Liquid composition for measuring ATP, and amp and / or ADP in sample

Abstract

In one embodiment, an object of the present invention is to provide a liquid cycling luminescent reagent having excellent stability.SOLUTION: In one embodiment, the present invention provides a liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising (I) luciferase, luciferin, an enzyme that catalyzes a reaction of generating ATP from AMP, a substrate for the enzyme that catalyzes a reaction of generating ATP from AMP, and a cofactor, or when at least one of these components is not contained in the liquid composition, the component not contained in the liquid composition is added to the liquid composition before or during the measurement, (ii) a relative light unit of the liquid composition during storage is equal to or less than 5500RLU, wherein the relative light unit is obtained by subtracting a control value from a measured value.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] In one embodiment, the present invention relates to a liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, a kit for measuring ATP in a sample comprising the liquid composition, and a method for measuring ATP, and AMP and / or ADP in a sample, comprising using the liquid composition. [Background technology]

[0002] Adenosine triphosphate (ATP) is a nucleotide found in all living organisms and is used by cells as a substrate for storing and releasing energy. Because ATP is contained in biologically derived substances, kits and methods for measuring the cleanliness of biological samples and biological instruments, as well as kits for measuring the contamination level of cooking instruments, have been reported (Patent Documents 1 and 2).

[0003] A typical ATP measurement method involves reacting ATP with the substrate luciferin in the presence of luciferase and measuring luminescence (Non-Patent Document 1). This reaction is catalyzed by luciferase and proceeds as follows in the presence of divalent metal ions: Luciferin + ATP + O2 → oxyluciferin + adenosine monophosphate (AMP) + pyrophosphate (PPi) + CO2 + light

[0004] ATP can be dephosphorylated relatively easily to form ADP, and ADP can also be dephosphorylated to form AMP in some cases. Therefore, by measuring ATP and ADP, or ATP, ADP, and AMP, rather than ATP alone, it is possible to stably measure ATP (or its degradation products) contained in biological substances and more accurately determine the cleanliness level. For example, Patent Document 1 describes that by measuring ATP and ADP, or ATP, ADP, and AMP, rather than ATP alone, it is possible to accurately detect residual blood or blood adhering to blood-related instruments, etc. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 147442 [Patent Document 2] International Publication No. 2018 / 147443 [Non-patent literature]

[0006] [Non-Patent Document 1] Marlene DeLuca, William D. McElroy, Biochemistry, 1974, 13 (5), pp 921-925 Summary of the Invention [Problem to be solved by the invention]

[0007] Luminescent reagents used in reaction systems (cycling reactions) that directly or indirectly convert AMP to ATP and measure ADP and / or AMP in addition to ATP are called "cycling luminescent reagents." Existing luminescent cycling reagents, such as Lucipack Pen (ATP + AMP measurement) (Kikkoman Biochemifa Corporation) and Lucipack A3 (ATP + ADP + AMP measurement) (Kikkoman Biochemifa Corporation), are designed and manufactured in powder form; no liquid cycling luminescent reagents exist. However, producing powdered reagents requires processes such as drying and grinding, and filling them quickly and accurately is difficult, making the manufacturing process complicated and costly. Powders also have the disadvantage of requiring measures to prevent moisture absorption.

[0008] Therefore, the present inventors attempted to design a new liquid cycling luminescent reagent, and discovered that the stability of the liquid cycling luminescent reagent was not necessarily sufficient.

[0009] In one embodiment, an object of the present invention is to provide a liquid cycling luminescent reagent with excellent stability. In one embodiment, an object of the present invention is to provide a kit including the cycling luminescent reagent, or a method for measuring ATP, and AMP and / or ADP in a sample using the cycling luminescent reagent or kit. [Means for solving the problem]

[0010] The present inventors have surprisingly found that in cycling luminescence reactions using luciferin-luciferase reactions, the stability of a liquid cycling luminescence reagent can be improved by reducing the relative luminescence intensity of the liquid cycling reagent during storage.

[0011] The present invention includes the following embodiments. [1] A liquid composition for measuring ATP, AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition contains luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added to the liquid composition before or during measurement; and (ii) the relative luminescence intensity of the liquid composition during storage is 5500 RLU or less; the relative light intensity is a value obtained by subtracting a control value from a measured value; The measurement was performed by adding 0.35 mL of the liquid composition to a measurement tube of Lucipack Pen (manufactured by Kikkoman Biochemifa Corporation), and then measuring 1 × 10 -7 0.01 mL of ATP (manufactured by Oriental Yeast Co., Ltd.) solution was added to M, and the mixture was left to stand at 25°C for 1 hour, after which the value was measured using a Lumitester Smart (manufactured by Kikkoman Biochemifa Corporation). The control value is a value obtained by measuring the amount of luminescence under the same conditions as those for obtaining the measured value, except that sterilized ultrapure water is added instead of the ATP solution. The liquid composition. [2] The liquid composition according to [1], further comprising at least one component selected from an enzyme that catalyzes a reaction to produce ATP from ADP, a substrate for the enzyme that catalyzes a reaction to produce ATP from ADP, an enzyme that catalyzes a reaction to produce AMP from ADP, and a substrate for the enzyme that catalyzes a reaction to produce AMP from ADP, or at least one of these is added before or during measurement. [3] A liquid composition for measuring ATP, AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition comprises luciferase, luciferin, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added before or during measurement; and (ii) the relative luminescence intensity of the liquid composition during storage is 5500 RLU or less; the relative light intensity is a value obtained by subtracting a control value from a measured value; The measurement was performed by adding 0.35 mL of the liquid composition to a measurement tube of Lucipack Pen (manufactured by Kikkoman Biochemifa Corporation), and then measuring 1 × 10 -7 0.01 mL of ATP (manufactured by Oriental Yeast Co., Ltd.) solution was added to M, and the mixture was left to stand at 25°C for 1 hour, after which the value was measured using a Lumitester Smart (manufactured by Kikkoman Biochemifa Corporation). The control value is a value obtained by measuring the amount of luminescence under the same conditions as those for obtaining the measured value, except that sterilized ultrapure water is added instead of the ATP solution. The liquid composition. [4] The liquid composition according to any one of [1] to [3], wherein the relative light emission amount is 2300 RLU or less. [5] A liquid composition for measuring ATP, AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition contains luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added to the liquid composition before or during measurement; and (ii) At least one of the following is true: the concentration of luciferin in the liquid composition is 0.4 mM or less; the concentration of luciferase in the liquid composition based on the Bradford assay is 0.3 mg / mL or less; the concentration of the enzyme that catalyzes the reaction of producing ATP from AMP in the liquid composition is 1 U / mL or less; the concentration of a substrate for an enzyme that catalyzes a reaction producing ATP from AMP in the liquid composition is 0.1 mM or less; the concentration of the cofactor in the liquid composition is 6 mM or less; The liquid composition. [6] The liquid composition according to [5], further comprising at least one component selected from an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, an enzyme that catalyzes a reaction that produces AMP from ADP, and a substrate for the enzyme that catalyzes a reaction that produces AMP from ADP, or at least one of these is added before or during measurement. [7] A liquid composition for measuring ATP, AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition comprises luciferase, luciferin, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added before or during measurement; and (ii) At least one of the following is true: the concentration of the enzyme that catalyzes the reaction of producing ADP from AMP in the liquid composition is 450 U / mL or less; the concentration of a substrate for an enzyme that catalyzes a reaction producing ADP from AMP in the liquid composition is 0.1 mM or less; the concentration of the enzyme that catalyzes the reaction of producing ATP from ADP in the liquid composition is 20 U / mL or less; the concentration of the substrate for the enzyme that catalyzes the reaction of producing ATP from ADP is 1.2 mM or less; the concentration of the cofactor in the liquid composition is 6 mM or less; The liquid composition. [8] The liquid composition according to any one of [1] to [7], which has a shelf life of one day or more. [9] The liquid composition according to [8], which has a shelf life of 30 days or more.

[10] The liquid composition according to any one of [1] to [9], which does not contain at least one component of luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, and the component not contained in the liquid composition is added before or during measurement.

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

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

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

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

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

[12] , wherein the concentration of luciferase in the liquid composition as determined by the Bradford method is 0.1 mg / mL or less.

[14] A kit for measuring ATP in a sample, comprising the liquid composition according to any one of [1] to

[13] .

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

[13] or the kit according to

[14] .

[16] The method described in

[15] , which does not use an ATP standard solution. This specification includes the disclosure of Japanese Patent Application No. 2020-023442, from which this application claims priority. [Effects of the Invention]

[0012] In one embodiment, the present invention provides a liquid cycling luminescent reagent with excellent stability. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the time course of luminescence intensity of the luminescent reagent without cycling. [Figure 2] FIG. 2 shows the stability of cycling luminescence reagents containing various concentrations of luciferin. [Figure 3] FIG. 3 shows the luminescence intensity after 9 hours of cycling the luminescence reagent containing luciferin at various concentrations in FIG. [Figure 4] FIG. 4 shows the stability of cycling luminescence reagents with and without luciferin. [Figure 5] Figure 5 shows the stability of the luminescence reagent without and with cycling. Results are shown both without and with luciferin. [Figure 6] FIG. 6 shows the stability of cycling luminescence reagents containing various concentrations of luciferase. [Figure 7] FIG. 7 shows the amount of luminescence after 9 hours from cycling luminescence reagents containing various concentrations of luciferase. [Figure 8] FIG. 8 shows the ATP concentration dependence of the decrease in luminescence intensity in the cycling luminescence reagent. [Figure 9] FIG. 9 shows the stability (change in luminescence intensity over time) of luminescence reagents containing luciferin at various concentrations when 0.01 mL of 1×10 −5 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 10] FIG. 10 shows the amount of luminescence after 1 hour from luminescence reagents containing luciferin at various concentrations when 0.01 mL of 1×10 −5 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 11] FIG. 11 shows the stability (change in luminescence intensity over time) of luminescence reagents containing luciferin at various concentrations when 0.01 mL of 1×10 −6 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 12] FIG. 12 shows the amount of luminescence after 1 hour from luminescence reagents containing luciferin at various concentrations when 0.01 mL of 1×10 −6 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 13] FIG. 13 shows the stability (change in luminescence intensity over time) of luminescence reagents containing luciferin at various concentrations when 0.01 mL of 1×10 −7 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 14] FIG. 14 shows the amount of luminescence after 1 hour from luminescence reagents containing luciferin at various concentrations when 0.01 mL of 1×10 −7 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 15] FIG. 15 shows the stability (changes in luminescence intensity over time) of luminescence reagents containing luciferase at various concentrations when 0.01 mL of 1×10 −5 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 16] FIG. 16 shows the amount of luminescence after 1 hour from luminescence reagents containing luciferase at various concentrations when 0.01 mL of 1×10 −5 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 17] FIG. 17 shows the stability (changes in luminescence intensity over time) of luminescence reagents containing luciferase at various concentrations when 0.01 mL of 1×10 −6 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 18] FIG. 18 shows the amount of luminescence after 1 hour from luminescence reagents containing luciferase at various concentrations when 0.01 mL of 1×10 −6 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 19]FIG. 19 shows the stability (changes in luminescence intensity over time) of luminescence reagents containing luciferase at various concentrations when 0.01 mL of 1×10 −7 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 20] FIG. 20 shows the amount of luminescence after 1 hour from luminescence reagents containing luciferase at various concentrations when 0.01 mL of 1×10 −7 M ATP was added to 0.35 mL of the luminescence reagent. [Figure 21] Figure 21 shows the luminescence intensity when each component was removed from the luminescent reagent, and the removed components were "mixed and stored" or "mixed after storage," and then 0.1 mL of 1 × 10 M ATP solution was added. The luminescence intensity (before storage) when 0.1 mL of 1 × 10 M ATP was added without storage is also shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Liquid composition] In a first aspect, the present invention provides a liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition contains luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added to the liquid composition before or during measurement; and (ii) A liquid composition having a relative luminescence intensity of 5500 RLU or less during storage, the relative luminescence intensity being the measured value minus the control value.

[0015] In a second aspect, the present invention provides a liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition comprises luciferase, luciferin, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added before or during measurement; and (ii) A liquid composition having a relative luminescence intensity of 5500 RLU or less during storage, the relative luminescence intensity being the measured value minus the control value.

[0016] In the compositions of the first and second aspects, the measurement was performed by adding 0.35 mL of the liquid composition to a measurement tube of Lucipack Pen (manufactured by Kikkoman Biochemifa Corporation), and then measuring 1 × 10 -7 The values ​​were measured using a Lumitester Smart (Kikkoman Biochemifa Corporation) after adding 0.01 mL of ATP (Oriental Yeast Co., Ltd.) solution to M and allowing to stand at 25°C for 1 hour. The control values ​​were measured under the same conditions as for obtaining the above measurements, except for adding sterile ultrapure water instead of the ATP solution. The Lucipack Pen (Kikkoman Biochemifa Corporation) measurement tube contains a luminescence reagent, which must be removed by washing to an extent that does not affect the measurement system before use. If it is difficult to obtain the Lucipack Pen (Kikkoman Biochemifa Corporation) measurement tube, an equivalent Lucipack A3 (Kikkoman Biochemifa Corporation) measurement tube may also be used. In principle, Oriental Yeast Co., Ltd. ATP is used, but if it is difficult to obtain, an equivalent ATP can be used. To avoid the background value from increasing and affecting the measurement value, it is preferable that the sterilized ultrapure water contains little ATP, ADP, and AMP. For example, the total concentration of ATP, ADP, and AMP is 1 × 10 -9 M or less, or 1 x 10 -10 The ATP may be diluted with sterile ultrapure water, which is the same as the sterile ultrapure water used for background measurement.

[0017] When measuring the relative luminescence intensity of a liquid composition during storage, if the total volume of the liquid composition (e.g., the liquid composition in one kit) is less than 0.35 mL, multiple equivalent liquid compositions (e.g., different liquid compositions in the same kit) may be mixed, and the relative luminescence intensity of the mixed liquid composition may be measured.

[0018] In this specification, the relative luminescence amount is, in principle, the value when the luminescence amount is measured under the above conditions. However, if the above Lucipack Pen is difficult to obtain, the following conditions can be used instead: the measured value is the value when 0.35 mL of the liquid composition is added to a Lumitube (manufactured by Kikkoman Biochemifa Corporation, 12φ×54 mm), and then 1×10 -7 0.01 mL of ATP solution (M) was added, and the mixture was allowed to stand at 25°C for 1 hour. The value measured using a Lumitester C-110 (Kikkoman Biochemifa Corporation) was then divided by 400. The control value was obtained by measuring the luminescence intensity under the same conditions as those used to obtain the above measurements, except that sterilized ultrapure water was added instead of the ATP solution, and dividing the value by 400.

[0019] In one embodiment, the relative light output of the liquid composition during storage may be 5500 RLU or less, 5000 RLU or less, 4500 RLU or less, 4000 RLU or less, 3500 RLU or less, or 3000 RLU or less. For example, the relative luminescence output of the liquid composition during storage may be 2900 RLU or less, 2800 RLU or less, 2700 RLU or less, 2600 RLU or less, 2500 RLU or less, 2400 RLU or less, 2300 RLU or less, 2200 RLU or less, 2100 RLU or less, 2000 RLU or less, 1900 RLU or less, 1800 RLU or less, 1700 RLU or less, 1600 RLU or less, 1500 RLU or less, 1400 RLU or less, 1300 RLU or less, 1200 RLU or less, 1100 RLU or less, 1000 RLU or less, 900 RLU or less, 800 RLU or less, 700 RLU or less, 600 RLU or less, 500 RLU or less, 400 RLU or less, 300 RLU or less, 200 RLU or less, or 100 RLU or less. The Examples show that the lower the relative luminescence intensity of a liquid composition during storage, the greater the stability of the liquid composition. Furthermore, when the relative luminescence intensity of a liquid composition during storage is 2500 RLU or less or 2300 RLU or less, the stability of the liquid composition may be particularly excellent. In this specification, "excellent stability" of a liquid composition means, for example, that there is little or no decrease in the luminescence intensity of the liquid composition.

[0020] Methods for reducing the relative luminescence intensity of a liquid composition during storage are not limited, and include changing the pH during storage from the optimal pH for the enzyme, adding an ATPase or the like to decompose ATP and prevent luminescence, adding a reaction inhibitor, reducing the concentration of components necessary for the cycling reaction or removing the components and then adding the removed components before or during measurement, or combinations of these.

[0021] For example, in a method of changing the pH during storage from the optimal pH, the pH of the liquid composition may be increased or decreased from the optimal pH to an extent that the enzymatic reaction does not proceed or is delayed, and the enzyme is not denatured, and the pH of the liquid composition may then be returned to the optimal pH before or during measurement. When acidic, the pH during storage may be, for example, 6 or less, 5 or less, or 4 or less, or 2 or more, or 3 or more. When basic, the pH during storage may 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 a pH that does not denature the enzyme and reduces the relative luminescence of the liquid composition, and can easily adjust the pH using an acid or base to achieve the determined pH. For example, in the case of the HLK described in JP 11-239493 A, the relative luminescence can be reduced to approximately one-third or less by adjusting the pH to 6.5 or less, and the relative luminescence can be reduced to approximately one-tenth or less by adjusting the pH to 6 or less. The pH before or during measurement may be 6 to 9, 7.5 to 8.5, or about 8. In another embodiment, the pH during storage and the pH before or during measurement are the same or almost the same (for example, the difference between the pH during storage and the pH before or during measurement may be within 1).

[0022] An example of a method for decomposing ATP by adding an ATPase is to add adenosine phosphate deaminase during storage. The ATPase may be used at a concentration that does not affect the ATP measurement system, and the influence of the ATPase may be reduced by diluting the liquid composition before or during measurement. The concentration and type of ATPase can be easily determined by those skilled in the art.

[0023] When a reaction inhibitor is added, examples of the reaction inhibitor include metal salts such as NaCl and surfactants such as benzalkonium chloride. The effect of the reaction inhibitor may be reduced by diluting the liquid composition before or during measurement. Alternatively, the reaction inhibitor may be removed before or during measurement; for example, metal salts can be removed with a chelating agent, and benzalkonium chloride can be removed with cyclodextrin. The concentration and type of the reaction inhibitor can be easily determined by one skilled in the art.

[0024] Methods for reducing the concentration of components required for the cycling reaction or for omitting components and then adding the removed components before or during the measurement are as described herein.

[0025] In a third aspect, the present invention provides a liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition contains luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added to the liquid composition before or during measurement; and (ii) At least one of the following is true: the concentration of luciferin in the liquid composition is 0.4 mM or less; the concentration of luciferase in the liquid composition based on the Bradford assay is 0.3 mg / mL or less; the concentration of an enzyme (e.g., PPDK) that catalyzes a reaction producing ATP from AMP in the liquid composition is 1 U / mL or less; the concentration of a substrate for an enzyme that catalyzes a reaction producing ATP from AMP in the liquid composition (e.g., when the enzyme is PPDK, pyrophosphate or a salt thereof and / or phosphoenolpyruvate or a salt thereof) is 0.1 mM or less; the concentration of a cofactor (e.g., a magnesium salt) in the liquid composition is 6 mM or less; The present invention relates to the liquid composition.

[0026] In a fourth aspect, the present invention provides a liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition comprises luciferase, luciferin, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added before or during measurement; and (ii) At least one of the following is true: the concentration of an enzyme (e.g., ADK) that catalyzes a reaction producing ADP from AMP in the liquid composition is 450 U / mL or less; the concentration of a substrate for an enzyme that catalyzes a reaction producing ADP from AMP in the liquid composition is 0.1 mM or less (for example, in the case of ADK, a substrate is not required); the concentration of an enzyme (e.g., PK) that catalyzes a reaction producing ATP from ADP in the liquid composition is 20 U / mL or less; the concentration of the substrate for the enzyme that catalyzes the reaction of producing ATP from ADP (e.g., phosphoenolpyruvate or a salt thereof when the enzyme is PK) is 1.2 mM or less; the concentration of a cofactor (e.g., a magnesium salt) in the liquid composition is 6 mM or less; The present invention relates to the liquid composition.

[0027] In one embodiment, the liquid compositions of the first and third aspects further comprise at least one component selected from an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, an enzyme that catalyzes a reaction that produces AMP from ADP, and a substrate for the enzyme that catalyzes a reaction that produces AMP from ADP, or at least one of these is added before or during measurement.

[0028] The liquid compositions of the first to fourth aspects (also collectively referred to herein as "the liquid compositions described herein") and their constituent components will be described in more detail below.

[0029] In one embodiment, the liquid composition described herein is for measuring AMP and / or ADP in addition to ATP (i.e., ATP and ADP; ATP and AMP; or ATP, ADP, and AMP). Biologically derived substances contain ATP, which can be relatively easily dephosphorylated to form ADP, and ADP can also be dephosphorylated to form AMP in some cases. Therefore, by measuring the two components, ATP and ADP or AMP, or the three components, ATP, ADP, and AMP, the ATP (or its degradation products) contained in biologically derived substances can be stably measured. Therefore, by measuring the two components, ATP and ADP or AMP, or the three components, ATP, ADP, and AMP, contamination is not overlooked and cleanliness can be more accurately determined.

[0030] [Luciferase] Luciferase is a general term for oxidases that produce bioluminescence. In one embodiment, luciferase catalyzes the conversion of ATP, O2, and luciferin into AMP, pyrophosphate, CO2, and oxyluciferin, resulting in the emission of light. The luciferase may be a naturally occurring luciferase or a genetically engineered recombinant luciferase mutant. The luciferase mutant may be one that has undergone site-directed or random mutation. It may also be a fusion protein with a protein having another function. The luciferase mutant may have desired properties, such as improved heat resistance or detergent resistance.

[0031] The amount of luminescence produced by luciferase can be evaluated using relative luminescence units (RLU) obtained using an appropriate luminometer, such as a luminometer (e.g., Lumitester Smart, Lumitester PD-20, or Lumitester PD-30, manufactured by Kikkoman Biochemifa) or a device equipped with a photodiode (e.g., SystemSURE Plus or EnSURE, manufactured by Hygiena, or AccuPoint Advanced, manufactured by Neogen). Luminescence generated during the conversion of luciferin to oxyluciferin is typically measured. High-sensitivity luminometers equipped with photomultiplier tubes (e.g., Cleantrace LM1 or Cleantrace UNG3, manufactured by 3M; Lumitester C-110 or Lumitester C-100, manufactured by Kikkoman Biochemifa; or Junior LB9509, CentroLB960, or Lumat3 LB9508, manufactured by Berthold) can also be used. An apparatus capable of highly sensitive measurements is useful for making accurate measurements, especially when the amount of luminescence is reduced.

[0032] Luciferases that use ATP as a substrate can be derived from bacteria, protozoa, animals, mollusks, or insects, but are not limited thereto. Insect-derived luciferases include beetle luciferases, such as those from the genus Photinus (e.g., Photinus pyralis), the genus Photuris (e.g., Photuris lucicrescens, Photuris pennsylvanica), the genus Luciola (e.g., Luciola cruciata, Luciola lateralis, Luciola parvula), the genus Pyrocoelia, and the genus Lucidina biplagiata, and click beetles (Pyrophorus). Many luciferase genes have been reported, and their nucleotide and amino acid sequences can be obtained from publicly known databases such as GeneBank.

[0033] The luciferase gene may be a wild-type gene or may have a mutation, which may be site-specifically introduced or may be a random mutation. Examples of known mutations include, but are not limited to, mutations that improve luminescence intensity as described in JP 2011-188787 A, mutations that improve luminescence persistence as described in JP 2000-197484 A, mutations that change luminescence wavelength as described in Japanese Patent No. 2666561 A or Japanese Translation of PCT International Publication No. 2003-512071 A, mutations that improve surfactant resistance as described in Japanese Patent No. 11-239493 A, mutations that improve substrate affinity as described in WO 99 / 02697, Japanese Translation of PCT International Publication No. 10-512750 A or Japanese Translation of PCT International Publication No. 2001-518799 A, and mutations that improve stability as described in Japanese Patent No. 3048466, Japanese Patent No. 2000-197487, Japanese Translation of PCT International Publication No. 9-510610 A and Japanese Translation of PCT International Publication No. 2003-518912 A.

[0034] Luciferase genes and their recombinant DNA can be prepared by conventional methods. For example, Japanese Patent Publication No. 7-112434 describes the Heike firefly luciferase gene. Japanese Patent Publication No. 1-51086 describes the Genji firefly luciferase gene.

[0035] The luciferase gene can be inserted into a vector such as a plasmid, bacteriophage, or cosmid, and then used to transform or transduce a suitable host. The host can be a microorganism, such as a bacterium such as E. coli, or a yeast. The transformed host capable of producing luciferase can be cultured by various known methods.

[0036] Examples of media include those containing one or more nitrogen sources such as tryptone, yeast extract, meat extract, peptone, corn steep liquor, or soybean or wheat bran infusion, to which one or more inorganic salts such as sodium chloride, potassium diphosphate, potassium diphosphate, magnesium chloride, ferric chloride, magnesium sulfate, or manganese sulfate have been added, and which may also contain carbohydrate raw materials, vitamins, etc., as necessary.

[0037] The initial pH of the medium can be, for example, 7 to 9. Cultivation can be carried out, for example, at 30 to 40°C for 2 to 24 hours by aeration and agitation culture, shaking culture, static culture, etc. After cultivation, luciferase is recovered from the culture by known techniques.

[0038] Specifically, the cells are subjected to ultrasonic disruption, grinding, etc. by conventional methods, or luciferase is extracted using a lytic enzyme such as lysozyme. The resulting extract is filtered, centrifuged, etc., and nucleic acids are removed, if necessary, with streptomycin sulfate, etc., and the extract is fractionated by adding ammonium sulfate, alcohol, acetone, etc. to obtain a crude enzyme.

[0039] The crude enzyme may be further purified by various gel filtration or chromatography techniques. Commercially available luciferases may also be used, such as the luciferase from Kikkoman Biochemifa Corporation, catalog number 61314. This luciferase is described in Japanese Patent Publication No. 11-239493 (Patent No. 3749628) (SEQ ID NO: 1 therein). Commercially available luciferases from Sigma-Aldrich, Promega, and Life Technologies, under the trademark Molecular Probes, may also be used.

[0040] In one embodiment, the luciferase concentration in the liquid compositions described herein may be, for example, 0.3 mg / mL or less, 0.25 mg / mL or less, 0.2 mg / mL or less, 0.1 mg / mL or less, 0.05 mg / mL or less, 0.01 mg / mL or less, 0.005 mg / mL or less, 0.001 mg / mL or less, or 0.0005 mg / mL or less, as measured by the Bradford assay. The Bradford assay concentration can be measured using a Coomassie (Bradford) Protein Assay Kit (Thermo Scientific) with a BSA solution as a standard, as described in the Examples. If this kit is difficult to obtain, the luciferase concentration can be measured by the Bradford assay, which provides equivalent values ​​known to those skilled in the art, using a BSA solution as a standard. Alternatively, the absorbance can be measured at 280 nm using the absorbance method described in the Examples, and then converted to obtain the Bradford assay concentration. A lower luciferase concentration can improve the stability of the liquid composition, but the overall amount of luminescence will decrease, so if the amount of luminescence is too low, luciferase may be added before or during measurement. The luciferase concentration in the liquid composition during storage or after adding luciferase before or during measurement may be, for example, 0.00001 mg / mL or more, 0.0001 mg / mL or more, 0.001 mg / mL or more, or 0.01 mg / mL or more, based on the Bradford method.

[0041] In this specification, the luciferase concentration is generally a value based on the Bradford assay. However, when a liquid composition is prepared containing proteins other than luciferase, the concentration of luciferase alone in the prepared liquid composition cannot be directly measured by the Bradford assay. In this case, the luciferase concentration based on the Bradford assay can be indirectly measured based on the luciferase activity as described below.

[0042] The liquid composition containing luciferase is diluted with enzyme diluent (5.0% glycerol, 1.0 mM EDTA·2Na·2H2O, 1.0 mM 2-mercaptoethanol, 50 mM tricine, 1.0% bovine serum albumin (BSA) (pH 7.8)) so that the luminescence intensity is 100,000 to 1,000,000 RLU. 100 μL of the liquid composition diluted with enzyme diluent was placed in a Laurent tube (Sarstedt) preheated to 25°C, and 100 μL of a luminescence reagent for measuring luciferase activity (50 mM Tricine, 4.0 mM ATP 2Na, 2.0 mM D-Luciferin, 10 mM MgSO4 7H2O (pH 7.8)) was added using an injector. The luminescence intensity for 20 seconds, starting from 0.5 seconds, was measured using a LUMAT LB9507 (Berthold). This luminescence intensity was designated the sample luminescence intensity (Es). Measurements were performed at 25°C. Similarly, the blank luminescence intensity (E0) was measured using enzyme diluent instead of luciferase solution. Luciferase activity (LU / mL) = (Es-E0) × dilution factor ÷ sample volume Sample volume: 0.1 mL

[0043] In addition to the above experiment, the luciferase activity and protein concentration were measured using luciferase that was confirmed to be a single band on SDS-PAGE. The luciferase activity was 8.4 × 10 14 The Bradford assay value for LU / mL was 39.4 mg / mL, i.e., the specific activity was 2.1 × 10 13 The specific activity is expressed as LU / mg, and by using this specific activity value, the luciferase activity can be converted into the protein concentration. Protein concentration (mg / mL) based on the Bradford assay = luciferase activity (LU / mL) ÷ (2.1 x 10 13 LU / mg)

[0044] This allows luciferase protein concentration to be determined even in reagents containing stabilizer proteins such as BSA or cycling enzymes (e.g., PPDK or PK). Note that this is a converted value when a specific luciferase (HLK described in JP-A-11-239493) is used, but a similar converted value can be calculated using a different luciferase, and the luciferase concentration can be measured based on this.

[0045] When the liquid composition contains a protein other than luciferase, the luciferase concentration can be measured by fractionating the liquid composition by chromatography such as HPLC, and measuring the amount of luciferase protein fractionated by the Bradford method, in addition to the activity measurement described above.

[0046] [Luciferin] Any luciferin may be used as long as it is recognized as a substrate by the luciferase used, and may be natural or chemically synthesized. Known luciferin derivatives may also be used. The basic structure of luciferin is imidazopyrazinone, and there are many tautomers. An example of luciferin is firefly luciferin. Firefly luciferin is a substrate for firefly luciferase (EC 1.13.12.7). Luciferin derivatives may be those described in JP 2007-91695 A, JP 2010-523149 A (WO 2008 / 127677 A), etc.

[0047] In one embodiment, the concentration of luciferin or a derivative thereof in the liquid composition described herein may be, for example, 0.4 mM or less, 0.35 mM or less, 0.3 mM or less, 0.25 mM or less, 0.15 mM or less, 0.1 mM or less, 0.05 mM or less, 0.02 mM or less, 0.01 mM or less, 0.005 mM or less, 0.001 mM or less, or 0.0001 mM or less. A lower luciferin concentration may improve the stability of the liquid composition, but may also result in a lower overall luminescence. Therefore, if the luminescence is too low, luciferin or a derivative thereof may be added before or during measurement. The concentration of luciferin or a derivative thereof in the liquid composition during storage or after adding luciferin before or during measurement may be, for example, 0.00001 mM or more, 0.0001 mM or more, 0.001 mM or more, or 0.01 mM or more.

[0048] The concentration of luciferin or a derivative thereof can be measured based on the amount of luminescence in the same manner as described above for luciferase. For example, the luminescence reagent is diluted to eliminate the influence of other components such as enzymes, and reagents containing different concentrations of luciferin are prepared in excess of luciferase. The luciferin concentration can be measured by comparing the amount of luminescence with that of a standard substance containing luciferin of a known concentration. Alternatively, the liquid composition can be fractionated by chromatography such as HPLC, the peak corresponding to luciferin is detected, and the luciferin concentration can be measured by comparing the peak intensity with that of a standard substance containing luciferin of a known concentration.

[0049] It was unexpected, as described below, that a lower luciferin concentration can improve the stability of a liquid composition. That is, in measurement systems using enzymatic reactions, substrates are typically added at high concentrations so that the reaction rate is not affected even if the substrate is reduced as the reaction progresses. In particular, in cycling reactions, substrates are continuously consumed, so it is reasonable for those skilled in the art to believe that designing the substrate concentration at a higher concentration will lead to the sustainability of the luminescence output of the reagent and stability during storage. However, the results of the Examples described herein revealed that, in cycling luminescent reagents utilizing the luciferin-luciferase reaction, the luminescence output of the cycling luminescent reagent can be stabilized by lowering the luciferin concentration. Without being bound by theory, it is possible that the decrease in the stability of luminescence output that occurs when a liquid cycling luminescent reagent is stored is not primarily due to a decrease in the amount of luciferin caused by luciferin consumption, but rather due to luminescence inhibition by oxyluciferin produced in the luminescent reaction. In other words, this phenomenon differs from that occurring in cycling methods using other enzymes and may be unique to liquid cycling luminescent reagents utilizing the luciferin-luciferase reaction.

[0050] [An enzyme that catalyzes the reaction that produces ATP from AMP] In one embodiment, the liquid composition described herein contains an enzyme that catalyzes the reaction of generating ATP from AMP. The enzyme that catalyzes the reaction of generating ATP from AMP converts the AMP present in the system into ATP. Then, ATP is converted into AMP by luciferase, and luminescence is generated. Therefore, in this embodiment, AMP can be measured in addition to ATP. Known enzymes that catalyze the reaction of producing ATP from AMP can be used, including, but not limited to, pyruvate phosphate dikinase (PPDK), pyruvate water dikinase (PWDK), and combinations thereof.

[0051] [Pyruvate-phosphate dikinase (PPDK)] Pyruvate-phosphate dikinase (EC 2.7.9.1) catalyzes the reaction between ATP, pyruvate, and orthophosphate with adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), and pyrophosphate (PPi): ATP + pyruvate + phosphate ←→ AMP + PEP + PPi

[0052] Pyruvate phosphate dikinase (PPDK) is also known as ATP:pyruvate phosphate phosphotransferase, pyruvate orthophosphate dikinase, and pyruvate phosphate ligase. These terms are interchangeable herein. PPDK typically converts pyruvate to PEP, consuming one molecule of ATP in the process, which converts it to AMP. The reaction can be divided into three reversible reactions: 1. PPDK binds to ATP to produce AMP and diphosphorylated PPDK. 2. Diphosphorylated PPDK binds to inorganic phosphate to produce diphosphate and monophosphorylated PPDK. 3. Monophosphorylated PPDK binds to pyruvate to generate PEP and PPDK. If the PEP concentration in the system is high, the reaction will proceed in the opposite direction as follows:

[0053] [ka]

[0054] For convenience, the reaction steps are described with the same numbers as above. 3. PEP binds to PPDK, producing monophosphorylated PPDK and pyruvate. 2. Diphosphate and monophosphorylated PPDK produce diphosphorylated PPDK and inorganic phosphate. 1. Diphosphorylated PPDK and AMP produce PPDK and ATP.

[0055] PPDKs are not particularly limited, but examples include those derived from microorganisms such as Microbispora thermorosea, Propionibacterium shremanii, Bacteroides symbiosus, Entamoeba histolytica, Acetobacter xylinum, and Propionibacter shermanii, as described in Japanese Patent Application Laid-Open No. 8-168375, and those derived from plants such as corn and sugarcane.

[0056] [Pyruvate water dikinase (PWDK)] Pyruvate water dikinase (EC 2.7.9.2) catalyzes the following reaction: ATP + pyruvate + HO ←→ AMP + phosphoenolpyruvate (PEP) + phosphate (P) Pyruvate water dikinase is also known as phosphoenolpyruvate synthase, pyruvate water dikinase (phosphorylating), PEP synthetase, phosphoenolpyruvate synthetase, phosphoenolpyruvic synthetase, and phosphopyruvate synthetase, and these terms are used interchangeably herein.

[0057] Examples of PWDK include, but are not limited to, those derived from Escherichia coli, Pseudomonas fluorescens, Pyrococcus furiosus, Staphylothermus marinus, Sulfolobus solfataricus, Thermococcus kodakarensis, Thermoproteus tenax, and Zea mays.

[0058] Using PWDK together with PEP can promote ATP production from AMP and PEP. When an enzyme that catalyzes the reaction of producing ATP from AMP is combined with an enzyme that catalyzes the reaction of producing ATP from ADP described herein, ADP is converted to ATP, and as a result, ATP, ADP, and AMP can be measured.

[0059] [An enzyme that catalyzes the reaction that produces ATP from ADP] In one embodiment, the liquid composition described herein contains an enzyme that catalyzes the reaction of generating ATP from ADP. The enzyme that catalyzes the reaction of generating ATP from ADP converts the ADP present in the system into ATP. The ATP is then converted into AMP by luciferase, and luminescence is generated. Therefore, in this embodiment, ADP can be measured in addition to ATP.

[0060] The enzyme that catalyzes the reaction of producing ATP from ADP can be any known enzyme, including, but not limited to, kinases capable of producing ATP, such as pyruvate kinase, acetate kinase, creatine kinase, polyphosphate kinase, riboflavin kinase, phosphofructokinase, fructose bisphosphatase, hexokinase, glucokinase, glycerol kinase, fructokinase, and combinations thereof.

[0061] [Pyruvate kinase (PK)] Pyruvate kinase (EC 2.7.1.40) converts phosphoenolpyruvate to pyruvate during glycolysis, converting ADP to ATP. This reaction is exergonic with negative Gibbs energy and is irreversible under natural conditions: PEP + ADP → Pyruvate + ATP The reverse reaction, during gluconeogenesis, is catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase, producing PEP and ADP from ATP and pyruvate. When cell extracts are used, various enzymes are present in the system, and the above reaction can proceed in both directions. In this case, ADP can be converted to ATP if phosphoenolpyruvate is present at high concentrations. Furthermore, the presence of pyruvate kinase in addition to phosphoenolpyruvate is thought to increase the conversion of ADP to ATP. PKs ​​are not particularly limited, but can be derived from animals such as rabbits, rats, and chickens, or from microorganisms such as yeast and Bacillus stearothermophilus.

[0062] [Acetate kinase (AK)] Acetate kinase (EC 2.7.2.1) catalyzes the conversion between ATP and acetate and ADP and acetylated phosphate in the presence of cations: ATP + acetate ←→ ADP + acetylated phosphate Acetate kinase (AK) is also known as ATP:acetate phosphotransferase or acetyl kinase. These terms are interchangeable herein. In vivo, it catalyzes the reaction from ATP and acetate to produce ADP and acetylated phosphate, ultimately producing acetyl-CoA. When acetylated phosphate and ADP, generated from acetyl-CoA, are present in the system, they can be converted to acetate and ATP. AK can be derived from microorganisms such as, but not limited to, Escherichia coli, Bacillus stearothermophilus, Costridium pasteurianum, Lactobacillus delbruckii, and Veillonella alcalescence.

[0063] [Creatine kinase (CK)] Creatine kinase (EC 2.7.3.2) mediates the conversion reactions 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 interchangeable herein. Normally, animal muscles convert creatine and ATP to creatine phosphate and ADP. However, this reaction is reversible; when high concentrations of creatine phosphate and ADP are present in the system, the reaction can proceed in the reverse direction, producing creatine and ATP. In vivo, cytoplasmic CK is composed of two subunits, B and M. Therefore, depending on the combination of subunits, three isozymes, CK-MM, CK-BB, and CK-MB, can exist. Although the isozyme pattern varies depending on the tissue, any combination can be used in the present invention. CK is not particularly limited, and animal-derived CK can be used, including those derived from rabbit, chicken, cow, pig, carp, catfish, and frog.

[0064] [Polyphosphate kinase (PPK)] Polyphosphate kinase (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 Polyphosphate kinase (PPK) is also known as ATP:polyphosphate phosphotransferase. These terms are interchangeable herein. PPK is involved in oxidative phosphorylation in vivo. When polyphosphate (n) and ADP are present in the system, they can be converted to polyphosphate (n-1) and ATP. There are no particular limitations on PPK, but PPKs derived from microorganisms such as Escherichia coli, yeast, and Corynebacterium xerosis can be used.

[0065] [Riboflavin kinase (FMNK)] Riboflavin kinase (EC 2.7.1.26), also known as FMNK, catalyzes the conversion of riboflavin and ATP to riboflavin phosphate (FMN) and ADP: ATP + riboflavin ←→ ADP + FMN Riboflavin kinase belongs to the ATP:riboflavin 5'-phosphotransferase (also called flavokinase) family. FMNK is not particularly limited, but can be derived from microorganisms or animals, such as yeast, rat, or bean (Phaseolus radiatus).

[0066] [Phosphofructokinase 1 (PFK1)] 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 belongs to the phosphofructokinase family. Phosphofructokinase 1 is sometimes referred to as Fru-1,6BPK herein. PFK1 can be derived from animals or microorganisms, but is not limited to these. Examples of PFK1 derived from microorganisms include baker's yeast, brewer's yeast, Clostridium pasteurianum, Escherichia coli, and Bacillus licheniformis.

[0067] [Fructose bisphosphatase (FBPase)] 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 may also be referred to as FBP or FBP1. FBPase is not particularly limited, and may be derived from animals, plants, or microorganisms, for example, rabbit or chicken.

[0068] [An enzyme that catalyzes the reaction of ADP to AMP] In one embodiment, the liquid composition described herein comprises an enzyme that catalyzes a reaction that produces AMP from ADP. The enzyme that catalyzes the reaction that produces AMP from ADP converts ADP present in the system to AMP. When the enzyme that produces AMP from ADP and the enzyme that produces ATP from AMP (e.g., PPDK) are combined, ADP is converted into AMP and AMP is converted into ATP, and as a result, ATP, ADP, and AMP can be measured. Known enzymes that catalyze the reaction of producing AMP from ADP can be used, including, but not limited to, ADP-dependent hexokinase, apyrase, and combinations thereof.

[0069] [ADP-dependent hexokinase] 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

[0070] [Apyrase] Apyrase (EC 3.6.1.5), also known as adenosine diphosphatase, ADPase, ATP diphosphatase, or ATP diphosphohydrolase, catalyzes the following two reactions: ATP + HO → ADP + phosphate (P) ADP + HO → AMP + phosphate (P)

[0071] [An enzyme that catalyzes the reaction of generating ADP from AMP] In one embodiment, the liquid composition described herein contains an enzyme that catalyzes a reaction that generates ADP from AMP. The enzyme that catalyzes the reaction that generates ADP from AMP converts AMP present in the system to ADP. Furthermore, when the enzyme that generates ADP from AMP is combined with an enzyme (e.g., PK) that generates ATP from ADP, AMP is converted into ADP and ADP is converted into ATP, and as a result, ATP, ADP, and AMP can be measured. The enzyme that catalyzes the reaction of producing ADP from AMP can be any known enzyme, including, but not limited to, adenylate kinase (ADK).

[0072] [Adenylate kinase (ADK)] 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 This reaction is reversible. ADK is an example of an enzyme that catalyzes the reaction of producing ADP from AMP. There are no particular limitations on the ADK, but examples include those derived from microorganisms such as yeast, and those derived from animals such as rabbits, pigs, cows, rats, and pigs.

[0073] [RNA degrading enzyme] In one embodiment, the kit of the present invention may include an RNase. Also, in one embodiment, the method of the present invention may use an RNase. Note that the RNase referred to here means an RNase that is not derived from the sample.

[0074] In one embodiment, the liquid composition described herein may contain RNase. In this specification, RNase refers to an RNase that is not derived from the sample. By using RNase, RNA can be decomposed into AMP, and the purity of RNA can be measured widely.

[0075] As used herein, RNase refers to an enzyme that catalyzes the synthesis of 5'-mononucleotides (AMP, GMP, CMP, and UMP) from RNA, including, for example, the following: (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). Endonuclease S1 includes Nuclease P1, Mung bean nuclease, and Neurospora crassa nuclease.

[0076] In this specification, the above-mentioned enzymes that catalyze the reaction of producing ATP from AMP (e.g., PPDK and PWDK), enzymes that catalyze the reaction of producing ATP from ADP, enzymes that catalyze the reaction of producing AMP from ADP, and enzymes that catalyze the reaction of producing ADP from AMP (e.g., ADK) may be collectively referred to as enzymes capable of producing ATP.

[0077] The ATP-producing enzyme may be any known enzyme derived from microorganisms, bacteria, eukaryotes, protists, plants, animals, etc., and may be, for example, a commercially available enzyme. The amount of enzyme to be added may be appropriately determined depending on the target concentration and reaction system.

[0078] Various enzymes are known to have the ability to generate ATP. In this specification, we focus on the ATP-generating ability of enzymes and define the activity unit (U) of an enzyme capable of generating ATP as the amount of enzyme that converts 1.0 μmol of substrate to ATP per minute at 37°C and pH 7.8 (1 U = 1 μmol ATP / min, pH 7.8, 37°C). The activity unit (U) of an enzyme capable of generating ATP is generally defined as above, except for enzymes that catalyze the reaction of generating ATP from ADP (e.g., PK), whose 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 (1 U = 1 μmol ATP / min, pH 7.4, 25°C). In one embodiment, the enzyme capable of generating ATP can be added so that its activity unit in the measurement system is 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 one embodiment, the enzyme capable of generating ATP can be added so that its activity unit in the measurement system is 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. Those skilled in the art can appropriately determine the amount of the enzyme to be added. In one embodiment, the concentration of the enzyme capable of generating ATP in the liquid composition described herein (if multiple enzymes are included, the concentration of each enzyme or the total concentration of the enzymes, the same applies hereinafter) may 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 an enzyme (e.g., PPDK) that catalyzes the reaction of producing ATP from AMP may be 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, and the concentration of an enzyme (e.g., PK) that catalyzes the reaction of producing ATP from ADP may be 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.A lower concentration of the enzyme capable of generating ATP can improve the stability of the liquid composition, but since the overall amount of luminescence decreases, if the amount of luminescence is too low, an enzyme capable of generating ATP may be added before or during measurement. The concentration of the enzyme capable of generating ATP (e.g., an enzyme that catalyzes the reaction of producing ATP from AMP (e.g., PPDK) and / or an enzyme that catalyzes the reaction of producing ATP from ADP (e.g., PK)) in the liquid composition during storage or after adding the enzyme capable of generating ATP before or during measurement 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.

[0079] When an enzyme capable of generating ATP is used, a substrate for each enzyme (e.g., a substrate for an enzyme that catalyzes the reaction of generating ATP from AMP, a substrate for an enzyme that catalyzes the reaction of generating ATP from ADP, a substrate for an enzyme that catalyzes the reaction of generating AMP from ADP, or a substrate for an enzyme that catalyzes the reaction of generating ADP or ATP from AMP) can be added. Note that different enzymes (e.g., an enzyme that catalyzes the reaction of generating ATP from AMP and an enzyme that catalyzes the reaction of generating ATP from ADP) may share a substrate. Furthermore, depending on the type of enzyme, it may not be necessary to add an additional component corresponding to the substrate. For example, when the enzyme that catalyzes the reaction of generating ADP from AMP is ADK, the enzymatic reaction is a reaction that generates ADP from ATP and AMP, and therefore, there is no need to include an additional substrate for the enzymatic reaction in the liquid composition. Substrates for PPDK include, but are not limited to, phosphoenolpyruvate or a salt thereof and pyrophosphate or a salt thereof; and for PK, AK, CK, PPK, and FMNK, substrates include phosphoenolpyruvate or a salt thereof, acetylphosphate or a salt thereof, creatine phosphate or a salt thereof, polyphosphate or a salt thereof, and riboflavin phosphate or a salt thereof, respectively. Substrates for PFK1 and FBPase include fructose-1,6-bisphosphate or a salt thereof. Substrates for PWDK include phosphoenolpyruvate or a salt thereof and phosphoric acid or a salt thereof, and substrates for ADP-dependent hexokinase include glucose. 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 (when multiple substrates are included, the concentration of each substrate or the total concentration of the substrates) may be, for example, 4 mM or less, 3 mM or less, 2.5 mM or less, 2 mM or less, 1.5 mM or less, 1.2 mM or less, 1 mM or less, 0.5 mM or less, 0.1 mM or less, 0.05 mM or less, 0.01 mM or less, 0.001 mM or less, or 0.0001 mM or less. A lower substrate concentration may improve the stability of the liquid composition, but will result in a lower overall luminescence level. Therefore, if the luminescence level is too low, additional substrate may be added before or during measurement.The concentration of the substrate in the liquid composition during storage or after adding the substrate before or during measurement may 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.

[0080] The concentration of a substrate (e.g., phosphoenolpyruvate, pyrophosphate, or a salt thereof) contained in a liquid composition can be measured based on an enzymatic reaction. For example, the concentrations of phosphoenolpyruvate and pyrophosphate can be measured by using PPDK to generate pyruvate from AMP, phosphoenolpyruvate, and pyrophosphate as substrates, reacting with lactate dehydrogenase and β-NADH, and measuring the absorbance at 340 nm. Alternatively, the concentrations of phosphoenolpyruvate and pyrophosphate can be measured by using PPDK to generate ATP from AMP, phosphoenolpyruvate, and pyrophosphate as substrates, and measuring luminescence using luciferase. For example, the concentration of pyrophosphate can be measured by using ATP sulfurylase to generate ATP from pyrophosphate as a substrate, and measuring the amount of luminescence using luciferase.

[0081] [Phosphoenolpyruvate (PEP)] In one embodiment, the liquid compositions described herein include phosphoenolpyruvate (PEP) or a salt thereof. Optionally, adding an excess of PEP or a salt thereof to the system can facilitate measuring the ATP and AMP present in the system.

[0082] [Pyrophosphate (PPi)] In one embodiment, the liquid compositions described herein include pyrophosphate (PPi) or a salt thereof. Optionally, adding an excess of PPi or a salt thereof to the system can facilitate the measurement of ATP and AMP present in the system.

[0083] [Cofactor] In one embodiment, the liquid compositions described herein include a cofactor. A cofactor refers to a chemical substance other than protein that is necessary for the catalytic activity of an enzyme. Non-limiting examples of cofactors include metal salts, vitamins and their derivatives, non-vitamin coenzymes, and organic prosthetic molecule groups, such as metal salts.

[0084] For example, cofactors for luciferase include metal salts, such as divalent metal ions such as magnesium and calcium, or manganese salts (e.g., magnesium acetate). Cofactors for PPDK include metal salts, such as magnesium. Those skilled in the art can determine the type and concentration of cofactors (e.g., metal salts) depending on the enzyme used.

[0085] In one embodiment, the concentration of a cofactor (e.g., a metal salt such as magnesium) in the liquid composition described herein may be, for example, 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, 8 mM or less, 6 mM or less, 4 mM or less, 2 mM or less, 1 mM or less, 0.5 mM or less, 0.1 mM or less, or 0.05 mM or less. A lower cofactor concentration may improve the stability of the liquid composition, but may also result in a lower overall luminescence. Therefore, if the luminescence is too low, additional cofactor may be added before or during measurement. The concentration of the cofactor in the liquid composition during storage or after addition of the cofactor before or during measurement may be, for example, 0.0001 mM or more, 0.001 mM or more, 0.01 mM or more, 0.1 mM or more, 1 mM or more, or 5 mM or more.

[0086] The concentration of the cofactor contained in the liquid composition can be measured by a general method known to those skilled in the art. For example, the liquid composition can be fractionated by chromatography such as HPLC, a peak corresponding to the cofactor is detected, and the peak intensity is compared with that of a standard substance containing a known concentration of the cofactor to measure the cofactor concentration. Alternatively, when the cofactor is a metal salt, the concentration can be measured by ICP atomic emission spectroscopy.

[0087] In one embodiment, the liquid compositions described herein contain an enzyme stabilizer, such as bovine serum albumin or gelatin, which protects reporter molecules such as luciferase from degradation. In one embodiment, the liquid compositions described herein contain a substance that adjusts pH or improves storage stability. Examples of such substances include pH buffers (e.g., HEPES, Tricine, Tris, phosphate buffer, acetate buffer), reducing agents (e.g., dithiothreitol (DTT), 2-mercaptoethanol), and sugars (e.g., glucose, sucrose, trehalose).

[0088] [Addition of ingredients before or during measurement] In one embodiment, the liquid composition does not contain at least one component necessary for the cycling reaction, and the component not contained in the liquid composition is added to the liquid composition before or during measurement. In another embodiment, the liquid composition contains at least one component necessary for the cycling reaction at a concentration so low that the cycling reaction proceeds little or not at all, and the component contained in the liquid composition at a low concentration is added to the liquid composition before or during measurement.

[0089] As used herein, the term "cycling 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.

[0090] In the present specification, "components necessary for the cycling reaction" include, for example, luciferase, luciferin, an enzyme that catalyzes the reaction of producing ATP from AMP or its substrate, and cofactors in the liquid compositions of the first and third embodiments. "components necessary for the cycling reaction" include, for example, luciferase, luciferin, an enzyme that catalyzes the reaction of producing ADP from AMP or its substrate, an enzyme that catalyzes the reaction of producing ATP from ADP or its substrate, and cofactors in the liquid compositions of the second and fourth embodiments.

[0091] In one embodiment, the compositions described herein do not contain at least one (e.g., one, two, three, four, five, or all) of these components, and contain other components, and the components not included in the liquid composition are added to the liquid composition before or during the measurement.

[0092] As used herein, "before measurement" is not limited as long as the stability of the liquid composition is improved, but may be, for example, 30 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, 10 seconds, or immediately before measuring ATP, and AMP and / or ADP. "At the time of measurement" means simultaneously with measurement. The shorter the time between adding the components necessary for the cycling reaction and measurement, the shorter the time for the cycling reaction to occur, and the more likely the stability of the liquid composition will be improved.

[0093] [sample] In this specification, the type of "sample" is not limited, but may be, for example, a biological sample or biological instrument, a blood sample or blood instrument, or a cooking instrument.

[0094] As used herein, the term "biologically relevant sample" encompasses any sample to which a biologically derived substance may be attached. As used herein, the term "biologically relevant instrument" refers to any instrument to which a biologically derived substance may be attached or remain. As used herein, the environment from which a biologically relevant sample or biologically relevant instrument originates includes an environment to which a biologically derived liquid may be attached or remain. Such environments include, but are not limited to, clothing, protective equipment such as gloves, hands, fingers, beds, switches, doorknobs, bed rails, nurse call buttons, handrails, washrooms, washbasins, toilets, and lavatories. The biologically derived substance may be derived from humans or animals. In one embodiment, the biologically derived substance is derived from humans. In one embodiment, the biologically relevant sample does not include a non-human animal-derived sample but includes a human-derived sample. In one embodiment, the biologically relevant instrument does not include a non-human animal-related instrument but includes a human body-related instrument.

[0095] Substances of biological origin include liquids and solids. Liquids include, but are not limited to, body fluids, blood, lymph, sweat, nasal mucus, tears, saliva, digestive fluid, tissue fluid, ascites, amniotic fluid, cerebrospinal fluid, urine, feces, vomit, and sebum. Solids include, but are not limited to, originally solid substances such as tissue fragments, flesh fragments, and cells, as well as solidified liquids, coagulated blood, excrement, dirt, eye mucus, and scabs.

[0096] Examples of biological instruments include medical instruments, such as surgical instruments, endoscopes (e.g., upper endoscopes used to examine the esophagus, stomach, and duodenum, lower endoscopes used to examine the rectum and large intestine, or double-balloon enteroscopes, preferably lower endoscopes), catheters, scalpels, tubes inserted into a patient's body, instruments inserted into a patient's body, surgical instrument washing tanks, and medical instrument washing environments.

[0097] As used herein, the term "blood-related sample" encompasses any sample to which blood may be attached. As used herein, the term "blood-related instrument" refers to any instrument to which blood may be attached or remain. Examples of blood-related instruments include medical instruments to which blood may be attached or remain. Examples of such instruments include surgical instruments, endoscopes (e.g., upper endoscopes used to examine the esophagus, stomach, and duodenum, lower endoscopes used to examine the rectum and large intestine, or double-balloon enteroscopes, preferably lower endoscopes), catheters, scalpels, tubes inserted into a patient's body, instruments inserted into a patient's body, surgical instrument washing tanks, and medical instrument washing environments. As used herein, the environment from which blood-related samples or blood-related instruments originate includes environments to which blood may be attached or remain. Examples of such environments include operating tables, washing tanks, protective equipment such as clothing and gloves, hands, fingers, beds, handrails, washrooms, washbasins, and medical facilities. Other examples include accident or injury scenes, and sites where blood is searched for. Blood may originate from humans or animals. In one embodiment, the blood is derived from humans. In one embodiment, blood does not include blood from animal or fish meat associated with food products.

[0098] Examples of blood include whole blood, serum, plasma, blood for transfusion, collected primary blood, diluted solutions of primary blood, etc. Blood-related samples also include solutions containing blood cells (white blood cells, red blood cells, platelets) or samples to which such solutions may be attached.

[0099] In one embodiment, the blood-related sample does not include the collected blood itself (for convenience, referred to as a primary sample). For example, in this embodiment, the "solution containing blood cells" included in the blood-related sample does not include blood itself. In one embodiment, the blood-related sample refers to a secondary sample derived from an instrument or environment that has come into contact with the primary sample. A secondary sample may be obtained by wiping an instrument or 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 the secondary sample for the presence or residual blood. In one embodiment, the blood-related sample may be a sample in which the existing blood has been diluted by a washing process or the like.

[0100] As used herein, "cooking-related utensils" refers to cooking utensils and the environment of a cooking site or items in that environment that may be contaminated. Examples of cooking utensils include, but are not limited to, cutting boards, pots, frying pans, pressure cookers, griddles, plates, knives, chopsticks, chopsticks, spoons, forks, knives, and other tableware; ladles, strainers, colanders, racks, cutting board stands, containers for storing cooking utensils, packaging containers, and packaging sheets; and other cooking utensils and related utensils. The cooking site environment refers to the environment of a cooking site, food processing plant, food service facility, etc., and examples of items in that environment include, but are not limited to, equipment such as blending tanks, piping, filling nozzles, and conveyor belts in food processing plants, as well as containers, doorknobs, handles, switches, and telephone receivers of appliances such as refrigerators and ovens that are frequently touched by human hands.

[0101] [Storage period] The liquid compositions described herein are intended for measuring ATP, AMP, and / or ADP in a sample after storage. The term "storage" as used herein refers not only to the period from preparation of a liquid composition until its use for measurement, but also to the storage period of the liquid composition when a liquid composition is produced during the production of a powder composition. In this case, the liquid composition described herein may be stored for a certain period after production, converted into a powder composition by a drying process, and then converted back into a liquid composition (optionally after being stored again) and used to measure ATP, AMP, and / or ADP in a sample. The storage period of the liquid composition (or the total period if the liquid composition is stored multiple times) is not limited, and may be, for example, 6 hours or more, 12 hours or more, 18 hours or more, 1 day or more, 2 days or more, 3 days or more, 7 days or more, 14 days or more, 30 days or more, 60 days or more, 90 days or more, 120 days or more, 150 days or more, 180 days or more, 210 days or more, 240 days or more, 270 days or more, or 300 days or more. The storage period may be 600 days or less, 500 days or less, or 400 days or less. The liquid compositions described herein may have improved stability, and the effect of the improved stability may be more clearly demonstrated over a longer storage period.

[0102] [kit] In a fifth aspect, the present invention relates to a kit for measuring ATP in a sample, comprising the liquid composition described herein. In addition to the liquid composition described herein, the kit of this aspect may also comprise at least one of 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, an instrument required for the test, a control, and an instruction manual.

[0103] In one embodiment, the kit may include a sample collection unit and a reaction unit. The sample collection unit is not particularly limited as long as it can collect a sample, and examples thereof include a cotton swab, a sponge, porous plastic, filter paper, nonwoven fabric, and a dropper. From the viewpoint of ease of sample collection, the sample collection unit is preferably, for example, rod-shaped, and particularly preferably has a rod-shaped shape with a fibrous or sponge-like wiping portion, such as a shape like a cotton swab.

[0104] The reaction zone is a site where a reaction occurs when ATP or its degradation products are present in the sample collected by the sample collection zone. In one embodiment, the reaction zone contains a liquid composition described herein. The reaction zone is preferably a transparent container, which allows the amount of luminescence to be measured directly.

[0105] In one embodiment, the kit may include other sites, such as a storage site or extraction site, in addition to the sample collection site and the reaction site. The storage site is a site for storing at least one component (e.g., luciferin) necessary for the cycling reaction when the liquid composition described herein does not contain the component. The extraction site is a site for extracting ATP or its degradation products into an extraction solution when ATP or its degradation products are present in the sample collected by the sample collection site. In one embodiment, the extraction site may contain an extraction solution. The sample extracted in the extraction site can be transferred to the reaction site, where the reaction can be carried out.

[0106] If the liquid composition described herein does not contain at least one component necessary for the cycling reaction, the 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 a reaction section, and the component not included in the liquid composition may be contained in a sampling section, storage section, extraction section, or other section isolated from the liquid composition.

[0107] [method] In a sixth aspect, the present invention relates to a method for measuring ATP, as well as AMP and / or ADP in a sample, comprising using a liquid composition as described herein, or a kit as described herein, or to the use of a liquid composition as described herein, or a kit as described herein, for measuring ATP, as well as AMP and / or ADP in a sample.

[0108] For example, but not limited to, ATP, AMP, and / or ADP in a sample can be measured by adding a sample solution containing ATP to the liquid composition described herein and measuring luminescence. The liquid composition and the sample solution may be the same or different in volume. The volumes of the liquid composition and the sample solution are not limited, but may 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. The sample solution may be, but is not limited to, a suspension of a sample wiped from a cotton swab or the like in an extracting solution.

[0109] The amount of luminescence can be measured using a known luminometer (e.g., Lumitester Smart, Lumitester PD-20, Lumitester PD-30, manufactured by Kikkoman Biochemifa Corporation), a device equipped with a photodiode (e.g., SystemSURE Plus, EnSURE, manufactured by Hygiena, AccuPoint Advanced, manufactured by Neogen), or a device equipped with a photomultiplier tube (e.g., Cleantrace LM1, Cleantrace UNG3, manufactured by 3M Corporation; Lumitester C-110, Lumitester C-100, manufactured by Kikkoman Biochemifa Corporation; or Junior LB9509, CentroLB960, or Lumat3 LB9508, manufactured by Berthold). Luminescence can be expressed as relative luminescence units (RLU) based on a specific standard. The relative luminescence unit value can be used directly for cleanliness control (e.g., when samples are collected using a rod-shaped sampler with a wipe-off portion). 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 an ATP standard solution, an ADP standard solution, and an AMP standard solution. When the relative luminescence units obtained by measuring only a sample without using a standard solution of ATP, ADP, and / or AMP (hereinafter also referred to as ATP, etc.) are directly utilized for cleanliness control or the like, a decrease in luminescence intensity due to the instability of the liquid composition directly affects the test results, so the use of the composition described herein, which has excellent stability, is particularly advantageous.

[0110] In another embodiment, a calibration curve may be prepared using a substrate solution containing a known concentration of ATP or the like, and then the liquid composition described herein may be added to a sample solution containing an unknown concentration of ATP or the like, and the luminescence may be measured under the same conditions to measure the concentration of ATP or the like in the sample solution. In this embodiment, even if the amount of luminescence decreases due to instability of the liquid composition, the degree to which the decrease in luminescence becomes a problem can be relatively reduced by preparing a calibration curve for ATP or the like using a liquid composition under the same conditions each time. [Example]

[0111] The present invention will be explained in more detail below using examples, but the technical scope of the present invention is not limited to these examples in any way.

[0112] <Example 1: Luciferase concentration measurement> (280nm absorbance method) The absorbance at 280 nm of the sample solution appropriately diluted with PBS was measured using an absorption spectrophotometer U-3900 (Hitachi High-Tech Science Corporation) and was taken as the luciferase concentration (mg protein / mL). (Bradford Act) Protein concentration was measured using a Coomassie (Bradford) Protein Assay Kit (Thermo Scientific) based on the Bradford method. Specifically, 100 μL of protein assay reagent (Quick Start Bradford 1× Dye Reagent, Thermo Scientific) was added to 100 μL of sample solution appropriately diluted with PBS, and the absorbance at 595 nm was measured using a Microplate Reader SH-9000 (Corona Electric). Measurements were performed using 2 mg / mL Albumin Standard Ampules included with the kit as a standard protein instead of the sample solution, and a calibration curve was created to determine the amount of luciferase. (Conversion between 280nm absorbance method and Bradford method) As a result of the measurement, 1 mg protein / mL in the 280 nm absorbance method was equivalent to 1.77 mg / mL in the Bradford method.

[0113] <Example 2: Preparation of cycling luminescence reagent and background luminescence amount> A new solution with a basic composition was designed based on an alternative composition for Lucipack Pen (a kit manufactured by Kikkoman Biochemifa Corporation (hereinafter the same), which is used by adding an extraction reagent (solution) to a powdered luminescence reagent) described in paragraph

[0094] of International Publication No. 2018 / 147443. The alternative composition of Lucipack Pen is as follows:

[0114] <Alternative composition of Lucipack Pen> 7 mM magnesium acetate, 0.5 mM luciferin, 25 mM tricine, 0.2 mg protein / mL luciferase (value at absorbance at 280 nm. Hereinafter, this will be expressed as 0.35 mg / mL, which is the value obtained by the Bradford method based on Example 1), 0.2 mM potassium pyrophosphate, 1.4 mM potassium phosphoenolpyruvate, 1.3 U / mL PPDK According to the Lucipack Pen instruction manual, the stainless steel surface was wiped with a cotton swab moistened with water, and then the cotton swab was pressed into the surface, allowing the powdered luminescent reagent to dissolve in the extracted reagent (solution). The volume of the solution was 0.35 mL, and the pH was 7.8. Based on these results and the composition of the Lucipack Pen described above, a new solution with the basic composition was designed as follows:

[0115] <Basic composition> 7 mM magnesium acetate, 0.5 mM luciferin (Biosynth, same below), 25 mM tricine, 0.35 mg / mL (Bradford method) luciferase (HLK described in JP-A-11-239493, same below), 0.2 mM potassium pyrophosphate, 1.4 mM potassium phosphoenolpyruvate, 1.3 U / mL PPDK (PPDK described in JP-A-8-168375, Kikkoman Biochemifa, same below), pH 7.8, liquid volume per luminescence measurement container: 0.35 mL A luminescence reagent with the above basic composition was prepared, and 0.35 mL was measured into a Lucipack Pen luminescence reagent container (measurement tube, upper part 10φ x height 35 mm, same below). This was attached to the main body with a cotton swab holder, and measured with a Lumitester Smart (manufactured by Kikkoman Biochemifa, same below). The luminescence at this time was taken as the background luminescence. To convert the background luminescence into an ATP concentration, a control of 1 x 10 -5 0.01 mL of ATP (manufactured by Oriental Yeast Co., Ltd.; the same applies hereinafter) solution was added to M, and the luminescence intensity was measured 10 seconds after addition using a Lumitester Smart. The luminescence intensity at this time was defined as the luminescence intensity upon addition of ATP. The delta luminescence intensity was calculated by subtracting the background luminescence intensity from the luminescence intensity upon addition of ATP.

[0116] 1×10 -5 When 0.01 mL of M ATP solution is added, the ATP concentration (M) in the solution (total 0.36 mL) is 2.77 × 10 -7 Therefore, the background ATP+AMP concentration was calculated based on the following formula: Background ATP + AMP concentration (M) = Background luminescence (RLU) / Δ luminescence (RLU) × 2.77 × 10 -7 (M) Table 1 shows the background light unit (RLU), light unit upon addition of ATP (RLU), delta light unit (RLU), and background ATP + AMP concentration (M). RLU is an abbreviation for Relative Light Units.

[0117] [Table 1]

[0118] The background luminescence is thought to be due to ATP or AMP contaminated from the reagents, containers, and equipment during preparation. When prepared with the usual care of a person skilled in the art, the luminescence is approximately 500 RLU, which, as shown in Table 1, corresponds to an ATP + AMP concentration in the luminescence reagent of 2.7 × 10 -10This concentration corresponds to approximately 1 × 10 M (0.27 nM) in 0.35 mL of the reagent. -8 This corresponds to the concentration when 0.01 mL of ATP solution is added.

[0119] In addition, in reagents that measure the three components of ATP + ADP + AMP by adding an enzyme that converts ADP to ATP or AMP, ADP contaminated in the reagent is also measured, and since an enzyme that may cause contamination of ATP, ADP, and AMP is added, it is thought that the background luminescence will be even higher.

[0120] <Example 3: Change in luminescence amount over time in a luminescence reagent without cycling> To prepare the non-cycling luminescence reagent, the basic composition was prepared by removing PPDK, an enzyme required for cycling. 0.35 mL of the prepared solution was measured into the LuciPak Pen luminescence reagent container and diluted to 1 x 10 -5 0.01 mL of ATP solution was added to M, the cotton swab holder was attached to the main body, and measurements were taken using the Lumitester Smart. The time course of luminescence intensity for the non-cycling luminescence reagent is shown in Figure 1. As shown in Figure 1, in the non-cycling luminescence reagent, the added ATP was quickly consumed in the luminescence reaction caused by the luciferin-luciferase reaction, and the luminescence intensity rapidly decayed, with the basic composition not containing PPDK almost quenching in about 2 minutes.

[0121] <Example 4: Stability of cycling luminescence reagents containing various concentrations of luciferin> Following the basic composition, the cycling luminescence reagent containing luciferin at each concentration was prepared by varying only the luciferin concentration. -6 M ATP solution was added to 3.5 mL of cycling luminescence reagent (each luciferin concentration) and allowed to stand at 25°C. At each time point, 0.36 mL was measured into a LuciPak Pen luminescence reagent container, attached to the main unit with a cotton swab holder, and measured using a Lumitester Smart. The luminescence at this time was taken as the background luminescence. To further confirm the stability of the luminescence, 1 × 10-5 0.01 mL of ATP solution was added to the sample, and the luminescence intensity was measured 10 seconds after addition using a Lumitester Smart. The luminescence intensity at this time was defined as the luminescence intensity upon ATP addition, and the difference between this luminescence intensity and the background luminescence intensity was defined as the delta luminescence intensity.

[0122] The time course of the luminescence intensity of the cycling luminescence reagent containing luciferin at each concentration is shown in Figure 2. Furthermore, the luminescence intensity after 9 hours for the cycling reagent containing luciferin at each concentration is shown in Figure 3.

[0123] In the basic composition, a decrease in Δ luminescence intensity upon addition of ATP was observed over time. This indicated that the cycling reagent was unstable. Furthermore, the decrease in Δ luminescence intensity upon addition of ATP was greater with increasing luciferin concentration. Lowering the luciferin concentration below the basic composition tended to suppress the decrease in luminescence intensity over time. Typically, in measurement systems using enzymatic reactions, high concentrations of substrate are added to prevent the reaction rate from being affected even if the substrate decreases as the enzymatic reaction progresses. In particular, in cycling reactions, substrate is continuously consumed, so adding a higher concentration leads to reagent stability, and it is considered appropriate to add a higher concentration of substrate. However, surprisingly, the results of this example revealed that in cycling luminescent reagents utilizing the luciferin-luciferase reaction, the luminescence intensity of the cycling luminescent reagent can be stabilized by lowering the luciferin concentration.

[0124] Example 5: Stability of luciferin-free cycling luminescence reagent Next, we confirmed the effect of improving stability by storing the reagent without luciferin and adding luciferin immediately before the reaction. A luciferin-free cycling luminescence reagent was prepared according to the basic composition, and 0.1 mL of 1 × 10 ATP was added to 3.5 mL of the reagent, assuming ATP contamination. -6 M ATP solution was added (hereinafter referred to as the mixed solution).

[0125] (1) For "Storage with luciferin," 0.1 mL of 17.5 mM luciferin solution (pH 7.8) (final concentration 0.5 mM) was added to 3.6 mL of the above mixture, and the mixture was further stored at 25°C. After each storage time, 0.37 mL of the mixture was measured into the luminescence reagent container of the LuciPack Pen, which was then attached to the main body equipped with a cotton swab holder, and the background luminescence was measured using the Lumitester Smart. Furthermore, to confirm the stability of the luminescence intensity, 1 × 10 -5 0.01 mL of ATP solution (M) was added, and the luminescence intensity after 10 seconds was measured using a Lumitester Smart. This was defined as the luminescence intensity upon addition of ATP. The difference between the luminescence intensity upon addition of ATP and the background luminescence intensity was defined as the delta luminescence intensity.

[0126] (2) For "Stored without luciferin," 3.6 mL of the above mixture was stored at 25°C without adding luciferin solution. After each storage time, 0.36 mL was measured and placed in a LuciPack Pen luminescence reagent container, and 0.01 mL of 17.5 mM luciferin solution (pH 7.8) (final concentration 0.5 mM) stored at 25°C was added. The background luminescence was measured, and then 1 × 10 -5 0.01 mL of ATP solution was added, and the luminescence intensity 10 seconds after addition was measured using a Lumitester Smart. This was defined as the luminescence intensity upon ATP addition. The difference between the luminescence intensity upon ATP addition and the background luminescence intensity was defined as the delta luminescence intensity.

[0127] The stability of the luminescence reagent with and without luciferin is shown in Figure 4. As shown in Figure 4, in the reagent "stored with luciferin," the cycling reaction progressed during storage, and the luminescence intensity decreased, but in the reagent "stored without luciferin," the luminescence intensity did not decrease during storage and remained constant, greatly improving stability.

[0128] Example 6: Stability of luciferin-free luminescence reagents in non-cycling conditions For a reagent that does not contain PPDK in its basic composition, i.e., a luminescence reagent that is not cycled, the stability of the luminescence intensity when stored (1) with luciferin and (2) without luciferin was confirmed, as in Example 5, and the residual luminescence intensity after 3 hours of storage was shown. The residual luminescence intensity after 3 hours of storage with cycling (Example 5) was also shown. The results are shown in Figure 5. No decrease in luminescence intensity was observed in any of the luminescence reagents stored without luciferin and to which luciferin was added immediately before measurement. However, among the reagents stored with luciferin, a decrease in luminescence intensity was observed only in the case of the cycling luminescence reagent containing PPDK, but no decrease in luminescence intensity was observed in the non-cycling reagent containing PPDK. In other words, the decrease in luminescence intensity observed in this example is a phenomenon that does not occur with luminescence reagents that utilize a luciferin-luciferase reaction and that measure only non-cycling ATP, and is thought to occur only when luminescence reagents that utilize a cycling reaction are stored in liquid form.

[0129] <Example 7: Stability of luminescence intensity in cycling luminescence reagents containing luciferase at various concentrations> Examples 3 to 6 demonstrated that suppressing the luminescence intensity is important for improving the stability of the luminescence intensity of a cycling luminescent reagent. Since the luminescence intensity can also be suppressed by reducing the luciferase concentration, the relationship between the stability of the luminescence intensity and the luciferase concentration in a cycling luminescent reagent was investigated.

[0130] Prepare cycling luminescence reagents containing luciferase at various concentrations according to the basic composition, but varying the luciferase concentration. -6 M ATP solution was added to 3.5 mL of cycling luminescence reagent and allowed to stand at 25°C. At each time point, 0.36 mL was measured into a Lucipack Pen luminescence reagent container, attached to the main body with a cotton swab holder, and measured using a Lumitester Smart. The luminescence at this time was taken as the background luminescence. Furthermore, to confirm the stability of the luminescence, 1 × 10 ATP solution was used as a control.-5 0.01 mL of ATP solution was added to the sample, and the luminescence intensity was measured 10 seconds after addition using a Lumitester Smart. The luminescence intensity at this time was defined as the luminescence intensity upon ATP addition, and the difference between this luminescence intensity and the background luminescence intensity was defined as the delta luminescence intensity.

[0131] The residual luminescence intensity of the cycling luminescence reagent containing luciferase at each concentration is shown in Figure 6. Furthermore, the luminescence intensity after 9 hours for the cycling reagent with each luciferase concentration in Figure 6 is shown in Figure 7.

[0132] As shown in FIG. 6, the higher the luciferase concentration, the more the luminescence intensity decreased over time, and the decrease in luminescence intensity tended to be suppressed as the luciferase concentration was reduced below that of the basic composition.

[0133] Example 8: ATP concentration dependence of decrease in luminescence intensity in cycling luminescence reagent In the case of a cycling luminescent reagent, if ATP is present in the measurement system, luminescence will normally continue at a constant level unless the substrate luciferin is depleted. Similar experimental results can be obtained by adding ATP before storage and observing the change in luminescence over time, even without adding ATP after storage as in Examples 4 to 7, making it easy to examine the stability of the luminescent reagent. In this example, in order to construct an experimental system that does not require the addition of ATP after storage, we investigated the appropriate ATP concentration when no ATP solution is added after storage.

[0134] Prepare the cycling luminescence reagent according to the basic composition, measure 0.35 mL into the luminescence reagent container of the LuciPak Pen, and then add 1 x 10 -5 ~1×10 -8 0.01 mL of ATP solution or sterile ultrapure water was added, and the luminescence intensity was measured over time using a Lumitester Smart. The luminescence intensity when each concentration of ATP was added minus the luminescence intensity in the sample to which sterile ultrapure water was added was calculated as Δluminescence intensity, and this is shown in each graph.

[0135] The results are shown in Figure 8. The attenuation of luminescence intensity correlated with the ATP concentration, with a tendency for the attenuation to be greater as the ATP concentration increased and smaller as the ATP concentration decreased.

[0136] The amount of ATP assumed to be contaminated in the cycling luminescence reagent prepared in Example 2 was approximately 1 × 10 -8 This corresponds to the addition of 0.01 mL of ATP, and with the presence of ATP at that concentration, no decay in luminescence is observed within 2 hours. Therefore, if the cycling luminescence reagent is used up in a short period of time, the decay in luminescence is not a major problem, but it may become a greater problem if stored for a long period of time. For example, if the decay in luminescence is proportional to the amount of luciferin consumed, i.e., the amount of luminescence, then 1 × 10 -5 When ATP was added, the luminescence intensity decreased to 30% in 2 hours. -6 When M ATP was added, the reaction time was 20 h and 1 x 10 -7 When M ATP was added, the reaction time was 200 hours (8.3 days), 1 × 10 -8 When ATP from M is mixed in, the luminescence intensity is expected to decrease to the same level in 2000 hours (83 days).

[0137] In the examples described herein, a high concentration of ATP solution was added to obtain results in a short time. Although the stability of the assay kit may be affected by factors such as the stability of the enzyme and the stability of the substrate itself, the luminescence intensity did not decrease at low concentrations of ATP, suggesting that evaluation is possible, at least in a short time, without being affected by the stability of the enzyme or substrate.

[0138] <Example 9: Luminescence intensity and stability of cycling luminescence reagents containing various concentrations of luciferin during storage> As shown in Example 8, the stability of the cycling luminescence reagent can be examined by adding ATP to the reagent and examining the change in luminescence over time. It was also found that adding a high concentration of ATP allows results to be obtained in a short period of time. Furthermore, Examples 3 to 6 and others revealed the importance of suppressing the luminescence reaction during storage. In this example, experiments were conducted to define the luminescence level under storage conditions that serve as a benchmark for suppressing the luminescence reaction during storage. First, the definition of luminescence level under storage conditions is presented, followed by the experimental results that served as the basis for this definition.

[0139] The luminescence unit (RLU) under storage conditions was calculated by adding 0.35 mL of the storage luminescence reagent to a measurement tube and measuring 1 × 10 -7 0.01 mL of ATP solution of M was added, and the mixture was allowed to stand at 25°C for 1 hour. The luminescence unit (RLU) was measured using a Lumitester Smart (Kikkoman Biochemifa Corporation).

[0140] The experiment to determine this definition is as follows: A cycling luminescence reagent containing luciferin at each concentration (components other than luciferin are the same as the basic composition) was prepared, and 0.35 mL was measured into the luminescence reagent container of the Lucipack Pen. -5 ~1×10 -8 0.01 mL of ATP solution or sterile ultrapure water was added, and the luminescence intensity was measured over time using a Lumitester Smart. The luminescence intensity when each concentration of ATP was added minus the luminescence intensity in the sample to which sterile ultrapure water was added was calculated as the delta luminescence intensity, and is shown in Figures 9 to 14.

[0141] As shown in Figures 9 and 10, 1 × 10 -5 When ATP (M) was added, a decrease in luminescence intensity was observed, and results were obtained similar to those in Example 4. This method confirmed that a short-term test can estimate long-term storage stability without being affected by decomposition of the enzyme or substrate itself.

[0142] When defining the luminescence level during storage, for example, when cycling is stopped, the luminescence level immediately after adding ATP is high as shown in Figure 1 of Example 3, and the effect of stopping cycling cannot be properly evaluated by simply measuring the luminescence level immediately after adding ATP. In contrast, for example, if ATP is added one hour later, luciferase will decompose ATP, so by examining the luminescence level one hour later, the luminescence level during storage, including the effect of stopping cycling, can be determined. However, as shown in Figures 9 to 12, the luminescence level at 1 x 10 -5 M or 1×10 -6 When 1 × 10 ATP solution was added, the luminescence intensity decreased over time, making it impossible to accurately measure the luminescence intensity during storage. -7 At 1×10 M, the luminescence intensity does not decrease for about 2 hours, so it is possible to examine the luminescence intensity during stable storage. -8 In the case of M, the Δluminescence intensity is about the same as the background luminescence intensity shown in Example 2, and is susceptible to this influence, so it is thought to be inappropriate (data not shown).

[0143] Based on the above results, the luminescence unit (RLU) under storage conditions was defined as above. The luminescence unit (RLU) under storage conditions according to this definition is shown in Table 2 below.

[0144] [Table 2]

[0145] Figure 10 shows that if the luciferin concentration is lowered below 0.5 mM (6000 RLU in luminescence output when stored, as shown in Table 2), the decrease in luminescence output is less than with the basic composition, and stability tends to be improved. Figure 10 also shows that if the luciferin concentration is 0.1 mM or less (2300 RLU in luminescence output when stored, as shown in Table 2), stability can be further improved.

[0146] <Example 10: Luminescence intensity and stability during storage of cycling luminescence reagents containing luciferase at various concentrations> Prepare a cycling luminescence reagent containing luciferase at each concentration (components other than luciferase are the same as the basic composition), measure 0.35 mL into the luminescence reagent container of the LuciPack Pen, and then add 1 x 10 -5 ~1×10 -8 0.01 mL of ATP solution or sterile ultrapure water was added, and the luminescence intensity was measured over time using a Lumitester Smart. The luminescence intensity when each concentration of ATP was added minus the luminescence intensity in the sample to which sterile ultrapure water was added was calculated as the delta luminescence intensity, and is shown in Figures 15 to 20.

[0147] 1×10 -5 A decrease in luminescence intensity was observed when ATP solution of 1×10 was added, and the same tendency as in Example 7 was obtained. This method confirmed that it is possible to examine long-term storage stability in a short time without being affected by the decomposition of the enzyme or the substrate itself. Furthermore, as shown in Figures 19 and 20, -7 Since no decay in luminescence intensity was observed within about 2 hours after addition of the ATP solution of M, this example also supports the validity of the definition of luminescence intensity under storage conditions shown in Example 9. The luminescence intensity (U) under storage conditions according to this definition is shown in Table 3 below.

[0148] [Table 3]

[0149] From Figure 15, it can be seen that if the luciferase concentration is lowered below 0.35 mg / mL (Table 3 shows that the luminescence intensity during storage is 6000 RLU or less), the decrease in luminescence intensity is less than that of the basic composition, and stability tends to be improved. Also, from Table 3, at improved concentrations (0.07 mg / mL or less), the luminescence intensity is 1 x 10 -7 The luminescence intensity of M after 1 hour of addition of the ATP solution was shown to be below 2300 RLU.

[0150] Example 11: Stability of luminescent reagents without cycling components We investigated whether stability could be improved by partially modifying the basic composition, removing some of the cycling-related substrates and enzymes, and adding them during measurement.

[0151] The luminescence reagent was divided into solutions A and B, and one of the components required for the luminescence reaction and cycling reaction (magnesium acetate, potassium phosphoenolpyruvate, potassium pyrophosphate, luciferin, luciferase, PPDK) was transferred to solution B to prepare the reagents. These were stored separately, and immediately before the reaction, 0.05 mL each was mixed, 0.1 mL of ATP solution was added, and the amount of luminescence was measured.

[0152] Luminescent reagent solution A was prepared in 50 mM Tricine, containing 24 mM magnesium acetate, 1.6 mM luciferin, 4 mM potassium phosphoenolpyruvate (PEP), 0.4 mM potassium pyrophosphate (PPi), 1.0 mg / mL luciferase (Bradford method), and 4.3 U / mL PPDK (pH 7.8), with one of the following components removed. Luminescent reagent solution B was prepared in 50 mM Tricine, containing 24 mM magnesium acetate, 4 mM PEP, 0.4 mM PPi, 1.6 mM luciferin, 1.0 mg / mL luciferase (Bradford method), and 4.3 U / mL PPDK (pH 7.8), with one of the following components removed from solution A: 24 mM magnesium acetate, 4 mM PEP, 0.4 mM PPi, 1.6 mM luciferin, 1.0 mg / mL luciferase (Bradford method), and 4.3 U / mL PPDK (pH 7.8).

[0153] The compositions of solutions A and B for the luciferase-free reagent, the PPDK-free reagent, the luciferin-free reagent, the Mg acetate-free reagent, the PEP-free reagent, and the PPi-free reagent are shown in Tables 4 to 9 below, respectively.

[0154] [Table 4]

[0155] [Table 5]

[0156] [Table 6]

[0157] [Table 7]

[0158] [Table 8]

[0159] [Table 9]

[0160] In the "mix and store" method, 0.5 mL of solution A is added to 0.5 mL of solution B, and 1 × 10 ATP is added to 0.5 mL of solution A. -5 0.01 mL of 1 × 10 ATP solution was added and stored at 25°C for 40 hours. After storage, 0.1 mL of the sample was collected and 0.1 mL of 1 × 10 -6 M ATP was added, and the luminescence intensity was measured 10 seconds after addition using a Lumitester Smart.

[0161] In the "mix after storage" case, 1 × 10 ATP was added to 0.5 mL of solution A, assuming that ATP was mixed in during preparation. -5 0.01 mL of ATP solution was added to solution B, and nothing was added to solution B. The solution was stored at 25°C for 40 hours. After storage, 0.05 mL of solution B containing ATP was added, and 0.1 mL of 1 × 10 -6 M ATP was added, and the luminescence intensity was measured 10 seconds after addition using a Lumitester Smart.

[0162] For comparison, 0.5 mL of solution A and 0.5 mL of solution B were mixed to obtain a 1 × 10 luminescence intensity before storage. -5 Add 0.01 mL of ATP solution (1 × 10) to the sample. -6 The amount of luminescence when M ATP was added is also shown in FIG.

[0163] It was found that when luciferase, PPDK, luciferin, magnesium acetate (Mg acetate), PEP, and PPi were "mixed and stored," the amount of luminescence decreased when ATP was added, while when they were "mixed after storage," a high amount of luminescence was maintained.

[0164] When luciferase, which is involved in the luminescence reaction, or PPDK, which is involved in the cycling reaction, were removed, the cycling reaction did not proceed and stability improved. Furthermore, stability improved when cofactors or substrates such as magnesium acetate, phosphoenolpyruvate, and pyrophosphate were removed. The roles of magnesium acetate, phosphoenolpyruvate, and pyrophosphate in the cycling reaction are as follows:

[0165] The magnesium ions in magnesium acetate are necessary factors for the luciferase and PPDK reactions, and the luminescence and cycling reactions do not proceed without these factors. Phosphoenolpyruvate is a substrate necessary for the PPDK reaction, and pyrophosphate is also a substrate necessary for the PPDK reaction. If phosphoenolpyruvate or pyrophosphate is omitted, the luminescence reaction proceeds for the amount of ATP present, but the luminescence is quenched once the ATP is consumed, and no further luminescence occurs.

[0166] These results indicate that storing the components required for luciferase reaction and cycling reaction separately is also effective in improving stability by stopping the reaction. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition contains luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added to the liquid composition before or during measurement; and (ii) the relative luminescence output of the liquid composition during storage is 5500 RLU or less; the relative light intensity is a value obtained by subtracting a control value from a measured value; The measurement was performed by adding 0.35 mL of the liquid composition to a measurement tube of Lucipack Pen (manufactured by Kikkoman Biochemifa Corporation), and then measuring 1 × 10 -7 0.01 mL of ATP (manufactured by Oriental Yeast Co., Ltd.) solution was added to M, and the mixture was left to stand at 25°C for 1 hour. The values ​​were measured using a Lumitester Smart (manufactured by Kikkoman Biochemifa Corporation). The control value is a value obtained by measuring the amount of luminescence under the same conditions as those for obtaining the measured value, except that sterilized ultrapure water is added instead of the ATP solution. The liquid composition.

2. The liquid composition according to claim 1, further comprising at least one component selected from an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, an enzyme that catalyzes a reaction that produces AMP from ADP, and a substrate for the enzyme that catalyzes a reaction that produces AMP from ADP, or at least one of these is added before or during measurement.

3. A liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) comprising luciferase, luciferin, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added before or during measurement; and (ii) the relative luminescence output of the liquid composition during storage is 5500 RLU or less; the relative light intensity is a value obtained by subtracting a control value from a measured value; The measurement was performed by adding 0.35 mL of the liquid composition to a measurement tube of Lucipack Pen (manufactured by Kikkoman Biochemifa Corporation), and then measuring 1 × 10 -7 0.01 mL of ATP (manufactured by Oriental Yeast Co., Ltd.) solution was added to M, and the mixture was left to stand at 25°C for 1 hour. The values ​​were measured using a Lumitester Smart (manufactured by Kikkoman Biochemifa Corporation). The control value is a value obtained by measuring the amount of luminescence under the same conditions as those for obtaining the measured value, except that sterilized ultrapure water is added instead of the ATP solution. The liquid composition.

4. The liquid composition according to any one of claims 1 to 3, wherein the relative light emission amount is 2300 RLU or less.

5. A liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) the liquid composition contains luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ATP from AMP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added to the liquid composition before or during measurement; and (ii) At least one of the following is true: The concentration of luciferin in the liquid composition is 0.4 mM or less; the concentration of luciferase in the liquid composition based on the Bradford assay is 0.3 mg / mL or less; the concentration of the enzyme that catalyzes the reaction of producing ATP from AMP in the liquid composition is 1 U / mL or less; the concentration of a substrate for an enzyme that catalyzes a reaction producing ATP from AMP in the liquid composition is 0.1 mM or less; the concentration of the cofactor in the liquid composition is 6 mM or less; The liquid composition.

6. The liquid composition according to claim 5, further comprising at least one component selected from an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, an enzyme that catalyzes a reaction that produces AMP from ADP, and a substrate for the enzyme that catalyzes a reaction that produces AMP from ADP, or at least one of these is added before or during measurement.

7. A liquid composition for measuring ATP, and AMP and / or ADP in a sample after storage, comprising: (i) comprising luciferase, luciferin, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes a reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes a reaction that produces ATP from ADP, and a cofactor, or, if at least one of these components is not contained in the liquid composition, the component that is not contained in the liquid composition is added before or during measurement; and (ii) At least one of the following is true: the concentration of the enzyme that catalyzes the reaction of producing ADP from AMP in the liquid composition is 450 U / mL or less; the concentration of a substrate for an enzyme that catalyzes a reaction producing ADP from AMP in the liquid composition is 0.1 mM or less; the concentration of the enzyme that catalyzes the reaction of producing ATP from ADP in the liquid composition is 20 U / mL or less; the concentration of the substrate for the enzyme that catalyzes the reaction of producing ATP from ADP is 1.2 mM or less; the concentration of the cofactor in the liquid composition is 6 mM or less; The liquid composition.

8. The liquid composition according to any one of claims 1 to 7, which has a shelf life of one day or more.

9. 9. The liquid composition according to claim 8, which has a shelf life of 30 days or more.

10. The liquid composition according to any one of claims 1 to 9, which does not contain at least one component selected from the group consisting of luciferase, luciferin, an enzyme that catalyzes a reaction that produces ATP from AMP, a substrate for the enzyme that catalyzes the reaction that produces ATP from AMP, an enzyme that catalyzes a reaction that produces ADP from AMP, a substrate for the enzyme that catalyzes the reaction that produces ADP from AMP, an enzyme that catalyzes a reaction that produces ATP from ADP, a substrate for the enzyme that catalyzes the reaction that produces ATP from ADP, and a cofactor, and the component not contained in the liquid composition is added before or during measurement.

11. 11. The liquid composition according to claim 1, wherein the concentration of luciferin in the liquid composition is 0.4 mM or less and / or the concentration of luciferase measured by the Bradford assay is 0.3 mg / mL or less.

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

13. The liquid composition according to any one of claims 1 to 12, wherein the concentration of luciferase in the liquid composition as determined by the Bradford method is 0.1 mg / mL or less.

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

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

16. The method of claim 15, wherein an ATP standard solution is not used.

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