Liquid composition for determining ATP, and AMP and / or ADP in a sample

Abstract

In one embodiment, the objective of the present invention is to provide a liquid cyclic luminescent reagent with excellent stability. In one embodiment, the present invention provides a liquid composition for measuring ATP, AMP and / or ADP in a sample after preservation, (i) the liquid composition comprising luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the measurement, and (ii) the relative luminescence intensity of the liquid composition at preservation is 5500 RLU or less, the relative luminescence intensity being the value obtained by subtracting a control value from the measured value.

Description

[0001] This application is a divisional application of patent application filed on February 15, 2021, with application number 202180014495.1 and entitled "Liquid Composition for Determining ATP, AMP and / or ADP in a Sample", the entire contents of which are incorporated herein by reference. Technical Field

[0002] In one embodiment, the present invention relates to a liquid composition for measuring ATP, AMP and / or ADP in a sample after preservation, a kit for measuring ATP in a sample comprising the liquid composition, and a method for measuring ATP, AMP and / or ADP in a sample, the method comprising using the liquid composition. Background Technology

[0003] Adenosine triphosphate (ATP) is a nucleotide found in all living organisms and is used by cells as a substrate for storing and releasing energy. Since ATP is contained in substances derived from living organisms, kits and methods for determining the cleanliness of biological samples and biological instruments by measuring ATP, as well as kits for determining the contamination level of cooking-related instruments, have been reported (Patent Documents 1-2).

[0004] As a representative ATP assay, a method is known to react ATP with the substrate luciferin in the presence of luciferase to measure luminescence (Non-Patent Literature 1). This reaction is catalyzed by luciferase and proceeds in the presence of divalent metal ions as follows.

[0005] Luciferin + ATP + O2 → Oxidized Luciferin + Adenosine Monophosphate (AMP) + Pyrophosphate (PPi) + CO2 + Light

[0006] ATP is readily dephosphated to form ADP, and ADP can sometimes also be dephosphated to form AMP. Therefore, by measuring not only ATP, but also ATP and ADP, or ATP, ADP, and AMP, it is possible to stably measure the ATP (or its breakdown products) contained in substances from organisms, thereby enabling more accurate determination of cleanliness. For example, Patent Document 1 describes how, by measuring not only ATP, but also ATP and ADP, or ATP, ADP, and AMP, residual blood and adhering blood on blood-related instruments can be accurately detected.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: International Publication No. 2018 / 147442

[0010] Patent Document 2: International Publication No. 2018 / 147443

[0011] Non-patent literature

[0012] Non-patent literature 1: Marlene DeLuca, William D. McElroy, Biochemistry, 1974, 13 (5), pp 921-925 Summary of the Invention

[0013] The luminescent reagent used in a cyclic reaction system (circular reaction) that directly or indirectly converts AMP to ATP, and measures ADP and / or AMP in addition to ATP, is called a "cyclic luminescent reagent." Existing cyclic luminescent reagents, such as LuciPac Pen (for measuring ATP + AMP) (manufactured by Kikkoman Biochemifa Company) and LuciPacA3 (for measuring ATP + ADP + AMP) (manufactured by Kikkoman Biochemifa Company), are all designed and manufactured in powder form; liquid cyclic luminescent reagents are not available. However, manufacturing powder reagents requires drying and pulverizing processes, and rapid and accurate filling is difficult, resulting in complex manufacturing methods and high costs. Furthermore, powders have drawbacks such as the need to address moisture absorption.

[0014] Therefore, the inventors redesigned the liquid circulating luminescent reagent. Furthermore, the inventors recently discovered that the stability of the liquid circulating luminescent reagent is not sufficient.

[0015] In one embodiment, the objective of the present invention is to provide a liquid cyclic luminescent reagent with excellent stability. In another embodiment, the objective of the present invention is to provide a kit comprising the cyclic luminescent reagent or a method for measuring ATP, AMP, and / or ADP in a sample using the cyclic luminescent reagent or kit.

[0016] The inventors have surprisingly discovered that, in a cyclic luminescence reaction using a luciferin-luciferase reaction, the stability of the liquid cyclic luminescence reagent can be improved by reducing the relative luminescence amount of the liquid cyclic reagent during storage.

[0017] The present invention includes the following embodiments.

[0018] [1] A liquid composition for use in determining ATP, AMP and / or ADP in a sample after preservation. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the assay, and (ii) The relative luminescence intensity of the liquid composition during storage is 5500 RLU or less. The relative luminescence is the value obtained by subtracting the control value from the measured value. The measured value refers to the addition of 0.35 mL of the liquid composition to a test tube of a LuciPac Pen (manufactured by Kikkoman Bio-Kemifag Co., Ltd.), followed by the addition of 0.01 mL of 1×10⁻⁶ mol / L. -7 The value of M's ATP (manufactured by Oriental Yeast Co., Ltd.) solution, after standing at 25°C for 1 hour, was measured using a Lumitester Smart fluorescence detector (manufactured by Kikkoman Bio-Kemifa Co., Ltd.). The control value refers to the value measured under the same conditions as when the measured value was obtained, except that sterile ultrapure water was added to replace the ATP solution.

[0019] [2] The liquid composition according to [1] further comprises at least one component selected from an enzyme that catalyzes the reaction of ADP to ATP, a substrate of an enzyme that catalyzes the reaction of ADP to ATP, an enzyme that catalyzes the reaction of ADP to AMP, a substrate of an enzyme that catalyzes the reaction of ADP to AMP, or at least one of the above is added before or during the assay.

[0020] [3] A liquid composition for use in determining ATP, AMP and / or ADP in a sample after preservation. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ADP, a substrate of the enzyme catalyzing the reaction from AMP to ADP, an enzyme catalyzing the reaction from ADP to ATP, a substrate of the enzyme catalyzing the reaction from ADP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the component not contained in the liquid composition is added before or during the assay, and (ii) The relative luminescence intensity of the liquid composition during storage is below 5500 RLU. The relative luminescence is the value obtained by subtracting the control value from the measured value. The measured value refers to the addition of 0.35 mL of the liquid composition to a test tube of a LuciPac Pen (manufactured by Kikkoman Bio-Kemifag Co., Ltd.), followed by the addition of 0.01 mL of 1×10⁻⁶ mol / L. -7 The value of M's ATP (manufactured by Orient Yeast Industry Co., Ltd.) solution, after standing at 25°C for 1 hour, was measured using a Lumitester Smart fluorescence detector (manufactured by Kikkoman Bio-Kemifa Co., Ltd.). The control value refers to the value measured under the same conditions as when the measured value was obtained, except that sterile ultrapure water was added to replace the ATP solution.

[0021] [4] The liquid composition according to any one of [1] to [3], wherein the relative luminescence intensity is 2300 RLU or less.

[0022] [5] A liquid composition for use in determining ATP, AMP and / or ADP in a sample after preservation. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the assay, and (ii) Meets at least one of the following: The concentration of fluorescein in the liquid composition is below 0.4 mM; The concentration of the Bradford-based luciferase in the liquid composition is less than 0.3 mg / mL; The concentration of the enzyme catalyzing the reaction from AMP to ATP in the liquid composition is less than 1 U / mL; The concentration of the substrate of the enzyme catalyzing the reaction from AMP to ATP in the liquid composition is below 0.1 mM; The concentration of the cofactor in the liquid composition is below 6 mM.

[0023] [6] The liquid composition according to [5] further comprises at least one component selected from an enzyme that catalyzes the reaction of ADP to ATP, a substrate of an enzyme that catalyzes the reaction of ADP to ATP, an enzyme that catalyzes the reaction of ADP to AMP, a substrate of an enzyme that catalyzes the reaction of ADP to AMP, or at least one of the above is added before or during the assay.

[0024] [7] A liquid composition for use in determining ATP, AMP, and / or ADP in a sample after preservation, wherein, (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ADP, a substrate of the enzyme catalyzing the reaction from AMP to ADP, an enzyme catalyzing the reaction from ADP to ATP, a substrate of the enzyme catalyzing the reaction from ADP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the component not contained in the liquid composition is added before or during the assay, and (ii) Meets at least one of the following: The concentration of the enzyme catalyzing the reaction from AMP to ADP in the liquid composition is below 450 U / mL; The concentration of the substrate of the enzyme catalyzing the reaction from AMP to ADP in the liquid composition is below 0.1 mM; The concentration of the enzyme catalyzing the reaction from ADP to ATP in the liquid composition is below 20 U / mL; The concentration of the substrate for the enzyme that catalyzes the reaction from ADP to ATP is below 1.2 mM; The concentration of the cofactor in the liquid composition is below 6 mM.

[0025] [8] The liquid composition according to any one of [1] to [7], wherein the storage time is more than 1 day.

[0026] [9] The liquid composition according to [8], wherein the storage time is more than 30 days.

[0027]

[10] The liquid composition according to any one of [1] to [9], wherein the liquid composition does not contain at least one of the following components: luciferase, luciferin, enzyme catalyzing the reaction of AMP to ATP, substrate of enzyme catalyzing the reaction of AMP to ATP, enzyme catalyzing the reaction of AMP to ADP, substrate of enzyme catalyzing the reaction of AMP to ADP, enzyme catalyzing the reaction of ADP to ATP, substrate of enzyme catalyzing the reaction of ADP to ATP, and cofactor, and the component not contained in the liquid composition is added before or during the assay.

[0028]

[11] The liquid composition according to any one of [1] to

[10] , wherein the concentration of luciferin in the liquid composition is less than 0.4 mM and / or the concentration of luciferase based on the Bradford method is less than 0.3 mg / mL.

[0029]

[12] The liquid composition according to any one of [1] to

[11] , wherein the concentration of fluorescein in the liquid composition is 0.1 mM or less.

[0030]

[13] The liquid composition according to any one of [1] to

[12] , wherein the concentration of the Bradford-based luciferase in the liquid composition is less than 0.1 mg / mL.

[0031]

[14] A kit for determining ATP in a sample, the kit comprising any one of [1] to

[13] liquid composition.

[0032]

[15] A method for determining ATP, AMP and / or ADP in a sample, comprising: using the liquid composition described in any one of [1] to

[13] or the kit described in

[14] .

[0033]

[16] The method according to

[15] , wherein no ATP standard solution is used.

[0034] This specification contains the disclosure of Japanese Patent Application No. 2020-023442, which forms the basis of the priority claim of this application.

[0035] In one embodiment, the present invention provides a liquid circulating luminescent reagent with excellent stability. Attached Figure Description

[0036] Figure 1 The change in luminescence intensity over time is shown for a non-cyclic luminescent reagent.

[0037] Figure 2 The stability of cyclic luminescent reagents containing different concentrations of fluorescein is shown.

[0038] Figure 3 Show Figure 2 The luminescence levels of cyclic luminescent reagents containing different concentrations of fluorescein after 9 hours.

[0039] Figure 4 The stability of the cyclic luminescent reagent with or without fluorescein is shown.

[0040] Figure 5 The stability of the luminescent reagent is shown for both non-cyclic and cyclic formulations. Results are shown for both cases with and without fluorescein.

[0041] Figure 6 The stability of cyclic luminescent reagents containing different concentrations of luciferase is shown.

[0042] Figure 7 The luminescence levels of cyclic luminescent reagents containing different concentrations of luciferase are shown after 9 hours.

[0043] Figure 8 This demonstrates the ATP concentration dependence of the decrease in luminescence intensity of the cyclic luminescent reagent.

[0044] Figure 9 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -5 The stability of luminescent reagents containing different concentrations of luciferin at M ATP (changes in luminescence over time).

[0045] Figure 10 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -5 The amount of light emitted after 1 hour when M ATP contains different concentrations of luciferin luminescent reagent.

[0046] Figure 11 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -6 The stability of M ATP containing different concentrations of luciferin luminescent reagent (changes in luminescence over time).

[0047] Figure 12 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -6 The amount of light emitted after 1 hour when M ATP contains different concentrations of luciferin luminescent reagent.

[0048] Figure 13 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -7 The stability of M ATP containing different concentrations of luciferin luminescent reagent (changes in luminescence over time).

[0049] Figure 14 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -7 The amount of light emitted after 1 hour when M ATP contains different concentrations of luciferin luminescent reagent.

[0050] Figure 15 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -5 The stability of luminescent reagents containing different concentrations of luciferase at M ATP (changes in luminescence over time).

[0051] Figure 16 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -5 The amount of light emitted by a luminescent reagent containing different concentrations of luciferase after 1 hour at M ATP.

[0052] Figure 17 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -6 The stability of luminescent reagents containing different concentrations of luciferase at M ATP (changes in luminescence over time).

[0053] Figure 18 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -6 The amount of light emitted by a luminescent reagent containing different concentrations of luciferase after 1 hour at M ATP.

[0054] Figure 19 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -7 The stability of luminescent reagents containing different concentrations of luciferase at M ATP (changes in luminescence over time).

[0055] Figure 20 The diagram shows the addition of 0.01 mL of 1×10⁻⁶ to 0.35 mL of the luminescent reagent. -7 The amount of light emitted by a luminescent reagent containing different concentrations of luciferase after 1 hour at M ATP.

[0056] Figure 21 The diagram shows the removal of each component from the luminescent reagent, followed by "mixing and storing" or "storing and mixing" of the removed components, and the addition of 0.1 mL of 1×10⁻⁶ solution. -6 The luminescence intensity when M ATP solution was added directly was also shown. Additionally, the luminescence intensity when 0.1 mL of 1 × 10⁻⁶ ATP solution was added without preservation was also shown. -6 Luminescence at M ATP (before storage). Detailed Implementation

[0057] [Liquid Composition]

[0058] In the first embodiment, the present invention relates to a liquid composition for use, after preservation, in determining ATP, as well as AMP and / or ADP in a sample. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the assay, and (ii) The relative luminescence of the liquid composition during storage is less than 5500 RLU, where the relative luminescence is the value obtained by subtracting the control value from the measured value.

[0059] In the second embodiment, the present invention relates to a liquid composition for use in determining ATP, AMP, and / or ADP in a sample after preservation. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ADP, a substrate of the enzyme catalyzing the reaction from AMP to ADP, an enzyme catalyzing the reaction from ADP to ATP, a substrate of the enzyme catalyzing the reaction from ADP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the component not contained in the liquid composition is added before or during the assay, and (ii) The relative luminescence of the liquid composition during storage is less than 5500 RLU, where the relative luminescence is the value obtained by subtracting the control value from the measured value.

[0060] In the compositions of the first and second schemes, the measured value refers to adding 0.35 mL of the liquid composition to a LuciPac Pen (manufactured by Kikkoman Bio-Kemifag Co., Ltd.) test tube, and then adding 0.01 mL of 1×10 -7 The ATP (manufactured by Orient Yeast Industry Co., Ltd.) solution of M was allowed to stand at 25°C for 1 hour, and then measured using a Lumitester Smart fluorescence detector (manufactured by Kikkoman Co., Ltd.). The control value refers to the value measured under the same conditions as when the measured value was obtained, except that sterile ultrapure water was added to replace the ATP solution. It should be noted that since the LuciPac Pen (manufactured by Kikkoman Co., Ltd.) test tube contains luminescent reagent, it should be removed by washing until it does not affect the measurement system before use. In cases where LuciPac Pen (manufactured by Kikkoman Co., Ltd.) test tubes are difficult to obtain, equivalent LuciPac A3 (manufactured by Kikkoman Co., Ltd.) test tubes can be used. Furthermore, while products manufactured by Orient Yeast Industry Co., Ltd. are generally used for ATP, equivalent ATP can be used in cases where it is difficult to obtain. To avoid increasing background levels and affecting the measured values, water with minimal contamination of ATP, ADP, and AMP is preferred as sterile ultrapure water. For example, the combined concentration of ATP, ADP, and AMP could be 1 × 10⁻⁶. -9 Below M or 1×10 -10 Water below M. Use sterile ultrapure water for ATP dilution, using the same sterile ultrapure water used for background determination.

[0061] When determining the relative luminescence of a liquid composition during storage, if the total amount of the liquid composition (e.g., the liquid composition in a kit) is less than 0.35 mL, multiple equal liquid compositions (e.g., other liquid compositions from the same type of kit) can be mixed, and the relative luminescence of the mixed liquid composition can be determined.

[0062] In this specification, the relative luminescence intensity is, in principle, the value measured under the conditions described above. However, in cases where the aforementioned LuciPac Pen or similar devices are not readily available, the following conditions may be used instead: The measured value refers to the amount of 0.35 mL of the liquid composition described above added to a Lumi tube (manufactured by Kikkoman Bio-Kemi Co., Ltd., 12). (×54mm), then add 0.01mL of 1×10 -7 The value obtained by dividing the ATP solution of M, after standing at 25°C for 1 hour, by measuring it with a Lumitester C-110 (manufactured by Kikkoman Bio-Kemifa Co., Ltd.), by 400. The control value is the value obtained by dividing the luminescence amount measured under the same conditions as when the measured value was obtained, except that sterile ultrapure water was added to replace the ATP solution, by 400.

[0063] In one embodiment, the relative luminescence of the liquid composition during storage can be less than 5500 RLU, less than 5000 RLU, less than 4500 RLU, less than 4000 RLU, less than 3500 RLU, or less than 3000 RLU. For example, the relative luminescence of the liquid composition during storage can be below 2900 RLU, below 2800 RLU, below 2700 RLU, below 2600 RLU, below 2500 RLU, below 2400 RLU, below 2300 RLU, below 2200 RLU, below 2100 RLU, below 2000 RLU, below 1900 RLU, below 1800 RLU, below 1700 RLU, below 1600 RLU, below 1500 RLU, below 1400 RLU, below 1300 RLU, below 1200 RLU, below 1100 RLU, below 1000 RLU, below 900 RLU, below 800 RLU, below 700 RLU, below 600 RLU, below 500 RLU, below 400 RLU, below 300 RLU, below 200 RLU, or below 100 RLU. The examples show that the lower the relative luminescence of the liquid composition during storage, the greater the stability of the liquid composition. Furthermore, the stability of the liquid composition may be particularly excellent when the relative luminescence of the liquid composition during storage is below 2500 RLU or below 2300 RLU. In this specification, "excellent stability" of the liquid composition means, for example, that the reduction in the luminescence of the liquid composition is minimal or nonexistent.

[0064] There are no limitations on the methods for reducing the relative luminescence of the liquid composition during storage. Examples include: changing the storage pH relative to the optimal pH of the enzyme; adding ATP-degrading enzymes to decompose ATP so that it cannot luminesce; adding reaction inhibitors; reducing the concentration of components required for the cyclic reaction or removing components and then adding the removed components before or during the measurement; or combinations of these methods.

[0065] For example, in methods that change the storage pH relative to the optimal pH, the pH of the liquid composition can be increased or decreased relative to the optimal pH until the enzyme reaction does not occur or is delayed, and the enzyme does not denature. The pH of the liquid composition is then restored to the optimal pH before or during the measurement. When the composition is acidic, the storage pH can be, for example, 6 or less, 5 or less, or 4 or less, or 2 or more, or 3 or more. When the composition is alkaline, the storage pH can be, for example, 9 or more, 10 or more, or 11 or more, or 13 or less, or 12 or less. Those skilled in the art can easily determine the pH at which the enzyme does not denature and the relative luminescence of the liquid composition can be reduced. Furthermore, the pH can be easily adjusted using an acid or base to achieve the determined pH. For example, as described in Japanese Patent Application Publication No. 11-239493, HLK reduces the relative luminescence by about 1 / 3 or less by setting the pH to 6.5 or less, and reduces the relative luminescence by about 1 / 10 or less by setting the pH to 6 or less. The pH before or during the measurement can be 6 to 9, 7.5 to 8.5, or about 8. In other embodiments, the pH at storage is the same or nearly the same as the pH before or at the time of measurement (e.g., the difference between the pH at storage and the pH before or at the time of measurement is less than 1).

[0066] One method for adding ATP-degrading enzymes to break down ATP is to add adenosine monophosphate deaminase during storage. ATP-degrading enzymes can be used at concentrations that do not affect the ATP assay system. Alternatively, the influence of ATP-degrading enzymes can be reduced by adding a liquid composition before or during the assay. Those skilled in the art can easily determine the concentration and type of ATP-degrading enzyme.

[0067] When a reaction inhibitor is added, examples of reaction inhibitors include metal salts such as NaCl and surfactants such as benzalkonium chloride. The effect of the reaction inhibitor can be reduced by diluting the liquid composition before or during the assay. Alternatively, the reaction inhibitor can be removed before or during the assay; for example, metal salts can be removed by chelating agents, and benzalkonium chloride can be removed by cyclodextrins. Those skilled in the art can easily determine the concentration and type of reaction inhibitor.

[0068] The method for adding the component before or during the determination after reducing the concentration of the component required for the cyclic reaction or removing the component is as described in this specification.

[0069] In the third embodiment, the present invention relates to a liquid composition for use in determining ATP, as well as AMP and / or ADP in a sample after preservation. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the assay, and (ii) Meets at least one of the following: The concentration of fluorescein in the liquid composition is below 0.4 mM; The concentration of the Bradford-based luciferase in the liquid composition is less than 0.3 mg / mL; The concentration of the enzyme (e.g., PPDK) catalyzing the reaction from AMP to ATP in the liquid composition is less than 1 U / mL; The concentration of the substrate of the enzyme that catalyzes the reaction of AMP to ATP in the liquid composition (e.g., in the case of PPDK, the substrate is pyrophosphate or a salt thereof and / or phosphoenolpyruvate or a salt thereof) is less than 0.1 mM. The concentration of the cofactor (e.g., magnesium salt) in the liquid composition is below 6 mM.

[0070] In the fourth embodiment, the present invention relates to a liquid composition for use in determining ATP, as well as AMP and / or ADP in a sample after preservation. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ADP, a substrate of the enzyme catalyzing the reaction from AMP to ADP, an enzyme catalyzing the reaction from ADP to ATP, a substrate of the enzyme catalyzing the reaction from ADP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the component not contained in the liquid composition is added before or during the assay, and (ii) Meets at least one of the following: The concentration of the enzyme (e.g., ADK) catalyzing the reaction from AMP to ADP in the liquid composition is below 450 U / mL; The concentration of the substrate of the enzyme catalyzing the reaction from AMP to ADP in the liquid composition is below 0.1 mM (e.g., no substrate is required for ADK). The concentration of the enzyme (e.g., PK) catalyzing the reaction from ADP to ATP in the liquid composition is less than 20 U / mL; The concentration of the substrate of the enzyme that catalyzes the reaction of ADP to ATP (e.g., in the case of PK enzyme, the substrate is phosphoenolpyruvate or a salt thereof) is less than 1.2 mM; The concentration of the cofactor (e.g., magnesium salt) in the liquid composition is below 6 mM.

[0071] In one embodiment, the liquid composition of the first and third schemes further comprises at least one component selected from an enzyme that catalyzes the reaction of ADP to ATP, a substrate of an enzyme that catalyzes the reaction of ADP to ATP, an enzyme that catalyzes the reaction of ADP to AMP, and a substrate of an enzyme that catalyzes the reaction of ADP to AMP, or at least one of the above is added before or during the assay.

[0072] The liquid compositions of the first to fourth embodiments (which are sometimes collectively referred to as "the liquid compositions described herein") and their constituent components will now be described in more detail.

[0073] In one embodiment, the liquid composition described herein is used not only to determine ATP but also to determine AMP and / or ADP (i.e., ATP and ADP; ATP and AMP; or ATP, ADP, and AMP). Although substances from living organisms contain ATP, ATP is more readily dephosphorylated to form ADP. Furthermore, ADP can sometimes also be dephosphorylated to form AMP. Therefore, by measuring two components (ATP and ADP or AMP) or three components (ATP, ADP, and AMP), the amount of ATP (or its breakdown products) contained in substances from living organisms can be stably determined. Thus, by measuring two components (ATP and ADP or AMP) or three components (ATP, ADP, and AMP), cleanliness can be determined more accurately without missing contaminants.

[0074] [Luciferase]

[0075] Luciferase is a general term for oxidases that induce bioluminescence. In one embodiment, luciferase catalyzes the conversion of ATP, O2, and luciferin into AMP, pyrophosphate, CO2, and oxidized luciferin, thereby inducing luminescence. Luciferase can be a natural luciferase or a genetically engineered recombinant luciferase variant. Luciferase variants can be substances with site-specific mutations or random mutations. They can also be fusion proteins with other functional proteins. Luciferase variants can be variants with enhanced heat resistance, surfactant resistance, or other desired properties.

[0076] The relative luminescence intensity (RLU) obtained using appropriate luminescence measurement devices, such as photometers (e.g., Kikkoman Co., Ltd., Lumitester Smart fluorescence detector, Lumitester PD-20, Lumitester PD-30, etc.) or devices equipped with photodiodes (e.g., Hygiena SystemSURE Plus, EnSURE, Neogen AccuPoint Advanced, etc.), can be used as an indicator to evaluate the luminescence amount of luciferase. Typically, the luminescence produced during the conversion from luciferin to oxidized luciferin is measured. As luminescence measurement devices, those capable of high-sensitivity measurement and equipped with photomultiplier tubes can also be used (e.g., 3M Clean-Trace LM1, Clean-Trace UNG3; Kikkoman Co., Ltd., Lumitester C-110, Lumitester C-100; or Berthold, JuniorLB9509, CentroLB960, or Lumat3 LB9508, etc.). Especially when the amount of light emitted is reduced, devices that can perform highly sensitive measurements can be used for accurate measurements.

[0077] Luciferases are not particularly restricted in their use of ATP as a substrate and can utilize substances from bacteria, protozoa, animals, mollusks, and insects. Examples of insect-derived luciferases include beetle luciferases, such as those from the following genera: *Photinus* (e.g., *Photinus pyralis*); *Photuris* (e.g., *Photuris lucicrescens*, *Photuris pennsylvanica*); *Luciola* (e.g., *Luciola cruciata*, *Luciola lateralis*, *Luciola parvula*, *Pyrocoelia*, *Lucidina biplagiata*); and click beetles from the genus *Pyrophorus*. Many luciferase genes have been reported, and their base and amino acid sequences can be obtained from well-known databases such as GeneBank.

[0078] The luciferase gene can be a wild-type gene or a variant gene. The variant can be a site-specific mutation or a random mutation. Known variants include: variations that increase luminescence intensity, as described in Japanese Patent Application Publication No. 2011-188787; variations that increase luminescence persistence, as described in Japanese Patent Application Publication No. 2000-197484; variations that change the emission wavelength, as described in Japanese Patent Application Publication No. 2666561 or Japanese Patent Application Publication No. 2003-512071; and variations that increase surfactant resistance, as described in Japanese Patent Application Publication No. 11-239493. Variations that improve substrate affinity, such as those described in International Publication No. 99 / 02697, Japanese Patent Publication No. 10-512750, or Japanese Patent Publication No. 2001-518799, and variations that improve stability, such as those described in Patent Publication No. 3048466, Japanese Patent Application Publication No. 2000-197487, Japanese Patent Publication No. 9-510610, and Japanese Patent Publication No. 2003-518912, etc., but are not limited to these.

[0079] Luciferase genes and their recombinant DNA can be prepared using common methods. For example, Japanese Patent Application Publication No. 7-112434 discloses the Hirai-no-Fluorescent Luciferase gene. Additionally, Japanese Patent Application Publication No. 1-51086 discloses the Genji-no-Fluorescent Luciferase gene.

[0080] The luciferase gene can integrate into vectors such as plasmids, bacteriophages, and granules, thereby transforming or transducing a suitable host. Hosts can include microorganisms, bacteria such as Escherichia coli, and yeast. Transformed hosts capable of producing luciferase can be cultured using various well-known methods.

[0081] As a culture medium, examples include: a culture medium in which one or more nitrogen sources such as tryptone, yeast extract, meat extract, peptone, corn steep liquor or extract of soybean or wheat bran are added, along with one or more inorganic salts such as sodium chloride, potassium phosphate, potassium phosphate, magnesium chloride, ferric chloride, magnesium sulfate or manganese sulfate, and sugar raw materials, vitamins, etc. are added as needed.

[0082] The initial pH of the culture medium can be, for example, 7–9. Incubation can be carried out at, for example, 30–40°C, for 2–24 hours via aeration and stirring, shaking, or static incubation. After incubation, luciferase is recovered from the culture using known methods.

[0083] Specifically, bacterial cells are subjected to ultrasonic disruption or grinding using common methods, or luciferase is extracted using lysozymes such as lysozyme. The resulting extract is then filtered, centrifuged, and nucleic acids are removed as needed using streptomycin sulfate. Ammonium sulfate, alcohol, acetone, etc., are added for fractionation to obtain crude enzyme.

[0084] The crude enzyme can be further purified using various gel filtration and chromatography methods. Commercially available luciferases can also be used; for example, the luciferase manufactured by Kikkoman Bio-Kemifa Co., Ltd., catalog number 61314, can be used. This luciferase is described in Japanese Patent Application Publication No. 11-239493 (Patent No. 3749628) (serial number 1 in that document). Alternatively, commercially available molecular probe (registered trademark) luciferases from Sigma-Aldrich, Prometheus, and LifeScience Co., Ltd. can also be used.

[0085] In one embodiment, the luciferase concentration in the liquid composition described herein, measured using the Bradford method, can be, for example, below 0.3 mg / mL, below 0.25 mg / mL, below 0.2 mg / mL, below 0.1 mg / mL, below 0.05 mg / mL, below 0.01 mg / mL, below 0.005 mg / mL, below 0.001 mg / mL, or below 0.0005 mg / mL. The Bradford method-based concentration can be determined using a Coomassie (Bradford) Protein Assay Kit (Thermo Scientific), as described in the examples, with a BSA solution as a standard. It should be noted that in cases where this kit is unavailable, the luciferase concentration can still be determined using a Bradford method known to those skilled in the art, which yields equivalent values. Alternatively, as described in the examples, the absorbance can be obtained using the 280 nm absorbance method, and then converted to determine the Bradford method-based concentration. Lower concentrations of luciferase improve the stability of the liquid composition, but reduce the overall luminescence. Therefore, if the luminescence is too low, additional luciferase can be added before or during the assay. Based on the Bradford method concentration meter, the luciferase concentration in the liquid composition, whether stored or after adding luciferase before or during the assay, can be, for example, 0.00001 mg / mL or higher, 0.0001 mg / mL or higher, 0.001 mg / mL or higher, or 0.01 mg / mL or higher.

[0086] In this specification, the luciferase concentration is generally based on the Bradford method. However, if the liquid composition is prepared containing proteins other than luciferase, the concentration of luciferase alone in such a liquid composition cannot be directly determined by the Bradford method. In this case, as described below, the luciferase concentration based on the Bradford method can be determined indirectly based on the luciferase activity.

[0087] The liquid composition containing luciferase was diluted with enzyme dilution buffer (5.0% Glycerol, 1.0 mM EDTA). 2Na The enzyme was diluted with 2H₂O, 1.0 mM 2-mercaptoethanol, 50 mM Tricine (tris(hydroxymethyl)methylglycine), and 1.0% bovine serum albumin (BSA) (pH 7.8) to achieve a luminescence intensity of 100,000–1,000,000 RLU. 100 μL of the diluted liquid composition was collected into a Laurent tube (manufactured by Saster) preheated to 25°C. Using a syringe, 100 μL of the luminescent reagent (50 mM Tricine, 4.0 mM ATP) for measuring luciferase activity was added to the diluted mixture. 2Na, 2.0mM D-luciferin, 10mM MgSO4 The luminescence intensity was measured using a LUMAT LB9507 (Berthold) at 7H2O (pH 7.8) for 0.5 to 20 seconds, and this intensity was taken as the sample luminescence intensity (Es). Measurements were performed at 25°C. Similarly, the blank luminescence intensity (E0) was measured using enzyme dilution buffer instead of the luciferase solution.

[0088] Luciferase activity (LU / mL) = (Es - E0) × dilution ratio ÷ sample volume

[0089] Sample volume: 0.1 mL

[0090] In addition to the experiments described above, a luciferase that was identified as a single band by SDS-PAGE was used to determine its activity and protein concentration. The results showed that the relative luciferase activity was 8.4 × 10⁻⁶. 14 The LU / mL value, based on the Bradford method, is 39.4 mg / mL. That is, the specific activity is 2.1 × 10⁻⁶. 13 LU / mg, using this specific activity value, allows for the calculation of protein concentration based on luciferase activity. That is, Protein concentration (mg / mL) based on the Bradford method = luciferase activity (LU / mL) ÷ (2.1 × 10⁻⁶) 13 LU / mg) Therefore, even in reagents containing proteins as stabilizers such as BSA and enzymes used for cycling (such as PPDK and PK), the protein concentration of luciferase can be determined. It should be noted that although this concentration is a conversion value used when using a specific luciferase (HLK as described in Japanese Patent Application Publication No. 11-239493), the conversion value can be calculated using the same method even when using different luciferases, and the luciferase concentration can be determined based on this value.

[0091] In cases where the liquid composition contains proteins other than luciferase, in addition to the above-mentioned activity assays, the liquid composition can be further fractionated by chromatography methods such as HPLC, and the amount of luciferase protein prepared can be determined by the Bradford method, thereby determining the luciferase concentration.

[0092] [Fluorescence]

[0093] Any luciferin can be used, whether natural or chemically synthesized, as long as it is recognized as a substrate by the luciferase used. Alternatively, known luciferin derivatives can also be used. The basic skeleton of luciferin is imidazopyrazinone, which has many tautomers. Firefly luciferin is an example of a luciferin. Firefly luciferin is a substrate of firefly luciferase (EC 1.13.12.7). Luciferin derivatives can be substances described in Japanese Patent Application Publication No. 2007-91695, Japanese Patent Application Publication No. 2010-523149 (International Publication No. 2008 / 127677), etc.

[0094] In one embodiment, the concentration of fluorescein or its derivative in the liquid composition described herein can be, for example, below 0.4 mM, below 0.35 mM, below 0.3 mM, below 0.25 mM, below 0.15 mM, below 0.1 mM, below 0.05 mM, below 0.02 mM, below 0.01 mM, below 0.005 mM, below 0.001 mM, or below 0.0001 mM. Lower concentrations of fluorescein improve the stability of the liquid composition, but because the overall luminescence is reduced, fluorescein or its derivative can be added before or during measurement if the luminescence is too low. The concentration of fluorescein or its derivative in the liquid composition during storage, or after adding fluorescein before or during measurement, can be, for example, above 0.00001 mM, above 0.0001 mM, above 0.001 mM, or above 0.01 mM.

[0095] The concentration of luciferin or its derivatives can be determined based on the luminescence intensity, following the method described above for luciferase. For example, by diluting the luminescent reagent in a manner that does not affect other components such as the enzyme, a reagent containing an excess of luciferase and different concentrations of luciferin can be prepared. The luminescence intensity can then be compared with that of a standard containing a known concentration of luciferin, thereby determining the concentration of luciferin. Alternatively, the liquid composition can be fractionated using chromatography methods such as HPLC, the peak corresponding to luciferin can be detected, and the peak intensity can be compared with that of a standard containing a known concentration of luciferin, thereby also determining the concentration of luciferin.

[0096] It should be noted that, as described below, the unexpected finding that lower luciferin concentration improves the stability of the liquid composition is surprising. That is, typically in assay systems using enzyme reactions, high substrate concentrations are added to ensure that even as the reaction proceeds and substrate levels decrease, the reaction rate is not affected. In particular, the substrate is continuously consumed during the cyclic reaction; therefore, those skilled in the art would naturally consider that pre-designing a higher substrate concentration would benefit the sustainability of the reagent's luminescence and its stability during storage. However, the results of the embodiments described in this specification show that, in cyclic luminescent reagents utilizing a luciferin-luciferase reaction, it is actually by setting the luciferin concentration to a low level that the luminescence of the cyclic luminescent reagent can be stabilized. The decrease in luciferin quantity due to consumption is not the primary cause of the reduced luminescence stability when storing the cyclic luminescent reagent in liquid form; the inhibition of luminescence by oxidized luciferin generated during the luminescence reaction may be the main cause, but this is not theoretically confined. That is, this differs from the phenomenon observed in cyclic methods using other enzymes and may be a phenomenon unique to liquid cyclic luminescent reagents using a luciferin-luciferase reaction.

[0097] [Enzymes that catalyze the reaction from AMP to ATP]

[0098] In one embodiment, the liquid composition described herein includes an enzyme that catalyzes the reaction from AMP to ATP. The enzyme catalyzes the reaction from AMP to ATP converts AMP present in the system into ATP. Subsequently, ATP is converted back to AMP by luciferase, typically producing luminescence. Therefore, in this embodiment, AMP can be measured in addition to ATP.

[0099] The enzyme that catalyzes the reaction from AMP to ATP can be a known enzyme. Examples include, but are not limited to, pyruvate-phosphokinase dual kinase (PPDK), pyruvate-water dual kinase (PWDK), and combinations thereof.

[0100] [Pyruvate-phosphokinase (PPDK)]

[0101] Pyruvate-phosphokinase dual kinase (EC 2.7.9.1) catalyzes the reactions between ATP, pyruvate, and orthophosphate with adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), and pyrophosphate (PPi): ATP + pyruvate + phosphate ←→ AMP + PEP + PPi Pyruvate-phosphokinase (PPDK), also known as ATP, is pyruvate, phosphotransferase, pyruvate-phosphokinase, and pyruvate-phospholigase. These terms are used interchangeably in this specification. PPDK typically converts pyruvate to PEP, consuming one molecule of ATP in the process, which is then converted to AMP. The reaction proceeds in three reversible steps.

[0102] 1. PPDK binds to ATP to produce AMP and diphosphorylated PPDK.

[0103] 2. PPDK diphosphorylation combines with inorganic phosphate to produce diphosphoric acid and PPDK monophosphorylation.

[0104] 3. PPDK monophosphate binds to pyruvate to produce PEP, and PPDK is produced at the same time.

[0105] If the concentration of PEP in the system is high at this point, the reaction proceeds in reverse as follows.

[0106] [Chemical Formula 1]

[0107] For convenience, the same numbering system as above will be used to describe the reaction stages.

[0108] 3. PEP binds to PPDK to produce phosphorylated PPDK and pyruvate.

[0109] 2. PPDK diphosphorylated and inorganic phosphoric acid are produced from diphosphoric acid and PPDK monophosphorylated.

[0110] 1. PPDK and ATP are produced by diphosphorylation of PPDK and AMP.

[0111] PPDK may include substances from microorganisms such as Microbispora thermorosea, Propionibacterium shermanii, Bacteroides symbiosus, Entamoeba histolytica, Acetobacter xylinum, and Propionibacter shermanii as described in Japanese Patent Application Publication No. 8-168375, as well as substances from plants such as corn and sugarcane, but is not particularly limited thereto.

[0112] [Pyruvate water dual kinase (PWDK)]

[0113] Pyruvate-water dual kinase (EC 2.7.9.2) catalyzes the following reaction: ATP + Pyruvate + H₂O ↔ AMP + Phosphoenolpyruvate (PEP) + Phosphate (P) Pyruvate-water dual kinase is also known as phosphoenolpyruvate synthase; pyruvate-water dual kinase (phosphorylation); PEP synthase; phosphoenolpyruvate synthase; phosphoenolpyruvate synthase; and phosphoenolpyruvate synthase. In this specification, these terms are used interchangeably.

[0114] PWDK can include substances derived from, for example, Escherichia coli, Pseudomonas fluorescens, Pyrococcus furiosus, Staphylothermus marinus, Sulfolobus solfataricus, Thermococcus kodakarensis, Thermoproteus tenax, and Zea mays, but is not particularly limited thereto.

[0115] By using PWDK together with PEP, the generation of ATP from AMP and PEP can be promoted. If the enzyme catalyzing the reaction from AMP to ATP is combined with the enzyme catalyzing the reaction from ADP to ATP as described in this specification, ADP is converted to ATP, thereby enabling the determination of ATP, ADP, and AMP.

[0116] [Enzymes that catalyze the reaction from ADP to ATP]

[0117] In one embodiment, the liquid composition described herein contains an enzyme that catalyzes the reaction from ADP to ATP. The enzyme catalyzes the reaction from ADP to ATP converts ADP present in the system into ATP. Subsequently, ATP is converted to AMP by luciferase, typically producing luminescence. Therefore, in this embodiment, ADP can be measured in addition to ATP.

[0118] As the enzyme catalyzing the reaction from ADP to ATP, known enzymes can be used, such as kinases capable of ATP generation. Examples of kinases capable of ATP generation include pyruvate kinase, acetate kinase, creatine kinase, polyphosphate kinase, riboflavin kinase, phosphofructokinase, fructose bisphosphatase, hexokinase, glucokinase, glycerol kinase, fructose kinase, and combinations thereof, but are not limited thereto.

[0119] [Pyruvate kinase (PK)]

[0120] Pyruvate kinase (EC 2.7.1.40) converts phosphoenolpyruvate to pyruvate in glycolysis, during which ADP is converted to ATP. This reaction is an exothermic reaction with a negative Gibbs energy and is irreversible under natural conditions. PEP + ADP → Pyruvate + ATP In the reverse reaction, during gluconeogenesis, pyruvate carboxylase and phosphoenolpyruvate carboxylkinase catalyze the production of PEP and ADP from ATP and pyruvate. After cell extraction, the system contains a mixture of various enzymes, allowing the above reactions to proceed bidirectionally. If a high concentration of phosphoenolpyruvate is present, ADP can be converted to ATP. Furthermore, if both phosphoenolpyruvate and pyruvate kinase are present in the system, ADP is considered to be further converted to ATP. PK can utilize substances from animals such as rabbits, rats, and chickens, as well as microorganisms such as yeast and Bacillus stearothermophilus, but is not particularly restricted.

[0121] [Acetylkinase (AK)]

[0122] Acetylkinase (EC 2.7.2.1) catalyzes the conversion of ATP and acetic acid to ADP and acetylated phosphate in the presence of cations. ATP + Acetic acid ←→ ADP + Acetylated phosphoric acid Acetyl kinase (AK), also known as ATP, is an enzyme that transfers acetylphosphotransferase. These terms are used interchangeably in this specification. In vivo, it promotes the production of ADP and acetylated phosphate from ATP and acetic acid, ultimately leading to the formation of acetyl-CoA. In the presence of acetylated phosphate and ADP produced from acetyl-CoA in the system, it can be converted into acetic acid and ATP. AK can utilize substances from microorganisms, but is not particularly limited to, such as *Escherichia coli*, *Bacillus stearothermophilus*, *Costridium pasteurianum*, *Lactobacillus delbruckii*, and *Veillonella alcalescence*.

[0123] Creatine kinase (CK)

[0124] Creatine kinase (EC 2.7.3.2) mediates the conversion between creatine and ATP and creatine phosphate and ADP: Creatine + ATP ←→ Creatine Phosphate + ADP Creatine kinase (CK) is also known as creatine phosphokinase (CPK) or phosphocreatine kinase. These terms are used interchangeably in this specification. Normally, in animal muscles, creatine and ATP produce creatine phosphate and ADP. However, this reaction is reversible; if a high concentration of creatine phosphate and ADP is present in the system, the reaction will proceed in reverse, producing creatine and ATP. In vivo, cytoplasmic CK consists of two subunits, B or M. Therefore, depending on the combination of subunits, three isoenzymes can exist: CK-MM, CK-BB, and CK-MB. The isoenzyme pattern varies depending on the tissue, but in this invention, any combination can be used. CK can be derived from animal sources, such as those from rabbits, chickens, cattle, pigs, carp, catfish, and frogs, but is not particularly limited thereto.

[0125] Polyphosphate kinase (PPK)

[0126] Polyphosphokinase (EC 2.7.4.1) catalyzes the reaction that converts polyphosphate (PolyPn) and ADP to polyphosphate (PolyPn-1) and ATP: ADP + PolyPn → ATP + PolyPn-1 Polyphosphokinase (PPK), also known as ATP, is a polyphosphate phosphotransferase. These terms are used interchangeably in this specification. PPK participates in oxidative phosphorylation in vivo. In the presence of polyphosphate (n) and ADP in the system, it can be converted into polyphosphate (n-1) and ATP. PPK can utilize substances from microorganisms such as *Escherichia coli*, yeast, and *Corynebacterium xerosis*, but is not particularly limited thereto.

[0127] [Riboflavin kinase (FMNK)]

[0128] Riboflavin kinase (EC 2.7.1.26), also known as FMNK, catalyzes the conversion of riboflavin and ATP into phosphate riboflavin (FMN) and ADP. ATP + Riboflavin ←→ ADP + FMN Riboflavin kinase belongs to the ATP:riboflavin 5'-phosphotransferase family (also known as riboflavin kinase). FMNK can use substances from, for example, microorganisms and animals, but is not particularly limited to any of them. Examples include substances from yeast, rats, and mung beans (Phaseolus radiatus).

[0129] [Phosphofructokinase 1 (PFK1)]

[0130] Phosphofructokinase 1 (EC 2.7.1.11), also known as PFK1, catalyzes the conversion of fructose-6-phosphate (Fru6P) and ATP to fructose-1,6-bisphosphate (Fru1,6-BP) and ADP. Fru6P + ATP ←→ Fru1,6-BP + ADP Phosphofructokinase 1 (PFK1) belongs to the phosphofructokinase family. In this specification, PFK1 is sometimes referred to as Fru-1,6BPK. PFK1 can utilize substances derived from animals and microorganisms, but is not particularly limited thereto. Examples of microbial substances include those derived from baker's yeast, brewer's yeast, Clostridium pasteurianum, Escherichia coli, and Bacillus licheniformis.

[0131] [Fructose-2-bisphosphatase (FBPase)]

[0132] Fructose bisphosphatase (EC 3.1.3.11), also known as FBPase, catalyzes the conversion of fructose-1,6-bisphosphate (Fru1,6-BP) and ADP to fructose-6-phosphate (Fru6P) and ATP. Fru1,6-BP + ADP ←→ Fru6P + ATP FBPase is sometimes also abbreviated as FBP or FBP1. FBPase can use substances derived from animals, plants, and microorganisms, such as substances derived from rabbits and chickens, but there are no particular restrictions.

[0133] [Enzymes that catalyze the reaction from ADP to AMP]

[0134] In one embodiment, the liquid composition described herein contains an enzyme that catalyzes the reaction from ADP to AMP. The enzyme catalyzes the reaction from ADP to AMP converts ADP present in the system into AMP. If an enzyme that generates AMP from ADP is combined with an enzyme that generates ATP from AMP (e.g., PPDK), ADP is converted to AMP, and AMP is converted to ATP, thereby enabling the determination of ATP, ADP, and AMP.

[0135] Enzymes that catalyze the reaction from ADP to AMP can use known substances. Examples include, but are not limited to, ADP-dependent hexokinase, adenosine triphosphate diphosphatase, and combinations thereof.

[0136] [ADP-dependent hexokinase]

[0137] ADP-dependent hexokinase (EC 2.7.1.147), also known as ADP-specific hexokinase, catalyzes the following reaction: D-glucose + ADP ←→ D-glucose-6-phosphate + AMP [Adenosine triphosphate diphosphatase] Adenosine triphosphate diphosphatase (EC 3.6.1.5), also known as adenosine diphosphate oxidase, ADP enzyme, ATP diphosphatase, or ATP diphosphate hydrolase, catalyzes the following two reactions: ATP + H₂O → ADP + Phosphate (P) ADP + H₂O → AMP + Phosphoric acid (P) [Enzymes that catalyze the reaction from AMP to ADP] In one embodiment, the liquid composition described herein contains an enzyme that catalyzes the reaction from AMP to ADP. The enzyme catalyzing the reaction from AMP to ADP converts AMP present in the system into ADP. Alternatively, if the enzyme that generates ADP from AMP is combined with an enzyme that generates ATP from ADP (e.g., PK), AMP is converted to ADP, and ADP is converted to ATP, thereby enabling the determination of ATP, ADP, and AMP.

[0138] The enzyme that catalyzes the reaction from AMP to ADP can be a known enzyme. Examples include, but are not limited to, adenosine kinase (ADK).

[0139] [Adenosine kinase (ADK)]

[0140] Adenylate kinase (EC 2.7.4.3), also known as adenylate kinase, catalyzes the following reaction in the presence of metal ions: ATP + AMP ←→ 2ADP The reaction is reversible. ADK is an example of an enzyme that catalyzes the reaction from AMP to ADP.

[0141] ADK may include substances from microorganisms such as yeast, and substances from animals such as rabbits, pigs, cattle, rats, and so on, but is not subject to any particular limitation.

[0142] [RNA-degrading enzyme]

[0143] In one embodiment, the kit of the present invention may contain an RNA-degrading enzyme. Additionally, in one embodiment, the method of the present invention may use an RNA-degrading enzyme. It should be noted that the RNA-degrading enzyme referred to herein is not an RNA-degrading enzyme derived from a sample.

[0144] In one embodiment, the liquid composition described herein may contain an RNA-degrading enzyme. In this specification, "RNA-degrading enzyme" refers to an RNA-degrading enzyme not derived from a sample. By using an RNA-degrading enzyme, RNA can be broken down into AMP, and a wide range of cleanliness, including RNA, can be determined.

[0145] In this specification, RNA-degrading enzymes refer to enzymes that catalyze the reaction from RNA to 5'-mononucleotides (AMP, GMP, CMP, and UMP), and examples include: (1) endonuclease S1 (EC 3.1.30.1), (2) venom exonuclease (EC 3.1.15.1), and (3) phosphodiesterase 1 (EC 3.1.4.1). It should be noted that the aforementioned endonuclease S1 includes nuclease P1, mung bean nuclease, and Neurospora crassa nuclease.

[0146] In this specification, enzymes that catalyze the reaction of generating ATP from AMP (e.g., PPDK, PWDK), enzymes that catalyze the reaction of generating ATP from ADP, enzymes that catalyze the reaction of generating AMP from ADP, and enzymes that catalyze the reaction of generating ADP from AMP (e.g., ADK) are sometimes collectively referred to as enzymes with ATP generating capacity.

[0147] Enzymes capable of generating ATP can be any known enzyme, such as those derived from microorganisms, bacteria, eukaryotes, protozoa, plants, or animals, and can be commercially available products. The amount of enzyme added can be appropriately set according to the target concentration and the reaction system.

[0148] Various enzymes with ATP-generating capacity are known. In this specification, focusing on the ATP-generating capacity of an enzyme, the activity unit (U) of an enzyme with ATP-generating capacity is defined as the amount of enzyme that converts 1.0 μmol of substrate to ATP per minute at 37°C and pH 7.8 (1U = 1 μmol ATP / min, pH 7.8, 37°C). The activity unit (U) of an enzyme with ATP-generating capacity is generally defined as above, except for enzymes that catalyze only the reaction from ADP to ATP (e.g., PK), for which the activity unit (U) is defined as the amount of enzyme that converts 1.0 μmol of substrate to ATP per minute at 25°C and pH 7.4 (1U = 1 μmol ATP / min, pH 7.4, 25°C). In one embodiment, an enzyme with ATP-generating capacity can be added in an amount of 0.001 U or more, 0.01 U or more, 0.1 U or more, 1 U or more, 2 U or more, 3 U or more, 4 U or more, or 5 U or more in the assay system. In one embodiment, an enzyme capable of generating ATP can be added in an amount of 10,000 U or less, 1,000 U or less, 100 U or less, 50 U or less, 10 U or less, 9 U or less, 8 U or less, 7 U or less, or 6 U or less in the assay system. Those skilled in the art can appropriately determine the amount of enzyme added. In one embodiment, the concentration of the enzyme capable of generating ATP in the liquid composition described herein (where multiple enzymes are included, this concentration is the concentration of each enzyme or the total concentration of the enzymes, hereinafter the same) can be, for example, 20 U / mL or less, 10 U / mL or less, 5 U / mL or less, 1 U / mL or less, 0.5 U / mL or less, 0.1 U / mL or less, 0.05 U / mL or less, 0.01 U / mL or less, or 0.001 U / mL or less. For example, the concentration of enzymes catalyzing the reaction from AMP to ATP (e.g., PPDK) can be below 1 U / mL, 0.5 U / mL, 0.1 U / mL, 0.05 U / mL, 0.01 U / mL, or 0.001 U / mL; and the concentration of enzymes catalyzing the reaction from ADP to ATP (e.g., PK) can be below 20 U / mL, 10 U / mL, 5 U / mL, 1 U / mL, 0.5 U / mL, 0.1 U / mL, 0.05 U / mL, 0.01 U / mL, or 0.001 U / mL. Lower concentrations of enzymes capable of ATP generation improve the stability of the liquid composition, but because the overall luminescence is reduced, enzymes capable of ATP generation can be added before or during the assay if the luminescence is too low.The concentration of the enzyme capable of generating ATP (e.g., an enzyme that catalyzes the reaction of generating ATP from AMP (e.g., PPDK) and / or an enzyme that catalyzes the reaction of generating ATP from ADP (e.g., PK)) in the liquid composition after the addition of an enzyme capable of generating ATP during storage, or before or during the assay, may be, for example, 0.00001 U / mL or more, 0.0001 U / mL or more, 0.001 U / mL or more, 0.01 U / mL or more, 0.1 U / mL or more, 1 U / mL or more, or 10 U / mL or more.

[0149] When using enzymes capable of ATP generation, the substrate for each enzyme can be added (e.g., the substrate of an enzyme catalyzing the reaction from AMP to ATP, the substrate of an enzyme catalyzing the reaction from ADP to ATP, the substrate of an enzyme catalyzing the reaction from ADP to AMP, or the substrate of an enzyme catalyzing the reaction from AMP to ADP or ATP). It should be noted that sometimes different enzymes (e.g., enzymes catalyzing the reaction from AMP to ATP and enzymes catalyzing the reaction from ADP to ATP) may have the same substrate. Furthermore, it is not necessary to add additional substrate components depending on the type of enzyme. For example, if the enzyme catalyzing the reaction from AMP to ADP is ADK, since this enzyme reaction is a reaction from ATP and AMP to ADP, the liquid composition does not need to contain additional substrate for this enzyme reaction. The substrates are not particularly limited. For example, for PPDK, examples include phosphoenolpyruvate or its salts and pyrophosphate or their salts; for PK, AK, CK, PPK, and FMNK, examples include phosphoenolpyruvate or its salts, acetyl phosphate or its salts, creatine phosphate or its salts, polyphosphate or its salts, and riboflavin phosphate or its salts. As substrates for PFK1 and FBPase, fructose-1,6-bisphosphate or its salts are examples. Additionally, as substrates for PWDK, examples include phosphoenolpyruvate or its salts and phosphate or its salts; and as substrates for ADP-dependent hexokinase, glucose is an example. In one embodiment, the liquid composition described herein further comprises these substrates. In one embodiment, the concentration of the substrate (e.g., phosphoenolpyruvate, pyrophosphate, or a salt thereof) in the liquid composition described herein (in the case of multiple substrates, this concentration is the concentration of each substrate or the total concentration of the substrates) can be, for example, below 4 mM, below 3 mM, below 2.5 mM, below 2 mM, below 1.5 mM, below 1.2 mM, below 1 mM, below 0.5 mM, below 0.1 mM, below 0.05 mM, below 0.01 mM, below 0.001 mM, or below 0.0001 mM. Lower substrate concentrations improve the stability of the liquid composition, but because the overall luminescence is reduced, additional substrate may be added before or during measurement if the luminescence is too low. The concentration of the substrate in the liquid composition, whether during storage, before or after measurement, can be, for example, 0.00001 mM or more, 0.0001 mM or more, 0.001 mM or more, 0.01 mM or more, 0.1 mM or more, or 1 mM or more.

[0150] The concentration of substrates (e.g., phosphoenolpyruvate, pyrophosphate, or their salts) contained in a liquid composition can be determined based on an enzymatic reaction. For example, using PPDK with AMP, phosphoenolpyruvate, and pyrophosphate as substrates, pyruvate is generated, lactate dehydrogenase and β-NADH are activated, and the absorbance at 340 nm is measured, thereby determining the concentrations of phosphoenolpyruvate and pyrophosphate. Alternatively, using PPDK with AMP, phosphoenolpyruvate, and pyrophosphate as substrates, ATP is generated, and luminescence is measured using luciferase, thereby also determining the concentrations of phosphoenolpyruvate and pyrophosphate. For example, using ATP thioacylase with pyrophosphate as a substrate, ATP is generated, and the amount of luminescence is measured using luciferase, thereby determining the concentration of pyrophosphate.

[0151] Phosphoenolpyruvate (PEP)

[0152] In one embodiment, the liquid composition described herein comprises phosphoenolpyruvate (PEP) or a salt thereof. Sometimes, by adding an excess of PEP or a salt thereof to the system, the determination of ATP and AMP present in the system can be facilitated.

[0153] [Pyrophosphate (PPi)]

[0154] In one embodiment, the liquid composition described herein comprises pyrophosphate (PPi) or a salt thereof. Sometimes, by adding an excess of PPi or a salt thereof to the system, the determination of ATP and AMP present in the system can be facilitated.

[0155] [Auxiliary Factor]

[0156] In one embodiment, the liquid composition described herein contains a cofactor. A cofactor is a chemical substance other than a protein required for the catalytic activity of an enzyme. Examples of cofactors include: metal salts, vitamins and their derivatives, non-vitamin enzymes, and organic cofactors such as metal salts, but there are no limitations.

[0157] For example, cofactors for luciferase can include metal salts, such as divalent metal ions like magnesium and calcium, or salts of manganese (e.g., magnesium acetate). Additionally, metal salts such as magnesium can be used as cofactors for PPDK. Those skilled in the art can determine the type and concentration of cofactors (e.g., metal salts) based on the enzyme used.

[0158] In one embodiment, the concentration of the cofactor (e.g., a metal salt such as magnesium) in the liquid composition described herein can be, for example, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, less than 10 mM, less than 8 mM, less than 6 mM, less than 4 mM, less than 2 mM, less than 1 mM, less than 0.5 mM, less than 0.1 mM, or less than 0.05 mM. Lower concentrations of the cofactor improve the stability of the liquid composition, but because the overall luminescence is reduced, the cofactor can be added before or during measurement if the luminescence is too low. The concentration of the cofactor in the liquid composition during storage, or after adding the cofactor before or during measurement, can be, for example, more than 0.0001 mM, more than 0.001 mM, more than 0.01 mM, more than 0.1 mM, more than 1 mM, or more than 5 mM.

[0159] The concentration of cofactors contained in a liquid composition can be determined using common methods well known to those skilled in the art. For example, by fractionating the liquid composition using chromatography such as HPLC, detecting the peak corresponding to the cofactor, and comparing the peak intensity with a standard containing a known concentration of the cofactor, the concentration of the cofactor can be determined. Alternatively, when the cofactor is a metal salt, its concentration can be determined by ICP-based luminescence spectrophotometry.

[0160] In one embodiment, the liquid composition described herein contains an enzyme stabilizer such as bovine serum albumin or gelatin to prevent the degradation of reporter molecules such as luciferase. In one embodiment, the liquid composition described herein contains a substance for adjusting pH and improving shelf life. Examples of such substances include: pH buffers (HEPES, Tricine, Tris, phosphate buffer, acetate buffer, etc.), reducing agents (dithiothreitol (DTT), 2-mercaptoethanol, etc.), sugars (glucose, sucrose, trehalose, etc.).

[0161] [Add ingredients before or during the test]

[0162] In one embodiment, the liquid composition does not contain at least one component required for the cycling reaction, and the component not present in the liquid composition is added to the liquid composition before or during the determination. In other embodiments, the liquid composition contains at least one component required for the cycling reaction at a low concentration, so that the cycling reaction is almost or completely eliminated, and the component contained at a low concentration in the liquid composition is added to the liquid composition before or during the determination.

[0163] In this specification, "cyclic reaction" refers to a reaction system in which ADP and / or AMP are directly or indirectly converted into ATP, and ADP and / or AMP are measured in addition to ATP.

[0164] In this specification, as "components required for the cyclic reaction," for example in the liquid compositions of the first and third embodiments, the following may be listed: luciferase, luciferin, an enzyme or substrate thereof catalyzing the reaction from AMP to ATP, and cofactors thereof. As "components required for the cyclic reaction," in the liquid compositions of the second and fourth embodiments, the following may be listed: luciferase, luciferin, an enzyme or substrate thereof catalyzing the reaction from AMP to ADP, an enzyme or substrate thereof catalyzing the reaction from ADP to ATP, and cofactors thereof.

[0165] In one embodiment, the composition described herein does not contain at least one of these ingredients (e.g., one, two, three, four, five, or all of them), contains other ingredients, and the ingredients not present in the liquid composition are added to the liquid composition before or during the determination.

[0166] In this specification, "before assay" is not limited to improving the stability of the liquid composition and can be, for example, 30 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, 10 seconds, or immediately before the assay of ATP, AMP, and / or ADP. "At assay" means simultaneously with the assay. The shorter the time from the addition of the components required for the cycling reaction to the assay, the less time is available for the cycling reaction to occur, and the greater the improvement in the stability of the liquid composition.

[0167] [sample]

[0168] In this specification, the type of "sample" can be, for example, a biological sample or biological device, a blood sample or blood device, or a cooking device, but is not limited thereto.

[0169] In this specification, "biologically related samples" includes all samples that may have substances from living organisms attached to them. In this specification, "biologically related devices" refers to all devices that may have substances from living organisms attached to or remaining on them. In this specification, the environment from which biologically related samples or biologically related devices originate can be listed as an environment where liquids from living organisms may be attached to or remain on them. Examples of such environments include: clothing, gloves and other protective equipment, hands, fingers, beds, switches, door handles, bed rails, nurse call buttons, handrails, restrooms, washbasins, toilets, and toilet bowls, but are not limited to these. Substances from living organisms can be from humans or animals. In one embodiment, the substances from living organisms are substances from humans. In one embodiment, biologically related samples do not include samples from non-human animals, but include samples from humans. In one embodiment, biologically related devices do not include non-human animal-related devices, but include human-related devices.

[0170] As substances derived from living organisms, both liquids and solids can be listed. Examples of liquids include: bodily fluids, blood, lymph, sweat, nasal mucus, tears, saliva, digestive juices, tissue fluid, ascites, amniotic fluid, cerebrospinal fluid, urine, feces, vomit, sebum, etc., but are not limited to these. Examples of solids include, in addition to substances that are inherently solid such as tissue fragments, flesh fragments, and cells, substances that have solidified from liquids, coagulated blood, excrement, body grime, eye discharge, and scabs, but are not limited to these.

[0171] Examples of biological devices include medical devices, such as surgical instruments, endoscopes (e.g., upper endoscopes for examining the esophagus, stomach and duodenum, lower endoscopes or double-balloon small bowel endoscopes for examining the rectum and large intestine, preferably lower endoscopes), catheters, scalpels, tubes inserted into the patient's body, instruments inserted into the patient's body, surgical instrument cleaning tanks and medical device cleaning environments, etc.

[0172] In this specification, blood-related samples include all samples that may have blood attached to them. In this specification, blood-related devices refer to all devices that may have blood attached to or left behind. Examples of blood-related devices refer to medical devices that may have blood attached to or left behind. Examples include: surgical instruments, endoscopes (e.g., upper endoscopes for examination of the esophagus, stomach, and duodenum, lower endoscopes for examination of the rectum and large intestine, or double-balloon enteroscopes, preferably lower endoscopes), catheters, scalpels, tubes inserted into the patient's body, instruments inserted into the patient's body, surgical instrument cleaning tanks, and medical device cleaning environments. In this specification, the environment from which blood-related samples or blood-related devices originate can be described as an environment where blood may be attached to or left behind. Examples of such environments include: operating tables, cleaning tanks, clothing, gloves, and other protective equipment, hands, fingers, beds, handrails, toilets, washbasins, and medical-related facilities. Additionally, examples include: accident scenes, injury incident scenes, and scenes where bloodstain searches are conducted. The blood may originate from humans or animals. In one embodiment, the blood originates from humans. In one implementation, the blood does not include blood from food-related animal meat or fish.

[0173] Examples of blood include: whole blood, serum, plasma, transfusion blood, single-collection blood, and solutions of diluted single-collection blood. Blood-related samples also include solutions containing blood cells (white blood cells, red blood cells, platelets) or samples that may be attached to such solutions.

[0174] In one embodiment, the blood-related sample does not include the collected blood itself (referred to as a primary sample for convenience). For example, in this embodiment, the "solution containing blood cells" included in the blood-related sample does not include the blood itself. In one embodiment, the blood-related sample refers to a secondary sample from instruments and the environment that have come into contact with the primary sample. The secondary sample can be obtained by wiping instruments and the environment that may have come into contact with the primary sample with a cotton swab or the like. In one embodiment, the method of the present invention examines whether blood is attached to or remains on the secondary sample. In one embodiment, the blood-related sample can be a sample after the blood present has been diluted through a washing process or the like.

[0175] In this instruction manual, "cooking-related equipment" refers to cooking equipment and the cooking environment or items within that environment, which may be contaminated. Examples of cooking equipment include, but are not limited to: cutting boards, pots, frying pans, pressure cookers, iron plates, plates, knives, chopsticks, spoons, forks, and other tableware; rice scoops; strainers; colanders; racks; cutting board racks; containers for storing cooking equipment; packaging containers; wrapping paper; and other cooking equipment and related equipment. The cooking environment refers to the cooking site, food processing plant, food supply facilities, etc. Items within this environment include: mixing tanks, pipes, filling nozzles, conveyor belts, and other equipment in food processing plants; containers; door handles; handles and switches of appliances such as refrigerators and ovens; telephones; and other parts or places frequently touched by human hands.

[0176] [Save Time]

[0177] The liquid composition described in this specification is used for the determination of ATP, AMP, and / or ADP in a sample after preservation. Preservation as described in this specification includes not only the period from the preparation of the liquid composition to its use for determination, but also the preservation time of the liquid composition during the intermediate stages of manufacturing the powder composition. In this case, the liquid composition described in this specification can be preserved for a certain period after manufacturing, converted into a powder composition through a drying process, and then restored to a liquid composition (optionally preserved again) for the determination of ATP, AMP, and / or ADP in a sample. The preservation time of the liquid composition (if the preservation time is divided into multiple periods, this time is the total time) can be, for example, more than 6 hours, more than 12 hours, more than 18 hours, more than 1 day, more than 2 days, more than 3 days, more than 7 days, more than 14 days, more than 30 days, more than 60 days, more than 90 days, more than 120 days, more than 150 days, more than 180 days, more than 210 days, more than 240 days, more than 270 days, or more than 300 days, but is not limited thereto. In addition, the storage time can also be less than 600 days, less than 500 days, or less than 400 days. The liquid composition described in this specification can be a composition with improved stability, therefore, its stability improvement effect can be more clearly demonstrated when stored for a longer period of time.

[0178] [Reagent test kit]

[0179] In the fifth embodiment, the present invention relates to a kit for determining ATP in a sample, comprising the liquid composition described herein. In addition to the liquid composition described herein, the kit may also contain at least one of the following: an extraction solution (e.g., water or a buffer solution or water or a buffer solution containing a surfactant such as benzalkonium chloride), a buffer solution, instruments required for the experiment, a control, and instructions for use.

[0180] In one embodiment, the kit may include a sample collection section and a reaction section. The sample collection section is not particularly limited as long as it can collect samples, and examples include: cotton swabs, sponges, porous plastics, filter paper, non-woven fabric, and droppers. From the perspective of convenience in sample collection, the sample collection section is preferably, for example, rod-shaped, and particularly preferably has a rod-shaped form with fibrous or sponge-like wiping portions, such as a shape similar to a cotton swab.

[0181] The reaction section is the site where a reaction takes place when ATP or its decomposition products are present in the sample collected by the sample collection section. In one embodiment, the reaction section comprises the liquid composition described in this specification. The reaction section is preferably a transparent container, thereby enabling direct measurement of the luminescence amount.

[0182] In one embodiment, the kit may include other parts besides the sample collection section and the reaction section, such as a storage section or an extraction section. The storage section is used to store components not included in the liquid composition when the liquid composition described herein does not contain at least one component required for the cyclic reaction (e.g., fluorescein). The extraction section is used to extract ATP or its decomposition products from the sample collected by the sample collection section into an extraction solution. In one embodiment, the extraction section may also contain an extraction solution. The sample extracted by the extraction section can be transferred to the reaction section for reaction.

[0183] If the liquid composition described in this specification does not contain at least one component required for the cyclic reaction, this kit may contain the component not included in the liquid composition. In this case, the liquid composition and the component not included in the liquid composition can be stored separately; for example, the liquid composition may be contained in the reaction section, and the component not included in the liquid composition may be contained in the sample collection section, storage section, extraction section, or other locations isolated from the liquid composition.

[0184] [method]

[0185] In the sixth embodiment, the present invention relates to a method for determining ATP, as well as AMP and / or ADP in a sample, or the use of the liquid composition or kit described herein in determining ATP, as well as AMP and / or ADP in a sample, wherein the method comprises using the liquid composition or kit described herein.

[0186] For example, by adding a sample solution containing ATP to the liquid composition described in this specification and measuring luminescence, ATP, as well as AMP and / or ADP in the sample can be detected, but this is not a limitation. The amounts of the liquid composition and the sample solution can be the same or different. The amounts of the liquid composition and the sample solution can be, for example, 0.01 mL to 10 mL, 0.25 mL to 4 mL, 0.5 mL to 2 mL, or 0.1 mL, but this is not a limitation. The sample solution can be a solution made by suspending a cotton swab or similar object after wiping the sample in the extract, but this is not a limitation.

[0187] The luminescence intensity can be measured using existing photometers (such as the LumitesterSmart fluorescence detector, Lumitester PD-20, and Lumitester PD-30 manufactured by Kikkoman Co., Ltd.) or devices equipped with photodiodes (such as SystemSURE Plus and EnSURE manufactured by Hygiena, and AccuPoint Advanced manufactured by Neogen) or devices equipped with photomultiplier tubes (such as Clean-Trace LM1 and Clean-Trace UNG3 manufactured by 3M; Lumitester C-110 and Lumitester C-100 manufactured by Kikkoman Co., Ltd.; or Junior LB9509, CentroLB960, or Lumat3 LB9508 manufactured by Berthold). A standard can be set, and the luminescence is recorded as a relative luminescence unit (RLU) relative to that standard. (For example, in cases where samples are collected using a rod-shaped device with a wiping portion.) The value of this relative luminescence unit can be directly used for cleanliness management, etc. In this case, a standard solution with a known ATP concentration is not used. In one embodiment, the method described herein does not use any of the ATP, ADP, or AMP standard solutions. When the relative luminescence units measured only when the sample is not used are directly applied to cleanliness management, etc., the decrease in luminescence caused by the instability of the liquid composition can directly affect the test results. Therefore, the advantage of using the composition described herein, which has excellent stability, is particularly significant.

[0188] In other embodiments, a calibration curve is prepared using a substrate solution with a known concentration of ATP, etc. Next, the liquid composition described herein is added to a sample solution with an unknown concentration of ATP, etc., and luminescence is measured under the same conditions. This allows for the determination of the concentration of ATP, etc., in the sample solution. In this embodiment, even if the liquid composition is unstable, leading to a decrease in luminescence, the severity of the problem caused by the decrease in luminescence can be relatively reduced by using the same liquid composition to prepare calibration curves for ATP, etc., each time.

[0189] Example

[0190] The present invention will now be described in more detail using examples. However, the scope of the present invention is not limited to these examples.

[0191] <Example 1: Luciferase Concentration Measurement>

[0192] (280nm absorbance method)

[0193] The absorbance of the sample solution appropriately diluted with PBS at 280 nm was measured using an U-3900 spectrophotometer (manufactured by Hitachi High-Technologies Corporation), and this was taken as the luciferase concentration (mg protein / mL).

[0194] (Bradford method)

[0195] The protein concentration was measured using a Coomassie (Bradford) Protein Assay Kit (manufactured by Thermo Scientific) based on the Bradford method. Specifically, 100 μL of the protein assay reagent (Quick Start Bradford 1×Dye Reagent, manufactured by Thermo Scientific) was added to 100 μL of the sample solution moderately diluted with PBS, and the absorbance at 595 nm was measured using a MICROPLATE READER SH-9000 (manufactured by corona Electric Co., Ltd.). Albumin Standard Ampules (2 mg / mL) included in the kit were used as the standard protein in place of the sample solution for measurement, and a calibration curve was prepared to obtain the amount of luciferase.

[0196] (Conversion between 280 nm absorbance method and Bradford method)

[0197] The measurement results showed that 1 mg protein / mL in the 280 nm absorbance method corresponded to 1.77 mg / mL in the Bradford method.

[0198] <Example 2: Preparation of cyclic luminescent reagent and background luminescence amount>

[0199] Based on the alternative composition of LuciPac Pen (manufactured by Kikkoman BPF Kemin Foods Co., Ltd. (hereinafter the same), a kit used after adding an extraction reagent (solution) to a powdered luminescent reagent) described in paragraph

[0094] of International Publication No. 2018 / 147443, the solution of the basic composition was redesigned. The alternative composition of LuciPac Pen is as follows.

[0200] <Alternative composition of LuciPac Pen>

[0201] 7 mM magnesium acetate, 0.5 mM luciferin, 25 mM trimethylglycine, 0.2 mg protein / mL luciferase (value of absorbance at 280 nm. Hereinafter, this will be denoted as the value obtained by the Bradford method based on Example 1, i.e., 0.35 mg / mL), 0.2 mM potassium pyrophosphate, 1.4 mM potassium phosphoenolpyruvate, 1.3 U / mL PPDK

[0202] Following the LuciPac Pen's operating instructions, after wiping the stainless steel surface with a water-moistened cotton swab, the swab was squeezed, and the powdered luminescent reagent was dissolved using the dripping extraction reagent (solution). The volume of the solution at this point was 0.35 mL, and the pH was 7.8. Based on these results and the composition of the LuciPac Pen described above, the solution with the following basic composition was redesigned.

[0203] <Basic Components>

[0204] 7 mM magnesium acetate, 0.5 mM luciferin (manufactured by Biosynth, hereinafter the same), 25 mM trimethylglycine, 0.35 mg / mL (Bradford method) luciferase (HLK as described in Japanese Patent Application Publication No. 11-239493, hereinafter the same), 0.2 mM potassium pyrophosphate, 1.4 mM potassium phosphoenolpyruvate, 1.3 U / mL PPDK (PPDK as described in Japanese Patent Application Publication No. 8-168375, manufactured by Kikkoman Bio-Kemifag Co., Ltd., hereinafter the same), pH 7.8; the liquid volume of each luminescence assay container is 0.35 mL.

[0205] Prepare the luminescent reagent with the above basic composition by adding it to the luminescent reagent container (measuring tube, upper 10) of the LuciPac Pen. Weigh 0.35 mL of the sample into a container (35 mm high, the same below), and attach the container to the main body equipped with a cotton swab holder. Measure the fluorescence using a Lumitester Smart fluorescence detector (manufactured by Kikkoman Bio-Kemifa Co., Ltd., the same below). The fluorescence intensity at this point is taken as the background fluorescence intensity. To convert the background fluorescence intensity into ATP concentration, add 0.01 mL of 1×10⁻⁶ ATP. -5 A solution of ATP (manufactured by Orient Yeast Industry Co., Ltd., hereinafter the same) of M was used as a control. The amount of light emitted 10 seconds after the addition was measured using a Lumitester Smart fluorescence detector, and this amount of light emitted was taken as the amount of light emitted when ATP was added. The Δ light emission was calculated by subtracting the background light emission from the amount of light emitted when ATP was added.

[0206] Add 0.01 mL of 1×10 -5 When the ATP concentration in the solution is M, the ATP concentration (M) in the solution (total 0.36 mL) is 2.77 × 10⁻⁶. -7 Therefore, the background ATP+AMP concentration is obtained based on the following calculation formula.

[0207] Background ATP+AMP concentration (M) = Background luminescence (RLU) / Δluminescence (RLU) × 2.77 × 10 -7 (M)

[0208] Table 1 shows the background luminescence (RLU), luminescence (RLU) with added ATP, Δ luminescence (RLU), and background ATP+AMP concentration (M). It should be noted that RLU is an abbreviation for Relative Light Units.

[0209] [Table 1]

[0210] It is generally believed that background luminescence is caused by ATP or AMP mixed in from reagents, containers, or instruments during preparation. Under normal and careful preparation conditions by those skilled in the art, this luminescence is approximately 500 RLU, as shown in Table 1. After conversion to the ATP+AMP concentration in the luminescent reagent, this is equivalent to 2.7 × 10⁻⁶ RLU. -10 M (0.27 nM). It should be noted that this concentration is equivalent to adding approximately 1 × 10⁻⁶ mL of 0.01 mL of reagent to 0.35 mL of the solution. -8 The concentration of M in ATP solution.

[0211] It should be noted that when further adding an enzyme that converts ADP to ATP or AMP to measure the three components ATP+ADP+AMP, the ADP mixed in with the reagent is also measured. Since an enzyme that causes ATP, ADP and AMP to be mixed in is added, the background luminescence will be considered to be higher.

[0212] <Example 3: Time-dependent changes in luminescence intensity in a non-cyclic luminescent reagent>

[0213] To prepare a non-cyclic luminescent reagent, the reagent was prepared by removing the enzyme PPDK required for cycling from the basic composition. 0.35 mL of the prepared solution was weighed into the luminescent reagent container of the LuciPac Pen, and 0.01 mL of 1×10⁻⁶ mol / L phospholipid was added. -5 The ATP solution of M was measured using a Lumitester Smart fluorescence detector after the above container was installed on a main body equipped with a cotton swab holder.

[0214] The time-dependent changes in luminescence of non-cyclic luminescent reagents are shown in the figure. Figure 1 .like Figure 1 As shown, in non-cyclic luminescent reagents, the added ATP is rapidly consumed in the luminescence reaction produced by the luciferin-luciferase reaction, and the luminescence intensity decreases sharply. In the basic composition that does not contain PPDK, it is basically extinguished in about 2 minutes.

[0215] <Example 4: Stability of Cyclic Effractometers Containing Different Concentrations of Fluorescein>

[0216] Based on the basic composition, only the concentration of luciferin was changed to prepare cyclic luminescent reagents containing different concentrations of luciferin. Assuming ATP was mixed in, 0.1 mL of 1×10⁻⁶ luciferin was used. -6 M ATP solution was added to 3.5 mL of cyclic luminescent reagent (various concentrations of fluorescein) and incubated at 25°C. At each time point, 0.36 mL was weighed into the LuciPac Pen luminescent reagent container, and the container was attached to the main body equipped with a cotton swab holder. The luminescence was measured using a Lumitester Smart fluorescence detector. The luminescence intensity at this time was taken as the background luminescence intensity. To further confirm the stability of the luminescence intensity, 0.01 mL of 1×10⁻⁶ ATP solution was added. -5 The luminescence intensity of the ATP solution M was measured 10 seconds after its addition using a Lumitester Smart fluorescence detector. This luminescence intensity was taken as the luminescence intensity at the time of ATP addition, and the difference between this luminescence intensity and the background luminescence intensity was taken as the Δluminescence intensity.

[0217] The time-varying luminescence of cyclic luminescent reagents containing different concentrations of fluorescein is shown in the figure. Figure 2 Furthermore, regarding Figure 2 The luminescence levels after 9 hours of cycling with different concentrations of fluorescein were shown in the figure. Figure 3 .

[0218] In the basic composition, a decrease in Δluminescence intensity over time was observed upon the addition of ATP. This indicates low stability of the cyclic reagent. Furthermore, the higher the luciferin concentration, the greater the decrease in Δluminescence intensity upon ATP addition. By lowering the luciferin concentration below the basic composition, this trend of decreasing luminescence intensity over time was suppressed. Typically, in assay systems using enzyme reactions, high substrate concentrations are added to ensure that the reaction rate is not affected even as the substrate decreases during the enzyme reaction. Especially in cyclic reactions, since the substrate is continuously consumed, higher substrate concentrations are considered beneficial for reagent stability, thus adding higher substrate concentrations is considered appropriate. However, surprisingly, the results of this example show that in cyclic luminescent reagents utilizing the luciferin-luciferase reaction, it is actually by using a low luciferin concentration that the luminescence intensity of the cyclic luminescent reagent is stabilized.

[0219] <Example 5: Stability of Cyclic Emission Reagents Without Fluorescein>

[0220] Next, the effect of storing the reagent in a fluorescein-free state and adding the desired fluorescein just before the reaction was confirmed to improve stability. A cyclic luminescent reagent without fluorescein was prepared according to the basic composition. Assuming ATP contamination, 0.1 mL of 1×10⁻⁶ fluorescein was added to 3.5 mL of the reagent. -6 M ATP solution (hereinafter referred to as the mixture).

[0221] (1) When "containing fluorescein for preservation", add 0.1 mL of 17.5 mM fluorescein solution (pH 7.8) (final concentration 0.5 mM) to 3.6 mL of the above mixture, and then store at 25 °C. After different storage times, weigh 0.37 mL of the mixture into the luminescent reagent container of the LuciPac Pen, install it onto the main body equipped with a cotton swab holder, and measure the background luminescence using a Lumitester Smart fluorescence detector. To further confirm the stability of the luminescence, add 0.01 mL of 1×10⁻⁶ fluorescein solution as a control. -5 The luminescence intensity of the ATP solution M was measured after 10 seconds using a Lumitester Smart fluorescence detector, and this value was taken as the luminescence intensity upon ATP addition. The difference between the background luminescence intensity and the luminescence intensity upon ATP addition was taken as the Δ luminescence intensity.

[0222] (2) When "preservation without fluorescein" is performed, 3.6 mL of the above mixture is stored at 25°C without the addition of fluorescein solution. After different storage times, 0.36 mL is weighed into the LuciPac Pen luminescent reagent container, and 0.01 mL of 17.5 mM fluorescein solution (pH 7.8) (final concentration 0.5 mM) stored at 25°C is added. The background luminescence is measured, and then 0.01 mL of 1×10⁻⁶ fluorescein solution is added. -5 The luminescence intensity of the ATP solution M was measured 10 seconds after its addition using a Lumitester Smart fluorescence detector, and this was taken as the luminescence intensity at the time of ATP addition. The difference between the background luminescence intensity and the luminescence intensity at the time of ATP addition was taken as the Δ luminescence intensity.

[0223] The stability of luminescent reagents, with or without fluorescein, is shown in [the table / formula]. Figure 4 .like Figure 4 As shown, in reagents containing fluorescein for preservation, the cyclic reaction proceeds during preservation, and the luminescence decreases. However, in reagents without fluorescein for preservation, the luminescence does not decrease during preservation and remains stable, thus greatly improving stability.

[0224] <Example 6: Stability of non-fluorescein-containing reagents in non-cyclic luminescent reagents>

[0225] For reagents that do not contain PPDK in their basic composition, i.e., non-cyclic luminescent reagents, the stability of luminescence levels was confirmed, as in Example 5, when (1) stored with fluorescein and (2) stored without fluorescein, and the residual luminescence levels after 3 hours of storage were shown. The residual luminescence levels after 3 hours of storage with cyclic luminescence (Example 5) were also shown.

[0226] The results are shown in Figure 5In luminescent reagents that did not contain luciferin for preservation and were added just before the assay, no decrease in luminescence was observed. However, in reagents containing luciferin for preservation, a decrease in luminescence was observed in cyclic luminescent reagents containing only PPDK. No decrease in luminescence was observed in non-cyclic reagents that did not contain PPDK. In other words, the decrease in luminescence observed in this example is a phenomenon that would not occur in non-cyclic luminescent reagents using the luciferin-luciferase reaction that only measure ATP. It is considered to be a phenomenon that only occurs when cyclic luminescent reagents are preserved in liquid form.

[0227] Example 7: Luminescence stability of cyclic luminescent reagents containing different concentrations of luciferase

[0228] As demonstrated in Examples 3-6, suppressing luminescence is crucial for improving the stability of luminescence in cyclic luminescent reagents. Since luminescence can also be suppressed by inhibiting luciferase concentration, the correlation between the stability of luminescence in cyclic luminescent reagents and luciferase concentration was investigated.

[0229] By varying the concentration of luciferase, and otherwise preparing cyclic luminescent reagents containing different concentrations of luciferase according to the basic composition, assuming the presence of ATP, 0.1 mL of 1×10 -6 M ATP solution was added to 3.5 mL of the circulating luminescent reagent and incubated at 25°C. At different time points, 0.36 mL of the reagent was weighed into the LuciPac Pen luminescent reagent container, attached to the main body equipped with a cotton swab holder, and measured using a Lumitester Smart fluorescence detector. The luminescence intensity at this point was taken as the background luminescence intensity. To further confirm the stability of the luminescence intensity, 0.01 mL of 1×10⁻⁶ ATP solution was added as a control. -5 The ATP solution of M was added, and the amount of light emitted 10 seconds later was measured using a Lumitester Smart fluorescence detector. The amount of light emitted at this time was taken as the amount of light emitted when ATP was added, and the difference between this amount and the background light emitted was taken as the amount of light emitted Δ.

[0230] The residual luminescence of cyclic luminescent reagents containing different concentrations of luciferase is shown in... Figure 6 Furthermore, regarding Figure 6 The luminescence levels after 9 hours of cycling with different concentrations of luciferase are shown in the figure. Figure 7 .

[0231] like Figure 6 As shown, the following trend can be observed: the higher the concentration of luciferase, the longer the luminescence will decrease; the lower the concentration of luciferase is compared to the basic components, the more it will inhibit the decrease in luminescence.

[0232] <Example 8: ATP concentration dependence of decreased luminescence intensity of cyclic luminescent reagent>

[0233] As a cyclic luminescent reagent, it will continuously emit light at a certain intensity as long as the luciferin, which is originally the substrate, is not depleted, provided that ATP is present in the assay system. Even if ATP is not added after storage as in Examples 4-7, the same experimental results can be obtained by adding ATP before storage and observing the change in luminescence intensity over time. Therefore, the stability of the luminescent reagent can be easily examined. In this example, in order to construct an experimental system that can be stored without adding ATP, the appropriate ATP concentration when no ATP solution is added after storage was investigated.

[0234] Prepare a cyclic luminescent reagent according to the basic composition. Weigh 0.35 mL into the luminescent reagent container of the LuciPac Pen, and then add 0.01 mL of 1×10⁻⁶ mol / L. -5 ~1×10 -8 The luminescence intensity of the ATP solution or sterile ultrapure water containing M was measured over time using a LumitesterSmart fluorescence detector. The luminescence intensity Δ was obtained by subtracting the luminescence intensity of the sample containing sterile ultrapure water from the luminescence intensity of the sample with different concentrations of ATP, and is shown in the graphs.

[0235] The results are shown in Figure 8 The following trend can be observed: the decrease in luminescence is correlated with ATP concentration; the higher the ATP concentration, the greater the decrease in luminescence, and the lower the ATP concentration, the smaller the decrease.

[0236] Assuming the amount of ATP mixed in the cyclic luminescent reagent prepared in Example 2 is equivalent to adding approximately 0.01 mL of 1 × 10⁻⁶ ATP... -8 With ATP at the specified concentration, no decrease in luminescence was observed after 2 hours. Therefore, the decrease in luminescence is not a significant problem if the cyclic luminescent reagent is used up quickly, but it could become a major issue with long-term storage. For example, if the decrease in luminescence is proportional to the amount of luciferin consumed (i.e., the amount of luminescence), then adding 1×10... -5 In the case of M's ATP, the luminescence decreased to 30% within 2 hours. Therefore, calculations showed that mixing in 1×10 - 6 M's ATP level was the luminescence decay level over 20 hours, and it was mixed with 1×10 -7 The ATP content of M decreased by 200 hours (8.3 days), and the amount of light emitted decreased by 1×10⁻⁶. -8 The luminescence decay level of M over 2000 hours (83 days) was the same.

[0237] In the examples described in this specification, a high concentration of ATP solution was added to obtain results in a short time. Although the stability of the enzyme and the substrate itself can affect the stability of the assay kit, the luminescence intensity does not decrease at low concentrations of ATP. Therefore, it is believed that the evaluation can be performed, at least for a short time, without being affected by the stability of the enzyme and substrate.

[0238] <Example 9: Luminescence intensity and stability of fluorophore cyclic luminescent reagents containing different concentrations during storage>

[0239] As shown in Example 8, the stability of the reagent can be assessed by observing the change in luminescence intensity over time after adding ATP to the cyclic luminescent reagent. It is also evident that adding a high concentration of ATP allows for obtaining results in a short time. Furthermore, Examples 3-6 demonstrate that suppressing the luminescence reaction during storage is crucial. In this example, experiments were conducted to suppress the luminescence reaction during storage to define the luminescence intensity under storage conditions as a standard for luminescence reaction. First, the definition of the luminescence intensity under storage conditions is shown, followed by the experimental results upon which this definition is based.

[0240] Add the stored luminescent reagent to a 0.35 mL test tube, and add 0.01 mL of 1×10⁻⁶ mol / L solution. -7 The ATP solution of M was incubated at 25°C for 1 hour and then measured using a Lumitester Smart fluorescence detector (manufactured by Kikkoman Bio-Kemifag Co., Ltd.). The amount of light emitted in the stored state (RLU) was defined as the amount of light emitted at this time (RLU).

[0241] The experiment used to establish this definition is described below. Cyclic luminescent reagents containing different concentrations of fluorescein (the components other than fluorescein are the same as the basic composition) were prepared. 0.35 mL was weighed into the luminescent reagent container of a LuciPac Pen, and then 0.01 mL of 1×10⁻⁶ fluorescein was added. -5 ~1×10 -8 The luminescence intensity of ATP solution or sterile ultrapure water containing M was measured over time using a Lumitester Smart fluorescence detector. The luminescence intensity Δ was calculated by subtracting the luminescence intensity of the sample containing sterile ultrapure water from the luminescence intensity of samples with different concentrations of ATP, and this value was displayed as... Figures 9-14 .

[0242] like Figures 9-10 As shown, when adding 1×10 -5 In the case of ATP in M, a decrease in luminescence was observed, yielding results with the same trend as in Example 4. This method confirms that short-term experiments are not affected by enzyme or substrate degradation, thus suggesting long-term storage stability.

[0243] When defining the amount of light emitted during storage, for example in the case of stopping the cycle, as in Example 3. Figure 1 As shown, the luminescence immediately after ATP addition is high; therefore, measuring only the luminescence immediately after ATP addition cannot accurately evaluate the effect of cycle cessation. Conversely, for example, if luciferase decomposes ATP one hour after addition, examining the luminescence one hour later allows us to assess the luminescence during storage, including the effect of cycle cessation. However, as... Figures 9-12 As shown, when adding 1×10 -5 M or 1×10 -6 In the case of ATP solution containing M, the luminescence intensity decreases over time, therefore the luminescence intensity during storage cannot be accurately measured. On the other hand, as... Figures 13-14 As shown, add 1×10 -7 At time M, no decrease in luminescence was observed after approximately 2 hours, thus allowing for the assessment of stable luminescence levels during storage. On the other hand, adding 1×10... -8 When M, the Δ emission level is the same as the background emission level in Example 2, and is easily affected by it, so it is not appropriate to consider it (data not shown).

[0244] Based on the results above, the light emission level (RLU) in the storage state is defined as above. The light emission level (RLU) in the storage state according to this definition is shown in Table 2 below.

[0245] [Table 2]

[0246] Depend on Figure 10 The following trend is observed: if the luciferin concentration is kept below 0.5 mM (as shown in Table 2, the luminescence intensity in the stored state is 6000 RLU), the decrease in luminescence intensity compared to the basic composition is smaller, and the stability is improved. Furthermore, from Figure 10 It can be seen that, especially when the concentration of fluorescein is below 0.1 mM (as shown in Table 2, the luminescence amount in the storage state is below 2300 RLU), the stability can be further improved significantly.

[0247] <Example 10: Luminescence and stability of cyclic luminescent reagents containing different concentrations of luciferase during storage>

[0248] Prepare cyclic luminescent reagents containing different concentrations of luciferase (the components other than luciferase are the same as the basic composition). Weigh 0.35 mL into the luminescent reagent container of the LuciPac Pen, and then add 0.01 mL of 1×10⁻⁶ luciferase. -5 ~1×10 -8The luminescence intensity of ATP solution or sterile ultrapure water containing M was measured over time using a Lumitester Smart fluorescence detector. The luminescence intensity Δ was calculated by subtracting the luminescence intensity of the sample containing sterile ultrapure water from the luminescence intensity of samples with different concentrations of ATP, and is shown in the figure. Figures 15-20 .

[0249] Add 1×10 -5 When M was in ATP solution, a decrease in luminescence was observed, yielding the same trend as in Example 7. This method confirms that it is not affected by enzyme or substrate degradation in the short term, allowing for the examination of long-term storage stability. Furthermore, as... Figures 19-20 As shown, add 1×10 -7 When M was in an ATP solution, no decay in luminescence was observed after approximately 2 hours. Therefore, this embodiment also supports the appropriateness of the definition of luminescence under the storage condition shown in Example 9. The luminescence (U) under the storage condition according to this definition is shown in Table 3 below.

[0250] [Table 3]

[0251] Depend on Figure 15 The following trend can be observed: if the luciferase concentration is kept below 0.35 mg / mL (as shown in Table 3, the luminescence intensity in the stored state is below 6000 RLU), the decrease in luminescence intensity compared to the basic components is smaller, and the stability is improved. Furthermore, Table 3 shows that at the improved concentration (below 0.07 mg / mL), adding 1×10... -7 The luminescence of M in ATP solution was less than 2300 RLU after 1 hour.

[0252] <Example 11: Stability of a luminescent reagent without cyclic components>

[0253] By modifying some of the basic components, removing some cycle-related substrates and enzymes, and adding them during the assay, we explored whether this could improve stability.

[0254] The luminescent reagent was divided into solution A and solution B. One of the components required for the luminescent reaction and the cyclic reaction (magnesium acetate, potassium phosphoenolpyruvate, potassium pyrophosphate, luciferin, luciferase, PPDK) was transferred to solution B for preparation and stored separately. Before the reaction, 0.05 mL of each was mixed and 0.1 mL of ATP solution was added to it, and the amount of luminescence was measured.

[0255] Luminescent reagent A and luminescent reagent B were prepared separately. The composition of luminescent reagent A (pH 7.8) was: 50 mM Tricine containing 24 mM Mg acetate, 1.6 mM luciferin, 4 mM potassium phosphoenolpyruvate (PEP), 0.4 mM potassium pyrophosphate (PPi), 1.0 mg / mL (Bradford method) luciferase, and 4.3 U / mL PPDK, after removing any one of these components. The composition of luminescent reagent B was: 50 mM Tricine containing any one of the following components removed from solution A: 24 mM magnesium acetate, 4 mM PEP, 0.4 mM MPPi, 1.6 mM luciferin, 1.0 mg / mL (Bradford method) luciferase, and 4.3 U / mL PPDK.

[0256] The compositions of solutions A and B are shown in Tables 4-9 below for reagents for removing luciferase, removing PPDK, removing luciferin, removing Mg acetate, removing PEP, and removing PPi.

[0257] [Table 4]

[0258] [Table 5]

[0259] [Table 6]

[0260] [Table 7]

[0261] [Table 8]

[0262] [Table 9]

[0263] When storing after mixing, add 0.5 mL of solution B to 0.5 mL of solution A. Assuming ATP was mixed in during preparation, add 0.01 mL of 1×10⁻⁶ ATP. -5 The ATP solution of M was stored at 25°C for 40 hours. 0.1 mL of the stored sample was collected, and 0.1 mL of 1×10⁻⁶ ATP solution was added to it. -6 M ATP, the amount of light emitted 10 seconds after addition was measured using a Lumitester Smart fluorescence detector.

[0264] When mixing after storage, assuming ATP was mixed in during preparation, add 0.01 mL of 1×10⁻⁶ ATP to 0.5 mL of solution A. -5Solution M contains ATP, while solution B contains no added substances. Both solutions are stored at 25°C for 40 hours. After storage, 0.05 mL of solution B is added to 0.05 mL of solution A containing ATP, and 0.1 mL of 1×10⁻⁶ phosphate is added to the solution. -6 M ATP, the amount of light emitted 10 seconds after addition was measured using a Lumitester Smart fluorescence detector.

[0265] As the pre-storage luminescence level for comparison, 0.5 mL of solution A and 0.5 mL of solution B were mixed, and 0.01 mL μL of 1×10⁻⁶ solution was added. -5 For the ATP solution of M, without preservation, directly collect 0.1 mL of sample and add 0.1 mL of 1×10⁻⁶ ATP solution. -6 M ATP, which also shows the amount of light emitted at this time. Figure 21 .

[0266] It can be seen that when luciferase, PPDK, luciferin, magnesium acetate (Mg acetate), PEP, and PPi are all "mixed and stored", the luminescence level decreases when ATP is added, while when they are "mixed after storage", the luminescence level remains high.

[0267] Removing the luciferase involved in the luminescence reaction and the PPDK involved in the cycling reaction prevents the cycling reaction from occurring, thus improving stability. Furthermore, removing auxiliary factors or substrates such as magnesium acetate, phosphoenolpyruvate, and pyrophosphate also improves stability. The roles of magnesium acetate, phosphoenolpyruvate, and pyrophosphate in the cycling reaction are described below.

[0268] Magnesium acetate contains magnesium ions, which are essential for the reactions of luciferase and PPDK. In reagents without magnesium acetate, the luminescence and cyclic reactions will not occur. Phosphoenolpyruvate and pyrophosphate are both substrates required for the PPDK reaction. In the absence of phosphoenolpyruvate or pyrophosphate, the luminescence reaction proceeds in proportion to the amount of ATP introduced, but it ceases when ATP is depleted, and the luminescence reaction does not continue thereafter.

[0269] The results above show that stability can be improved by storing the components required for the luciferase reaction and the cycling reaction separately to stop the reaction.

[0270] All publications, patents and patent applications cited in this specification are incorporated herein by reference.

Claims

1. A liquid composition for use, after preservation, in determining ATP, AMP, and / or ADP in a sample. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the assay, and (ii) The relative luminescence intensity of the liquid composition during storage is below 5500 RLU. The relative luminescence is the value obtained by subtracting the control value from the measured value. The measured value refers to the addition of 0.35 mL of the liquid composition to a test tube of a LuciPac Pen manufactured by Kikkoman Co., Ltd., followed by the addition of 0.01 mL of 1×10⁻⁶ solution. -7 The ATP solution of M, manufactured by Oriental Yeast Industry Co., Ltd., was incubated at 25°C for 1 hour, and the value was measured using a Lumitester Smart fluorescence detector manufactured by Kikkoman Bio-Kemifa Co., Ltd. The control value refers to the value measured under the same conditions as when the measured value was obtained, except that sterile ultrapure water was added to replace the ATP solution.

2. The liquid composition according to claim 1, wherein, The liquid composition further comprises at least one component selected from an enzyme that catalyzes the reaction of ADP to ATP, a substrate of an enzyme that catalyzes the reaction of ADP to ATP, an enzyme that catalyzes the reaction of ADP to AMP, a substrate of an enzyme that catalyzes the reaction of ADP to AMP, or at least one of them added before or during the assay.

3. A liquid composition for use, after preservation, to determine ATP, as well as AMP and / or ADP in a sample. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ADP, a substrate of the enzyme catalyzing the reaction from AMP to ADP, an enzyme catalyzing the reaction from ADP to ATP, a substrate of the enzyme catalyzing the reaction from ADP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the component not contained in the liquid composition is added before or during the assay, and (ii) The relative luminescence intensity of the liquid composition during storage is below 5500 RLU. The relative luminescence is the value obtained by subtracting the control value from the measured value. The measured value refers to the addition of 0.35 mL of the liquid composition to a test tube of a LuciPac Pen manufactured by Kikkoman Co., Ltd., followed by the addition of 0.01 mL of 1×10⁻⁶ solution. -7 The ATP solution of M, manufactured by Oriental Yeast Industry Co., Ltd., was incubated at 25°C for 1 hour, and the value was measured using a Lumitester Smart fluorescence detector manufactured by Kikkoman Bio-Kemifa Co., Ltd. The control value refers to the value measured under the same conditions as when the measured value was obtained, except that sterile ultrapure water was added to replace the ATP solution.

4. The liquid composition according to any one of claims 1 to 3, wherein, The relative luminescence intensity is below 2300 RLU.

5. A liquid composition for use, after preservation, in determining ATP, AMP, and / or ADP in a sample. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ATP, a substrate of the enzyme catalyzing the reaction from AMP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the components not contained in the liquid composition are added to the liquid composition before or during the assay, and (ii) Meets at least one of the following: The concentration of fluorescein in the liquid composition is below 0.4 mM; The concentration of the Bradford-based luciferase in the liquid composition is less than 0.3 mg / mL; The concentration of the enzyme catalyzing the reaction from AMP to ATP in the liquid composition is less than 1 U / mL; The concentration of the substrate of the enzyme catalyzing the reaction from AMP to ATP in the liquid composition is below 0.1 mM; The concentration of the cofactor in the liquid composition is below 6 mM.

6. The liquid composition according to claim 5, wherein, The liquid composition further comprises at least one component selected from an enzyme that catalyzes the reaction of ADP to ATP, a substrate of an enzyme that catalyzes the reaction of ADP to ATP, an enzyme that catalyzes the reaction of ADP to AMP, a substrate of an enzyme that catalyzes the reaction of ADP to AMP, or at least one of them added before or during the assay.

7. A liquid composition for use, after preservation, in determining ATP, AMP, and / or ADP in a sample. (i) The liquid composition comprises luciferase, luciferin, an enzyme catalyzing the reaction from AMP to ADP, a substrate of the enzyme catalyzing the reaction from AMP to ADP, an enzyme catalyzing the reaction from ADP to ATP, a substrate of the enzyme catalyzing the reaction from ADP to ATP, and a cofactor, or, if the liquid composition does not contain at least one of these components, the component not contained in the liquid composition is added before or during the assay, and (ii) Meets at least one of the following: The concentration of the enzyme catalyzing the reaction from AMP to ADP in the liquid composition is below 450 U / mL; The concentration of the substrate of the enzyme catalyzing the reaction from AMP to ADP in the liquid composition is below 0.1 mM; The concentration of the enzyme catalyzing the reaction from ADP to ATP in the liquid composition is below 20 U / mL; The concentration of the substrate for the enzyme that catalyzes the reaction from ADP to ATP is below 1.2 mM; The concentration of the cofactor in the liquid composition is below 6 mM.

8. The liquid composition according to any one of claims 1 to 7, wherein, It should be stored for more than one day.

9. The liquid composition according to claim 8, wherein, It can be stored for more than 30 days.

10. The liquid composition according to any one of claims 1 to 9, wherein, The liquid composition does not contain at least one of the following: luciferase, luciferin, enzyme catalyzing the reaction of AMP to ATP, substrate of enzyme catalyzing the reaction of AMP to ATP, enzyme catalyzing the reaction of AMP to ADP, substrate of enzyme catalyzing the reaction of AMP to ADP, enzyme catalyzing the reaction of ADP to ATP, substrate of enzyme catalyzing the reaction of ADP to ATP, and cofactor. The components not contained in the liquid composition are added before or during the assay.

11. The liquid composition according to any one of claims 1 to 10, wherein, The concentration of luciferin in the liquid composition is less than 0.4 mM and / or the concentration of luciferase based on the Bradford method is less than 0.3 mg / mL.

12. The liquid composition according to any one of claims 1 to 11, wherein, The concentration of fluorescein in the liquid composition is below 0.1 mM.

13. The liquid composition according to any one of claims 1 to 12, wherein, The concentration of the Bradford-based luciferase in the liquid composition is below 0.1 mg / mL.

14. A kit for determining ATP in a sample, the kit comprising the liquid composition according to any one of claims 1 to 13.

15. A method for determining ATP, AMP, and / or ADP in a sample, comprising: Use the liquid composition according to any one of claims 1 to 13 or the kit according to claim 14.

16. The method according to claim 15, wherein, Do not use ATP standard solution.

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