JP2006280365A - Method for detecting adenosine triphosphate and reagent for detecting the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting adenosine triphosphate, having enough high sensitivity and accuracy. <P>SOLUTION: This method for detecting the adenosine triphosphate comprises an amplifying and light-emitting step for mixing a sample containing the adenosine triphosphate with polyphosphoric acid, adenosine monophosphate, adenylate kinase, polyphosphate kinase, luciferin, luciferase, and bivalent metal ions, so as to amplify the adenosine triphosphate and to make the luciferin emit light, and an emitted light intensity-measuring step for measuring intensity of the light emitted by the luciferin in the amplifying and light-emitting step, wherein the adenylate kinase, the polyphosphate kinase, the luciferin, the luciferase, and the bivalent metal ions are together mixed in the presence of oxygen, before they are mixed with the sample. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting adenosine triphosphate (hereinafter referred to as “ATP”) and a reagent for detection.

  In recent years, as a method for detecting microorganisms such as bacteria, a method for detecting ATP in microorganisms by measuring bioluminescence (luminescence based on luciferin-luciferase reaction) using ATP present in all living organisms as an index is used. It is like that.

  For example, Patent Documents 1 to 3 listed below disclose adding coenzyme A, polyphosphoric acid, a sulfhydryl compound, and the like to a luciferin-luciferase reaction system as a technique for enhancing the persistence and stability of bioluminescence.

Patent Document 4 below discloses adenosine monophosphate (hereinafter referred to as “AMP”), adenylate kinase (hereinafter referred to as “ADK”), polyphosphate and polyphosphate kinase (hereinafter referred to as “PPK”), A method is disclosed in which ATP is amplified by mixing with a sample containing ATP and then reacting with luciferin and luciferase to generate luminescence, and the amount of luminescence is measured to detect ATP.
Japanese Patent No. 3171595 JP-A-8-47399 JP 2002-191396 A JP 2001-299390 A

  However, the techniques disclosed in Patent Documents 1 to 3 have insufficient ATP detection sensitivity and cannot detect a very small amount of ATP.

  On the other hand, in the technique disclosed in Patent Document 4, although ATP detection sensitivity is improved, ATP is generated from ATP or adenosine diphosphate (hereinafter referred to as “ADP”) mixed as an impurity in the reaction system. Since ATP other than the target ATP is amplified and detected with high sensitivity, the target ATP cannot be detected with high accuracy.

  Accordingly, an object of the present invention is to provide an ATP detection method having sufficiently high ATP detection sensitivity and accuracy, and an ATP detection reagent capable of implementing such an ATP detection method.

In order to achieve the above object, the present invention provides the following ATP detection methods (1) to (3).
(1) An amplification luminescence step of mixing a sample containing ATP with polyphosphate, AMP, ADK, PPK, luciferin, luciferase, and divalent metal ions to amplify the ATP and emit the luciferin, and the amplified luminescence A luminescence measuring step for measuring the amount of light emitted by the luciferin in the step, wherein the ADK, the PPK, the luciferin, the luciferase and the divalent metal ion are the sample (2) ATP-containing sample is mixed with polyphosphate, AMP, ADK, PPK, luciferin, luciferase, and divalent metal ions. Amplification that amplifies the ATP and emits the luciferin A method for detecting ATP, comprising: a light step; and a light emission amount measurement step for measuring an amount of light emitted by the luciferin in the amplified light emission step, wherein the AMP, the ADK, the PPK, the luciferin, and the luciferase And a method of detecting ATP, wherein the divalent metal ion is mixed in the presence of oxygen before being mixed with the sample. (3) A sample containing ATP is treated with polyphosphate, AMP, ADK, Amplification emission step for mixing with PPK, luciferin, luciferase and divalent metal ions to amplify the ATP and emitting the luciferin, and a luminescence measurement for measuring the amount of light emitted by the luciferin in the amplification emission step A method for detecting ATP, comprising: the polyphosphoric acid, the ADK The PPK, the luciferin, the luciferase and the divalent metal ions, prior to being mixed with the sample, the detection method of ATP characterized in that it is mixed in the presence of oxygen

  As shown in FIG. 1, one molecule of ATP is amplified to two molecules of ATP by reaction with AMP, ADK, polyphosphate, and PPK. Therefore, in the ATP detection methods (1) to (3) above, ATP in the sample is amplified, and sufficiently high ATP detection sensitivity can be obtained.

  ATP reacts with luciferin, luciferase, and divalent metal ions in the presence of oxygen to generate luminescence by luciferin. In the above ATP detection methods (1) to (3), ADK, PPK, luciferin, luciferase and divalent metal ions, or these substances and AMP or polyphosphate are already mixed before mixing with the sample. Therefore, the ATP amplification reaction and the luminescence reaction proceed simultaneously, and the operation becomes simple.

  Furthermore, ATP, PPK and luciferase are likely to contain ATP as an impurity. In the methods for detecting ATP described in (1) to (3) above, ATP in the sample is used for ATP amplification reaction and luminescence reaction. Since the enzymes (ADK, PPK and luciferase) are already mixed with luciferin and divalent metal ions in the presence of oxygen before being provided, ATP in the enzyme is oxygen, luciferin, luciferase and divalent metal ions. It is consumed by the reaction with AMP and changes to AMP. In the ATP detection methods of (1) and (2) above, the polyphosphate necessary for ATP amplification is not mixed with the enzyme before ATP in the sample is subjected to ATP amplification reaction and luminescence reaction. Therefore, ATP in the enzyme is consumed exclusively without being amplified. Therefore, the ATP detection methods (1) and (2) can detect ATP in a sample with sufficiently high accuracy. In the ATP detection method of (3) above, polyphosphate is already mixed with ADK, PPK, luciferin, luciferase and divalent metal ions before ATP in the sample is subjected to ATP amplification reaction and luminescence reaction. Therefore, there is a possibility that ATP in the enzyme is amplified. However, since most of ATP in the enzyme is consumed before being amplified, detection of ATP in the sample is not affected. Therefore, ATP in the sample can be detected with sufficiently high accuracy even by the method for detecting ATP of (3) above.

  The amplification luminescence step in the ATP detection method of (1) includes a step of mixing a sample with polyphosphate and AMP, and a mixture of the obtained sample, polyphosphate and AMP in the presence of oxygen with ADK, PPK, luciferin, and luciferase. And a step of mixing with a divalent metal ion. The amplification luminescence step in the ATP detection method of (2) above comprises the steps of mixing a sample with polyphosphoric acid, and mixing the obtained sample and polyphosphoric acid in the presence of oxygen with AMP, ADK, PPK, luciferin, luciferase. And a step of mixing with a divalent metal ion. The amplification luminescence step in the method for detecting ATP of (3) includes a step of mixing a sample with AMP, and a mixture of the obtained sample and AMP in the presence of oxygen with polyphosphate, ADK, PPK, luciferin, luciferase and And a step of mixing with a divalent metal ion. The amplification luminescence step in the ATP detection method of (1) to (3) above is a step of simultaneously mixing a sample with polyphosphate, AMP, ADK, PPK, luciferin, luciferase and divalent metal ions in the presence of oxygen. It may be.

  In the ATP detection methods (1) to (3) above, it is preferable that ADK and PPK form a fusion protein. By using such a fusion protein, the amount of ADK and PPK can be easily controlled, the stability of the ATP amplification reaction is increased, and the reproducibility of the detection result is increased.

The present invention also provides an ATP detection reagent used in the ATP detection methods of (1) to (3) above. That is, the following ATP detection reagents (i) to (iii) are provided.
(I) ATP detection reagent obtained by mixing ADK, PPK, luciferin, luciferase and divalent metal ions in the presence of oxygen (ii) AMP, ADK, PPK, luciferin, luciferase and divalent A reagent for detecting ATP obtained by mixing metal ions in the presence of oxygen (iii) Obtained by mixing polyphosphate, ADK, PPK, luciferin, luciferase and divalent metal ions in the presence of oxygen ATP detection reagent characterized by the above

  When the reagent (i) is used with polyphosphoric acid and AMP, the ATP detection method (1) can be carried out. By using the reagent (ii) together with polyphosphoric acid and using the reagent (iii) together with AMP, it is possible to carry out the method for detecting ATP (2) and (3), respectively. To do.

  In the ATP detection reagents (i) to (iii) above, it is preferable that ADK and PPK form a fusion protein.

  According to the present invention, there are provided an ATP detection method having sufficiently high ATP detection sensitivity and accuracy, and an ATP detection reagent capable of implementing such an ATP detection method.

  Hereinafter, embodiments of the ATP detection method and detection reagent of the present invention will be described.

(ATP detection method)
The method for detecting ATP of the present invention comprises the steps of mixing a sample with polyphosphate, AMP, ADK, PPK, luciferin, luciferase and divalent metal ions, and measuring the amount of light emitted by luciferin, , PPK, luciferin, luciferase and divalent metal ions, or these substances and AMP or polyphosphoric acid are already mixed in the presence of oxygen before being mixed with the sample.

That is, the ATP detection method of the present invention is the following ATP detection method (1) to (3).
(1) An amplification luminescence step of mixing a sample containing ATP with polyphosphate, AMP, ADK, PPK, luciferin, luciferase, and divalent metal ions to amplify the ATP and emit the luciferin, and the amplified luminescence A luminescence measuring step for measuring the amount of light emitted by the luciferin in the step, wherein the ADK, the PPK, the luciferin, the luciferase and the divalent metal ion are the sample (2) ATP-containing sample is mixed with polyphosphate, AMP, ADK, PPK, luciferin, luciferase, and divalent metal ions. Amplification that amplifies the ATP and emits the luciferin A method for detecting ATP, comprising: a light step; and a light emission amount measurement step for measuring an amount of light emitted by the luciferin in the amplified light emission step, wherein the AMP, the ADK, the PPK, the luciferin, and the luciferase And a method of detecting ATP, wherein the divalent metal ion is mixed in the presence of oxygen before being mixed with the sample. (3) A sample containing ATP is treated with polyphosphate, AMP, ADK, Amplification emission step for mixing with PPK, luciferin, luciferase and divalent metal ions to amplify the ATP and emitting the luciferin, and a luminescence measurement for measuring the amount of light emitted by the luciferin in the amplification emission step A method for detecting ATP, comprising: the polyphosphoric acid, the ADK The PPK, the luciferin, the luciferase and the divalent metal ions, prior to being mixed with the sample, the detection method of ATP characterized in that it is mixed in the presence of oxygen

  In the method for detecting ATP of (1) to (3) above, the mixing of the sample with polyphosphate, AMP, ADK, PPK, luciferin, luciferase and divalent metal ions is divided into two steps. AMP, or polyphosphoric acid alone, or AMP alone may be mixed with the sample, and then the mixture and the remaining material may be mixed, or the sample and all materials may be mixed in one step.

  When the sample and all materials are mixed in one step, the mixing is performed in the presence of oxygen. In the case of dividing into two steps, the mixing in the first step is not necessarily performed in the presence of oxygen, but the mixing in the subsequent step is performed in the presence of oxygen.

  As shown in FIG. 1, one molecule of ATP is amplified to two molecules of ATP by reaction with AMP, ADK, polyphosphate, and PPK. Therefore, in the ATP detection methods (1) to (3) above, ATP in the sample is amplified, and sufficiently high ATP detection sensitivity can be obtained.

  ATP reacts with luciferin, luciferase, and divalent metal ions in the presence of oxygen to generate luminescence by luciferin. In the ATP detection method according to the above (1) to (3), a sample containing ATP and ADK, PPK, luciferin, luciferase and divalent metal ions, or these substances and AMP or polyphosphate are mixed at the same time, and ATP amplification is performed. Since the reaction and the luminescence reaction proceed simultaneously, the reaction conditions (reaction time, reaction temperature, etc.) need only be controlled once, and the operation becomes simple.

  Furthermore, ATP, PPK and luciferase are likely to contain ATP as an impurity. In the methods for detecting ATP described in (1) to (3) above, ATP in the sample is used for ATP amplification reaction and luminescence reaction. Since the enzymes (ADK, PPK and luciferase) are already mixed with luciferin and divalent metal ions in the presence of oxygen before being provided, ATP in the enzyme is oxygen, luciferin, luciferase and divalent metal ions. It is consumed by the reaction with AMP and changes to AMP. In the ATP detection methods of (1) and (2) above, the polyphosphate necessary for ATP amplification is not mixed with the enzyme before ATP in the sample is subjected to ATP amplification reaction and luminescence reaction. Therefore, ATP in the enzyme is consumed exclusively without being amplified. Therefore, the ATP detection methods (1) and (2) can detect ATP in a sample with sufficiently high accuracy. In the ATP detection method of (3) above, polyphosphate is already mixed with ADK, PPK, luciferin, luciferase and divalent metal ions before ATP in the sample is subjected to ATP amplification reaction and luminescence reaction. Therefore, there is a possibility that ATP in the enzyme is amplified. However, since most of ATP in the enzyme is consumed before being amplified, detection of ATP in the sample is not affected. Therefore, ATP in the sample can be detected with sufficiently high accuracy even by the method for detecting ATP of (3) above.

  When mixing the sample with polyphosphate, AMP, ADK, PPK, luciferin, luciferase and divalent metal ions in two steps, either the luciferin or the luciferase is not only the subsequent step, but also the first step. But it can be mixed with the sample. When luciferin or luciferase is mixed in the first step, luciferin or luciferase is mixed in both steps. In this case, since both luciferin and luciferase are not mixed in the first step, ATP in the sample does not cause luminescence. In addition, when luciferase is mixed in the first step, ATP mixed in luciferase may be amplified and detected in a later step, but most of them are subjected to a luminescence reaction before being amplified. As a result, the detection of ATP in the sample is not affected. Further, the divalent metal ion can be mixed with the sample not only in the later step but also in the first step.

  Any mixing in the ATP detection methods (1) to (3) is preferably performed in a liquid solvent, and particularly preferably in a buffer solution. By performing the mixing in the buffer, the pH is maintained in a certain range, and the stability of the ATP detection process and the reproducibility of the detection result are enhanced. The pH of the buffer is preferably 4-9, more preferably 7-8. The mixing performed in the presence of oxygen may be performed in a buffer solution in contact with air.

Polyphosphoric acid is a compound represented by the chemical formula (PO ₃ ⁻ ) _n (n ≧ 2), and in the ATP amplification reaction cycle shown in FIG. Play a role to generate. The number n of phosphoric acid groups contained in polyphosphoric acid is preferably 10 ≦ n ≦ 1000, more preferably 10 ≦ n ≦ 100. In the case of n <10, the number of phosphate groups is small and the phosphate groups are difficult to be donated continuously. On the other hand, in the case of n> 1000, the molecule is large and polyphosphoric acid hardly reacts with ADP and PPK. Therefore, ATP tends not to be sufficiently amplified. In the case of 10 ≦ n ≦ 100, polyphosphoric acid easily reacts with ADP and PPK, and ATP amplification is more reliably realized. A commercially available polyphosphoric acid can be used.

  ADK and PPK may exist as separate proteins from each other or may be fused to form a single fusion protein, but preferably form a fusion protein. Use of such a fusion protein makes it easy to control the amounts of ADK and PPK, increases the stability of the ATP amplification reaction, and increases the reproducibility of detection results.

  As ADK and PPK, those derived from any of animals, plants and microorganisms can be used, but those derived from Escherichia coli are preferable from the viewpoint of ease of preparation of the enzyme. ADK or PPK can be mass-produced in E. coli cells by, for example, incorporating an enzyme gene into an expression vector and introducing it into E. coli according to a known method. Enzymes produced in Escherichia coli cells are used after being disrupted according to a known method and then purified by chromatography, electrophoresis or the like. The purified ADK and PPK are preferably used after further treatment with an apyrase to remove ATP and ADP in the enzyme as much as possible. The fusion protein of ADK and PPK can also be prepared by the same method as described above except that a fusion gene in which the genes of both enzymes are linked is incorporated into the vector (Akio Kuroda et al., Biosci. Biotechnol. Biochem., 68 (6), 1216-1220 (2004)).

  Examples of luciferin and luciferase include luciferin and luciferase derived from fireflies (Genji firefly, Heike firefly, American firefly, etc.). As luciferin and luciferase, commercially available products can be used.

Examples of the divalent metal ion include Mg ²⁺ , Ca ²⁺ and Mn ²⁺ , and Mg ²⁺ is preferable.

  As long as the apparatus used in the ATP detection method of (1) to (3) above includes a container for mixing the sample and various substances, and means for measuring the amount of light generated by the mixing, Good.

  As a container for mixing, it is preferable to use a microtube, a microplate, or a microchannel chip from the viewpoint of increasing the degree of light integration.

  As means for measuring the amount of luminescence, a luminometer, germanium photodiode, gallium arsenide photodiode, phototransistor, CCD element, or the like can be used.

  When quantifying ATP in a sample using the ATP detection method of (1) to (3) above, the same amount of luminescence is changed over time by performing the same operation as the detection method used for standard solutions of various ATP concentrations. And this may be compared with the change over time of the light emission amount obtained for the sample.

(ATP detection reagent)
The ATP detection reagent of the present invention is one of the following ATP detection reagents (i) to (iii).
(I) ATP detection reagent obtained by mixing ADK, PPK, luciferin, luciferase and divalent metal ions in the presence of oxygen (ii) AMP, ADK, PPK, luciferin, luciferase and divalent A reagent for detecting ATP obtained by mixing metal ions in the presence of oxygen (iii) Obtained by mixing polyphosphate, ADK, PPK, luciferin, luciferase and divalent metal ions in the presence of oxygen ATP detection reagent characterized by the above

  The ATP detection reagents (i) to (iii) can be used in the ATP detection methods (1) to (3). When the reagent (i) is used with polyphosphoric acid and AMP, the ATP detection method (1) can be carried out. By using the reagent (ii) together with polyphosphoric acid and using the reagent (iii) together with AMP, it is possible to carry out the method for detecting ATP (2) and (3), respectively. To do.

  The reagents for detecting ATP of (i) to (iii) above can also be used in ATP detection methods other than (1) to (3) above. That is, for example, an ATP-containing sample is mixed with polyphosphate, AMP, ADK, PPK, luciferin, luciferase and divalent metal ions to amplify ATP, and luciferin is further added to the mixture obtained in the amplification step. And a luminescence step in which luciferase is mixed to cause luminescence, and a luminescence measurement step for measuring the amount of light emitted in the luminescence step can be used in an ATP detection method. Specifically, detection of ATP of any one of (i) to (iii) above as a part of polyphosphate, AMP, ADK, PPK, luciferin, luciferase and divalent metal ions mixed with the sample in the amplification step Reagents can be used.

  The mixing performed to obtain the ATP detection reagents (i) to (iii) is preferably performed in a liquid solvent in contact with the atmosphere, particularly in a buffer solution. The pH of the buffer is preferably 4-9, more preferably 7-8.

  ADK and PPK in the reagents for detecting ATP in the above (i) to (iii) may exist as separate proteins or may be fused to form a single fusion protein. It is preferable to form.

  Examples of the luciferin and luciferase in the ATP detection reagents of (i) to (iii) above include luciferin and luciferase derived from fireflies (Genji firefly, Heike firefly, American firefly, etc.).

Examples of the divalent metal ions in the ATP detection reagents (i) to (iii) above include Mg ²⁺ , Ca ²⁺ and Mn ²⁺ , and Mg ²⁺ is preferred.

  Examples of the present invention and comparative examples will be described below, but the present invention is not limited to these examples.

Example 1
First, two types of ATP solutions (ATP: 4.0E-13M, 4.0E-12M) in which ATP was dissolved in Tris-Mg buffer (pH 7.4) were prepared. Further, AMP and polyphosphoric acid ((PO ₃ ⁻ ) ₆₅ ) (Sodium Phosphate Glass Type 65 manufactured by Sigma) dissolved in Tris-Mg buffer (pH 7.4) (AMP: 4.0E-5M; polyphosphorus) Acid: 2 mM). Furthermore, a solution (fusion protein: 2.36 mg / mL) (1.7 μL) in which the fusion protein of ADK and PPK was dissolved in Tris-Mg buffer (pH 7.4), and Lucifer (trademark) 250 plus (by Kikkoman) A solution B mixed with luciferin, luciferase and Mg ²⁺ ) (98.3 μL) is prepared to obtain a solution of an ATP detection reagent comprising ADK, PPK, luciferin, luciferase and divalent metal ions. It was. The fusion protein of ADK and PPK was prepared according to the method described in the above literature (Akio Kuroda et al., Biosci. Biotechnol. Biochem., 68 (6), 1216-1220 (2004)).

Next, solution A (50 μL) was mixed with two ATP solutions (ATP: 4.0E-13M, 4.0E-12M) (50 μL each) in the wells of a 96-well microplate, and then solution B was further added. (100 μL) was mixed, and the amount of luminescence (CPS / 10) was measured over time. Moreover, Tris-Mg buffer solution (pH 7.4) which does not contain ATP was used as a control, and the same operation was performed for this. For the measurement of luminescence, a Wallac 1420 ARVO _MX multi-label microplate counter manufactured by Perkin-Elmer was used.

  Table 1 shows the change over time in the light emission amount in Example 1. FIG. 2 is a graph showing the change over time of the light emission amount in Example 1.

  As is clear from Table 1 and FIG. 2, in both the ATP solution and the control, only a light emission amount that was so low as to be difficult to distinguish from the dark count of the measuring instrument was measured until 4 to 5 minutes after the start of the reaction. However, over time, the amount of emitted light began to increase geometrically. The amount of luminescence and the amount of increase depended on the amount of ATP (ATP concentration). At 10 minutes after the start of the reaction, even when the initial concentration of ATP was 4.0E-13M, the amount of luminescence was sufficiently large and the difference from the control was also sufficiently large.

(Example 2)
First, two types of ATP solutions (ATP: 4.0E-13M, 4.0E-12M) in which ATP was dissolved in Tris-Mg buffer (pH 7.4) were prepared. Further, a solution C (AMP: 4.0E-5M) in which AMP was dissolved in a Tris-Mg buffer (pH 7.4) was prepared. Furthermore, a solution (polyphosphoric acid: 50 mM) (4.6 μL) of polyphosphoric acid ((PO ₃ ⁻ ) ₆₅ ) (Sodium Phosphate Glass Type 65 manufactured by Sigma) dissolved in Tris-Mg buffer (pH 7.4); A solution (fusion protein: 2.2 mg / mL) (1.8 μL) obtained by dissolving a fusion protein of ADK and PPK in Tris-Mg buffer (pH 7.4) and Lucifer (trademark) 250 plus (93. 6 μL) was prepared to obtain an ATP detection reagent solution comprising polyphosphate, ADK, PPK, luciferin, luciferase, and divalent metal ions. The fusion protein of ADK and PPK was prepared according to the method described in the above literature (Akio Kuroda et al., Biosci. Biotechnol. Biochem., 68 (6), 1216-1220 (2004)).

Next, after mixing two kinds of ATP solutions (ATP: 4.0E-13M, 4.0E-12M) (each 50 μL) in a well of a 96-well microplate, solution C (50 μL) was further added to solution D. (100 μL) was mixed, and the amount of luminescence (CPS / 10) was measured over time. Moreover, Tris-Mg buffer solution (pH 7.4) which does not contain ATP was used as a control, and the same operation was performed for this. For the measurement of luminescence, a Wallac 1420 ARVO _MX multi-label microplate counter manufactured by Perkin-Elmer was used.

  Table 2 shows the change over time in the light emission amount in Example 2. FIG. 3 is a graph showing the change over time of the light emission amount in Example 2.

  As is clear from Table 1 and FIG. 2, in both the ATP solution and the control, only a light emission amount that was so low as to be difficult to distinguish from the dark count of the measuring instrument was measured until 4 to 5 minutes after the start of the reaction. However, over time, the amount of emitted light began to increase geometrically. The amount of luminescence and the amount of increase depended on the amount of ATP (ATP concentration). At 10 minutes after the start of the reaction, even when the initial concentration of ATP was 4.0E-13M, the amount of luminescence was sufficiently large and the difference from the control was also sufficiently large.

(Example 3)
First, an ATP solution (ATP: 4.0E-13M) in which ATP was dissolved in Tris-Mg buffer (pH 7.4) was prepared. Further, AMP and polyphosphoric acid ((PO ³⁻ ) ₆₅ ) (Sodium Phosphate Glass Type 65 manufactured by Sigma) dissolved in Tris-Mg buffer (pH 7.4) (AMP: 4.0E-5M; polyphosphorus) Acid: 2 mM). Further, ADK and PPK fusion protein, luciferin and luciferase (luciferase recombinant manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in Tris-Mg buffer (pH 7.4) (fusion protein: 40 μg / mL; luciferin: 2.7 μM; luciferase: 0.45 μg / mL) was prepared to obtain a solution of an ATP detection reagent comprising ADK, PPK, luciferin, luciferase and divalent metal ions. The fusion protein of ADK and PPK was prepared according to the method described in the above literature (Akio Kuroda et al., Biosci. Biotechnol. Biochem., 68 (6), 1216-1220 (2004)).

  Next, after the solution A (100 μL) was mixed with the ATP solution (ATP: 4.0E-13M) (100 μL) in an Eppendorf tube, the solution E (200 μL) was further mixed. After mixing the solution E with the mixed solution of the ATP solution and the solution A, 10 μL of the mixed solution is taken into a Lumi tube every minute and further mixed with Kikkoman's Lucifer® 250 Plus (50 μL). The light emission amount (RLU) was measured. Moreover, Tris-Mg buffer solution (pH 7.4) which does not contain ATP was used as a control, and the same operation was performed for this. Lumitester C-1000 manufactured by Kikkoman Corp. was used for the measurement of the amount of luminescence.

  Table 3 shows the change over time of the light emission amount in Example 3. FIG. 4 is a graph showing the change over time of the light emission amount in Example 3.

  As is apparent from Table 3 and FIG. 4, for both the ATP solution and the control, only a low luminescence amount was measured so that it was difficult to distinguish from the dark count of the measuring instrument until 2 minutes after the start of the reaction. Over time, the amount of emitted light began to increase geometrically. The amount of luminescence and the amount of increase depended on the amount of ATP (ATP concentration). At 6 minutes after the start of the reaction, even when the initial concentration of ATP was 4.0E-13M, the amount of luminescence was sufficiently large and the difference from the control was also sufficiently large.

(Comparative Example 1)
First, ATP, AMP and polyphosphoric acid ((PO ₃ ⁻ ) ₆₅ ) (Sodium Phosphate Glass Type 65 manufactured by Sigma) were dissolved in Tris-Mg buffer (pH 7.4) in three types of solutions F (ATP: 2. 0E-11M, 2.0E-10M, 2.0E-9M; AMP: 4.0E-5M; polyphosphoric acid: 2 mM). Further, a solution G (fusion protein: 40 μg / mL) in which a fusion protein of ADK and PPK was dissolved in a Tris-Mg buffer (pH 7.4) was prepared. The fusion protein of ADK and PPK was prepared according to the method described in the above literature (Akio Kuroda et al., Biosci. Biotechnol. Biochem., 68 (6), 1216-1220 (2004)).

Next, the solution G (100 μL) was mixed with three kinds of solutions F (ATP: 2.0E-11M, 2.0E-10M, 2.0E-9M) (each 100 μL) in an Eppendorf tube, and then constant. Every time, 10 μL of the mixed solution of solutions F and G was put in a Lumi tube, and Lucifer (trademark) 250 plus (50 μL) manufactured by Kikkoman was further mixed, and the light emission amount (RLU) was measured over time. Further, AMP and polyphosphate - solution in ^{_{((PO 3) 65) (}} Sigma Co. Sodium Phosphate Glass Type 65) and Tris-Mg buffer (pH7.4) (AMP: 4.0E- 5M; polyphosphate : 2 mM) was used as a control, and the same operation was performed for this. A Lumitester C-1000 manufactured by Kikkoman Corp. was used for the measurement of the amount of luminescence.

  Table 4 shows the change over time in the light emission amount in Comparative Example 1. FIG. 5 is a graph showing the change over time in the light emission amount in Comparative Example 1.

  As is clear from Table 4 and FIG. 5, as in Examples 1 to 3, the amount of luminescence and the amount of increase depended on the amount of ATP (ATP concentration).

  However, the results of Examples 1 and 2 (particularly, the amount of luminescence 10 minutes after the start of the reaction when the initial concentration of ATP is 4.0E-12M) and the result of Comparative Example 1 (particularly the initial concentration of ATP is 2). (Emission amount of 10 minutes after the start of reaction in the case of 0.0E-12M) clearly shows that Examples 1 and 2 have a larger emission amount and a larger difference from the control. In addition, the results of Example 3 (particularly, the amount of luminescence 6 minutes after the start of the reaction when the initial concentration of ATP is 4.0E-13M) and the results of Comparative Example 1 (particularly the initial concentration of ATP is 2.0E). Comparing with the amount of luminescence of 10 minutes after the start of the reaction in the case of −12M, it is clear that Example 3 has a larger amount of luminescence and a larger difference from the control.

  Examples 1 to 3 and Comparative Example 1 show that even if a very small amount of ATP can be detected with sufficiently high sensitivity and accuracy, the ATP detection method or detection reagent of the present invention can be used. .

  The ATP detection method of the present invention can be used as a method for detecting microorganisms such as bacteria using ATP as an index. It can also be used to measure the activity of enzymes that produce ATP (such as acetate kinase). Furthermore, ATP is produced simultaneously in the production system of glycerin, glucose, creatine, creatinine, etc., but can also be used for detection of these compounds.

It is a figure which shows an ATP amplification process. 4 is a graph showing a change with time of light emission amount in Example 1. 6 is a graph showing a change with time of light emission amount in Example 2. 6 is a graph showing a change with time in light emission amount in Example 3. 6 is a graph showing a change with time in light emission amount in Comparative Example 1;

Claims (12)

A sample containing adenosine triphosphate is mixed with polyphosphate, adenosine monophosphate, adenylate kinase, polyphosphate kinase, luciferin, luciferase and divalent metal ions to amplify the adenosine triphosphate, and the luciferin An amplified light emission step for emitting light;
A step of measuring the amount of light emitted from the luciferin in the amplified luminescence step, and a method for detecting adenosine triphosphate comprising:
The adenylate kinase, the polyphosphate kinase, the luciferin, the luciferase, and the divalent metal ion are mixed in the presence of oxygen before being mixed with the sample. Detection method. A sample containing adenosine triphosphate is mixed with polyphosphate, adenosine monophosphate, adenylate kinase, polyphosphate kinase, luciferin, luciferase and divalent metal ions to amplify the adenosine triphosphate, and the luciferin An amplified light emission step for emitting light;
A step of measuring the amount of light emitted from the luciferin in the amplified luminescence step, and a method for detecting adenosine triphosphate comprising:
The adenosine monophosphate, the adenylate kinase, the polyphosphate kinase, the luciferin, the luciferase, and the divalent metal ion are mixed in the presence of oxygen before being mixed with the sample. A method for detecting adenosine triphosphate. A sample containing adenosine triphosphate is mixed with polyphosphate, adenosine monophosphate, adenylate kinase, polyphosphate kinase, luciferin, luciferase and divalent metal ions to amplify the adenosine triphosphate, and the luciferin An amplified light emission step for emitting light;
A step of measuring the amount of light emitted from the luciferin in the amplified luminescence step, and a method for detecting adenosine triphosphate comprising:
The adenosine, wherein the polyphosphate, the adenylate kinase, the polyphosphate kinase, the luciferin, the luciferase, and the divalent metal ion are mixed in the presence of oxygen before being mixed with the sample. Method for detecting triphosphate.   The amplified luminescence step comprises mixing the sample with the polyphosphate and the adenosine monophosphate, and mixing the obtained sample, the polyphosphate and the adenosine monophosphate in the presence of oxygen with the adenylate kinase. The method for detecting adenosine triphosphate according to claim 1, comprising the step of mixing with the polyphosphate kinase, the luciferin, the luciferase and the divalent metal ion.   The amplified luminescence step comprises mixing the sample with the polyphosphate, and mixing the obtained sample and the polyphosphate in the presence of oxygen with the adenosine monophosphate, the adenylate kinase, the polyphosphate kinase, The method for detecting adenosine triphosphate according to claim 2, comprising the step of mixing with the luciferin, the luciferase and the divalent metal ion.   The amplification luminescence step comprises mixing the sample with the adenosine monophosphate, and mixing the obtained sample and the adenosine monophosphate in the presence of oxygen with the polyphosphate, the adenylate kinase, and the polyphosphate. The method for detecting adenosine triphosphate according to claim 3, comprising a step of mixing with a kinase, the luciferin, the luciferase and the divalent metal ion.   The amplification luminescence step comprises the step of simultaneously mixing the sample with the polyphosphate, the adenosine monophosphate, the adenylate kinase, the polyphosphate kinase, the luciferin, the luciferase, and the divalent metal ion in the presence of oxygen. The method for detecting adenosine triphosphate according to any one of claims 1 to 3, wherein:   The method for detecting adenosine triphosphate according to any one of claims 1 to 7, wherein the adenylate kinase and the polyphosphate kinase form a fusion protein.   A reagent for detecting adenosine triphosphate, which is obtained by mixing adenylate kinase, polyphosphate kinase, luciferin, luciferase and divalent metal ions in the presence of oxygen.   A reagent for detecting adenosine triphosphate, which is obtained by mixing adenosine monophosphate, adenylate kinase, polyphosphate kinase, luciferin, luciferase and divalent metal ions in the presence of oxygen.   A reagent for detecting adenosine triphosphate, obtained by mixing polyphosphate, adenylate kinase, polyphosphate kinase, luciferin, luciferase and divalent metal ions in the presence of oxygen.   The reagent for detecting adenosine triphosphate according to any one of claims 9 to 11, wherein the adenylate kinase and the polyphosphate kinase form a fusion protein.

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