Method for preventing deterioration of lactate dehydrogenase activity measurement reagents after opening.

Abstract

To provide a method for preventing deterioration after opening an activity-measuring reagent of lactic acid dehydrogenase, in which accurate measurement result (measured value) not affected by carbon dioxide in the atmosphere even after opening can be obtained, a measured value not deviated from an IFCC SOP method-measuring reagent can be shown, and then, reagent blank does not rise with time.SOLUTION: In an activity-measuring reagent of lactic acid dehydrogenase, 2-amino-2-methyl-1,3-propanediol is used as buffer solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for preventing deterioration of lactate dehydrogenase activity measurement reagents after opening. This invention is useful in fields such as clinical testing, clinical pathology, and medicine, as well as in fields of chemistry, such as analytical chemistry. [Background technology]

[0002] Lactate dehydrogenase (L-lactate dehydrogenase; LD) [EC 1.1.1.27] produces pyruvate using L-lactate as a substrate, and simultaneously produces oxidized β-nicotinamide adenine dinucleotide (NAD). + This reaction converts ) to reduced β-nicotinamide adenine dinucleotide (NADH) [forward reaction].

[0003] Conversely, lactate dehydrogenase uses pyruvate as a substrate to produce L-lactate, and at the same time converts reduced β-nicotinamide adenine dinucleotide (NADH) into oxidized β-nicotinamide adenine dinucleotide (NADH). + [Reverse reaction] causes a change to ).

[0004] The clinical significance of lactate dehydrogenase lies in its widespread distribution throughout the body's tissues and its function in the final stage of anaerobic glycolysis. Elevated levels in the blood are observed in various diseases of the heart, liver, and kidneys, as well as in malignant tumors, leukemia, and pernicious anemia, serving as an indicator for the diagnosis and monitoring of these conditions (see Non-Patent Literature 1 and Non-Patent Literature 2). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Practical Clinical Chemistry, Enlarged Edition, pp. 394-401, Motoji Kitamura et al., Ishiyaku Publishers, 1982. [Non-Patent Document 2] Clinical Laboratory Methods Handbook, 32nd Revised Edition, pp. 615-621, Masamitsu Kanai et al., Kinbara Publishing, 2005. [Overview of the project] [Problems that the invention aims to solve]

[0006] The most widely used method for measuring lactate dehydrogenase activity is the measurement reagent based on the International Federation of Clinical Chemistry (IFCC) Standard Operating Procedure (SOP method), which is a measurement method based on the IFCC's recommended method. (This is sometimes referred to as the "IFCC-SOP method measurement reagent.")

[0007] When IFCC-SOP method reagents are left in an automated analyzer for one month after opening, the measured lactate dehydrogenase activity decreases by approximately 10% compared to the measured value of unopened reagents. This is because carbon dioxide from the atmosphere dissolves into the measuring reagent, causing it to deteriorate.

[0008] One possible countermeasure to prevent carbon dioxide from dissolving into the measuring reagent is to increase the concentration of the buffer in the measuring reagent. However, it was found that increasing the buffer concentration in the measurement reagent caused the measured values ​​to deviate from those obtained using the IFCC-SOP method reagent, and therefore this was not an effective solution.

[0009] Furthermore, it is known that the reagent blank rate (reagent blinding) increases over time with IFCC-SOP method reagents.

[0010] Thus, with IFCC-SOP method reagents, after opening, carbon dioxide from the atmosphere dissolves into the reagent, causing it to deteriorate and resulting in negative errors in the measurement results (measured values). Therefore, for example, in clinical testing in a medical setting, if the measurement results (measured values) of lactate dehydrogenase activity in a sample were to be used for diagnosis, there was a possibility of misdiagnosis of diseases, etc.

[0011] Therefore, in reagents for measuring the activity level of lactate dehydrogenase in a sample, there was a need for a method to prevent the reagent from degrading after opening due to carbon dioxide from the atmosphere dissolving into the reagent.

[0012] In contrast, the object of the present invention is to provide a method for preventing deterioration of lactate dehydrogenase activity measurement reagents after opening.

[0013] Furthermore, the objective is to provide a method for preventing deterioration of the activity measurement reagent after opening, which will result in measurement values ​​that do not deviate from those obtained using the IFCC-SOP method.

[0014] Furthermore, the objective is to provide a method for preventing the reagent blank from increasing over time and for preventing deterioration of the activity measurement reagent after opening. [Means for solving the problem]

[0015] The inventors of the present invention have conducted extensive research on methods to prevent deterioration after opening of a reagent for measuring the activity of lactate dehydrogenase. They have found that the above problem can be solved by using a specific compound as a buffer solution, and have completed the present invention.

[0016] In other words, the present invention consists of the following inventions. (1) A method for preventing deterioration of a lactate dehydrogenase activity assay reagent after opening, characterized by using 2-amino-2-methyl-1,3-propanediol as a buffer in the lactate dehydrogenase activity assay reagent. [Effects of the Invention]

[0017] The present invention provides a method for preventing deterioration of a lactate dehydrogenase activity measurement reagent after opening, thereby preventing deterioration of the reagent due to the dissolution of carbon dioxide from the atmosphere into the reagent after opening, and preventing errors in the measurement results (measured values). That is, in the method for preventing deterioration after opening of the reagent for measuring the activity of lactate dehydrogenase of the present invention, accurate measurement results (measured values) that are not affected by carbon dioxide in the atmosphere can be obtained.

[0018] In addition, the method for preventing deterioration after opening of the reagent for measuring the activity of lactate dehydrogenase of the present invention can show measurement values that do not deviate from the IFCC·SOP method measurement reagent.

[0019] Furthermore, the method for preventing deterioration after opening of the reagent for measuring the activity of lactate dehydrogenase of the present invention has a very small increase in the reagent blank over time.

Brief Description of the Drawings

[0020] [Figure 1] Figure 1 is a graph confirming the stability after opening of the measurement reagent in the present invention. [Figure 2] Figure 2 is a graph confirming the reagent blank of the measurement reagent in the present invention over time. [Figure 3] Figure 3 is a graph confirming the correlation of the measurement in the present invention.

Modes for Carrying Out the Invention

[0021] 〔1〕Buffer solution The method for preventing deterioration after opening of the reagent for measuring the activity of lactate dehydrogenase of the present invention is characterized in that 2-amino-2-methyl-1,3-propanediol (AMPdiol) is used as a buffer solution in the reagent for measuring the activity of lactate dehydrogenase.

[0022] Furthermore, in the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening according to the present invention, by using 2-amino-2-methyl-1,3-propanediol as a buffer solution, accurate measurement results (measured values) can be obtained that are not affected by carbon dioxide in the atmosphere even after opening, and the measured values ​​can be obtained that do not deviate from those of the IFCC-SOP method measurement reagent, and the reagent blank does not increase over time.

[0023] In the present invention, if the lactate dehydrogenase activity measurement reagent consists of a single measurement reagent, the buffer solution of the measurement reagent is 2-amino-2-methyl-1,3-propanediol buffer solution.

[0024] Furthermore, in the case where the lactate dehydrogenase activity measurement reagent of the present invention consists of two measurement reagents, the buffer of the first reagent may be 2-amino-2-methyl-1,3-propanediol buffer, or the buffer of the second reagent may be 2-amino-2-methyl-1,3-propanediol buffer, or the buffers of both the first and second reagents may be 2-amino-2-methyl-1,3-propanediol buffer. In the present invention, it is preferable that the buffer solutions for the first and second reagents be 2-amino-2-methyl-1,3-propanediol buffer solutions.

[0025] There are no particular limitations on the concentration of 2-amino-2-methyl-1,3-propanediol in the 2-amino-2-methyl-1,3-propanediol buffer in the lactate dehydrogenase activity measurement reagent of the present invention, but it is preferable that the lower limit be 10 mM or more, more preferably 100 mM or more, even more preferably 200 mM or more, and particularly preferably 300 mM or more.

[0026] Furthermore, regarding the upper limit of the 2-amino-2-methyl-1,3-propanediol concentration in the 2-amino-2-methyl-1,3-propanediol buffer in the lactate dehydrogenase activity measurement reagent of the present invention, considering costs and other factors, up to 2,000 mM is sufficient. This upper limit is preferably 1,600 mM or less, more preferably 1,200 mM or less, and particularly preferably 1,000 mM or less.

[0027] In the lactate dehydrogenase activity measurement reagent of the present invention, the preferred concentration of 2-amino-2-methyl-1,3-propanediol in the 2-amino-2-methyl-1,3-propanediol buffer is, for example, 10 mM to 2,000 mM, more preferably 10 mM to 1,600 mM, even more preferably 10 mM to 1,200 mM, and particularly preferably 10 mM to 1,000 mM, provided that the lower limit is 10 mM. Furthermore, if the lower limit is 100 mM, then 100 mM to 2,000 mM is preferred, more preferably 100 mM to 1,600 mM, even more preferably 100 mM to 1,200 mM, and particularly preferably 100 mM to 1,000 mM. Furthermore, if the lower limit is set to 200 mM, then 200 mM to 2,000 mM is preferred, 200 mM to 1,600 mM is more preferred, 200 mM to 1,200 mM is even more preferred, and 200 mM to 1,000 mM is particularly preferred. And if the lower limit is set to 300 mM, then 300 mM to 2,000 mM is preferred, 300 mM to 1,600 mM is more preferred, 300 mM to 1,200 mM is even more preferred, and 300 mM to 1,000 mM is particularly preferred.

[0028] The pH of the 2-amino-2-methyl-1,3-propanediol buffer in the lactate dehydrogenase activity measurement reagent of the present invention is such that lactate dehydrogenase produces pyruvate using L-lactate as a substrate, and at the same time oxidized β-nicotinamide adenine dinucleotide (NAD) + Since the optimal pH for the reaction that converts ) to reduced β-nicotinamide adenine dinucleotide (NADH) is pH 8.0 to pH 11.0, the pH during the above reaction is preferably pH 8.3 to pH 10.7, more preferably pH 8.6 to pH 10.4, even more preferably pH 8.9 to pH 10.1, particularly preferably pH 9.2 to pH 9.8, and especially preferably pH 9.4.

[0029] [2] Lactic acid In the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening according to the present invention, the lactate dehydrogenase activity measurement reagent must contain lactate as a substrate.

[0030] Examples of lactic acid include L(+)-lactic acid as an acid or salts of L(+)-lactic acid.

[0031] Examples of L(+)-lactic acid salts include lithium salts, sodium salts, potassium salts, or calcium salts. In the present invention, lithium salts are preferred as the salts of L(+)-lactic acid.

[0032] In the present invention, if the reagent for measuring the activity of lactate dehydrogenase consists of a single measuring reagent, lactic acid is included in the measuring reagent.

[0033] Furthermore, in the case where the lactate dehydrogenase activity measurement reagent of the present invention consists of two measurement reagents, the first reagent may contain lactic acid, or the second reagent may contain lactic acid, or both the first and second reagents may contain lactic acid. In the present invention, it is preferable to include lactic acid in the first reagent.

[0034] The concentration of lactate in the lactate dehydrogenase activity measurement reagent of the present invention is not particularly limited, but it is important that the lactate dehydrogenase produces pyruvate using L-lactate as a substrate, and at the same time oxidized β-nicotinamide adenine dinucleotide (NAD) + The concentration at the time of the reaction that converts ) to reduced β-nicotinamide adenine dinucleotide (NADH) is preferably 10 mM or higher as the lower limit, more preferably 20 mM or higher, even more preferably 30 mM or higher, and particularly preferably 40 mM or higher.

[0035] Furthermore, regarding the upper limit of the lactate concentration in the lactate dehydrogenase activity measurement reagent of the present invention, considering the cost and other factors, a concentration of up to 200 mM is sufficient at the concentration used in the above reaction. This upper limit is preferably 160 mM or less, more preferably 120 mM or less, and particularly preferably 80 mM or less.

[0036] In the case where the lactate dehydrogenase activity measurement reagent of the present invention consists of two measurement reagents, the first reagent and / or the second reagent should contain lactic acid so that the concentration of lactic acid is the same as the concentration at the time of the above reaction.

[0037] [3] Oxidized β-nicotinamide adenine dinucleotide In the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening according to the present invention, the lactate dehydrogenase activity measurement reagent includes oxidized β-nicotinamide adenine dinucleotide (NAD) as a coenzyme. + It is necessary to include )

[0038] In the present invention, if the reagent for measuring lactate dehydrogenase activity consists of a single reagent, the reagent contains oxidized β-nicotinamide adenine dinucleotide.

[0039] Furthermore, in the present invention, if the lactate dehydrogenase activity measurement reagent consists of two reagents, the first reagent may contain oxidized β-nicotinamide adenine dinucleotide, or the second reagent may contain oxidized β-nicotinamide adenine dinucleotide, or both the first and second reagents may contain oxidized β-nicotinamide adenine dinucleotide. In the present invention, it is preferable to include oxidized β-nicotinamide adenine dinucleotide in the second reagent.

[0040] The concentration of oxidized β-nicotinamide adenine dinucleotide in the lactate dehydrogenase activity measurement reagent in this invention is not particularly limited, but it is important that when lactate dehydrogenase produces pyruvate using L-lactate as a substrate, oxidized β-nicotinamide adenine dinucleotide (NAD) is present. +The concentration at the time of the reaction that converts ) to reduced β-nicotinamide adenine dinucleotide (NADH) is preferably 1 mM or higher as the lower limit, more preferably 2 mM or higher, even more preferably 4 mM or higher, and particularly preferably 6 mM or higher.

[0041] Furthermore, regarding the upper limit of the concentration of oxidized β-nicotinamide adenine dinucleotide in the lactate dehydrogenase activity measurement reagent in the present invention, considering the cost and other factors, a concentration of up to 100 mM is sufficient at the concentration used in the above reaction. This upper limit is preferably 80 mM or less, more preferably 60 mM or less, and particularly preferably 40 mM or less.

[0042] In the present invention, if the lactate dehydrogenase activity measurement reagent consists of two reagents, the first and / or second reagents should contain oxidized β-nicotinamide adenine dinucleotide so that the concentration of oxidized β-nicotinamide adenine dinucleotide is the same as the concentration at the time of the above reaction.

[0043] [4] Sample The sample in this invention is a sample that may contain lactate dehydrogenase, and the purpose is to measure the activity level of this lactate dehydrogenase.

[0044] As long as the sample meets the above criteria, there are no particular limitations, but examples of samples include biological samples.

[0045] For example, examples of biological samples include human or animal blood, serum, plasma, cerebrospinal fluid, organs such as the heart, tissues such as muscles or nerves, and cells.

[0046] In this invention, the sample is preferably a liquid. If the sample containing lactate dehydrogenase is not in liquid form, pretreatment such as extraction or solubilization may be performed according to known methods to incorporate lactate dehydrogenase into a liquid.

[0047] In this invention, biological samples are preferred as the sample, and serum or plasma is more preferred as the sample.

[0048] [5] Other ingredients In the present invention, the lactate dehydrogenase activity measurement reagent may optionally contain metal ions or metal salts containing them, chelating agents, stabilizers such as proteins like albumin, sugars or polymer compounds, preservatives such as sodium azide or antibiotics, scavenging agents or inhibitors of interfering substances contained in other samples (for example, potassium ferrocyanide for bilirubin, or ascorbate oxidase for ascorbic acid), surfactants, or activators.

[0049] While there are no particular limitations on the concentration of these substances present, it is preferable that the concentration be 0.001% (w / v) or higher during the measurement reaction.

[0050] Furthermore, the lower limit of the concentration when these are present is more preferably 0.01% (w / v) or higher, and particularly preferably 0.1% (w / v) or higher, at the time of the measurement reaction.

[0051] While there is no particular upper limit to the concentration of these substances, considering costs and other factors, 10% (w / v) is usually sufficient.

[0052] Furthermore, the upper limit of the concentration of these substances present is more preferably 5% (w / v) or less, and particularly preferably 2.5% (w / v) or less, during the measurement reaction.

[0053] Furthermore, it is preferable that the lactate dehydrogenase activity measurement reagent in the present invention contains the aforementioned components at a concentration such that the concentration is reached during the measurement reaction.

[0054] [6] Preserve In the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening according to the present invention, there are no limitations on the temperature at which the lactate dehydrogenase activity measurement reagent is stored, but it is preferably 20°C or lower, more preferably 10°C or lower, and particularly preferably 2°C to 8°C.

[0055] [7] Measurement In the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening according to the present invention, when measuring the activity of lactate dehydrogenase in a sample, it is preferable to perform the measurement by the reaction rate method.

[0056] Furthermore, when optically measuring the reduced β-nicotinamide adenine dinucleotide produced in this measurement reaction, it is preferable to measure the absorbance at a wavelength of 340 nm or nearby.

[0057] Furthermore, in this measurement, the temperature during the measurement reaction should be set to a temperature within the range where the measurement reaction proceeds without the enzymes or coenzymes involved in the measurement reaction being inactivated, denatured, or altered by heat, such as 30°C or 37°C.

[0058] Furthermore, in this measurement, the method for initiating the measurement reaction may be either by adding a substrate or by adding a sample.

[0059] Furthermore, in this measurement, the measurement may be performed by manual methods or by using equipment such as an automated analyzer.

[0060] [8] Specific examples of measuring lactate dehydrogenase activity in a sample A specific example of the measurement of lactate dehydrogenase activity using the activity measurement reagent in this invention is described below.

[0061] (a) Preparation of measurement reagents The first and second reagents for measuring lactate dehydrogenase activity were prepared by dissolving a fixed amount of each of the following components in pure water and adjusting the pH to a constant pH.

[0062] (i) Reagent 1 L(+)-Lithium lactate 2-amino-2-methyl-1,3-propanediol [buffering agent]

[0063] (ii) Second reagent Oxidized β-nicotinamide adenine dinucleotide 2-amino-2-methyl-1,3-propanediol [buffering agent]

[0064] (b) Sample Human serum was used as a sample.

[0065] (c) Measurement (i) Stage 1 The sample and the first reagent are mixed to prepare a mixture.

[0066] The amounts of the sample and the first reagent to be mixed should be determined appropriately according to the amount of the second reagent, the activity level of lactate dehydrogenase contained in the sample, the specifications of the analytical instrument used, and other conditions. Generally, it is preferable that the sample volume be in the range of 0.5 to 100 μL, and the volume of the first reagent be in the range of 20 to 1,000 μL.

[0067] After preparing this mixture, incubate it. There are no particular restrictions on the incubation time, but it is usually preferably 20 minutes or less, more preferably 10 minutes or less, and especially preferably 5 minutes or less.

[0068] Furthermore, the incubation temperature should be above the freezing point of the aforementioned mixture. Generally speaking, a higher temperature during the measurement reaction is preferable because it results in a higher reaction rate. However, if the temperature is too high, the enzymes or coenzymes involved in the reaction will denature or become inactive. Therefore, the incubation temperature must be below the temperature at which the enzymes or coenzymes involved in the measured reaction denature or become inactive. The incubation temperature is usually 2 to 70°C, but 20 to 37°C is preferred, and 30 to 37°C is more preferred.

[0069] The preparation and incubation of a mixture of this sample and the first reagent brings into contact between the lactate dehydrogenase contained in the sample and the L-lactic acid contained in the first reagent.

[0070] (ii) Second stage The second reagent is mixed with the "mixture of sample and first reagent" prepared in the first step described above. This is the final reaction solution.

[0071] The amount of the second reagent to be mixed should be determined appropriately depending on the amount of sample, the amount of the first reagent, the activity level of lactate dehydrogenase contained in the sample, the specifications of the analytical instrument used, and other conditions. Generally, it is preferable that the amount of the second reagent be in the range of 10 to 1,000 μL.

[0072] After preparing this final reaction solution, incubation is performed. There are no particular restrictions on the incubation time, but it is usually preferably 20 minutes or less, more preferably 10 minutes or less, and especially preferably 5 minutes or less.

[0073] Furthermore, the incubation temperature should be above the temperature at which the final reaction solution freezes. Generally speaking, a higher temperature during the measurement reaction is preferable because it results in a higher reaction rate. However, if the temperature is too high, the enzymes or coenzymes involved in the measurement reaction will denature and become inactive. Therefore, the incubation temperature must be below the temperature at which the enzymes or coenzymes involved in the measurement reaction denature and become inactive. The incubation temperature is usually 2 to 70°C, but 20 to 37°C is preferred, and 30 to 37°C is more preferred.

[0074] The preparation and incubation of this final reaction solution allows lactate dehydrogenase to produce pyruvate using L-lactic acid as a substrate, while simultaneously producing oxidized β-nicotinamide adenine dinucleotide (NAD). + ) is converted to reduced β-nicotinamide adenine dinucleotide (NADH). The amount of reduced β-nicotinamide adenine dinucleotide (NADH) produced is calculated based on the molar extinction coefficient from the absorbance at 340 nm, and the activity value of the lactate dehydrogenase contained in the sample is determined.

[0075] Furthermore, in the present invention, it is preferable to calculate the activity value of lactate dehydrogenase contained in the sample by subtracting the reagent blank (reagent blind) from the absorbance obtained by measuring the sample.

[0076] The procedure for measuring the activity of lactate dehydrogenase in the sample may be performed manually by the person performing the measurement, or it may be performed using an automated analyzer or other device. [Examples]

[0077] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0078] [Example 1] (Preparation of measurement reagent) Each of the measurement reagents used in the study was prepared. 1. Preparation of IFCC-SOP method reagents (1) Preparation of IFCC-SOP method reagent (reagent 1) The reagent components listed below were dissolved in pure water to the concentrations indicated, and the pH was adjusted to 9.4 (37°C) to prepare the first reagent for lactate dehydrogenase activity measurement [IFCC-SOP method measurement reagent (first reagent)]. N-methyl-D-glucamine (Fujifilm Wako Pure Chemical Industries) 373.8 mM L(+)-Lithium lactate 57.5 mM

[0079] (2) Preparation of IFCC-SOP method reagent (second reagent) The following reagent components were dissolved in pure water to the described concentrations to prepare the second reagent of the lactic dehydrogenase activity measurement reagent [IFCC·SOP method measurement reagent (second reagent)]. Oxidized β-nicotinamide adenine dinucleotide [NAD + (Oriental Yeast Co., Ltd.) 115.01 mM

[0080] 2. Preparation of the measurement reagent of the method of the present invention (1) Preparation of the reagent of the method of the present invention (first reagent) The following reagent components were dissolved in pure water to the described concentrations respectively, and the pH was adjusted to 9.7 (20 °C) to prepare the first reagent of the lactic dehydrogenase activity measurement reagent [reagent of the method of the present invention (first reagent)]. 2-Amino-2-methyl-1,3-propanediol (FUJIFILM Wako Pure Chemical Corporation) 890 mM L(+)-Lithium lactate 68.5 mM

[0081] (2) Reagent of the method of the present invention (second reagent) The following reagent components were dissolved in pure water to the described concentrations to prepare the second reagent of the lactic dehydrogenase activity measurement reagent [reagent of the method of the present invention (second reagent)]. Oxidized β-nicotinamide adenine dinucleotide [NAD + (Oriental Yeast Co., Ltd.) 61.7 mM

[0082] 3. Preparation of the IFCC method·SOP equivalent reagent·formulation measurement reagent (1) Preparation of the IFCC method·SOP equivalent reagent·formulation (first reagent) The following reagent components were dissolved in pure water to the described concentrations respectively, and the pH was adjusted to 9.4 (37 °C) to prepare the first reagent of the lactic dehydrogenase activity measurement reagent [IFCC method·SOP equivalent reagent·formulation (first reagent)]. N-Methyl-D-glucamine (FUJIFILM Wako Pure Chemical Corporation) 445 mM L(+)-Lithium lactate 68.5 mM

[0083] (2) Preparation of the IFCC method·SOP equivalent reagent·formulation (second reagent) The following reagent components were dissolved in pure water to the concentrations indicated, and the second reagent for lactate dehydrogenase activity measurement [IFCC method, SOP equivalent reagent, formulation (second reagent)] was prepared. Oxidized β-nicotinamide adenine dinucleotide [NAD + (Oriental Yeast Co., Ltd.) 41.1mM

[0084] [Example 2] (Study of stability after opening) The stability of the measurement reagent after opening in the method of the present invention was investigated. 1. Measurement reagents As measurement reagents, the IFCC-SOP method measurement reagent (first reagent) prepared in Example 1, 1(1) above, the IFCC-SOP method measurement reagent (second reagent) prepared in Example 1, 1(2) above, the method of the present invention (first reagent) prepared in Example 1, 2(1) above, and the method of the present invention (second reagent) prepared in Example 1, 2(2) above were used, respectively.

[0085] 2. Sample Human pooled serum was used as the sample.

[0086] 3. Examination of the stability of the measurement reagent after opening. Each of the measurement reagents described in item 1 was stored in its opened state for one month in the reagent compartment (temperature: 6°C) of a Hitachi 7180 automatic analyzer.

[0087] On the day of opening and storage (day 0), and 7, 14, 24, 28, and 35 days after opening and storage, the same frozen pooled serum sample was thawed and used as a sample, and the lactate dehydrogenase activity level of this sample was measured using the above-mentioned measurement reagents.

[0088] The measurements were performed using a Hitachi 7180 automatic analyzer.

[0089] When using the IFCC-SOP method reagent (first reagent) prepared in 1(1) of Example 1 above, and the IFCC-SOP method reagent (second reagent) prepared in 1(2) of Example 1 above, the measurement was performed by adding 200 μL of the IFCC-SOP method reagent (first reagent) on the day of opening and storage (day 0) or after opening and storage to 10 μL of the sample, reacting at 37°C for 5 minutes, and then adding 20 μL of the IFCC-SOP method reagent (second reagent) on the day of opening and storage (day 0) or after opening and storage, and reacting at 37°C for 5 minutes.

[0090] The absorbance was measured at the primary wavelength of 340 nm and the secondary wavelength of 405 nm, and the change in absorbance per minute was determined. The lactate dehydrogenase activity levels in the samples were calculated using a calibration curve created with the measurement reagent used on the day of opening and storage (day 0).

[0091] On each measurement day, five consecutive measurements were taken, and the average value is shown in Figure 1.

[0092] Furthermore, when using the method of the present invention (reagent 1) prepared in Example 1, 2 (1) above, and the method of the present invention (reagent 2) prepared in Example 1, 2 (2) above, as measurement reagents, 150 μL of the method of the present invention (reagent 1) prepared on the day of opening and storage (day 0) or after opening and storage was added to 4 μL of the sample, reacted at 37°C for 5 minutes, then 50 μL of the method of the present invention (reagent 2) prepared on the day of opening and storage (day 0) or after opening and storage was added, and reacted at 37°C for 5 minutes.

[0093] The absorbance was measured at the primary wavelength of 340 nm and the secondary wavelength of 405 nm, and the change in absorbance per minute was determined. The lactate dehydrogenase activity levels in the samples were calculated using a calibration curve created with the measurement reagent used on the day of opening and storage (day 0).

[0094] On each measurement day, five consecutive measurements were taken, and the average value is shown in Figure 1.

[0095] 4.Measurement results The graph of the measurement results described above is shown in Figure 1. In this graph, the horizontal axis represents the time elapsed (days) since opening each measurement reagent, and the vertical axis represents the relative value (%) of the lactate dehydrogenase activity in the sample after opening and storage, with the measured value on the first day of opening and storage (day 0) set as 100%.

[0096] In this graph, the dashed line represents the values ​​obtained using the IFCC-SOP method reagent (reagent 1) and the IFCC-SOP method reagent (reagent 2) (indicated as "IFCC-SOP method"), while the solid line represents the values ​​obtained using the present invention method (reagent 1) and the present invention method (reagent 2) (indicated as "present invention method").

[0097] This graph shows that when using the present invention method (reagent 1) and the present invention method (reagent 2), the decrease in measured values ​​after opening is suppressed compared to when using the IFCC-SOP method measurement reagent (reagent 1) and the IFCC-SOP method measurement reagent (reagent 2).

[0098] This confirms that the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening, as described in the present invention, prevents deterioration of the lactate dehydrogenase activity measurement reagent after opening due to the dissolution of carbon dioxide from the atmosphere into the reagent, thereby preventing errors in the measurement results (measured values).

[0099] In other words, it has been confirmed that the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening, according to the present invention, allows for accurate measurement results (measured values) that are not affected by carbon dioxide in the atmosphere.

[0100] [Example 3] (Accuracy check) The accuracy of the measurements in the present invention was examined. 1. Measurement reagents As measurement reagents, the IFCC-SOP method measurement reagent (first reagent) prepared in Example 1, 1(1) above, the IFCC-SOP method measurement reagent (second reagent) prepared in Example 1, 1(2) above, the method of the present invention (first reagent) prepared in Example 1, 2(1) above, and the method of the present invention (second reagent) prepared in Example 1, 2(2) above were used, respectively.

[0101] 2. Sample The following samples containing lactate dehydrogenase were used as specimens. (1) The Japanese Society of Clinical Chemistry's commonly used enzyme CRM001d (Japanese Clinical Laboratory Standards Council) [hereinafter referred to as "CRM"] (2) Aalto EC (Sinotest Corporation) (3) Aalto Control LEVEL I (Sinotest Corporation) (4) Aalto Control LEVEL II (Sinotest Corporation) (5) Suitrol I (Nissui Pharmaceutical Co., Ltd.) (6) Suitrol II (Nissui Pharmaceutical Co., Ltd.) (7) Suitrol IIEX (Nissui Pharmaceutical Co., Ltd.) (8) QAP Troll 1X (Sysmex Corporation) (9) QAP Troll 2X (Sysmex Corporation) (10) Human pooled serum

[0102] 3. Examination of accuracy The lactate dehydrogenase activity levels in each of the samples in section 2 were measured using each of the measurement reagents described in section 1. Five consecutive measurements were performed, and the average value was calculated.

[0103] When using the IFCC-SOP method reagent (first reagent) prepared in Example 1, 1(1) and the IFCC-SOP method reagent (second reagent) prepared in Example 1, 1(2) as the measurement reagents, the measurements were performed using a Hitachi 7180 automatic analyzer. 200 μL of the IFCC-SOP method reagent (first reagent) was added to 10 μL of the sample, reacted at 37°C for 5 minutes, then 20 μL of the IFCC-SOP method reagent (second reagent) was added, and reacted at 37°C for 5 minutes.

[0104] The absorbance was measured at the primary wavelength of 340 nm and the secondary wavelength of 405 nm, and the change in absorbance per minute was determined. Lactate dehydrogenase activity in the samples was calculated using CRM as a calibrator.

[0105] Furthermore, when using the method of the present invention (first reagent) prepared in Example 1, 2 (1) and the method of the present invention (second reagent) prepared in Example 1, 2 (2) as measurement reagents, 150 μL of the method of the present invention (first reagent) was added to 4 μL of the sample, reacted at 37°C for 5 minutes, then 50 μL of the method of the present invention (second reagent) was added, and reacted at 37°C for 5 minutes.

[0106] The absorbance was measured at the primary wavelength of 340 nm and the secondary wavelength of 405 nm, and the change in absorbance per minute was determined. Lactate dehydrogenase activity in the samples was calculated using CRM as a calibrator.

[0107] 4.Measurement results The results of the above measurements are shown in Table 1.

[0108] [Table 1]

[0109] In this table, measurements using the IFCC-SOP method reagent (reagent 1) and the IFCC-SOP method reagent (reagent 2) are indicated as "IFCC-SOP," and measurements using the present invention method (reagent 1) and the present invention method (reagent 2) are indicated as "present invention method."

[0110] The left column shows the activity value (measured value) of lactate dehydrogenase contained in each sample [U (unit) / L], and the right column shows the relative value (%) of the lactate dehydrogenase activity value (measured value) when measured using the present invention method (reagent 1) and the present invention method (reagent 2), with the lactate dehydrogenase activity value (measured value) when measured using the IFCC-SOP method reagent (reagent 1) and the IFCC-SOP method reagent (reagent 2) for each sample set to 100%.

[0111] This table shows that the measured values ​​obtained using the present invention method (reagent 1) and the present invention method (reagent 2) are 99-100% of the measured values ​​obtained using the IFCC-SOP method reagent (reagent 1) and the IFCC-SOP method reagent (reagent 2).

[0112] This confirms that the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening, as described in the present invention, can produce measurement values ​​that do not deviate from those of the IFCC-SOP method measurement reagent.

[0113] [Example 4] (Confirmation of reagent blank over time) The reagent blank for measurement in the present invention was confirmed over time. 1. Measurement reagents As measurement reagents, the following were used: the method of the present invention (reagent 1) prepared in Example 1-2(1), the method of the present invention (reagent 2) prepared in Example 1-2(2), the IFCC method / SOP equivalent reagent / formulation (reagent 1) prepared in Example 1-3(1), and the IFCC method / SOP equivalent reagent / formulation (reagent 2) prepared in Example 1-3(2).

[0114] 2. Sample Physiological saline (0.9% sodium chloride aqueous solution) was used as the sample.

[0115] 3. Measurement of reagent blank Each of the measurement reagents described in item 1 was stored in a sealed state in a constant temperature bath (temperature: 10°C) for 6 months.

[0116] Using a combination of the above reagents prepared on the day of preparation (day 0) [5°C] and after 6 months of storage [10°C], this reagent blank (reagent blind) was measured using physiological saline as the sample.

[0117] More specifically, the combinations of measurement reagents are as follows: (1) to (4). (1) A combination of the method of the present invention (first reagent) after storage for 6 months [10℃] and the method of the present invention (second reagent) on the day of reagent preparation (day 0) [5℃]. (2) A combination of the method of the present invention (first reagent) on the day of reagent preparation (day 0) [5℃] and the method of the present invention (second reagent) after 6 months of storage [10℃]. (3) A combination of IFCC method / SOP equivalent reagent / formulation (Reagent 1) stored for 6 months [10℃] and IFCC method / SOP equivalent reagent / formulation (Reagent 2) on the day of reagent preparation (Day 0) [5℃]. (4) A combination of IFCC method / SOP equivalent reagent / formulation (1st reagent) on the day of reagent preparation (day 0) [5℃] and IFCC method / SOP equivalent reagent / formulation (2nd reagent) after 6 months of storage [10℃].

[0118] For reagent blank measurements, a Hitachi 7180 automated analyzer was used. 150 μL of each first reagent was added to 4 μL of sample, reacted at 37°C for 5 minutes, and then 50 μL of each second reagent was added and reacted at 37°C for 5 minutes.

[0119] The absorbance at the primary wavelength of 340 nm and the secondary wavelength of 405 nm was measured to determine the reagent blank.

[0120] 4.Measurement results The graph of the measurement results described above is shown in Figure 2. In this figure, measurements using the present invention method (reagent 1) and the present invention method (reagent 2) are indicated as "present invention method," and measurements using the IFCC method / SOP equivalent reagent / formulation (reagent 1) and the IFCC method / SOP equivalent reagent / formulation (reagent 2) are indicated as "SOP equivalent reagent."

[0121] In this figure, the measurement results for the combination of Reagent 1 after 6 months of storage [10°C] and Reagent 2 on the day of reagent preparation (0 days) [5°C] (R2 5°C 0 months) are shown as black bar graphs, and the measurement results for the combination of Reagent 1 on the day of reagent preparation (0 days) [5°C] and Reagent 2 after 6 months of storage [10°C] (R2 10°C 6 months) are shown as gray bar graphs.

[0122] This figure shows that, in the measurement results (R2 10℃ 6 months) of the combination of the first reagent on the day of reagent preparation (day 0) [5℃] and the second reagent after 6 months of storage [10℃], the reagent blank when using the present invention method (second reagent) is significantly lower than the reagent blank when using the IFCC method / SOP equivalent reagent / formulation (second reagent).

[0123] This confirms that the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening, as described in the present invention, results in an extremely small increase in the reagent blank over time.

[0124] [Example 5] (Investigation of correlation) The correlation of measurements in the present invention was examined. 1. Measurement reagents As measurement reagents, the IFCC-SOP method measurement reagent (first reagent) prepared in Example 1, 1(1) above, the IFCC-SOP method measurement reagent (second reagent) prepared in Example 1, 1(2) above, the method of the present invention (first reagent) prepared in Example 1, 2(1) above, and the method of the present invention (second reagent) prepared in Example 1, 2(2) above were used, respectively.

[0125] 2. Sample One hundred human serum samples were used as the test material.

[0126] 3. Examination of correlation The lactate dehydrogenase activity levels contained in each of the samples in the above 2 were measured using each of the measurement reagents described in 1 above.

[0127] When using the IFCC-SOP method reagent (first reagent) prepared in Example 1, 1(1) and the IFCC-SOP method reagent (second reagent) prepared in Example 1, 1(2) as the measurement reagents, the measurements were performed using a Hitachi 7180 automatic analyzer. 200 μL of the IFCC-SOP method reagent (first reagent) was added to 10 μL of the sample, reacted at 37°C for 5 minutes, then 20 μL of the IFCC-SOP method reagent (second reagent) was added, and reacted at 37°C for 5 minutes.

[0128] The absorbance was measured at the primary wavelength of 340 nm and the secondary wavelength of 405 nm, and the change in absorbance per minute was determined. The lactate dehydrogenase activity levels in the samples were calculated using a calibration curve created with the above-mentioned measurement reagents.

[0129] Furthermore, when using the method of the present invention (first reagent) prepared in Example 1, 2 (1) and the method of the present invention (second reagent) prepared in Example 1, 2 (2) as measurement reagents, 150 μL of the method of the present invention (first reagent) was added to 4 μL of the sample, reacted at 37°C for 5 minutes, then 50 μL of the method of the present invention (second reagent) was added, and reacted at 37°C for 5 minutes.

[0130] The absorbance was measured at the primary wavelength of 340 nm and the secondary wavelength of 405 nm, and the change in absorbance per minute was determined. The lactate dehydrogenase activity levels in the samples were calculated using a calibration curve created with the above-mentioned measurement reagents.

[0131] 4.Measurement results The graph of the measurement results described above is shown in Figure 3.

[0132] In this figure, measurements using the IFCC-SOP method reagent (first reagent) and the IFCC-SOP method reagent (second reagent) are indicated as "IFCC-SOP method," while measurements using the present invention method (first reagent) and the present invention method (second reagent) are indicated as "present invention method."

[0133] The horizontal axis (x) shows the lactate dehydrogenase activity value (measured value) [U (unit) / L] of each serum sample when measured using the IFCC-SOP method reagent (first reagent) and the IFCC-SOP method reagent (second reagent), and the vertical axis (y) shows the lactate dehydrogenase activity value (measured value) [U (unit) / L] of the sample when measured using the present invention method (first reagent) and the present invention method (second reagent).

[0134] The correlation regression equation in this figure is y = 1.0026x - 2.6562(R 2 (=0.9987), which shows that it exhibits an excellent correlation. This confirms that the method for preventing deterioration of the lactate dehydrogenase activity measurement reagent after opening, as described in the present invention, shows excellent correlation with the IFCC-SOP method.

Claims

[Claim 1] A method for measuring the activity of lactate dehydrogenase using L-lactic acid as a substrate, characterized by using 2-amino-2-methyl-1,3-propanediol as a buffer, which provides measurement values ​​that do not deviate from the standard procedure of IFCC, and prevents negative errors from occurring in the measurement results after opening of the lactate dehydrogenase activity measuring reagent, in which the pH during the reaction in which lactate dehydrogenase uses L-lactic acid as a substrate and simultaneously converts oxidized β-nicotinamide adenine dinucleotide (NAD+) to reduced β-nicotinamide adenine dinucleotide (NADH) is 8.6 to 10.4.

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