual NADH rate

[0073] The NADH content of each solution was measured before and after performing step 2. The NADH content after performing step 2 was divided by the NADH content before performing step 2, and the resulting value was multiplied by 100 to determine the NADH residual rate (percent). The higher the NADH residual rate, the better the stability of NADH. The values ​​are shown in Table 3. The NADH content was measured as follows: a. Preparation of NADH content measurement reagent: Potassium dihydrogen phosphate (Kanto Chemical) was dissolved in pure water to a concentration of 0.1 M, and dipotassium phosphate (Fujifilm Wako Pure Chemical Industries) was dissolved in pure water to a concentration of 0.1 M. These were mixed to a pH of 7.0 (25°C), and Triton (registered trademark) X-100 (Nacalai Tesque) was added to this to a concentration of 1 g / L to prepare reagent α. Furthermore, reagent β was prepared by adding sodium pyruvate (Kanto Chemical) to reagent α to a concentration of 15.5 mM and rLDH (PH) (Oriental Yeast) to a concentration of 46.5 kU / L. b. Method for Measuring NADH Content Measurements were performed using a Canon Medical Systems Corporation TBA-120FR Sora Edition automated analyzer. 4 μL of reagent α and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was then measured. 4 μL of reagent β and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was then measured. The difference between the absorbance measured using reagent α and that measured using reagent β was taken as the NADH content.

[0074]

[0075] 4. Summary

[0076] The NADH residual rates of solutions F-1 to F-4 were higher than 60%, but the NADH residual rates of solutions F-5 to F-7 were lower than 60%. The NADH residual rates of solutions G-1 to G-4 were higher than 60%, but the NADH residual rates of solutions G-5 to G-7 were lower than 60%. The NADH residual rates of solutions H-1 to H-3 were higher than 60%, but the NADH residual rates of solutions H-4 to H-7 were lower than 60%.

[0077] The NADH residual rate in solutions F-1 to F-4, which contained 50 mM or less aspartic acid, was higher than 60%, whereas the NADH residual rate in solutions F-5 to F-7, which contained 100 mM or more aspartic acid, was lower than 60%, indicating that the addition of a certain amount of aspartic acid reduced the stability of NADH. Furthermore, the NADH residual rate was lower in solution F-6 than in solution F-5, and in solution F-7 than in solution F-6, indicating that the stability of NADH decreased depending on the concentration of added aspartic acid.

[0078] The NADH residual rate in solutions G-1 to G-4, which contained 50 mM or less sodium aspartate, was higher than 60%, whereas the NADH residual rate in solutions G-5 to G-7, which contained 100 mM or more sodium aspartate, was lower than 60%, indicating that the addition of a certain amount of sodium aspartate reduced the stability of NADH. Furthermore, the NADH residual rate was lower in solution G-6 than in solution G-5, and in solution G-7 than in solution G-6, indicating that the reduction in NADH stability depended on the concentration of added sodium aspartate.

[0079] The NADH residual rate in solutions H-1 to H-3, which contained 20 mM or less alanine, was higher than 60%, whereas the NADH residual rate in solutions H-4 to H-7, which contained 50 mM or more alanine, was lower than 60%, indicating that the addition of a certain amount of alanine reduced the stability of NADH. Furthermore, the NADH residual rate was lower in solution H-5 than in solution H-4, solution H-6 than in solution H-5, and solution H-7 than in solution H-6, indicating that the stability of NADH decreased depending on the concentration of alanine added.

[0080] This confirmed that the stability of NADH was reduced by the presence of amino acids in a solution that did not contain pyridoxal phosphate.

[0081] Example 4 Confirmation of the Influence of Amino Acids on NADH Stability The influence of amino acids on NADH stability was confirmed by measuring the residual rate of NADH in a solution containing amino acids and NADH.

[0082] 1. Preparation of solutions

[0083] (57) Preparation of Solution I-1 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution I-1. NADH disodium 0.2 mM (Oriental Yeast Co., Ltd.), Tris-HCl 100 mM (MP Biomedicals), Glycerol 5% (v / v) (Kanto Chemical Co., Ltd.), rLDH (PH) 3458 U / L (Oriental Yeast Co., Ltd.), Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries, Ltd.). (58) Preparation of Solution I-2 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution I-2. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH(PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) L(−)-phenylalanine 100 mM (Fujifilm Wako Pure Chemical Industries) (59) Preparation of Solution I-3 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare Solution I-3. NADH disodium 0.2 mM (Oriental Yeast) Tris-HCl 100 mM (MP Biomedicals) Glycerol 5% (v / v) (Kanto Chemical) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical) L-asparagine monohydrate 100 mM (Fujifilm Wako Pure Chemical)

[0084] 2. Storage of the solution

[0085] Each of the solutions prepared in (57) to (59) of 1 above was stored in an incubator at 30° C. for 7 days.

[0086] 3. Measurement of residual NADH rate

[0087] The NADH content of each solution was measured before and after performing step 2. The NADH content after performing step 2 was divided by the NADH content before performing step 2, and the resulting value was multiplied by 100 to determine the NADH residual rate (percent). The higher the NADH residual rate, the better the stability of NADH. The values ​​are shown in Table 4. The NADH content was measured as follows: a. Preparation of NADH content measurement reagent: Potassium dihydrogen phosphate (Kanto Chemical) was dissolved in pure water to a concentration of 0.1 M, and dipotassium phosphate (Fujifilm Wako Pure Chemical Industries) was dissolved in pure water to a concentration of 0.1 M. These were mixed to a pH of 7.0 (25°C), and Triton (registered trademark) X-100 (Nacalai Tesque) was added to this to a concentration of 1 g / L to prepare reagent α. Furthermore, reagent β was prepared by adding sodium pyruvate (Kanto Chemical) to reagent α to a concentration of 15.5 mM and rLDH (PH) (Oriental Yeast) to a concentration of 46.5 kU / L. b. Method for Measuring NADH Content Measurements were performed using a Canon Medical Systems Corporation TBA-120FR Sora Edition automated analyzer. 4 μL of reagent α and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was then measured. 4 μL of reagent β and 120 μL of the solution to be measured were mixed and reacted at 37°C for 10 minutes. The absorbance at 340 nm was then measured. The difference between the absorbance measured using reagent α and that measured using reagent β was taken as the NADH content.

[0088]

[0089] 4. Summary

[0090] The residual NADH rate in solution I-1 was higher than 90%, while the residual NADH rates in solutions I-2 and I-3 were lower than 90%.

[0091] The residual NADH rate in solution I-1, which did not contain any amino acids, was higher than 90%, whereas the residual NADH rates in solutions I-2 and I-3, which contained 100 mM phenylalanine or asparagine, were lower than 90%, indicating that the addition of amino acids reduced the stability of NADH.

[0092] This confirmed that the stability of NADH was reduced by amino acids.

[0093] [Example 5] (Confirmation of measurement values ​​in AST measurement reagent) Among AST measurement reagents, the effect of amino acids on the stability of NADH was confirmed by measuring the enzymatic activity of AST in a measurement reagent containing the amino acids of the present invention and NADH.

[0094] 1. Preparation of reagents

[0095] (60) AST Measurement Reagents Preparation of Reagent J-1 The following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent, Reagent J-1: Tris-HCl 80 mM (MP Biomedicals), NADH 0.336 mM (Oriental Yeast), rLDH (PH) 1050 U / L (Oriental Yeast), MDH 1050 U / L (Oriental Yeast), Triton X-100 1 g / L (Nacalai Tesque). Furthermore, the following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 6.0 (20°C) to prepare the second reagent, Reagent J-1. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 420 mM (Actec) α-Ketoglutaric acid 21 mM (Actec) (61) AST Measurement Reagent Preparation of Reagent J-2 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent J-2. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 50 mM (Actec) NADH 0.336 mM (Oriental Yeast) rLDH (PH) 1050 U / L (Oriental Yeast) MDH 1050 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 5.1 (20°C) to prepare a second reagent, Reagent J-2. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 370 mM (Actec) α-Ketoglutaric acid 21 mM (Actec) (62) AST Measurement Reagent Preparation of Reagent J-3 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent J-3.Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 210 mM (Actec) NADH 0.336 mM (Oriental Yeast) rLDH (PH) 1050 U / L (Oriental Yeast) MDH 1050 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in purified water to the concentrations listed, and the pH was adjusted to 4.6 (20°C) to prepare the second reagent, Reagent J-3. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 210 mM (Actec) α-Ketoglutaric acid 21 mM (Actec).

[0096] (63) AST Measurement Reagents Preparation of Reagent K-1 The following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent, Reagent K-1: Tris-HCl 80 mM (MP Biomedicals), NADH 0.336 mM (Oriental Yeast), Pyridoxal phosphate 0.21 mM (Fujifilm Wako Pure Chemical Industries, Ltd.), rLDH (PH) 1050 U / L (Oriental Yeast), MDH 1050 U / L (Oriental Yeast), Triton X-100 1 g / L (Nacalai Tesque). The following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 6.0 (20°C) to prepare the second reagent, Reagent K-1. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 420 mM (Actec) α-Ketoglutaric acid 21 mM (Actec) (64) AST Measurement Reagent Preparation of Reagent K-2 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent K-2. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 20 mM (Actec) NADH 0.336 mM (Oriental Yeast) Pyridoxal phosphate 0.21 mM (Fujifilm Wako Pure Chemical Industries) rLDH (PH) 1050 U / L (Oriental Yeast) MDH 1050 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 5.2 (20°C) to prepare Reagent K-2, a second reagent. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 400 mM (Actec) α-Ketoglutaric acid 21 mM (Actec) (65) AST Measurement Reagent Preparation of Reagent K-3 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent K-3.Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 210 mM (Actec) NADH 0.336 mM (Oriental Yeast) Pyridoxal phosphate 0.21 mM (Fujifilm Wako Pure Chemical Industries) rLDH (PH) 1050 U / L (Oriental Yeast) MDH 1050 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 4.6 (20°C) to prepare Reagent K-3, a second reagent. Tris-HCl 80 mM (MP Biomedicals), sodium L-aspartate 210 mM (Actec), α-ketoglutaric acid 21 mM (Actec).

[0097] 2. Storage of reagents

[0098] The reagents prepared in (60) to (62) of 1 above were stored in an incubator at 37°C for 21 days. The reagents prepared in (63) to (65) of 1 above were stored in an incubator at 30°C for 7 days.

[0099] 3. Confirmation of AST enzyme activity

[0100] The AST enzyme activity of the samples was measured using each reagent before and after performing step 2. The activity value after performing step 2 was divided by the activity value before performing step 2, and the result was multiplied by 100 to determine the percent change in AST measurement value. The higher the percent change in AST measurement value, the better the stability of lactate dehydrogenase. The values ​​are shown in Table 5. AST enzyme activity was measured as follows: a. Sample preparation: Serum with high AST values ​​was diluted with purified water. Different samples were prepared for reagents J-1 to J-3 and reagents K-1 to K-3. b. Method for measuring AST enzyme activity: A Hitachi High-Tech 7180 automatic analyzer was used for measurements. 10 μL of the sample was mixed with 100 μL of the first reagent of Reagent J-1 and incubated at 37°C for 5 minutes. Then, 100 μL of the second reagent of Reagent J-1 was further mixed and incubated at 37°C for 5 minutes. The change in absorbance per minute at 340 nm measured after mixing the second reagent was proportional to the change in absorbance per minute at 340 nm measured when Enzyme Calibrator S (Shinotest), which displays the enzyme activity of AST, was used instead of the sample. The enzyme activity of AST was measured using the sample. Similar measurements were also performed with Reagents J-2, J-3, K-1, K-2, and K-3.

[0101]

[0102] 4. Summary

[0103] The rate of change in AST measurement values ​​for reagent J-3 was lower than 95%, but the rate of change in AST measurement values ​​for reagents J-1 and J-2 was higher than 95%. The rate of change in AST measurement values ​​for reagent K-3 was lower than 95%, but the rate of change in AST measurement values ​​for reagents K-1 and K-2 was higher than 95%.

[0104] The rate of change in AST measurement value for reagent J-3, which is a first reagent containing NADH and has a sodium aspartate concentration of 210 mM, was lower than 95%, whereas the rate of change in AST measurement value for reagents J-1 and J-2, which have sodium aspartate concentrations lower than 210 mM, was higher than 95%, indicating that the stability of NADH is reduced by adding a certain amount of sodium aspartate.

[0105] The rate of change in AST measurement value for reagent K-3, which is a first reagent containing NADH and has a sodium aspartate concentration of 210 mM, was lower than 95%, whereas the rate of change in AST measurement value for reagents K-1 and K-2, which have sodium aspartate concentrations lower than 210 mM, was higher than 95%, indicating that the stability of NADH is reduced by adding a certain amount of sodium aspartate.

[0106] This confirmed that amino acids reduce the stability of NADH. This phenomenon was observed regardless of the presence or absence of pyridoxal phosphate. In the AST measurement reagent, reducing the amino acid concentration of the reagent containing NADH was effective in improving the stability of the AST measurement reagent.

[0107] [Example 6] (Confirmation of measurement values ​​in ALT measurement reagent) Among ALT measurement reagents, the enzyme activity of ALT in a sample containing the amino acid of the present invention and NADH was measured to confirm the effect of the amino acid on the stability of NADH.

[0108] 1. Preparation of reagents

[0109] (66) ALT Measurement Reagent Preparation of Reagent L-1 The following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent, Reagent L-1: Tris-HCl 100 mM (MP Biomedicals), NADH 0.336 mM (Oriental Yeast), rLDH (PH) 4200 U / L (Oriental Yeast), Triton X-100 1 g / L (Nacalai Tesque). Furthermore, the following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 3.9 (20°C) to prepare the second reagent, Reagent L-1. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1050 mM (Actec) α-Ketoglutaric acid 31.5 mM (Actec) (67) ALT Measurement Reagent Preparation of Reagent L-2 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent L-2. Tris-HCl 100 mM (MP Biomedicals) L-alanine 20 mM (Actec) NADH 0.336 mM (Oriental Yeast) rLDH (PH) 4200 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 4.0 (20°C) to prepare a second reagent, Reagent L-2. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1030 mM (Actec) α-Ketoglutaric acid 31.5 mM (Actec) (68) ALT Measurement Reagent Preparation of Reagent L-3 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent L-3.Tris-HCl 100 mM (MP Biomedicals), L-alanine 525 mM (Actec), NADH 0.336 mM (Oriental Yeast), rLDH (PH) 4200 U / L (Oriental Yeast), Triton X-100 1 g / L (Nacalai Tesque). Furthermore, the following reagent components were dissolved in purified water to the indicated concentrations, and the pH was adjusted to 3.1 (20°C) to prepare the second reagent, Reagent L-3. Tris-HCl 100 mM (MP Biomedicals), L-alanine 525 mM (Actec), α-ketoglutaric acid 31.5 mM (Actec).

[0110] (69) ALT Measurement Reagent Preparation of Reagent M-1 The following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent, Reagent M-1: Tris-HCl 100 mM (MP Biomedicals) NADH 0.336 mM (Oriental Yeast) Pyridoxal phosphate 0.21 mM (Fujifilm Wako Pure Chemical Industries) rLDH (PH) 4200 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 3.9 (20°C) to prepare the second reagent, Reagent M-1. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1050 mM (Actec) α-Ketoglutaric acid 31.5 mM (Actec) (70) ALT Measurement Reagent Preparation of Reagent M-2 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent M-2. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1 mM (Actec) NADH 0.336 mM (Oriental Yeast) Pyridoxal phosphate 0.21 mM (Fujifilm Wako Pure Chemical Industries) rLDH (PH) 4200 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Furthermore, the following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 4.0 (20°C) to prepare a second reagent, Reagent M-2. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1049 mM (Actec) α-Ketoglutaric acid 31.5 mM (Actec) (71) ALT Measurement Reagent Preparation of Reagent M-3 The following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare the first reagent of Reagent M-3.Tris-HCl 100 mM (MP Biomedicals), L-alanine 525 mM (Actec), NADH 0.336 mM (Oriental Yeast), Pyridoxal phosphate 0.21 mM (Fujifilm Wako Pure Chemical Industries, Ltd.), rLDH (PH) 4200 U / L (Oriental Yeast), Triton X-100 1 g / L (Nacalai Tesque). Furthermore, the following reagent components were dissolved in purified water to the indicated concentrations, and the pH was adjusted to 3.1 (20°C) to prepare the second reagent, Reagent M-3. Tris-HCl 100 mM (MP Biomedicals), L-alanine 525 mM (Actec), α-ketoglutaric acid 31.5 mM (Actec).

[0111] 2. Storage of reagents

[0112] The reagents prepared in (66) to (68) of 1 above were stored in an incubator at 37°C for 21 days. The reagents prepared in (69) to (71) of 1 above were stored in an incubator at 30°C for 7 days.

[0113] 3. Confirmation of ALT enzyme activity

[0114] The ALT enzyme activity of the samples was measured using each reagent before and after performing step 2. The activity value after performing step 2 was divided by the activity value before performing step 2, and the result was multiplied by 100 to determine the percent change in ALT measurement value. The higher the percent change in ALT measurement value, the better the stability of lactate dehydrogenase. The values ​​are shown in Table 6. The ALT enzyme activity was measured as follows. a. Sample preparation: Serum with high ALT levels was diluted with purified water. Different samples were prepared for reagents L-1 to L-3 and reagents M-1 to M-3. b. Method for measuring ALT enzyme activity: A Hitachi High-Tech 7180 automatic analyzer was used for measurements. 10 μL of the sample was mixed with 100 μL of the first reagent of Reagent L-1 and incubated at 37°C for 5 minutes. Then, 100 μL of the second reagent of Reagent L-1 was further mixed and incubated at 37°C for 5 minutes. The change in absorbance per minute at 340 nm measured after mixing the second reagent was proportionally calculated to the change in absorbance per minute at 340 nm measured when Enzyme Calibrator S (Shinotest), which displays the ALT enzyme activity value, was used instead of the sample. The ALT enzyme activity of the sample was measured. Similar measurements were also performed with Reagents L-2, L-3, M-1, M-2, and M-3.

[0115]

[0116] 4. Summary

[0117] The rate of change in ALT measurement values ​​for reagent L-3 was lower than 95%, but the rate of change in ALT measurement values ​​for reagents L-1 and L-2 was higher than 95%. The rate of change in ALT measurement values ​​for reagent M-3 was lower than 95%, but the rate of change in ALT measurement values ​​for reagents M-1 and M-2 was higher than 95%.

[0118] The rate of change in ALT measurement value for reagent L-3, which is a first reagent containing NADH and has an alanine concentration of 525 mM, was lower than 95%, whereas the rate of change in ALT measurement value for reagents L-1 and L-2, which have an alanine concentration lower than 525 mM, was higher than 95%, indicating that the stability of NADH was reduced by adding a certain amount of alanine.

[0119] The rate of change in ALT measurement value for reagent M-3, which is a first reagent containing NADH and has an alanine concentration of 525 mM, was lower than 95%, whereas the rate of change in ALT measurement value for reagents M-1 and M-2, which have an alanine concentration lower than 525 mM, was higher than 95%, indicating that the stability of NADH is reduced by adding a certain amount of alanine.

[0120] This confirmed that amino acids reduce the stability of NADH. This phenomenon was observed regardless of the presence or absence of pyridoxal phosphate. In the ALT measurement reagent, reducing the amino acid concentration of the reagent containing NADH was effective in improving the stability of the ALT measurement reagent.

Claims

1. A measurement reagent that is composed of two or more reagents and contains coenzymes and amino acids, characterized in that the amino acid concentration in the reagent containing the highest amount of coenzymes is lower than the amino acid concentrations in the other reagents.

2. The measuring reagent according to claim 1, wherein the coenzyme is NADH.

3. The measuring reagent according to claim 2, wherein the amino acid is at least one of alanine, phenylalanine, asparagine, aspartic acid, and glycine.

4. The measuring reagent according to claim 3, wherein the concentration of amino acids in the reagent containing the highest amount of coenzyme is 50 mM or less.

5. The measuring reagent according to claim 3, wherein the concentration of amino acids in the reagent containing the highest amount of coenzyme is 20 mM or less.

6. The measuring reagent according to claim 3, wherein the concentration of amino acids in the reagent containing the highest amount of coenzyme is 1 mM or less.

7. The measuring reagent according to claim 3, wherein the reagent containing the highest amount of coenzyme does not contain any amino acids.

8. The measuring reagent according to any one of claims 3 to 7, wherein the coenzyme is contained in only one reagent.

9. The measuring reagent according to any one of claims 3 to 7, which is an AST measuring reagent.

10. The measuring reagent according to any one of claims 3 to 7, which is an ALT measuring reagent.

11. The measuring reagent according to claim 8, which is an AST measuring reagent.

12. The measuring reagent according to claim 8, which is an ALT measuring reagent.

13. The measuring reagent according to claim 9, wherein the measuring reagent contains pyridoxal phosphate.

14. The measuring reagent according to claim 10, wherein the measuring reagent contains pyridoxal phosphate.

15. The measuring reagent according to claim 11, wherein the measuring reagent contains pyridoxal phosphate.

16. The measuring reagent according to claim 12, wherein the measuring reagent contains pyridoxal phosphate.

17. A method for improving the stability of a coenzyme contained in a measurement reagent, which is composed of two or more reagents and contains a coenzyme and an amino acid, characterized in that the amino acid concentration of the reagent containing a higher amount of coenzyme is made lower than the amino acid concentration of the other reagents.

18. The method of claim 17, wherein the coenzyme is NADH.

19. The method of claim 18, wherein the amino acid is at least one of alanine, phenylalanine, asparagine, aspartic acid, and glycine.

20. The method of claim 19, wherein the coenzyme is contained in only one reagent.

21. The method according to claim 19 or 20, wherein the measuring reagent is an AST or ALT measuring reagent.

22. The method according to claim 21, wherein the assay reagent contains pyridoxal phosphate.

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