JPH09107994A - Assay of intracellular atp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly assay in high sensitivity intracellular ATP for bacteria tests, etc., by subjecting a cell-contg. specimen to extraction with an ATP extractive reagent followed by adding a biochemiluminescent reagent to the resultant liquid extract to conduct a biochemiluminescence in the presence of an enzymatic reaction inhibition-suppressive reagent. SOLUTION: An ATP extractive reagent containing a surfactant (e.g. polyoxyethylene-10-octylphenyl ether) is added to a specimen containing cells (e.g. bacteria) to extract the ATP in the cells, and a luminescent reagent containing luctiferin and luciferase is then added to the resultant liquid extract to conduct a biochemiluminescence by enzymatic reaction, and based on the resultant luminescence, the ATP in the cells is assayed. In this intracellular ATP assay, the biochemiluminescence is conducted in the presence of an enzymatic reaction inhibition-suppressive reagent containing a silicone (e.g. dimethylpolysiloxane) to suppress the inhibition of the enzymatic reaction for the biochemiluminescence by the extractive reagent, thus quickly assaying in high sensitivity the target intracellular ATP for bacteria tests etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring intracellular ATP using a biochemiluminescence method.

[0002]

2. Description of the Related Art Escherichia coli, yeast, lactic acid bacterium and other viable cell counts are measured by food hygiene, biotechnology, clinical testing, medicine,
It is very important in fields such as ultrapure water and the environment. Generally, cell number is measured by counting colonies in the growth medium,
It is performed by measuring with a hemocytometer under a microscope and measuring turbidity.

The method of counting colonies in a growth medium is as follows:
It is excellent in that it is highly sensitive, that only live cells can be measured, and that microbial cell types can be identified by using a selective medium.
However, since it requires culturing of cells, the measurement usually takes one day or more, which is not suitable when a quick result is desired.

The method using a hemocytometer has drawbacks such as complicated operation due to the use of a microscope and difficulty in automation.

The method of measuring turbidity is excellent in that it is rapid and easy to automate, but it has low sensitivity, the number of living cells and dead cells cannot be distinguished, and the turbidity of bases such as fermented milk is high. There is a problem that it cannot be applied to samples.

By the way, rapid and highly sensitive measurement is required for the cell number measurement in the above fields. For example, in the field of food hygiene, inspection of products for microbial contamination is essential for product shipment. Conventionally, this inspection is performed by the colony counting method, but since the inspection requires more than one day, the product must be stored in the warehouse until the result is obtained. Therefore, not only is there a problem in terms of distribution efficiency, but the possibility of microbial contamination increases in products such as milk as the storage time increases. In addition, since the concentration of microorganisms that poses a problem of contamination in food is generally low, a highly sensitive test is required.

As a method for measuring the concentration of microorganisms satisfying the above requirements, there is known a method for measuring adenosine triphosphate (ATP) which is always present in living microorganisms by using a biochemiluminescence method. This method utilizes the fact that the concentration of ATP contained in a microorganism is proportional to the concentration of the microorganism, and is a very effective method because the measurement time is short and the sensitivity is high.

In the measurement by the biochemiluminescence method, luciferin, which is a substrate for fluorescence emission, and luciferase, which is its enzyme, are used. In order to measure the ATP concentration of microbial cells using this method, it is necessary to extract ATP contained in the cells.

As the ATP extraction method for cells, a method in which an aqueous solution of trichloroacetic acid (TCA) is added to a microorganism for extraction (TCA method), a method of extracting with an aqueous surfactant solution (surfactant method), and a temperature of about 90 ° C. There are a method of extracting with a heated Tris buffer (Tris buffer method), a method of using ethanol (ethanol method), a method of using a lysing enzyme such as lysozyme (enzyme method), and the like.

However, the TCA method, the ethanol method,
The surfactant method often inhibits the enzymatic reaction of biochemiluminescence, and requires pretreatment such as dilution and pH adjustment, which often complicates the operation and lowers the sensitivity. In the Tris buffer method, the extraction solution needs to be heated to a high temperature of 90 ° C., which is troublesome in terms of operation. The enzymatic method also has a problem in that extraction takes time, reagents are expensive and unstable.

[0011]

As described above, all of the methods have advantages and disadvantages, and at present, they are used properly according to the case, but the surfactant method is relatively frequently used. Generally, a cationic surfactant is used.

As described above, this method often inhibits the enzymatic reaction of biochemiluminescence, and the degree of inhibition increases as the concentration of the surfactant increases. The higher the concentration of the surfactant is, the larger the extraction ability becomes. Therefore, if the concentration of the surfactant is low, the inhibition of the enzyme reaction can be reduced, but the extraction ability becomes insufficient. Therefore, the surfactant method is desired to have a high extraction ability without inhibiting the enzyme reaction.

Therefore, an object of the present invention is to measure intracellular ATP by extracting intracellular ATP from a sample containing cells with an extraction reagent containing a surfactant and applying a biochemiluminescence method using luciferase and luciferin to this. When doing
An object of the present invention is to provide a method for measuring intracellular ATP, which can suppress inhibition of an enzymatic reaction of biochemiluminescence by an extraction reagent and which does not reduce the extraction ability of intracellular ATP.

[0014]

The above objects can be achieved by the method for measuring intracellular ATP according to the present invention. In summary, the present invention provides AT containing a detergent in a sample containing cells.
A cell in which PTP extraction reagent is added to extract intracellular ATP, and a luminescent reagent containing luscheferin and lucheferase is added to the extract to cause biochemiluminescence due to an enzymatic reaction, and ATP is measured by the luminescence The method for measuring intracellular ATP is characterized in that the biochemiluminescence is performed in the presence of an enzyme reaction inhibition inhibitor containing silicone.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for measuring intracellular ATP by extracting intracellular ATP with an extraction reagent containing a surfactant and applying a biochemiluminescence method thereto.
This has been achieved by the discovery of an enzyme reaction inhibition suppressing reagent (inhibition suppressing reagent) capable of suppressing the inhibition of enzyme reaction without reducing the extraction ability of the extraction reagent. That is, although a surfactant is used to extract ATP from cells, it often inhibits an enzymatic reaction of biochemiluminescence, and a feature of the present invention is that silicone, particularly an enzyme, is used to suppress this inhibition. The contact is with an inhibition inhibitor containing silicone used as a foaming agent.

The type of silicone used as an antifoaming agent has the effect of suppressing the action of the surfactant and preventing the surfactant from denaturing lucheferase and inhibiting the enzymatic reaction. Furthermore, the fact that silicone is chemically inactive and there is no risk of itself inhibiting an enzymatic reaction or denaturing lusferin or lusfererase, is also suitable for use as an inhibitor.

The measurement of intracellular ATP using the biochemiluminescence method is carried out by contacting a sample containing cells with an extraction reagent containing a surfactant to extract intracellular ATP from the outside of the cell.
In general, the extracted ATP is brought into contact with a luminescent reagent containing luciferin, which is a substrate for fluorescence emission, and luciferase, which is an enzyme, to cause biochemical luminescence by an enzymatic reaction by luciferin, luciferase, and ATP, and the produced light is generally measured. Is.

In the present invention, when the extraction reagent is added to the sample containing cells and then the inhibitor containing silicone is added, or when the luminescence reagent is added to the extraction liquid in which intracellular ATP is extracted, It is possible to suppress the enzymatic reaction of biochemiluminescence by adding the suppressor reagent at the same time as the addition or by adding the suppressor reagent and then adding the luminescent reagent to perform biochemiluminescence in the presence of the suppressor inhibitor. become.

When the inhibitory reagent is added at the same time as the addition of the luminescent reagent, the inhibitory reagent can be added in advance to the luminescent reagent. Preferably, the step of adding and contacting the inhibitory reagent is desirable at the same time as contacting the extracted ATP with the luminescent reagent or before contacting with the luminescent reagent in order to suppress inhibition of the enzymatic reaction of biochemiluminescence.

These reagents can be added by a manual method, a method using a device such as a pump, a flow injection analysis method (FIA method) or a combination thereof, and the amount of luminescence can be measured. It can be performed by an analytical method utilizing a flow such as a batch method or FIA method. In the FIA method, the object of the present invention can also be achieved by adding an inhibitory reagent to the carrier liquid.

Surfactants used for the extraction reagent include alkyldimethylbenzylammonium chloride (benzalkonium chloride), benzethonium chloride, cetyltrimethylammonium chloride and polyoxyethylene-10.
-Octyl phenyl ether (trade name: TritonX
-100) and the like can be preferably used.

The amount of the surfactant added is preferably 0.005 to 0.05 wt% of the total amount of the sample and the extraction reagent.
It is. If the amount of the surfactant added is less than 0.005 wt%, the extraction of intracellular ATP will be insufficient, and conversely, it will be 0.
If it exceeds 05 wt%, the extraction capacity will be saturated and only the inhibition of the luminescence reaction will increase.

The silicone used as the suppressing reagent may be any one that has been used as an antifoaming agent, and may be in the form of silicone resin, silicone oil or a modified product thereof. Any one or more kinds can be selected and used.

Specifically, for example, dimethylpolysiloxane can be used as the silicone resin. As the silicone oil, for example, linear dimethylpolysiloxane can be used. As the modified silicone resin or oil, for example, a modified dimethylpolysiloxane (modified dimethylpolysiloxane) can be used. These silicones are
Any one kind or plural kinds can be selected and used.

The amount of silicone added is preferably about 0.3 to 7 wt% of the total amount of the extraction liquid from which ATP is extracted and the luminescent reagent. If the amount of silicone added is less than 3 wt%,
The effect of suppressing the inhibition of the luminescence reaction by the surfactant is insufficient, and if it exceeds 7 wt%, the luminescence reaction may be inhibited on the contrary.

The amount of the luminescent reagent added is usually 25 times that of the extract.
About 50 Vol% is used.

[0028]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, specific examples of the present invention will be described.

Example 1 First, a test showing the inhibitory effect of silicone on the chemiluminescent reaction inhibition of the extraction reagent will be described. A batch-type biochemiluminescence measuring device was used for measuring luminescence. Dodecyldimethylbenzylammonium chloride (benzalkonium chloride) was used as the surfactant of the extraction reagent, and KM-72 (trade name, containing 30% of silicone resin by Shin-Etsu Chemical Co., Ltd.) was used as the silicone of the inhibition reagent. used.

The above-mentioned benzalkonium chloride is known as a bactericidal agent, and has the ability to act on the cell wall of cells to allow the penetration of relatively small molecules such as ATP. Therefore, the ability to extract ATP is very high, and it is an excellent extraction reagent. Extraction of ATP is accomplished by adding a fixed amount of a suspension of cells to a fixed amount of the extraction reagent and stirring. In order to express a sufficient extraction ability, it is preferable that the concentration of benzalkonium chloride in the mixed solution (extract solution) when the suspension and the extraction reagent are mixed is 0.05% or more. . However, when the concentration of benzalkonium chloride is 0.05% or more, the enzymatic reaction is significantly inhibited in the step of measuring the ATP concentration by biochemiluminescence, so that it is necessary to dilute it with a buffer solution or the like after extraction.

In the present invention, since the inhibitory reagent containing KM-72 is added, it is possible to inhibit the above inhibition without diluting after extraction.

The measurement operation is as follows. 100nm
100 μl of ol / l ATP standard solution was placed in a cuvette, 0.1% benzalkonium chloride and 1 mM EDTA were added.
100 μl of an extraction reagent (hereinafter referred to as extraction reagent A) dissolved in pH 7.75 HEPES buffer is added and stirred to extract ATP. 20% KM-72 and 1m
After adding 50 μl of an inhibitory reagent in which M EDTA was dissolved in a pH 7.75 HEPES buffer (this is referred to as inhibitory reagent a) and stirring, a luminescence reagent containing firefly luciferin and luciferase (Toa Denpa Kogyo ATPA-1L1) was added. 1
Add 00 μl and stir. Then, the cuvette is set in the biochemiluminescence measurement device, and the light generated by the biochemiluminescence reaction is measured to obtain the luminescence amount. The amount of light emission was determined by setting the cuvette, starting 10 seconds later, measuring the light emission at 0.1 second intervals for 10 seconds, and integrating the results.

EDTA added to extraction reagent A and inhibition reagent a
Is used for the purpose of suppressing the enzymatic action such as alkaline phosphatase, which decomposes ATP when cells are contacted with an extraction reagent. pH 7.75 HEPES buffer
It is necessary to keep the enzyme that measures ATP using the biochemiluminescence method in an optimal state.

Table 1 shows the relative luminescence amount in the ATP measurement. For comparison, KM-
Excluding 72 (suppression reagent b) and 1 mM EDTA
Was used in the case of using a reagent (extraction reagent B) dissolved in pH 7.75 HEPES buffer.

[0035]

[Table 1]

Since the combination of the inhibition reagent b and the extraction reagent B used as a blank did not contain KM-72 and benzalkonium chloride, it is considered that there is no inhibition on the enzymatic reaction of biochemiluminescence. Therefore, this is selected on the basis of 100% relative light emission. As shown in Table 1, in the combination of the inhibition reagent a and the extraction reagent A according to the present invention, the relative amount of luminescence is 70 when the combination of the inhibition reagent b and the extraction reagent B is obtained.
%, And inhibition of the luminescence reaction is suppressed. KM-7
In the case of the combination of the suppression reagent b not containing 2 and the extraction reagent A, the relative amount of luminescence is only 9%, which shows that the inhibition of the luminescence reaction is extremely large.

As described above, it was confirmed that the addition of the inhibitory reagent containing KM-72 can substantially inhibit the inhibition of the biochemiluminescent enzyme reaction.

Next, microbial intracellular ATP according to the method of the present invention
Is shown in comparison with the measurement by the conventional method. The conventional method relied on the TCA extraction method using trichloroacetic acid (TCA) for the extraction of ATP. The TCA extraction method is generally used when extracting ATP from microorganisms, and has a very excellent ATP extraction ability. A lactic acid bacterium suspension was used as a measurement sample.

The procedure for measuring ATP in microorganisms using the TCA extract is as follows. Sample 100 of lactic acid bacteria suspension
1 ml of 5% trichloroacetic acid solution is added to μl and stirred for 60 seconds to extract ATP from lactic acid bacteria. This sample is
00 μl was collected and pH was 7.75 HEPES buffer 3.9.
Add ml and stir well. 100 μl of this sample is placed in a cuvette, 100 μl of a luminescent reagent is added and stirred, and the sample is set in the above-mentioned biochemiluminescence measuring device to determine the luminescence amount.
ATP standard solution of 100 μl of known concentration was compared with the amount of luminescence measured by a biochemiluminescence measuring device,
The concentration was determined.

The operation of adding the pH 7.75 HEPES buffer solution and stirring the mixture is necessary to prevent trichloroacetic acid from significantly inhibiting the enzymatic reaction of biochemiluminescence. However, the sample solution is greatly diluted (80 times in this example) with trichloroacetic acid and a buffer solution, and the measurement sensitivity is lowered, which is a problem of the TCA extraction method.

The procedure for measuring the intracellular ATP of a microorganism using the inhibitory reagent a according to the present invention is as follows. A 100 μl sample of the lactic acid bacterium suspension is placed in a cuvette, 100 μl of the extraction reagent A is added, and the mixture is stirred for 20 seconds to extract ATP from the lactic acid bacterium. Further, 50 μl of the suppressing reagent a is added and stirred, and then 100 μl of the luminescent reagent is added and stirred, and the cuvette is set in the biochemiluminescence measurement device, and the luminescence amount of light generated by the biochemiluminescence reaction is measured. As described above, the amount of light emission was obtained by setting the cuvette, starting 10 seconds later, measuring the light emission every 0.1 second for 10 seconds, and integrating the results. With respect to the combination of the suppression reagent b and the extraction reagent A, and the combination of the suppression reagent b and the extraction reagent B, the same operation was performed and the luminescence amount was measured. And 100 μl of ATP standard solution of known concentration
Was compared with the amount of luminescence measured with a biochemiluminescence measuring device to determine the ATP concentration. Table 2 shows these results and the results by the TCA extraction method.

[0042]

[Table 2]

As shown in Table 2, the combination of the inhibitory reagent a and the extraction reagent A according to the present invention gave almost the same results as those measured by the TCA method, but the inhibitory reagent without KM-72 was added. The combination of b with the extraction reagent A inhibits the enzymatic reaction of luminescence, and therefore has a lower AT than the TCA method.
Only P concentration can be obtained. Further, in the case of the combination of the inhibitory reagent b and the extraction reagent B containing no surfactant, since the extraction does not occur at all, the measured value of the ATP concentration is zero. As described above, the combination of the suppression reagent a containing silicone according to the present invention and the extraction reagent A showed excellent results.

In the measurement according to this embodiment, the sample solution is diluted only slightly more than 3 times by adding the extraction reagent and the inhibition reagent. If the conventional method is used without adding the inhibitory reagent, it is necessary to dilute the sample solution by 10 times or more.
Considering that the method requires dilution as much as 80 times, the method of the present invention is very advantageous in terms of sensitivity.

In the above, the concentration of benzalkonium chloride is preferably 0.1% under the condition that 1 volume of the sample containing the microorganism to 1 volume of the extraction reagent is used, but it is not always 0% when the concentration of the microorganism is low. It does not have to be 1%,
Within the range of 0.01 to 0.1%, the concentration of benzalkonium chloride can be appropriately lowered. The concentration of benzalkonium chloride is preferably selected depending on the type of cells to be extracted.

The concentration of KM-72 added to the inhibitory reagent was
20% is preferable with respect to 1 volume of the extracted sample under the condition of 0.5 volume of the added reagent, but it is not necessarily 20% when the concentration of benzalkonium chloride is 0.1% or less. The higher the KM-72 concentration is to some extent, the greater the effect of suppressing the inhibition of the luminescence reaction is. However, if the KM-72 concentration is unnecessarily high, the enzyme reaction is inhibited on the contrary. . The concentrations of benzalkonium chloride and KM-72 are not limited to the above examples,
It is necessary to appropriately set the amount and type of the suppression reagent, the extraction reagent, and the luminescence reagent.

The EDTA and the pH buffer solution to be added to the suppression reagent and the extraction reagent do not necessarily have to be added to both reagents, but may be added to either one. When the extraction time is short, it is not always necessary to add EDTA, and the pH buffer solution need not be used if the pH of the sample to be measured is near neutral.

The time required for extracting ATP varies depending on the type of microorganism, but is preferably 10 seconds or longer for E. coli, lactic acid bacteria and general bacteria, and 30 seconds or longer for yeast. If the sample is left standing after extraction, the ATP concentration will gradually decrease, so it is preferable to measure the luminescence within 5 minutes after extraction, especially when using an extraction reagent that does not contain EDTA. Is preferably measured immediately after the extraction is complete. Further, although the reagent is manually added in this example, the reagent may be automatically added by using a pump or an automatic syringe, which allows the intracellular ATP to be measured more easily.

Example 2 In this example, the case where an inhibitory reagent is added to a luminescent reagent will be described. Benzethonium chloride is used as the surfactant of the extraction reagent, and KS-502 is used as the polymer compound of the inhibition reagent.
(Brand name: Shin-Etsu Chemical Co., Ltd., containing 100% modified silicone oil). As the measuring device, a batch type biochemiluminescence measuring device was used.

Luminescent reagent containing firefly lusferin and luciferase (Toa Denpa Kogyo Co., Ltd. ATPA-1L1)
Luminescent reagent (luminous reagent X) to which 10% of KS-502 as a suppressive reagent was added, 0.1% benzethonium chloride and 1 m
An extraction reagent (extraction reagent C) in which M EDTA was dissolved in pH 7.75 HEPES buffer was used.

The measurement operation is as follows. 100 nmo
Add 100 μl of 1 / l ATP standard solution to the cuvette,
100 μl of extraction reagent C is added and stirred to extract ATP. Next, 100 μl of the luminescent reagent X according to the present invention is added and stirred, and the cuvette is set in the biochemiluminescence measurement device, and the amount of light emitted by the biochemiluminescence reaction is measured. The amount of light emission was determined by setting the cuvette, starting 10 seconds later, measuring and measuring the light emission at 0.1 second intervals for 10 seconds, and integrating the results.

Table 3 shows the ATP of each luminescent reagent and extraction reagent.
The relative luminescence amount in the measurement is shown. For comparison, the above luminescent reagent composition excluding KS-502 (luminescent reagent Y) and 1 mM EDTA were added to pH 7.75 HEPES.
The case of using the extraction reagent dissolved in the buffer solution (extraction reagent B described above) is also shown.

[0053]

[Table 3]

In the combination of the luminescent reagent Y and the extraction reagent B, K
Since S-502 and the surfactant are not included, there is no inhibition to the luminescence reaction, and it is considered that the relative luminescence amount is 100%.
In the combination of the luminescent reagent X and the extraction reagent C according to the present invention, as shown in Table 3, the relative luminescence amount is 81% of that in the combination of the luminescent reagent Y and the extraction reagent B, and there is no significant inhibition. On the other hand, in the combination of the luminescence reagent Y and the extraction reagent C based on the conventional method not containing KS-502, the relative luminescence amount is 15%.
It turns out that it is low and the inhibition is large. As described above, it is understood that the effect of the present invention can be obtained even if silicone is added to the luminescent reagent.

Next, the microbial intracellular ATP measurement according to the present invention will be shown in comparison with the measurement by the conventional method. A sample was a yeast suspension. The conventional method is the same as in the first embodiment.
The TCA extraction method was used to extract P, and the procedure for measuring the intracellular ATP of the microorganism was as described in Example 1.

The procedure for measuring ATP in microbial cells using the luminescent reagent X according to the present invention is as follows. 100 μl of the yeast suspension sample was placed in a cuvette and the extraction reagent C was added.
Add 100 μl of the mixture and stir for 30 seconds
Is extracted. Add 100 μl of luminescent reagent and stir,
A cuvette is set in the biochemiluminescence measurement device, and the amount of light emitted by the biochemiluminescence reaction is measured. The amount of light emission was obtained by setting the cuvette and starting 10 seconds later, measuring the light emission every 0.1 second for 10 seconds, and integrating it.

With respect to the combination of the luminescent reagent Y and the extraction reagent C and the combination of the luminescent reagent Y and the extraction reagent B, the luminescence amount was measured by the same operation. Then, the ATP concentration was determined by comparing the amount of luminescence measured with a biochemiluminescence measuring device with 100 μl of the ATP standard solution having a known concentration. Table 4 shows the results of these and the TCA method.

[0058]

[Table 4]

As shown in Table 4, in the combination of the luminescent reagent X and the extraction reagent C based on the present invention, the result equivalent to the value measured by the TCA method was obtained. On the other hand, KS-
The combination of the luminescent reagent Y and the extraction reagent C without the addition of 502 cannot prevent the inhibition of the enzymatic reaction of luminescence.
Only ATP concentration lower than that of Method A can be obtained. When the extraction reagent B containing no surfactant is used, almost no extraction occurs, so that the measured value of ATP is almost zero. Therefore, it is clear that the combination of the luminescent reagent X and the extraction reagent C according to the present invention is excellent.

Regarding the concentration of the surfactant, the concentration of the silicone and the extraction time, the same precautions as in Example 1 are required.

Example 3 In this example as well, a suppressing reagent was added to the luminescent reagent for use. Triton X-100 (trade name; polyoxyethylene-10-octylphenyl ether, Wako Pure Chemical Industries, Ltd.) as a surfactant, and KS-506 (trade name; Shin-Etsu Chemical Co., Ltd.) modified silicone oil 100 as a silicone of the suppressing reagent. % Content) was used. The measuring device is a batch-type biochemiluminescence measuring device as before.

It is known that Triton X-100 has no ability to extract ATP from most microbial cells, and has the ability to extract ATP from only somatic cells of animals. For that reason, it may be used when separately measuring ATP of animal cells and microbial cells, but since it inhibits biochemiluminescence reaction like cationic surfactants such as benzalkonium chloride, it is also used. There is a problem such as a decrease in ATP measurement sensitivity.

In this example, a luminescent reagent containing firefly luciferase and luciferin (ATPA-manufactured by Toa Denpa Kogyo) was used.
1L1) a luminescence reagent (luminescence reagent Z) obtained by adding 5% of a suppressing reagent, KS-506, and 0.1% Triton X-1.
An extraction reagent (extraction reagent D) prepared by dissolving 00 and 1 mM EDTA in a pH 7.75 HEPES buffer was used. For comparison, the luminescent reagent obtained by removing KS-506 from the above luminescent reagent composition (the luminescent reagent Y described above) and 1 mM EDTA.
Extraction Reagent B prepared by dissolving EDTA in pH 7.75 HEPES buffer
Was also tested.

The inhibition inhibition was measured in the same manner as in Example 2. Table 5 shows the relative luminescence amount of each luminescence reagent and the extraction reagent in the ATP measurement.

[0065]

[Table 5]

When the combination of the luminescent reagent Y and the extraction reagent B is K,
Since S-506 and Triton X-100 are not contained, there is no inhibition to the luminescence reaction, and it is considered that the relative luminescence amount is 100%. As shown in Table 5, the relative amount of luminescence in the case of the combination of the luminescence reagent Z and the extraction reagent C according to the present invention is 91% of the case of the combination of the luminescence reagent Y and the extraction reagent B, and there is almost no inhibition. It can be seen that the combination of the luminescence reagent Y and the extraction reagent D based on the conventional method not containing KS-506 has a low relative luminescence amount of 35%, which shows a large inhibition. As described above, it is understood that the effect of the present invention can be obtained also by adding KS-506 to the luminescent reagent.

Next, the measurement of ATP in animal somatic cells according to the present invention will be shown in comparison with the conventional method. A mouse lung cell suspension was used as a sample. The conventional method is the same as the conventional method.
This is a method of extracting ATP by the extraction method. TCA
The extraction method and the procedure for measuring intracellular ATP using the luminescent reagent Z and the extraction reagent D according to the present invention are as described in Example 2.

With respect to the combination of the luminescent reagent Y and the extraction reagent D and the combination of the luminescent reagent Y and the extraction reagent B, the luminescence amount was measured by the same operation, and the luminescence amount measured with a biochemiluminescence measuring device for 100 μl of ATP standard solution of known concentration. Compared with AT
The P concentration was determined. Table 6 shows the results.

[0069]

[Table 6]

As shown in Table 6, in the combination of the luminescent reagent Z and the extraction reagent D according to the present invention, the same result as the value measured by the TCA method was obtained, but the luminescent reagent without KS-506 was added. The combination of Y and extraction reagent D has a lower ATP than the TCA method because it inhibits the enzymatic reaction of luminescence.
Only concentration can be obtained. Moreover, since the extraction reagent B containing no Triton X-100 does not cause any extraction,
The measured value of P concentration is almost zero. It is clear that the combination of luminescent reagent Z and extraction reagent D according to the invention is excellent.

Regarding the concentration of the surfactant, the concentration of the silicone and the extraction time, the same precautions as in Example 1 are required.

[0072]

As described above, according to the present invention, the AT
In contrast to the inhibition of the biochemical chemiluminescence enzyme reaction by the surfactant of the P extraction reagent, the inhibition reagent containing silicone was used, so that the inhibition of the enzyme reaction can be suppressed and light emission can be achieved.
The ATP concentration can be accurately measured by detecting the luminescence. Further, since the measurement sample does not need to be diluted, the measurement sensitivity can be improved, the extraction ability is not lowered, and the ATP measurement method which is simpler in operation can be constructed. Therefore, it exerts great power when testing microorganisms such as food hygiene and biotechnology.

Claims (10)

[Claims] 1. AT containing a surfactant in a sample containing cells
A cell in which PTP extraction reagent is added to extract intracellular ATP, and a luminescent reagent containing luscheferin and lucheferase is added to the extract to cause biochemiluminescence due to an enzymatic reaction, and ATP is measured by the luminescence In the method for measuring intracellular ATP, the method for measuring intracellular ATP is characterized in that the biochemiluminescence is performed in the presence of an enzyme reaction inhibition inhibitor containing silicone. 2. The method for measuring intracellular ATP according to claim 1, wherein the silicone is selected from the group consisting of silicone resin, silicone oil, and modified products thereof. 3. The method for measuring intracellular ATP according to claim 2, wherein the silicone resin is dimethylpolysiloxane. 4. The method for measuring intracellular ATP according to claim 2, wherein the silicone oil is linear dimethylpolysiloxane. 5. The method for measuring intracellular ATP according to claim 2, wherein the modified product is a modified product of dimethylpolysiloxane. 6. The amount of silicone added is 0.3 to 7 wt% of the total amount of the extract and the luminescent reagent.
2, 3, 4 or 5 intracellular ATP measurement methods. 7. The method for measuring intracellular ATP according to claim 1, 2, 3, 4, 5 or 6, wherein the inhibitory reagent is added after the extraction reagent is added to the sample. 8. The method for measuring intracellular ATP according to claim 1, 2, 3, 4, 5 or 6, wherein a suppressive reagent is added when the luminescent reagent is added to the extract. 9. The surfactant is benzalkonium chloride,
2. A compound selected from the group consisting of benzethonium chloride, cetyltrimethylammonium chloride and polyoxyethylene-10-octylphenyl ether.
2, 3, 4, 5, 6, 7 or 8 intracellular ATP measurement method. 10. The addition amount of the surfactant is 0.005 to 0.05 wt% of the total amount of the sample and the extraction reagent, and the addition amount is preferably 1, 2, 3, 4, 5, 6, 7, 8, or 9. Intracellular A
Method for measuring TP.