Measurement reagent containing lactate dehydrogenase, and method for stabilizing the same

Abstract

[Problem] To provide a measurement reagent which contains lactate dehydrogenase, wherein the stability of the lactate dehydrogenase is improved in order to enable the measurement reagent to be used for a long period of time; and to provide a method for improving the stability of the lactate dehydrogenase. [Solution] An alkali metal compound is included in a reagent containing lactate dehydrogenase.

Description

Lactate dehydrogenase-containing assay reagent and method for stabilizing the same

[0001] The present invention relates to a measurement reagent containing an alkali metal to improve the stability of lactate dehydrogenase in solution. The present invention also relates to a method for improving the stability of lactate dehydrogenase contained in a measurement reagent containing an alkali metal. 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 being stored for a long period of time in a refrigerator, etc., and it is known that making measurement reagents usable for a long period of time is a problem. To address this problem, there is a demand for measurement reagents that do not lose enzyme activity, such as lactate dehydrogenase, even after long-term storage, i.e., that are stable. Conventional techniques include a method of coexisting an alcohol compound and / or a chelating reagent with an enzyme (see Patent Document 1).

[0005] WO2007 / 148451 publication

[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 alcohol compounds or chelating reagents to measurement reagents has been considered for reasons such as enabling the reagents to be used for extended periods of time. However, alcohol compounds, due to their denaturing properties, and chelating reagents, due to their metal chelating properties, 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 inability to perform measurements at all. The inability to obtain accurate measurements or the inability to perform measurements at all 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 enzyme stability other than by adding alcohol compounds or chelating reagents.

[0008] As a result of intensive research to solve the above problems, the inventors discovered that the stability of the enzyme can be improved by adding a certain compound to the measurement reagent, and thus completed the present invention.

[0009] That is, the present invention is as follows. (1) A measuring reagent characterized by containing an alkali metal compound in a reagent containing lactate dehydrogenase. (2) The measuring reagent according to (1) above, wherein the alkali metal compound is at least one of a sodium compound, a lithium compound, and a potassium compound. (3) The measuring reagent according to (1) above, wherein the alkali metal compound is at least one of sodium chloride, lithium chloride, potassium chloride, sodium acetate, lithium acetate, and potassium acetate. (4) The measuring reagent according to any one of (1) to (3) above, wherein the concentration of the alkali metal compound is 55 mM or more. (5) The measuring reagent according to any one of (1) to (3) above, wherein the concentration of the alkali metal compound is 110 mM or more. (6) The measuring reagent according to any one of (1) to (3) above, wherein the measuring reagent is a measuring reagent for measuring AST or ALT. (7) The measuring reagent according to (4) above, wherein the measuring reagent is a measuring reagent for measuring AST or ALT. (8) The measuring reagent according to (5) above, wherein the measuring reagent is a measuring reagent for measuring AST or ALT. (9) The measuring reagent according to (6) above, wherein the measuring reagent contains pyridoxal phosphate. (10) The measuring reagent according to (7) above, wherein the measuring reagent contains pyridoxal phosphate. (11) The measuring reagent according to (8) above, wherein the measuring reagent contains pyridoxal phosphate. (12) A method for improving the stability of lactate dehydrogenase contained in a measuring reagent, characterized by adding an alkali metal compound to the reagent containing lactate dehydrogenase. (13) The method according to (12) above, wherein the alkali metal compound is at least one of a sodium compound, a lithium compound, and a potassium compound. (14) The method according to (12) above, wherein the alkali metal compound is at least one of sodium chloride, lithium chloride, potassium chloride, sodium acetate, lithium acetate, and potassium acetate. (15) The method according to any one of (12) to (14) above, wherein the measurement reagent is a measurement reagent for measuring AST or ALT. (16) The method according to (15) above, wherein the measurement reagent contains pyridoxal phosphate.

[0010] According to the present invention, the stability of lactate dehydrogenase 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, there is a need for enzymes that do not lose their activity even after long-term storage. Conventional techniques, such as a method of coexisting an alcohol compound and / or a chelating reagent with an enzyme (see Patent Document 1), exist. However, alcohol compounds, due to their denaturing properties, and chelating reagents, due to their metal chelating properties, 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 an inaccurate measurement value or the inability to perform the measurement at all. The inability to obtain an accurate measurement value or the inability to perform the measurement at all is a serious problem that can lead to incorrect or delayed diagnosis of disease. A measurement reagent that can obtain accurate measurement values ​​is desirable. Therefore, there is a need for methods to improve enzyme stability other than by adding an alcohol compound or a chelating reagent. As a result of intensive research to solve the above problems, the present inventors have found that the stability of enzymes can be improved by adding a certain compound to a measurement reagent. The present invention was made based on this finding. In this specification, "stabilization" is synonymous with "improving stability."

[0013] 2. Lactate dehydrogenase The lactate dehydrogenase contained in the measuring reagent of the present invention is not particularly limited for its purpose. For example, for AST measuring reagent, it can be contained to avoid the influence of pyruvic acid originally contained in the measuring sample on the measurement. For ALT measuring reagent, it can be contained to make the pyruvic acid resulting from the reaction between the measuring sample and the measuring reagent react with lactate dehydrogenase in the presence of NADH to convert it into lactic acid and NAD.When lactate dehydrogenase is contained for the above purpose, it is not particularly limited as long as it has this function, for example, it can be derived from pig heart, pig muscle, rabbit muscle, microorganism, etc.In addition, when lactate dehydrogenase is contained for the above purpose, it is not particularly limited as long as it has this function, for example, it can be derived from purified products such as organs, purified products from gene-transfected E. coli, etc. Furthermore, when lactate dehydrogenase is contained for the above-mentioned purpose, it is not particularly limited as long as it has the function, but examples thereof include lactate dehydrogenases composed of four cardiac muscle-type subunits, three cardiac muscle-type subunits and one skeletal muscle-type subunit, two cardiac muscle-type subunits and two skeletal muscle-type subunits, one cardiac muscle-type subunit and three skeletal muscle-type subunits, four skeletal muscle-type subunits, etc. Furthermore, when lactate dehydrogenase is contained for the above-mentioned purpose, it is not particularly limited as long as it has the function, but examples thereof include D-type lactate dehydrogenase that reacts with D-type lactic acid, L-type lactate dehydrogenase that reacts with L-type lactic acid, and mixtures thereof.

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

[0015] The concentration of lactate dehydrogenase contained in the measurement reagent is not particularly limited.For example, in the AST measurement reagent, in order to avoid the influence of pyruvic acid originally contained in the measurement sample on the measurement, in the ALT measurement reagent, lactate dehydrogenase is contained in order to convert the pyruvic acid resulting from the reaction between the measurement sample and the measurement reagent into lactic acid and NAD by reacting it with lactate dehydrogenase in the presence of NADH, so that the concentration of lactate dehydrogenase 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 lactate dehydrogenase concentration is preferably 10 U / L, particularly preferably 250 U / L, and more preferably 500 U / L.In addition, the upper limit is preferably 100,000 U / L, particularly preferably 30,000 U / L, and more preferably 15,000 U / L. With regard to the concentration of lactate dehydrogenase, for example, when the lower limit is 10 U / L, examples thereof include 10 U / L to 15,000 U / L, 10 U / L to 30,000 U / L, or 10 U / L to 100,000 U / L; when the lower limit is 250 U / L, examples thereof include 250 U / L to 15,000 U / L, 250 U / L to 30,000 U / L, or 250 U / L to 100,000 U / L; and when the lower limit is 500 U / L, examples thereof include 500 U / L to 15,000 U / L, 500 U / L to 30,000 U / L, or 500 U / L to 100,000 U / L.

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

[0017] 3. Alkali Metal Compound The alkali metal compound contained in the measurement reagent of the present invention is not particularly limited as long as it has the effect of improving the stability of lactate dehydrogenase, and examples thereof include alkali metal compounds such as sodium, lithium, and potassium. Among these, sodium chloride, lithium chloride, potassium chloride, sodium acetate, lithium acetate, and potassium acetate are particularly preferred. Furthermore, alkali metal compounds may be contained for purposes other than obtaining the above-mentioned effects.

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

[0019] The concentration of the alkali metal compound contained in the assay reagent is not particularly limited as long as it is a concentration that improves the stability of lactate dehydrogenase. When lactate dehydrogenase and the alkali metal compound are mixed, the lower limit of the alkali metal compound concentration is preferably 11 mM, particularly preferably 55 mM, and more preferably 110 mM. The upper limit is preferably 220 mM, particularly preferably 440 mM, and more preferably 880 mM. Regarding the concentration of this alkali metal compound, for example, when the lower limit is 11 mM, the concentration can be 11 mM to 220 mM, 11 mM to 440 mM, or 11 mM to 880 mM; when the lower limit is 55 mM, the concentration can be 55 mM to 220 mM, 55 mM to 440 mM, or 55 mM to 880 mM; and when the lower limit is 110 mM, the concentration can be 110 mM to 220 mM, 110 mM to 440 mM, or 110 mM to 880 mM.

[0020] When the measurement reagent is composed of multiple reagents, the alkali metal compound may be contained in the reagent in which the stability of lactate dehydrogenase is to be improved, and typically, the alkali metal compound is contained in the reagent containing lactate dehydrogenase. When the alkali metal compound is contained in the reagent containing lactate dehydrogenase, the alkali metal compound may also be contained in the reagent not containing lactate dehydrogenase.

[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 one-reagent method in which a measurement sample is mixed with one reagent to perform measurement, a two-reagent method in which a measurement sample is mixed with two reagents simultaneously or in an appropriate order to perform measurement, or a multi-reagent method in which a measurement sample is mixed with three or more reagents 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 alkali metal compound and lactate dehydrogenase, 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 an Alkali Metal Compound on Improving the Stability of Lactate Dehydrogenase) The effect of an alkali metal compound on improving the stability of lactate dehydrogenase was confirmed by measuring the residual rate of lactate dehydrogenase in a solution containing an alkali metal compound and lactate dehydrogenase.

[0036] 1. Preparation of solutions

[0037] (1) Preparation of Control A-0 The following components were dissolved in pure water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Control A-0. Tris-HCl 100 mM (MP Biomedicals) Triton (registered trademark) X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) (2) Preparation of Invention 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 a solution of Invention A-1. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium chloride 11 mM (Kanto Chemical) (3) Preparation of Present Invention A-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 a solution of Present Invention A-2. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium chloride 55 mM (Kanto Chemical) (4) Preparation of Present Invention 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 a solution of Present Invention A-3. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium chloride 110 mM (Kanto Chemical) (5) Preparation of Present Invention 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 a solution of Present Invention A-4.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium chloride 220 mM (Kanto Chemical) (6) Preparation of Present Invention 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 a solution of Present Invention A-5. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium chloride 440 mM (Kanto Chemical) (7) Preparation of Present Invention 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 a solution of Present Invention A-6. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium chloride 880 mM (Kanto Chemical) (8) Preparation of Present Invention 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 a solution of Present Invention A-7. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Lithium chloride 110 mM (Fujifilm Wako Pure Chemical Industries) (9) Preparation of Present Invention A-8 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention A-8. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Potassium chloride 110 mM (Kanto Chemical) (10) Preparation of the present invention A-9 The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of the present invention A-9.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Sodium acetate trihydrate 110 mM (Kanto Chemical) (11) Preparation of Present Invention A-10 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention A-10. Tris-HCl 100 mM (MP Biomedicals), Triton X-100 1 g / L (Nacalai Tesque), rLDH (PH) 3458 U / L (Oriental Yeast), Lithium acetate 110 mM (Fujifilm Wako Pure Chemical Industries). (12) Preparation of Invention A-11 The following components were dissolved in purified water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Invention A-11. Tris-HCl 100 mM (MP Biomedicals), Triton X-100 1 g / L (Nacalai Tesque), rLDH (PH) 3458 U / L (Oriental Yeast), Potassium acetate 110 mM (Fujifilm Wako Pure Chemical Industries).

[0038] (13) Preparation of Control B-0 The following components were dissolved in purified water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Control B-0. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) (14) Preparation of Present Invention B-1 The following components were dissolved in purified water to the concentrations listed, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention B-1. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 11 mM (Kanto Chemical) (15) Preparation of Present Invention B-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 a solution of Present Invention B-2. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 55 mM (Kanto Chemical) (16) Preparation of Present Invention 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 a solution of Present Invention B-3. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 110 mM (Kanto Chemical) (17) Preparation of Present Invention 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 a solution of Present Invention B-4.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 220 mM (Kanto Chemical) (18) Preparation of Present Invention 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 a solution of Present Invention B-5. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 440 mM (Kanto Chemical) (19) Preparation of Present Invention 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 a solution of Present Invention B-6. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 880 mM (Kanto Chemical) (20) Preparation of Present Invention 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 a solution of Present Invention B-7. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Lithium chloride 110 mM (Fujifilm Wako Pure Chemical Industries) (21) Preparation of Present Invention B-8 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of the present invention B-8.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Potassium chloride 110 mM (Kanto Chemical) (22) Preparation of Present Invention B-9 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of the present invention B-9. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium acetate trihydrate 110 mM (Kanto Chemical) (23) Preparation of Present Invention B-10 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention B-10. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) rLDH (PH) 3458 U / L (Oriental Yeast) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Lithium acetate 110 mM (Fujifilm Wako Pure Chemical Industries) (24) Preparation of Present Invention B-11 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention B-11. Tris-HCl 100 mM (MP Biomedicals), Triton X-100 1 g / L (Nacalai Tesque), rLDH (PH) 3458 U / L (Oriental Yeast), pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), potassium acetate 110 mM (Fujifilm Wako Pure Chemical).

[0039] (25) Preparation of Control C-0 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Control C-0. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) (26) Preparation of Invention C-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 a solution of Invention C-1. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium chloride 11 mM (Kanto Chemical) (27) Preparation of Present Invention C-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 a solution of Present Invention C-2. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium chloride 55 mM (Kanto Chemical) (28) Preparation of Present Invention C-3 The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention C-3. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium chloride 110 mM (Kanto Chemical) (29) Preparation of Present Invention 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 a solution of Present Invention C-4. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium chloride 220 mM (Kanto Chemical) (30) Preparation of Present Invention 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 a solution of Present Invention C-5.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium chloride 440 mM (Kanto Chemical) (31) Preparation of Present Invention C-6 The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention C-6. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium chloride 880 mM (Kanto Chemical) (32) Preparation of Present Invention C-7 The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention C-7. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Lithium chloride 110 mM (Fujifilm Wako Pure Chemical Industries) (33) Preparation of Present Invention C-8 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of the present invention C-8. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Potassium chloride 110 mM (Kanto Chemical) (34) Preparation of Present Invention C-9 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of the present invention C-9. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Sodium acetate trihydrate 110 mM (Kanto Chemical) (35) Preparation of Present Invention C-10 The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention C-10.Tris-HCl 100 mM (MP Biomedicals), Triton X-100 1 g / L (Nacalai Tesque), D-LDH 2155 U / L (Toyobo), Lithium acetate 110 mM (Fujifilm Wako Pure Chemical Industries, Ltd.) (36) Preparation of Present Invention C-11 The following components were dissolved in purified water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Present Invention C-11. Tris-HCl 100 mM (MP Biomedicals), Triton X-100 1 g / L (Nacalai Tesque), D-LDH 2155 U / L (Toyobo), Potassium acetate 110 mM (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0040] (37) Preparation of Control D-0 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of Control D-0. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries, Ltd.) (38) Preparation of Present Invention D-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 a solution of Present Invention D-1. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 11 mM (Kanto Chemical) (39) Preparation of D-2 of the Present Invention The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-2 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 55 mM (Kanto Chemical) (40) Preparation of D-3 of the present invention The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-3 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 110 mM (Kanto Chemical) (41) Preparation of D-4 of the Present Invention The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-4 of the present invention.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 220 mM (Kanto Chemical) (42) Preparation of D-5 of the present invention The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-5 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 440 mM (Kanto Chemical) (43) Preparation of D-6 of the present invention The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-6 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium chloride 880 mM (Kanto Chemical) (44) Preparation of D-7 of the Present Invention The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-7 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Lithium chloride 110 mM (Fujifilm Wako Pure Chemical Industries) (45) Preparation of D-8 of the Present Invention The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-8 of the present invention.Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Potassium chloride 110 mM (Kanto Chemical) (46) Preparation of D-9 of the Present Invention The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-9 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Sodium acetate trihydrate 110 mM (Kanto Chemical) (47) Preparation of D-10 of the present invention The following components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 9.0 (30°C) to prepare a solution of D-10 of the present invention. Tris-HCl 100 mM (MP Biomedicals) Triton X-100 1 g / L (Nacalai Tesque) D-LDH 2155 U / L (Toyobo) Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical Industries) Lithium acetate 110 mM (Fujifilm Wako Pure Chemical Industries) (48) Preparation of Present Invention D-11 The following components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 9.0 (30°C) to prepare a solution of the present invention D-11. Tris-HCl 100 mM (MP Biomedicals), Triton X-100 1 g / L (Nacalai Tesque), D-LDH 2155 U / L (Toyobo), Pyridoxal phosphate monohydrate 0.12 mM (Fujifilm Wako Pure Chemical), Potassium acetate 110 mM (Fujifilm Wako Pure Chemical).

[0041] 2. Storage of the solution

[0042] Each of the solutions prepared in (1) to (24) of 1 above was stored in an incubator at 5°C for 7 days. Also, each of the solutions prepared in (25) to (48) of 1 above was stored in an incubator at 30°C for 7 days.

[0043] 3. Measurement of lactate dehydrogenase residual rate

[0044] The enzyme activity of lactate dehydrogenase in each solution was measured 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 obtain the lactate dehydrogenase residual rate (percent). The higher the lactate dehydrogenase residual rate, the better the stability of lactate dehydrogenase. The values ​​are shown in Table 1. The enzyme activity of lactate dehydrogenase was measured as follows: a. Preparation of lactate dehydrogenase activity measurement reagent The following reagent components were dissolved in pure water to the concentrations shown, respectively, to prepare solutions X and Y. Solution X (pH 7.8 (25°C)) Tris-HCl 100mM (MP Biomedicals) Solution Y Tris 10mM (MP Biomedicals) NADH 21mM (Oriental Yeast) Furthermore, potassium dihydrogen phosphate (Kanto Chemical) dissolved in pure water to a concentration of 0.1M and dipotassium phosphate (Fujifilm Wako Pure Chemical Industries) dissolved in pure water to a concentration of 0.1M were mixed to a pH of 7.0 (25°C), and Triton X-100 (Nacalai Tesque) was added to this to a concentration of 1g / L to prepare solution Z. Reagent α was prepared by mixing 25mL of solution X and 0.5mL of solution Y, and reagent β was prepared by mixing 8mL of solution X and 1.5mL of solution Z. b. Measurement of lactate dehydrogenase activity: A Hitachi High-Tech 7180 automatic analyzer was used for the measurement. 2.5 μL of the solution to be measured was mixed with 200 μL of reagent α and reacted at 37°C for 5 minutes, after which 50 μL of reagent β was added and reacted at 37°C for another 5 minutes. The change in absorbance at 340 nm per minute after mixing with reagent β was measured, and the enzyme activity of lactate dehydrogenase was calculated using the following formula: Lactate dehydrogenase enzyme activity (U / L) = change in absorbance per minute × (total reaction solution volume (μL) ÷ measurement sample solution volume (μL)) ÷ molar extinction coefficient of NADH (L mol^-1 cm^-1) ÷ optical path length (cm) × 10^6

[0045]

[0046] 4. Summary

[0047] The lactate dehydrogenase residual rate of the control A-0 was 21%, but the lactate dehydrogenase residual rates of the present invention A-1 to A-11 were higher than 21%. The lactate dehydrogenase residual rate of the control B-0 was 19%, but the lactate dehydrogenase residual rates of the present invention B-1 to B-11 were higher than 19%. The lactate dehydrogenase residual rate of the control C-0 was 64%, but the lactate dehydrogenase residual rates of the present invention C-1 to C-11 were higher than 64%. The lactate dehydrogenase residual rate of the control D-0 was 64%, but the lactate dehydrogenase residual rates of the present invention D-1 to D-11 were higher than 64%.

[0048] The lactate dehydrogenase residual rate of the control A-0, which did not contain an alkali metal compound, was 21%, while the lactate dehydrogenase residual rates of the present invention A-1 to A-11, to which an alkali metal compound was added, were higher than 21%, indicating that the addition of an alkali metal compound improved the stability of lactate dehydrogenase.Furthermore, the lactate dehydrogenase residual rate was higher in the present invention A-2 than in the present invention A-1, in the present invention A-3 than in the present invention A-2, in the present invention A-4 than in the present invention A-3, in the present invention A-5 than in the present invention A-4, and in the present invention A-6 than in the present invention A-5, indicating that the stability of lactate dehydrogenase improved depending on the concentration of the added alkali metal compound.

[0049] The lactate dehydrogenase residual rate of the control B-0, which did not contain an alkali metal compound, was 19%, while the lactate dehydrogenase residual rates of the present invention B-1 to B-11, which had alkali metal compounds added to this, were higher than 19%, indicating that the addition of an alkali metal compound improved the stability of lactate dehydrogenase. Furthermore, the lactate dehydrogenase residual rate was higher in the present invention B-2 than in the present invention B-1, in the present invention B-3 than in the present invention B-2, in the present invention B-4 than in the present invention B-3, in the present invention B-5 than in the present invention B-4, and in the present invention B-6 than in the present invention B-5, indicating that the stability of lactate dehydrogenase improved depending on the concentration of the added alkali metal compound.

[0050] The lactate dehydrogenase residual rate of the control C-0, which did not contain an alkali metal compound, was 64%, while the lactate dehydrogenase residual rates of the present inventions C-1 to C-11, to which an alkali metal compound was added, were higher than 64%, indicating that the addition of an alkali metal compound improved the stability of lactate dehydrogenase.Furthermore, the lactate dehydrogenase residual rate was higher in present invention C-2 than in present invention C-1, in present invention C-3 than in present invention C-2, and in present invention C-4 than in present invention C-3, indicating that the stability of lactate dehydrogenase improved depending on the concentration of the added alkali metal compound.

[0051] The lactate dehydrogenase residual rate of the control D-0, which did not contain an alkali metal compound, was 64%, while the lactate dehydrogenase residual rates of the present invention D-1 to D-11, which had alkali metal compounds added to them, were higher than 64%, indicating that the addition of an alkali metal compound improved the stability of lactate dehydrogenase. Furthermore, the lactate dehydrogenase residual rate was higher in the present invention D-2 than in the present invention D-1, in the present invention D-3 than in the present invention D-2, in the present invention D-4 than in the present invention D-3, and in the present invention D-5 than in the present invention D-4, indicating that the stability of lactate dehydrogenase improved depending on the concentration of the added alkali metal compound.

[0052] This confirmed that alkali metal compounds have the effect of improving the stability of lactate dehydrogenase, and this effect was observed for both D- and L-type lactate dehydrogenase, regardless of the presence or absence of pyridoxal phosphate.

[0053] [Example 2] (Confirmation of pyruvate elimination ability in AST measurement reagent) Among AST measurement reagents, the pyruvate elimination ability of a measurement reagent containing the alkali metal compound of the present invention and lactate dehydrogenase was measured to confirm the effect of the alkali metal compound in improving the stability of lactate dehydrogenase.

[0054] 1. Preparation of reagents

[0055] (49) Preparation of AST Measurement Reagent Control E-0 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 a first reagent for Control E-0: Tris-HCl 80 mM (MP Biomedicals), NADH 0.336 mM (Oriental Yeast), D-LDH 1392 U / L (Toyobo), 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 5.9 (20°C) to prepare a second reagent for Control E-0. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 420 mM (Actec) α-Ketoglutaric acid 21 mM (Actec) (50) Preparation of AST Measurement Reagent E-1 of the Present Invention 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 a first reagent of the present invention E-1. Tris-HCl 80 mM (MP Biomedicals) NADH 0.336 mM (Oriental Yeast) D-LDH 1392 U / L (Toyobo) MDH 1050 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Sodium chloride 110 mM (Kanto Chemical) Furthermore, the following reagent components were dissolved in purified water to the concentrations shown, and the pH was adjusted to 5.9 (20°C) to prepare the second reagent of the present invention, E-1. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 420 mM (Actec) α-Ketoglutaric acid 21 mM (Actec)

[0056] (51) Preparation of AST Measurement Reagent Control F-0 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 a first reagent for Control F-0. Tris-HCl 80 mM (MP Biomedicals) NADH 0.336 mM (Oriental Yeast) Pyridoxal phosphate monohydrate 0.21 mM (Fujifilm Wako Pure Chemical Industries) D-LDH 1392 U / L (Toyobo) 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 5.9 (20°C) to prepare a second reagent for Control F-0. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 420 mM (Actec) α-Ketoglutaric acid 21 mM (Actec) (52) Preparation of AST Measurement Reagent F-1 of the Present Invention 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 a first reagent of the present invention F-1. Tris-HCl 80 mM (MP Biomedicals) NADH 0.336 mM (Oriental Yeast) Pyridoxal phosphate monohydrate 0.21 mM (Fujifilm Wako Pure Chemical Industries) D-LDH 1392 U / L (Toyobo) MDH 1050 U / L (Oriental Yeast) Triton X-100 1 g / L (Nacalai Tesque) Sodium chloride 110 mM (Kanto Chemical) Furthermore, the following reagent components were dissolved in pure water to the concentrations shown, and the pH was adjusted to 5.9 (20°C) to prepare a second reagent of the present invention, F-1. Tris-HCl 80 mM (MP Biomedicals) Sodium L-aspartate 420 mM (Actec) α-Ketoglutaric acid 21 mM (Actec)

[0057] 2. Storage of reagents

[0058] The reagents prepared in (49) to (52) of 1 above were stored in an incubator at 30° C. for 7 days.

[0059] 3. Confirmation of pyruvate elimination ability

[0060] The pyruvate scavenging ability of each reagent was confirmed before and after step 2, and the pyruvate scavenging ability after step 2 was subtracted from the pyruvate scavenging ability before step 2 to determine the change in pyruvate scavenging ability. The smaller the change in pyruvate scavenging ability, the better the stability of lactate dehydrogenase. The change in pyruvate scavenging ability of each reagent is shown in Table 2. The pyruvate scavenging ability was confirmed as follows: a. Preparation of pyruvate aqueous solution A 5 mg / dL aqueous solution of sodium pyruvate (Kanto Chemical) was diluted with pure water to 4 mg / dL to prepare a pyruvate aqueous solution. b. Method for measuring pyruvate scavenging ability A Hitachi High-Tech 7180 automatic analyzer was used for the measurement. 10 μL of the pyruvic acid aqueous solution to be measured was mixed with 100 μL of the first reagent (control E-0) and incubated at 37°C for 5 minutes. Then, 100 μL of the second reagent (control E-0) 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 using Enzyme Calibrator S (Shinotest), which displays the AST enzyme activity value, instead of the pyruvic acid aqueous solution. The AST enzyme activity (U / L) was measured using the pyruvic acid aqueous solution. Similar measurements were also performed using the combination of the first and second reagents (control E-1), the combination of the first and second reagents (control F-0), and the combination of the first and second reagents (control F-1).

[0061] In the AST measurement reagent of this example, pyruvic acid contained in the pyruvic acid aqueous solution is converted to lactic acid by the first reagent, so AST enzymatic activity is not observed when measuring the pyruvic acid aqueous solution. This conversion rate depends on the enzymatic activity of lactate dehydrogenase in the first reagent. If the enzymatic activity of lactate dehydrogenase is insufficient, pyruvic acid will remain without being converted to lactic acid, resulting in the observation of AST enzymatic activity despite the measurement of the pyruvic acid aqueous solution. This results in falsely high values. The ability to convert pyruvic acid to lactic acid is called pyruvic acid scavenging ability, which is quantified as the enzymatic activity of AST when measuring the pyruvic acid aqueous solution. A higher value indicates a lower pyruvic acid scavenging ability, while a lower value indicates a higher pyruvic acid scavenging ability. Low pyruvic acid scavenging ability also indicates low lactate dehydrogenase enzymatic activity. Although the pyruvic acid scavenging ability is high immediately after preparation of the reagent, it may decrease over time. This is thought to be primarily due to a decrease in the enzymatic activity of lactate dehydrogenase in the reagent over time. AST assay reagents with a large decrease in pyruvate scavenging ability in a short period of time, i.e., large changes in pyruvate scavenging ability, are prone to falsely high values ​​due to poor lactate dehydrogenase stability, meaning that the AST assay reagent is also poorly stable.On the other hand, AST assay reagents with a small changes in pyruvate scavenging ability have good lactate dehydrogenase stability, meaning that the AST assay reagent is also stable.

[0062]

[0063] 4. Summary

[0064] The change in pyruvate scavenging ability of the control E-0 was 13, but the change in pyruvate scavenging ability of the present invention E-1 was lower than 13. The change in pyruvate scavenging ability of the control F-0 was 13, but the change in pyruvate scavenging ability of the present invention F-1 was lower than 13.

[0065] The change in pyruvate scavenging ability of the control E-0, which did not contain an alkali metal compound, was 13, while the change in pyruvate scavenging ability of the present invention E-1, to which an alkali metal compound was added, was lower than 13, indicating that the stability of lactate dehydrogenase was improved by the addition of an alkali metal compound.

[0066] The change in pyruvate scavenging ability of the control F-0, which did not contain an alkali metal compound, was 13, while the change in pyruvate scavenging ability of the present invention F-1, to which an alkali metal compound was added, was lower than 13, indicating that the stability of lactate dehydrogenase was improved by the addition of an alkali metal compound.

[0067] This confirmed that alkali metal compounds have the effect of improving the stability of lactate dehydrogenase. This effect was observed regardless of the presence or absence of pyridoxal phosphate. In the case of AST measurement reagents, adding alkali metal compounds to reagents containing lactate dehydrogenase was effective in improving the stability of the AST measurement reagents.

[0068] [Example 3] (Confirmation of measurement values ​​in ALT measurement reagent) Among ALT measurement reagents, in a measurement reagent containing the alkali metal compound of the present invention and lactate dehydrogenase, the enzyme activity of ALT in a sample was measured to confirm the effect of the alkali metal compound in improving the stability of lactate dehydrogenase.

[0069] 1. Preparation of reagents

[0070] (53) Preparation of ALT Measurement Reagent Control G-0 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 a first reagent for Control G-0. Tris-HCl 100 mM (MP Biomedicals) NADH 0.168 mM (Oriental Yeast) D-LDH 1086 U / L (Toyobo) 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 4.0 (20°C) to prepare a second reagent for Control G-0. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1050 mM (Actec) α-Ketoglutaric acid 31.5 mM (Actec) (54) Preparation of ALT Measurement Reagent of the Present Invention G-1 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 a first reagent of the present invention G-1. Tris-HCl 100 mM (MP Biomedicals) NADH 0.168 mM (Oriental Yeast) D-LDH 1086 U / L (Toyobo) Triton X-100 1 g / L (Nacalai Tesque) Sodium chloride 110 mM (Kanto Chemical) The following reagent components were dissolved in purified water to the concentrations shown, and the pH was adjusted to 4.0 (20°C) to prepare the second reagent of the present invention, G-1. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1050 mM (Actec) α-ketoglutaric acid 31.5 mM (Actec)

[0071] (55) Preparation of ALT Measurement Reagent Control H-0 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 a first reagent for Control H-0. Tris-HCl 100 mM (MP Biomedicals) NADH 0.168 mM (Oriental Yeast) Pyridoxal phosphate monohydrate 0.21 mM (Fujifilm Wako Pure Chemical Industries) D-LDH 1086 U / L (Toyobo) 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 4.0 (20°C) to prepare a second reagent for Control H-0. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1050 mM (Actec) α-Ketoglutaric acid 31.5 mM (Actec) (56) Preparation of ALT Measurement Reagent H-1 of the Present Invention 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 a first reagent of the present invention H-1. Tris-HCl 100 mM (MP Biomedicals) NADH 0.168 mM (Oriental Yeast) Pyridoxal phosphate monohydrate 0.21 mM (Fujifilm Wako Pure Chemical Industries) D-LDH 1086 U / L (Toyobo) Triton X-100 1 g / L (Nacalai Tesque) Sodium chloride 110 mM (Kanto Chemical) Furthermore, the following reagent components were dissolved in purified water to the concentrations shown, and the pH was adjusted to 4.0 (20°C) to prepare the second reagent of the present invention, H-1. Tris-HCl 100 mM (MP Biomedicals) L-alanine 1050 mM (Actec) α-ketoglutaric acid 31.5 mM (Actec)

[0072] 2. Storage of reagents

[0073] The reagents prepared in (53) to (56) of 1 above were stored in an incubator at 37° C. for 7 days.

[0074] 3. Confirmation of ALT enzyme activity

[0075] The ALT enzyme activity of the sample 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 ALT measurement change rate (percent). The higher the ALT measurement change rate, the better the stability of lactate dehydrogenase. The values ​​are shown in Table 3. The ALT enzyme activity was measured as follows: a. Sample preparation: Aalto Control LEVEL IIα (Shinotest) was prepared according to the instruction manual. b. Method for measuring ALT enzyme activity: A Hitachi High-Tech 7180 automatic analyzer was used for measurements. 8 μL of the Aalto Control LEVEL IIα to be measured was mixed with 80 μL of the first reagent of control G-0 and incubated at 37°C for 5 minutes. Then, 80 μL of the second reagent of control G-0 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 Aalto ECα (Shinotest), which displays the enzyme activity of ALT, was used as the measurement target instead of Aalto Control LEVEL IIα. The ALT enzyme activity (U / L) when measuring Aalto Control LEVEL IIα was measured. Similar measurements were also performed for present invention G-1, control H-0, and present invention H-1.

[0076]

[0077] 4. Summary

[0078] The rate of change in ALT measurement values ​​for the control G-0 was 45%, but the rate of change in ALT measurement values ​​for the present invention G-1 was higher than 45%. The rate of change in ALT measurement values ​​for the control H-0 was 46%, but the rate of change in ALT measurement values ​​for the present invention H-1 was higher than 46%.

[0079] The control G-0, which did not contain an alkali metal compound, had a change rate of 45% in ALT measurement values, while the invention G-1, to which an alkali metal compound was added, had a change rate of more than 45%, indicating that the addition of an alkali metal compound improved the stability of lactate dehydrogenase.

[0080] The change rate of ALT measurement value for the control H-0 containing no alkali metal compound was 46%, while the change rate of ALT measurement value for the present invention H-1 to which an alkali metal compound was added was higher than 46%, indicating that the stability of lactate dehydrogenase was improved by adding an alkali metal compound.

[0081] This confirmed that alkali metal compounds have the effect of improving the stability of lactate dehydrogenase. This effect was observed regardless of the presence or absence of pyridoxal phosphate. In the ALT measurement reagent, adding an alkali metal compound to a reagent containing lactate dehydrogenase was effective in improving the stability of the ALT measurement reagent.

Claims

1. A measuring reagent characterized by containing an alkali metal compound in a reagent containing lactate dehydrogenase.

2. The measuring reagent according to claim 1, wherein the alkali metal compound is at least one of a sodium compound, a lithium compound, and a potassium compound.

3. The measuring reagent according to claim 1, wherein the alkali metal compound is at least one of sodium chloride, lithium chloride, potassium chloride, sodium acetate, lithium acetate, and potassium acetate.

4. The measuring reagent according to any one of claims 1 to 3, wherein the concentration of the alkali metal compound is 55 mM or more.

5. The measuring reagent according to any one of claims 1 to 3, wherein the concentration of the alkali metal compound is 110 mM or more.

6. The measuring reagent according to any one of claims 1 to 3, which is a measuring reagent for measuring AST or ALT.

7. The measuring reagent according to claim 4, which is a measuring reagent for measuring AST or ALT.

8. The measuring reagent according to claim 5, which is a measuring reagent for measuring AST or ALT.

9. The measuring reagent according to claim 6, wherein the measuring reagent contains pyridoxal phosphate.

10. The measuring reagent according to claim 7, wherein the measuring reagent contains pyridoxal phosphate.

11. The measuring reagent according to claim 8, wherein the measuring reagent contains pyridoxal phosphate.

12. A method for improving the stability of lactate dehydrogenase contained in a measurement reagent, characterized by adding an alkali metal compound to the reagent containing lactate dehydrogenase.

13. The method according to claim 12, wherein the alkali metal compound is at least one of a sodium compound, a lithium compound, and a potassium compound.

14. The method of claim 12, wherein the alkali metal compound is at least one of sodium chloride, lithium chloride, potassium chloride, sodium acetate, lithium acetate, and potassium acetate.

15. The method according to any one of claims 12 to 14, wherein the measuring reagent is a measuring reagent for measuring AST or ALT.

16. The method according to claim 15, wherein the measurement reagent contains pyridoxal phosphate.

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