JP2000189197A - Atp eliminant, atp elimination and measurement of atp in cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an ATP eliminant for eliminating ATP of measurement system, useful in food sanitation, clinical examination, medicine, etc., suitable for rapidly measuring ATP in a cell in high sensitivity by making the ATP eliminant include a luciferase as an active ingredient. SOLUTION: A luciferase (e.g. luciferase of Luciola cruciata), a luciferin (e.g. a luciferin of firefly) and further an ATPase as active ingredients are dissolved in a buffer solution containing magnesium acetate, bovine serum albumin(BSA), dithiothreitol sucrose, etc., to give the objective ATP eliminant for rapidly measuring ATP in cells in high selectivity, useful for efficiently eliminating ATP of measurement system, especially free ATP, useful in the field of food sanitation, biotechnology, clinical examination, medicine, etc., suitable for judging the presence of cells in a sample and measuring the number of living cells in the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an ATP scavenger containing luciferase as an active ingredient, an ATP scavenging method for a measuring system using the ATP scavenger, and a method for measuring intracellular ATP using the ATP scavenger.

[0002]

2. Description of the Related Art The measurement of ATP to be measured in a sample (hereinafter referred to as "target ATP") is widely performed in various industrial fields. In the fields of food hygiene, biotechnology, clinical testing, medicine, etc., intracellular A is used for the purpose of determining the presence or absence of cells in a sample, measuring the number of living cells in a sample, and the like.
The measurement of TP has been performed.

[0003] As a method for measuring intracellular ATP, a method utilizing a bioluminescence method is generally used. Bioluminescence is a luminescence phenomenon seen in the luminous organs of luminous organisms (luminous bacteria, firefly squid, insect fireflies, etc.). Bioluminescence is generated by a reaction between luciferin, which is a luminescent substrate, and luciferase, which is an enzyme. In insect firefly systems, light is generated by the reaction of luciferin with luciferase in the presence of ATP and magnesium ions. Firefly luciferase is widely used as a reagent for measuring intracellular ATP.

In the method for measuring intracellular ATP using the bioluminescence method, first, a sample containing cells is brought into contact with an ATP extraction reagent to extract intracellular ATP outside the cells. As the ATP extraction reagent, a surfactant, trichloroacetic acid (TCA),
Those containing tris buffer, ethanol, lytic enzymes and the like are used. Next, the extracted ATP is brought into contact with a bioluminescent reagent containing luciferin and luciferase (luciferin-luciferase luminescent reagent), and the amount of luminescence generated at this time is measured to determine the amount of intracellular ATP.
The method for measuring the number of cells in the above field is required to be quick and highly sensitive. For example, in the field of food hygiene, testing for microbial contamination of products is essential for shipping products.

Conventionally, this test is performed by the pour culture method. However, since the test requires one day or more, the product must be stored in a warehouse until a result is obtained. For this reason, not only is there a problem in terms of distribution efficiency, but the possibility of microbial contamination increases as the storage time of products such as milk increases. In addition, since the concentration of microorganisms that cause contamination in food is generally low, high-sensitivity testing is required. For this reason, the method using the bioluminescent reagent is widely used as a rapid and highly sensitive method for measuring the number of cells.

[0006] By the way, ATP is contained in the cells of all living organisms, albeit with different contents. For this reason, in the method of measuring target ATP, ATP other than target ATP (hereinafter referred to as “free ATP”) uses a measurement system (eg, sample, medium, various reagents, containers, measuring instruments and devices, etc.).
However, there has been a problem that the measurement sensitivity is reduced due to the contamination of the sample. That is, when free ATP is mixed into the measurement system, the free ATP is measured simultaneously with the target ATP.
It becomes difficult to accurately measure only TP. ATP adhering to the operator's fingers or airborne dust or the like can be free ATP, but it is difficult to prevent the ATP from being mixed into the measurement system. Further, when measuring the number of living cells in a sample, there is a problem that it is difficult to accurately measure the number of living cells because ATP released from dead cells acts as free ATP. Furthermore, when a reagent with a low degree of purification is used in the measurement system, there is a possibility that free ATP is mixed in the reagent. For example, when counting the number of microbial cells present in a medium containing a yeast extract based on the amount of intracellular ATP, accurate measurement of the amount of intracellular ATP is required because a large amount of free ATP derived from yeast extract is present. There was a problem that it became difficult. Especially intracellular ATP using bioluminescent reagents
The measurement method described in (1) above has a problem that the measurement sensitivity is reduced by simultaneously measuring free ATP as a background light emission amount (noise).

[0007] Since ATP exists in a place where an organism exists, it is extremely difficult to completely prevent free ATP from being mixed into the measurement system. For this reason, in the method of measuring the target ATP, it has been required to establish a method for efficiently removing free ATP mixed into the measurement system. As a method for removing free ATP, a membrane filter method for removing a sample from a sample using a membrane filter and a centrifugal separation method for separating by centrifugation are known. However, these methods have problems that the operation is very complicated and the ATP removal rate is not sufficient. Other than the above,
A method for eliminating free ATP using an ATP scavenger is known. The “ATP scavenger” is a reagent having the following activity (1) or (2). (1) Activity for decomposing ATP molecules. (2) An activity of converting an ATP molecule into another molecule that does not reduce the measurement sensitivity of the target ATP.

[0008] A method (enzymatic method) for eliminating free ATP with an ATP scavenger containing an ATPase as an active ingredient is known. The enzymatic method is simple in operation and free ATP
Is excellent in that the erasing efficiency is high. As the ATPase, for example, apyrase, adenosine triphosphatase (ATPase), hexokinase or A
TP pyrophosphatase and the like (Monthly Food Chemical, published by Shokuhin Kagaku Shimbun, May 1995, 55-55).
63 page; JP-A-2-65800; USP 5,31
6,907, Analytical Biochemi
The Story Vol. 218, pp. 20-25 1994
Bulletin of the Japans
e Society of Scientific F
isheries Vol. 52, No. 9, page 1695
1986, Marine Ecology-Prog
Less Series 13, 305-309 (1983)). A method for measuring intracellular ATP by combining an enzymatic method and a bioluminescent reagent is also known.

The conventional enzymatic method has a problem in that the operation must be performed without knowing how much free ATP has been eliminated. That is, free ATP
In some cases, the erasing state of free ATP could not be confirmed, and the erasing time of free ATP was insufficient. In addition, if the erasing time is too long, cells in the sample proliferate, so that the number of living cells cannot be accurately measured.

[0010]

The objects of the present invention are mainly (1) an ATP scavenger capable of efficiently eliminating ATP in a measuring system, particularly free ATP, and (2) erasing ATP in a measuring system, particularly free ATP. And (3) to provide a rapid and highly sensitive method for measuring intracellular ATP by combining the ATP elimination method and the bioluminescence method.

[0011] The term "deletion of ATP" means not only erasing all ATP molecules in the measurement system but also target ATP.
ATP of the measurement system to the extent that it does not substantially affect the measurement of
It also means reducing the amount of (especially free ATP).

[0012]

Means for Solving the Problems The present inventors have found that luciferase, which has been conventionally used as a component of a bioluminescent reagent, is useful as an ATP scavenger, and completed the present invention based on the findings.

[0013] The present invention provides the following ATP scavenger:
The present invention relates to an ATP elimination method and a method for measuring intracellular ATP. 1. An ATP scavenger containing luciferase as an active ingredient. 2. AT containing luciferin and luciferase
P scavenger. 3. Further containing an ATPase as an active ingredient,
3. The ATP scavenger according to claim 1 or 2. 4. An ATP elimination method for a measurement system, comprising adding the ATP elimination agent according to any one of Items 1 to 3 to the measurement system.

5. It is characterized by including the following steps,
Method for measuring intracellular ATP. (1) A first step of adding the ATP scavenger according to any one of Items 1 to 3 to a sample containing cells to eliminate free ATP in the sample. (2) The second step of inactivating the enzyme activity in the ATP scavenger added in the first step. (3) Third step of extracting intracellular ATP. (4) Fourth step of measuring extracted ATP Intracellular ATP, comprising the following steps:
Measurement method. (1) A first step of adding the ATP scavenger according to any one of Items 1 to 3 to a sample containing cells to eliminate free ATP in the sample. (2) ATP extraction reagent is added to the sample, and intracellular ATP
6. The second step of extracting the ATP and inactivating the enzyme activity in the ATP scavenger added in the first step (3) The third step of measuring the extracted ATP 7. The method for measuring intracellular ATP according to item 6, wherein the ATP extraction reagent is a surfactant. 8. 8. The method for measuring intracellular ATP according to any one of claims 5 to 7, wherein the method for measuring the extracted ATP is a method using a luciferin-luciferase luminescence reagent. 9. 9. The method for measuring intracellular ATP according to item 8, wherein the luciferase contained in the luciferin-luciferase luminescence reagent has resistance to the ATP extraction reagent.

[0015]

DETAILED DESCRIPTION OF THE INVENTION Regarding the ATP scavenger of the present invention The ATP scavenger of the present invention contains luciferase as an active ingredient. The luciferase may be derived from any luminescent organism as long as it has an activity of eliminating ATP in the measurement system. In the present invention, for example, luciferase derived from firefly squid, insects and the like can be used. As the luciferase, natural luciferase purified from the luminous organ of the above organism, recombinant luciferase prepared by genetic engineering techniques, and addition or deletion of one or more amino acids in the amino acid sequence of the natural luciferase,
Mutant luciferase into which a mutation such as substitution has been introduced can be used.

Among insect luciferases, particularly fireflies,
For example, Genji firefly (Luciola cruciata), Heike firefly (Luciola lateralis), and Hime firefly (Hotaria pa
rvula), American fireflies (Photinus pyralis) and the like are easily available and suitable as the luciferase of the present invention.

The ATP scavenger may contain components other than luciferase. Examples of other components include components necessary for bioluminescence, specifically, luciferin. A whose active ingredient is firefly luciferase
TP scavenger, as other components, firefly luciferin,
It preferably contains magnesium ions.

When an ATP scavenger containing a component necessary for bioluminescence is used, the change in the amount of luminescence is measured,
It is possible to check the erasure status of ATP.

Firefly luciferase eliminates ATP by the following reaction in the presence of firefly luciferin, magnesium ions and the like.

[0020]

Embedded image In order to further improve the ATP elimination efficiency, the ATP elimination agent preferably contains luciferase and ATPase as active ingredients. ATPases include, for example, adenosine phosphate deaminase,
, Alkaline phosphatase, acid phosphatase, hexokinase and adenosine triphosphatase can be used. The ATPase can be used alone or in combination of two or more. In addition, as other components other than the active ingredient, excipients, dispersants, emulsifiers, preservatives, preservatives, pH
Buffers, hygroscopic agents and the like can be used. The type of other components contained in the ATP scavenger, the mixing ratio with the active ingredient, and the like are not particularly limited, and are appropriately set in accordance with the use, operability, type of measurement system, state of the sample, and the like of the ATP scavenger. Just do it. The shape of the ATP scavenger is not particularly limited, and may be solid (eg, granule, powder, tablet, etc.) or liquid.

For simplicity of operation, the ATP scavenger is preferably in liquid form. In that case, luciferase or ATPase, which is an active ingredient, may be dissolved in an appropriate buffer (for example, phosphate buffer, HEPES buffer, scalpel (MES) buffer). II. About ATP elimination method of measurement system The ATP elimination method of the present invention is characterized by adding the above-mentioned ATP elimination agent to the measurement system. The present invention relates to a measurement system (for example, a sample, a culture medium, various reagents, a container, a measuring instrument or device, etc.)
It is useful as a method for erasing ATP (especially free ATP) mixed or adhered to the DNA. The final concentration of luciferase added to the measurement system, the conditions for contacting the ATP scavenger with the measurement system, and the like may be appropriately set according to the type and properties of the measurement system. The addition of the ATP scavenger can be carried out, for example, by the following method. (1) An ATP scavenger is added to a sample, a medium, various reagents, and the like. (2) The containers, measuring instruments and devices, etc. are washed with a solution-state ATP scavenger. Hereinafter, a method of adding an ATP scavenger to a sample will be described.

The sample is not particularly limited as long as it contains the target ATP.
Industrial products such as cosmetics, semi-finished products, raw materials, seawater,
River water, industrial water, sewage, soil, urine, feces, blood, sputum, pus, cell cultures, etc. In addition, samples in various tests such as a bacterial contamination test, a cleanliness test, and a wiping test can also be used as the sample of the present invention. Further, the sample is prepared by using a suitable solvent (for example, distilled water, physiological saline,
A solution suspended in a phosphate buffer, a Tris buffer, a sodium acetate buffer, or the like) may be used as a sample. When the sample contains a solid content, the sample can be handled in the same manner as a solution by suspending the sample in the above solvent or homogenizing with a mixer or the like.

When a specimen is used as a sample in a wiping test, a wiping stick such as a cotton swab is moistened with sterile water, the test portion is wiped off, and then the test sample is rinsed with sterile water or a reagent solution. The part is used as a sample.

When the target ATP is intracellular ATP,
A solution supposed to contain cells or a culture solution of cells may be used as a sample. Further, the above solution or culture solution,
After capturing cells by filtration through a hydrophilic or hydrophobic filtration membrane, the filtration membrane may be used as a sample. In addition, the cell here means, for example, yeast, mold, mushroom, bacteria, actinomycetes,
It refers to unicellular algae, viruses, protozoa, animal or plant cells and tissue cultures, and the like.

The method of the present invention is excellent as an ATP erasing method because it is easy to confirm the erasing state of ATP.
In particular, when an ATP scavenger containing components required for bioluminescence (for example, an ATP scavenger containing firefly luciferase as an active ingredient and firefly luciferin and magnesium ions as other components) is used, the change in the amount of luminescence is measured. Thus, the erasure status of ATP can be confirmed.
In this case, after the ATP scavenger is added to the measurement system, the time-dependent change in the amount of emitted light may be measured using a luminometer (luminometer).

When the measurement system is in liquid form (sample, medium, various reagents, etc.) and the volume is small, the whole amount may be supplied to a luminometer to measure the amount of luminescence. When the capacity is large, a part of the sample or the like may be collected and the luminescence amount may be measured. III. Method for measuring intracellular ATP The method for measuring intracellular ATP of the present invention includes the following steps. (1) A first step of adding the ATP scavenger of the present invention to a sample containing cells to eliminate free ATP in the sample. (2) The second step of inactivating the enzyme activity in the ATP scavenger added in the first step. (3) Third step of extracting intracellular ATP. (4) A fourth step of measuring the extracted ATP. 2. In the first step, free ATP is eliminated by reacting luciferase in the ATP scavenger with free ATP. When using firefly luciferase as the active ingredient, it is preferable to use an ATP scavenger containing firefly luciferase, firefly luciferin, and magnesium ion.

The final concentration of luciferase added to the sample, the conditions for contacting the ATP scavenger with the sample, and the like may be appropriately set according to the type and properties of the sample. When using an ATP scavenger containing firefly luciferase as an active ingredient,
The first step may be performed, for example, under the following conditions. (1) Luciferase final concentration: 0.001 GLU / m
1 or more (particularly preferably 0.01 to 10 GLU / ml).

GLU is a unit of the amount of light emission obtained by the following method. First, the luciferase preparation was appropriately diluted with an enzyme diluent (1 mM EDTA, 1 mM 2-mercaptoethanol, 1% BSA, 50 mM HEPES, (pH 7.5)), and 100 μl of the diluted solution was added to 100 μl of the substrate solution (1.4 μl). mM luciferin, 40 mM ATP, 300 mM MgSO4.7H2O, 50 mM HEPES,
(pH 7.5)). Next, the amount of emitted light is measured using a BLR-201 Luminescence reader (manufactured by Aloka) under the following setting conditions. Measurement range: x100, display value: x1000, measurement temperature: 30
° C, measurement time: 20 seconds When the measured value under these conditions is 1,000,000 counts, the activity value is 1 GLU (capillary light unit) / ml. (2) pH condition: weakly acidic to weakly alkaline (particularly preferably pH 5.0 to 8.5) (3) Reaction temperature: 20 to 50 ° C (particularly preferably 30 to 45 ° C) (4) Reaction time: about 2 Within hours (especially preferably 1 to 60 minutes) Confirmation of the elimination state of free ATP may be performed by subjecting the whole or a part of the sample to a luminometer and measuring the change with time of the luminescence. 3. In the second step, the enzyme activity in the ATP scavenger is deactivated. The enzyme referred to here is luciferase which is an active ingredient. When the ATP scavenger contains an ATPase, the enzyme means luciferase and ATPase. The method of inactivating the enzyme activity is not particularly limited, and includes, for example, a method of inactivating the enzyme by adding an activity inhibitor. As the enzyme activity inhibitor, an ATP extraction reagent described later can be used. As another method, there is a method of inactivating the enzyme by heating a sample containing an ATP scavenger. Further, a method of changing the pH of the sample to a range in which the enzyme is inactivated by adding an acid or an alkali can be used. 4. As a method of extracting intracellular ATP in the third step, a method of adding an ATP extraction reagent to a sample is preferable. ATP extraction reagents include ATP
The extraction method is not particularly limited. For example, a method of adding an ATP extraction reagent to a sample is employed. Examples of the ATP extraction reagent include a mixture of ethanol and ammonia,
Methanol, ethanol, surfactant, trichloroacetic acid, perchloric acid and the like can be used. Surfactants are suitable as ATP extraction reagents because of their high ATP extraction efficiency.
Examples of the surfactant include anionic surfactants (for example, sodium dodecyl sulfate (SDS), potassium lauryl sulfate, sodium monolauroyl phosphate, sodium alkylbensulfonate), and cationic surfactants (for example,
Benzalkonium chloride (BAC), benzethonium chloride (B
ZC), cetylpyridinium chloride, cetyltrimethylammonium bromide, myristyldimethylbenzylammonium chloride), amphoteric (Twitter) surfactants (eg Twit)
tergent Detergent 3-08, 3-10, 3-12, 3-14, 3-16, Te
go), nonionic surfactants (eg Tween 20, 60,
80, Span 60, 80, Triton X-45, X-100, polyoxyethylene ether, polyoxyethylene lauryl ether).

In the present invention, the second step and the third step may be performed simultaneously. A simple method of simultaneously carrying out the two steps is a method of adding an ATP extraction reagent also serving as an enzyme activity inhibitor to a sample. In this case, AT
A P extraction reagent is added to extract intracellular ATP and to inactivate the enzyme activity in the ATP scavenger added in the first step. The above-mentioned ATP extraction reagent can be used as an enzyme activity inhibitor by appropriately setting the final concentration to be added to the sample. For example, firefly luciferase
It can be deactivated by a surfactant. In the presence of 0.01% benzalkonium chloride, wild-type Genji firefly luciferase has a catalytic activity of about 2
It drops to 0%.

The method for measuring intracellular ATP in the fourth step is not particularly limited, but a method utilizing a bioluminescence method is preferable. In this case, a sample containing the extracted ATP is brought into contact with a luciferin-luciferase luminescence reagent, and the amount of luminescence generated is measured to determine the amount of intracellular ATP. When the luciferase in the bioluminescent reagent is firefly luciferase, the bioluminescent reagent preferably contains luciferin, luciferase and magnesium ion. The amount of light generated is determined by a luminometer, such as a Lumitester.
C-100, K-100, K-200, K-210 (all manufactured by Kikkoman), luminescence reader B
LR-201 improved type (Aloka), Lumat LB9501
(Manufactured by Berthold). Further, when a filter membrane capturing cells is used as a sample, a bioluminescence image analysis system device, for example, ARGUS-50 / CL
[With taper fiber: Hamamatsu Photonics Co., Ltd.]
The number of cells can be measured by imaging the bright spots on the filtration membrane using.

In general, the higher the concentration of the ATP extraction reagent added to a sample, the higher the intracellular ATP extraction efficiency. However, when the concentration of the ATP extraction reagent is too high, the ATP extraction reagent may inhibit the step of measuring intracellular ATP, and the measurement sensitivity may be reduced. For this reason, the reagent used in the step of measuring intracellular ATP is a high concentration of AT.
It is preferable that it is not inhibited even in the presence of the P extraction reagent. For example, when a luciferin-luciferase luminescence reagent is used in the step of measuring intracellular ATP, it is preferable that the luciferase in the luminescence reagent has resistance to the ATP extraction reagent added to the sample. In this case, it is preferable that luciferase, which is an active ingredient of the ATP scavenger, be inactivated by the ATP extraction reagent. By doing so, the step of measuring intracellular ATP can be performed without removing or neutralizing the ATP extraction reagent, so that the operation can be simplified.

As the luciferase having resistance to a surfactant, two kinds of mutant luciferases ("HLK" and "HIK") described in Japanese Patent Application No. 9-36122 can be used. In addition, a polypeptide having an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence of the mutant luciferase and having a luciferase activity resistant to a surfactant can also be used.

"HLK" and "HIK" are those prepared by introducing a mutation into the amino acid sequence of thermotolerant Heike firefly luciferase. Recombinant Escherichia coli containing the gene encoding HLK was delivered to the Institute of Biotechnology,
Deposited as RM BP-6147. Recombinant Escherichia coli containing the gene encoding HIK has been deposited with the National Institute of Bioscience and Human Technology as FERM BP-6146.

The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to these. [Example 1] A tryptic soy broth medium was used as a sample, and free ATP in the sample was eliminated using an ATP scavenger containing luciferase as an active ingredient. 1. Preparation of reagents and the like (1) Preparation of ATP scavenger ATP scavenger of the present invention: 25 mM containing 150 GLU / ml thermostable Genji firefly luciferase (described in JP-A-5-244942), 1.5 mM firefly luciferin (manufactured by YMC). Tricine buffer (15 mM magnesium acetate, 0.2% BS
A, 0.2 mM dithiothreitol (DTT);
37% sucrose (Sucrose). ATP scavenger of Comparative Example: 25 mM tricine buffer (15 mM magnesium acetate, 0.2% BSA, containing 150 mU / ml apyrase enzyme solution (manufactured by Sigma)
0.2 mM DTT, containing 0.37% sucrose). (2) Preparation of sample: Tryptic soy broth medium (1.7
% Tripeptone, 0.3% soypeptone, 0.25% glucose, 0.25% dipotassium phosphate, 0.5% sodium chloride, pH 7.3 ± 0.2) (Eiken Chemical Co., Ltd.) 3 g
Was dissolved in 100 ml of purified water to prepare a tryptic soy broth medium. The medium was sterilized at 120 ° C. for 15 minutes, cooled to room temperature, and used as a sample. 2. Elimination of free ATP in sample Elimination of free ATP in the sample is performed by the following method.
The effect of the ATP scavenger of the present invention was confirmed. First step: Elimination of free ATP in the sample Second step: Measurement of luminescence amount Third step: Creation of ATP calibration curve Fourth step: Calculation of ATP concentration in the sample (First step: Free ATP in the sample Erasing) 0.6 ml of each ATP erasing agent was collected, 1.2 ml of a sample was added thereto, and ATP erasing was started at room temperature. 0, 10, 3
After 0 and 60 minutes, 0.1 ml of the sample was collected. (Second step: measurement of luminescence amount) The luminescence amount was measured using a lucifer ATP extraction reagent (manufactured by Kikkoman) and a lucifer luminescence reagent-HS (manufactured by Kikkoman, which was dissolved in an attached luminescence reagent dissolving solution). I went. 0.1 ml of the ATP extraction reagent was added to 0.1 ml of the collected sample, and 0.1 ml of lucifer luminescence reagent-HS was added. The generated luminescence amount was measured by a luminescence measuring device “Lumitester C-100” manufactured by Kikkoman Co. The luminescence value at this time was S.

On the other hand, in the above measurement method, “0.1 ml of ultrapure water” was used instead of “0.1 ml of collected sample”.
Other than that, the luminescence amount was measured in exactly the same manner. This value was used as the blank value R of the luminescent reagent.

Then, SR was obtained and the net light emission value A was obtained. (Third step: preparation of ATP calibration curve) ATP dissolved in lucifer ATP extraction reagent to 1 × 10 ⁻⁸ M
Is further diluted by 5 steps in 10-fold increments, and 1 × 10 ⁻⁸ to
A 1 × 10 ⁻¹³ M dilution train was prepared.

To 0.1 ml of ultrapure water, 0.1 ml of ATP extraction reagent containing ATP at each concentration was added, and 0.1 ml of lucifer luminescent reagent-HS was added. The luminescence was measured using a Lumitester C-100 manufactured by Kikkoman Corporation.

Further, 0.1 ml of ATP extraction reagent containing no ATP, 0.1 ml of ultrapure water, luminescence reagent-HS0.1
The luminescence was measured using the luminescence reagent as a blank value of the luminescence reagent in the ATP calibration curve. The standard value of the luminescence amount for the added ATP was obtained by subtracting the blank value from the luminescence amount. FIG. 1 shows a calibration curve in Example 1.

(Fourth Step: Amount of ATP Concentration in Sample) In the bioluminescence reaction, the luminescence is inhibited by the components in the sample, so the luminescence amount A obtained in the second step is an apparent value. Therefore, the light emission amount A is corrected by the following method, and A in the sample is corrected.
The TP concentration was calculated. First, the light emission rate K was obtained, and then the correction value Y (true light emission amount) was obtained by dividing the light emission amount A by the light emission rate K. Using the calibration curve (FIG. 1), the ATP concentration corresponding to the correction value Y was calculated (corrected light emission values in the examples described later were similarly obtained).

How to determine the correction value Y.

Y (correction value) = A / KK = {HA} / {GR} H (internal standard value): 0.1 of each sample collected over time
ATP extraction reagent containing 1 × 10 ^-9 MATP in ml
The value of the luminescence amount obtained by adding 0.1 ml and 0.1 ml of the luminescence reagent. A (apparent value): 0.1 ml of the ATP extraction reagent and 0.1 ml of the luminescence reagent containing no ATP were added to 0.1 ml of each sample.
The value of the amount of luminescence obtained by adding ml. G (standard value): ATP standard solution 1 in 0.1 ml of ultrapure water
The value of the amount of luminescence obtained by adding 0.1 ml of × 10 ⁻⁹ M and 0.1 ml of a luminescent reagent. R (blank value): 0.1 ml of ATP extraction reagent not containing ATP and 0.1 m of luminescence reagent in 0.1 ml of ultrapure water
The value of the luminescence amount obtained by adding l. In Example 1, G = 38201 and R = 20. Table 1 shows the results of obtaining H, A, and K. Further, numerical values other than the above are shown in Table 2 (the present invention) and Table 3 (Comparative Example).

[0041]

[Table 1]

[Table 2]

[Table 3] FIG. 2 shows the change over time in the ATP concentration in the sample calculated by the above method. By using the ATP scavenger of the present invention, free AT contained in tryptic soy broth medium
It can be seen that the P concentration could be reduced to 1/100 or less in 60 minutes. In addition, the effect of the ATP scavenger of the present invention was found to be equivalent to that of the ATP scavenger of Comparative Example (containing apyrase as an active ingredient). In addition, the unit of thermostable firefly luciferase contained in the ATP scavenger of the present invention is 0, 30, 60, 150, 300G.
FIG. 3 shows the results of a preliminary test in which the same operation was performed at LU / ml. FIG. 3 shows that as the number of luciferase units added increases, free ATP is eliminated in a shorter time. However, the results for 150 GLU and 300 GLU were almost the same. From this, in the first embodiment,
The number of luciferase units in the ATP scavenger is 150 GL
U / ml. [Example 2] Using a tryptic soy broth medium as a sample, free AT in the sample was measured using an ATP scavenger containing luciferase and ATPase as active ingredients.
P was erased. 1. Preparation of reagents and the like (1) Preparation of ATP scavenger ATP scavenger of the present invention: a 25 mM tricine buffer containing 15 GLU / ml thermostable firefly luciferase, 1.5 mM firefly luciferin, 150 mM adenosine phosphate deaminase (15 mM magnesium acetate, 0 mM .2% BSA, 0.2 mM DTT, 0.37
% Including sucrose). ATP scavenger of Comparative Example 1: 25 mM Tricine buffer containing 150 mU / ml adenosine phosphate deaminase (15 mM magnesium acetate, 0.2% BSA,
0.2 mM DTT, containing 0.37% sucrose). ATP scavenger of Comparative Example 2: 25 mM tricine buffer containing 300 mU / ml adenosine phosphate deaminase (15 mM magnesium acetate, 0.2% BSA,
0.2 mM DTT, containing 0.37% sucrose). (2) Sample: tryptic soy bouillon medium prepared in Example 1. 2. Elimination of free ATP in sample Elimination of free ATP in the sample, measurement of luminescence, creation of ATP calibration curve, and ATP in the same manner as in Example 1.
The concentration was calculated.

As in Example 1, H (internal standard value), A
(Apparent value) G (this standard value) R (blank value)
The correction value Y (true light emission amount) was calculated. In the second embodiment, G
= 38201 and R = 20. Table 4 (ATP scavenger of the present invention) and Table 5 (Comparative Example 1) show the results of the determination of H, A, and K.
It was shown to. The luminous efficiency K in Comparative Example 2 was the same as in Comparative Example 1. Table 6 shows the values other than the above.
(Invention), Table 7 (Comparative Example 1) and Table 8 (Comparative Example 2).

[0043]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8] FIG. 4 shows the change over time in the ATP concentration in the sample calculated by the above method. At an ATP concentration at an erasing time of 60 minutes, Comparative Example 1 (ATP scavenger containing 150 mM adenosine phosphate deaminase as an active ingredient) and Comparative Example 2 (3
ATP scavenger containing 00 mM adenosine phosphate deaminase as an active ingredient) does not show a large difference. However, 15 mM adenosine phosphate deaminase was not added.
By using 0 GLU luciferase together, 30
It can be seen that free ATP can be eliminated to a lower value than in the case of 0 mM adenosine phosphate deaminase. [Example 3] Using a tryptic soy broth medium as a sample, elimination of free ATP and confirmation of the elimination state were performed using two ATP elimination agents of the present invention. 1. Preparation of reagents and the like (1) Preparation of ATP scavenger ATP scavenger 1: 25 mM tricine buffer (15 mM magnesium acetate, 0.2% BSA, 0.2 mM, containing 150 GLU / ml heat-resistant firefly luciferase and 1.5 mM firefly luciferin) DTT, 0.
37% sucrose). ATP scavenger 2: 150 GLU / ml thermostable Genji firefly luciferase, 1.5 mM firefly luciferin,
25 containing 150 mM adenosine phosphate deaminase
mM Tricine buffer (15 mM magnesium acetate,
0.2% BSA, 0.2 mM DTT, 0.37% sucrose). (2) Sample: Tryptic soy broth medium prepared in Example 1. 2. Elimination of Free ATP in Sample and Confirmation of Elimination Status The above ATP elimination agents 1 and 2 were added to test tubes for 0.1
ml was collected, and 0.2 ml of each sample was added. The test tube for measurement was set in Lumitester C-100 (manufactured by Kikkoman Corporation), and the amount of emitted light was measured over time for 60 minutes. The results are shown in Table 9 and FIG.

[0044]

[Table 9] As can be seen from FIG.
2 showed the same tendency as the change with time of the ATP concentration in the sample. It was shown that the elimination status of free ATP can be confirmed by measuring the amount of luminescence generated from the sample.

Example 4 Free ATP in a sample containing cells was eliminated using the ATP scavenger of the present invention, and then the amount of luminescence derived from intracellular ATP was measured. The correlation was examined. 1. Preparation of reagents and the like (1) Preparation of ATP scavenger ATP scavenger of the present invention 1: 25 mM tricine buffer (15 mM magnesium acetate, 0.2% BSA, containing 150 GLU / ml thermostable firefly luciferase and 1.5 mM firefly luciferin) 0.2 mM DT
T, containing 0.37% sucrose). ATP scavenger of the present invention 2: 150 mM GLU / ml thermostable firefly luciferase, 1.5 mM firefly luciferin, 25 mM tricine buffer containing 150 mM adenosine phosphate deaminase (15 mM magnesium acetate, 0.2% BSA, 0.2 mM DTT, 0.1 mM 3
7% sucrose). (2) Sample preparation: Escherichia coli (Escheri)
chia coli: ATCC 25922) and Bacillus subtilis.
is: ATCC 9372) was used as the test microorganism. A diluted solution of the culture solution of the test microorganism was prepared by the following method and used as a sample. 10 ml of each of the tryptic soy broth media prepared in Example 1 was put into two sterilized test tubes.
After dispensing, a loopful of the test microorganism was inoculated at 35 ° C.
And cultured overnight with shaking to obtain a culture solution. The above culture solution was diluted 10000-fold using a tryptic soy broth medium,
From this, five 10-fold dilutions were performed to prepare a total of six dilutions.

2. Elimination of Free ATP in Sample The number of viable cells in the sample was measured by the following method. First step: Elimination of free ATP in the sample Second step: Extraction of intracellular ATP Third step: Measurement of luminescence amount Fourth step: Calculation of CFU of the sample by the pour culture method (First step: Elimination of free ATP) 1.2m sample
After adding 0.6 ml of the ATP scavenger to
After leaving it for 0 minutes, the free ATP in the sample was eliminated. (Second step: extraction of intracellular ATP) 0.1 ml of a sample from which free ATP had been eliminated was collected, and 0.1 ml of ATP extraction reagent manufactured by Kikkoman was added thereto.
The ATP was extracted from the cells by leaving it for 2 seconds. (Third step: measurement of luminescence amount) To 0.2 ml of the sample subjected to ATP extraction, 0.1 ml of Lucifer luminescent reagent-HS manufactured by Kikkoman Corporation was added, and after stirring, using Lumitester C-100 manufactured by Kikkoman Corporation. The luminescence was measured. The amount of luminescence at this time was calculated by comparing the amount of free ATP
And the total luminescence (T) of intracellular ATP.

Using a tryptic soy broth medium containing no test microorganism instead of the sample, elimination of free ATP,
The ATP extractant was added and the amount of luminescence was measured. The amount of luminescence at this time was defined as the amount of free ATP (free value) F that could not be erased.

By subtracting the free value F from the total value T, an accurate luminescence amount of intracellular ATP can be obtained. The corrected light emission amount was determined in the same manner as in Example 1. (Fifth step: Calculation of CFU of sample by pour culture method) Using the pour culture method, the CF of the test microorganism contained in the sample is calculated.
U (colony forming unit) was determined. The obtained CFU was used as the measured value of the number of living cells.

First, 0.1 ml of a sample was aseptically collected in a sterile petri dish with a sterile pipette, and was previously stored at 121 ° C.
Sterilization is performed for 20 minutes, and then, the melted standard agar medium (0.25% yeast extract,
0.5% tryptone, 0.1% glucose, pH 7.1,
2.0% agar), mixed uniformly, and solidified as a flat plate. Next, the plate was cultured at 35 ° C. overnight, and the number of colonies that appeared was measured. The number of the colonies was defined as the CFU of the microorganism contained in 0.1 ml of the sample. Note that the sampling and pour-out of the sample at the time of calculating the CFU were performed immediately before the measurement of the light emission amount.

The correlation between the CFU / ml and the corrected light emission amount (RLU) obtained as described above is shown in FIG. 6 (result of the erasing agent 1 of the present invention) and FIG. 7 (result of the erasing agent 1 of the present invention). Show. In the figure, "E. coli" and "Bacillu"
"s" means "Escheric" used as a test microorganism.
hia "and" Bacillus subtili "
s ".

FIGS. 6 and 7 show that by eliminating free ATP using the ATP scavenger of the present invention, the background luminescence can be suppressed to a low level, and the measured values of the amount of luminescence derived from intracellular ATP and the number of living cells It has been found that it is possible to have a high correlation with CFU that is The ATP scavenger and the ATP scavenging method of the present invention can be used for intracellular ATP.
It was shown that it can be suitably used in the measurement method.

[0052]

Industrial Applicability According to the present invention, an ATP scavenger containing luciferase as an active ingredient, an ATP scavenging method for a measurement system using the ATP scavenger, and intracellular A using the ATP scavenger
A method for measuring TP was provided. When the ATP erasing agent of the present invention is used, it is possible to easily confirm the erasure state of ATP by measuring the luminescence generated from the measurement system. The present invention is suitable as a method for eliminating free ATP. In addition, the ATP scavenger of the present invention can be suitably used particularly in a method for measuring intracellular ATP by scavenging free ATP in a sample.

[Brief description of the drawings]

FIG. 1 is a calibration curve in Example 1.

FIG. 2 is a diagram showing a change over time in ATP concentration in Example 1.

FIG. 3 is a diagram showing the results of a preliminary test in Example 1.

FIG. 4 is a diagram showing a change over time in ATP concentration in Example 2.

FIG. 5 is a diagram showing a change over time in the amount of light emission in Example 3.

FIG. 6 is a diagram showing a correlation between CFU / ml and a corrected light emission amount in Example 4.

FIG. 7 is a diagram showing a correlation between CFU / ml and a corrected light emission amount in Example 4.

Front page of the continued F-term (reference) 2G045 AA13 AA16 AA28 BA01 BB20 BB29 CA25 CB01 CB03 CB04 CB08 CB17 CB21 DA15 FB13 GC15 2G054 AA07 AA08 BA01 CA21 CE02 EA02 4B063 QA01 QA19 QQ03 QQ06 QQ07 QQ08 QQ15 QQ16 QQ17 QQ18 QQ19 QQ63 QR02 QR13 QR14 QR41 QR66 QS20 QX02

Claims (9)

[Claims] 1. An ATP scavenger containing luciferase as an active ingredient. 2. An ATP scavenger containing luciferin and luciferase. 3. The ATP scavenger according to claim 1, further comprising an ATPase as an active ingredient. 4. An ATP elimination method for a measurement system, comprising adding the ATP elimination agent according to claim 1 to the measurement system. 5. A method for measuring intracellular ATP, comprising the following steps: A first step of adding the ATP scavenger according to any one of claims 1 to 3 to a sample containing cells to eliminate free ATP in the sample. ATP added in the first step
The second step of inactivating the enzyme activity in the erasing agent. Intracellular AT
Third step of extracting P. Fourth step of measuring the extracted ATP 6. A method for measuring intracellular ATP, comprising the following steps: A first step of adding the ATP scavenger according to any one of claims 1 to 3 to a sample containing cells to eliminate free ATP in the sample. ATP extraction reagent is added to the sample to extract intracellular ATP, and the enzyme activity in the ATP scavenger added in the first step is deactivated. The second step is to measure the extracted ATP. 7. The method for measuring intracellular ATP according to claim 6, wherein the ATP extraction reagent is a surfactant. 8. The method for measuring intracellular ATP according to claim 5, wherein the method for measuring the extracted ATP is a method using a luciferin-luciferase luminescence reagent. 9. The intracellular ATP according to claim 8, wherein the luciferase contained in the luciferin-luciferase luminescence reagent has resistance to the ATP extraction reagent.
Measurement method.