WO2001081618A1

WO2001081618A1 - Method of measuring biological luminescence

Info

Publication number: WO2001081618A1
Application number: PCT/JP2001/003459
Authority: WO
Inventors: Noriaki Hattori; Yasuhiro Harada; Tatsuya Sakakibara; Toshinori Igarashi; Shigeya Suzuki; Seiji Murakami
Original assignee: Kikkoman Corporation
Priority date: 2000-04-26
Filing date: 2001-04-23
Publication date: 2001-11-01
Also published as: AU4883401A

Abstract

A method of measuring biological luminescence characterized in that, in measuring biological luminescence induced by brining a sample into contact with a biological luminescent reagent and an ATP regenerating enzyme, a photosensitive material is exposed to the biological luminescence. This method involves: the first step of bringing a sample into contact with luciferin, luciferase, pyruvate o-phosphate dikinase, phosphoenolpyruvic acid and pyrophosphoric acid in the presence of magnesium ion; and the second step of exposing a photosensitive material to the biological luminescence induced in the first step. Examples of the above-described biological luminescent reagent include luciferin, luciferase and magnesium ion. Examples of the above-described ATP regenerating enzyme include pyruvate o-phosphate dikinase, phosphoenolpyruvic acid synthase and a mixture of adenylate kinase with pyruvate kinase. Examples of the above-described photosensitive material include photographic films, instant films and photographic papers.

Description

9 Description Bioluminescence measurement technology Field of technology

The present invention relates to a method for measuring bioluminescence. Background art

It has been known that luminescence is generated when adenosine triphosphate (hereinafter referred to as “AT PJ”) reacts with insect luciferin and luciferase in the presence of magnesium ions. Using this principle, a method for measuring ATP in a sample or a method for measuring the number of cells by measuring intracellular ATP has been developed using this principle. Many reagents involved in luminescence (hereinafter referred to as "bioluminescent reagents") have also been developed.

In addition, various methods for measuring a specific substance in a sample using a bioluminescent reagent have been developed. A method of mixing a sample and a bioluminescent reagent in a test tube or cell, a method of adding a bioluminescent reagent after extracting ATP from cells captured on a filtration membrane, and a method of using a carrier on which a bioluminescent reagent is immobilized as a sample. And the like. In addition, a method has been developed for detecting a band of proteins or nucleic acids separated by electrophoresis using a bioluminescent reagent as a probe. In these measurement methods, it is an issue to measure the generated bioluminescence simply and with high sensitivity.

When the amount of ATP in the reaction system, for example, the amount of ATP in the sample or the amount of ATP contained in the cells, is small, conventional bioluminescent reagents and methods may not provide sufficient measurement results. As a method for improving the sensitivity of a measurement method using a bioluminescent reagent, a method of adding pyruvate orthophosphate dikinase (hereinafter referred to as “PPDK”) to a reaction system is known (Japanese Unexamined Patent Publication No. 9-23). No. 409, Japanese Unexamined Patent Application Publication No. 11-69994). When ATP, luciferin and luciferase react, ATP is consumed and the luminescence rapidly attenuates. AMP and pyrophosphate are generated by this bioluminescence reaction I do. PPDI catalyzes the reaction that regenerates ATP (ATP regeneration reaction) by acting on these products in the presence of magnesium ions, so that the luminescence in the bioluminescence reaction is not attenuated and the luminescence is maintained for a long time. be able to. For this reason, the measurement sensitivity can be improved.

As an enzyme that acts on a substance produced when ATP is consumed in a bioluminescence reaction to regenerate ATP (hereinafter referred to as “regenerating enzyme”), phosphoenolpyruvate synthetase is known in addition to PPDK. . Also, Adenire Tokina

A method for regenerating ATP using a combination of -ze and pyruvate kinase is also known. Thus, the method of combining the bioluminescent reagent and the ATP regenerating enzyme is useful for improving the measurement sensitivity.

In the case of measuring bioluminescence, a luminometer such as a luminescence reader BLR-201 manufactured by Aloka, a luminometer G-100 manufactured by Kikkoman, or the like is usually used. Also, a method for measuring a microorganism captured on a filtration membrane with high sensitivity using a bioluminescent reagent, PPDK, and an image analysis system device (ARGUS-50 / CL manufactured by Hamamatsu Photonics Co., Ltd.) is known (Japanese Patent Laid-Open Publication No. 1 1—69994).

As another method, a method of measuring bioluminescence by exposing bioluminescence to photographic film is known, and an apparatus for carrying out the method has been developed (Japanese Patent Publication No. 2000-500568). U.S. Patents US3666631, US5188965, etc.). The method using a photographic film is also used to measure a luminescence reaction other than bioluminescence (Japanese Patent Laid-Open No. 35147/1991), which eliminates the need for expensive measurement equipment such as a luminometer. Excellent in point. With regard to the method using photographic film, higher sensitivity is also required. Disclosure of the invention

An object of the present invention is to provide a simple and highly sensitive method for measuring bioluminescence. The present inventors have conducted intensive studies on the above problems, and as a result, by combining a bioluminescent reaction system using a bioluminescent reagent and an ATP regenerating enzyme with a method using a photosensitive substrate such as a photographic film, Easy and highly sensitive measurement of luminescence I found what I could do. The present invention has been completed based on this finding.

That is, the present invention relates to the following method.

(1) A method for measuring bioluminescence, which comprises sensitizing a substrate to bioluminescence when measuring bioluminescence generated by contacting a sample, a bioluminescent reagent, and an ATP regenerating enzyme.

(2) The method for measuring bioluminescence according to (1), wherein the bioluminescence reagent comprises luciferin, luciferase, and magnesium ion.

(3) The method for measuring bioluminescence according to (1), wherein the ATP regenerating enzyme is pyruvate orthophosphate dikinase or phosphoenolpyruvate synthetase, or a mixture of adenylate kinase and pyruvate tokinase. .

(4) The method for measuring bioluminescence according to (1), wherein the photosensitive substrate is a photographic film, an instant film, or a photographic paper.

(5) A method for measuring bioluminescence including the following steps.

First step: a step of contacting a sample, luciferin, luciferase, pyruvate orthophosphate dikinase, phosphoenolpyruvate and pyrophosphate in the presence of magnesium ions.

2nd step ′: The 2nd step of exposing the bioluminescence generated in the 1st step to a photosensitive substrate.

(6) The method for measuring bioluminescence according to (5), wherein the photosensitive substrate is a photographic film, an instant film, or a photographic paper. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram illustrating a device for carrying out the present invention, FIG. 2 is a diagram illustrating a device for carrying out the present invention, and FIG. 3 is a diagram illustrating a device for carrying out the present invention. FIG. 4 is a diagram showing a part of an apparatus for carrying out the present invention, and FIG. FIG. 6 is a view showing a measurement result of bioluminescence (5 minutes exposure) using a photographic film of FIG. 6, and FIG. 6 is a view showing a measurement result of bioluminescence by a photographic film of the example (15 minutes exposure). In the above figure, 1 is a photosensitive substrate, 2 is a dark box-shaped main body, 3 is a test tube which is a bioluminescence reaction container, etc., 4 is a lid, 5 is an inlet, 6 is a film holder, and 7 is a lid. Illum pack, 8 is a support for holding test tubes, etc., 10 is an instant film, 1 is an instant film take-out port, 12 is a light shielding cover, 13 is a CCD, 14 is a data output device, Numeral 15 denotes bioluminescent light, numeral 16 denotes a condenser lens, and numeral 17 denotes a structure of a test tube or the like which functions as a condenser lens. BEST MODE FOR CARRYING OUT THE INVENTION

The method for measuring bioluminescence of the present invention is characterized in that, when measuring bioluminescence generated by contacting a sample, a bioluminescent reagent and an ATP regenerating enzyme, the bioluminescence is exposed to a photosensitive substrate.

1. How to generate bioluminescence

In the method of the present invention, the method of contacting the sample, the bioluminescent reagent and the ATP regenerating enzyme is not limited, and a known method can be used.

Samples include, for example, foods and drinks, pharmaceuticals, cosmetics, seawater, river water, industrial water, sewage, soil, urine, feces, blood, sputum, pus, cultures of cells, and wiping off deposits on machinery and equipment And the like. The above sample may be diluted with an appropriate solvent (for example, distilled water, physiological saline, phosphate buffer, Tris buffer, sodium acetate buffer, etc.). In the present invention, a filter membrane capturing cells such as microorganisms may be used as the sample.

As the bioluminescent reagent, for example, a reagent containing luciferin, luciferase, and magnesium ion can be used. In this case, as the ATP regenerating enzyme, for example, PPDK and phosphoenolpyruvate synthetase can be used. Also, two or more enzymes involved in the ATP regeneration reaction, such as a mixture of adenylate kinase and pyruvate kinase, may be used in combination and used as the ATP regeneration enzyme. The bioluminescent reagent contains other reagents necessary for the bioluminescence reaction and the ATP regeneration reaction, or reagents (preservatives, pH adjusters, antioxidants, etc.) that allow each reaction to proceed stably. Is also good.

As the PPDK, any enzyme can be used as long as it is an enzyme that acts on AMP and pyrophosphate to generate ATP, for example, from corn leaves [Biochemistr y 12, 2862-2867 (1973)] and enzymes derived from sugar cane leaves [The Biochemical Journal 114, I 17-125 (1969)]. Microbial sources include, for example, Propionibacterium shermani i) Enzymes derived from microorganisms belonging to [Biochemistry 10, 721-729 (1971)], the genus Microbispora (eg, Microbispora thermorosea IFO 14047) and the like. As the luciferin and luciferase, for example, those derived from insects (genji firefly, Heike firefly, North American firefly, Japanese click beetle, glow firefly, etc.) can be used.

PPDK and luciferase are added to one or more amino acids in the amino acid sequence of natural enzymes purified from cultures and tissues of the above organisms, recombinant enzymes prepared by genetic engineering techniques, and natural enzymes. Mutant enzymes into which mutations such as deletion, substitution, etc. have been introduced can be used.

Examples of specific methods for generating bioluminescence by contacting a sample, a bioluminescent reagent, and an ATP regenerating enzyme include, in the presence of magnesium ions, a sample, luciferin, luciferase, pi-reversion orthophosphate dikinase. , Phosphoeno- rupyruvic acid and pyrophosphoric acid.

2. Photosensitive substrate

In the present invention, bioluminescence is exposed to a photosensitive substrate. As the photosensitive substrate, a material in which a material whose physical properties change upon irradiation with light is fixed on a suitable carrier can be used. As the photosensitive substrate, any material having a property capable of sensitizing bioluminescence can be used, and for example, photosensitive films such as photographic films, photographic paper, photosensitive resin, CCD (Charge Coupled Divice) and the like can be used. .

3. Measurement method using photographic film or photographic paper

Photographic films or photographic papers are easily available as commercial products having various characteristics and are excellent in operability, portability, and economy, and thus are suitable as the photosensitive substrate of the present invention. As a photographic film, a high-sensitivity film for black-and-white photographs or color photographs, a high-sensitivity X-ray film, a negative type and a positive type film, and the like can be used. In the following, bioluminescence is referred to as photographic film or photographic paper (hereinafter referred to as "photographic film, etc." An example of a method for exposing to ()) will be described.

First, a test tube, cell, filter membrane, carrier, etc. that captures bioluminescence (hereinafter referred to as “test tube, etc.”) is brought into contact with or approached a photographic film. The two may be in direct contact, or may be in contact via a thin glass plate or plastic plate. Similar results can be obtained by re-photographing the test tubes and the like in a dark room with a camera loaded with photographic film. It is preferable that the photosensitive operation is performed in a dark room or a dark box.

If photographic film is used, after exposure, develop and fix the film and, if necessary, print it on photographic paper. When photographic paper is used, development and fixing are performed. The resulting bioluminescence image appears as a container silhouette, dots, bands, etc. By observing these, it becomes possible to calculate the amount of ATP and the number of cells in the sample. The sensitivity (Iso speed) and type of the photographic film used, the exposure time, the aperture of the camera, and the speed of the camera are appropriately determined according to the type of sample, the type of bioluminescent reagent, and the equipment used. Just set it. As the photographic film, those having an ISO speed of 3000 or more can be preferably used.

4. Measurement method using instant film

It is unique in that it can save development time.

Product name: Instant B & W Film FP-3000B (manufactured by Fuji Photo Film Co., Ltd.) Product name: Boraroid Coreless Black and White

6 7 (Bolaroid) can be used. Hereinafter, an example of a method for exposing bioluminescence is described.

First, instance ^f 'attached to one (Bora port id, Inc., etc.). After bringing the test tube, etc. that is producing bioluminescence into contact with the film holder with the shutter cover closed as much as possible, open the shutter cover in the dark room. After exposing the bioluminescence to the film for an appropriate time, close the shirt cover. Remove the film from the film holder.

5. Measurement method using CCD CCDs are widely used as photosensitive substrates in digital cameras and can store optical information as digital signals, making them excellent at processing, recording, and transmitting measurement results. When a CCD is used as the photosensitive substrate, bioluminescence can be exposed according to a method using a photographic film or the like. That is, a test tube or the like that is producing bioluminescence is brought into direct or indirect contact with the CCD. Alternatively, the above test tube or the like may be photographed using a digital camera using a CCD. The method of using a digital camera is excellent in that the obtained image can be checked immediately by the built-in monitor screen or the monitor of a connected computer.

6. Advantages of the present invention

Compared to the method of measuring bioluminescence generated by a bioluminescent reagent containing ATP regenerating enzyme using a luminometer (conventional method), the method of measuring using a photographic film or photographic paper (the present invention) Advantages include:

(1) No expensive luminometer is required.

(2) The measurement result can be stored in a state that can be seen as a photograph.

(3) Used equipment · The equipment is compact and highly portable.

(4) No electrical energy is required for measurement.

Compared to the conventional method of measuring bioluminescence generated by only a bioluminescent reagent using a photographic film or photographic paper (conventional method), the bioluminescence generated by a bioluminescent reagent containing an ATP regenerating enzyme is measured using a photographic film or photographic paper. Advantages of the measuring method (the present invention) include the following.

(5) Since bioluminescence lasts longer than conventional methods, photographic films can be sufficiently sensitized and clear images can be obtained. Therefore, it is possible to increase the sensitivity of the measurement.

7. Application examples of the present invention

The present invention is particularly useful as a method for counting the number of living cells in a sample. One example for counting the number of cells is shown below.

First, a sample that is thought to contain cells is liquefied, and then filtered through a filtration membrane. Then, in order to extract ATP from cells, filter membrane and ATP extractant (surfactant etc.) Make contact. Next, the filter membrane, the ATP regenerating enzyme, and the bioluminescent reagent are brought into contact, and the resulting bioluminescence is exposed to the photosensitive substrate. If cells are present on the filtration membrane, the cells are observed on the photosensitive substrate as light spots. Thus, the number of living cells in the sample can be counted.

The cells to be measured include animal, plant and cell cells, as well as mold, yeast and bacterial cells.

8. Apparatus for implementing the present invention

When carrying out the method of the present invention, known devices and instruments for measuring bioluminescence or chemiluminescence using a photographic film or the like can be used as they are or partially modified.

Examples of an apparatus for performing the method of the present invention are shown in FIGS.

The apparatus shown in FIG. 1 (1) comprises a dark box-shaped main body 2 for housing a photosensitive substrate 1. A test tube 3 which is a reaction vessel for bioluminescence is rushed in from an inlet 5 with a lid 4. It is preferable that the dark box block light when the lid is closed. However, if the device is used in a darkroom, the lid is not required. (2) is a perspective view of the apparatus. FIG. 2 is an example of the apparatus when the photosensitive substrate is an instant film. In this apparatus, a film pack 7 for accommodating a plurality of instant films is attached to a film holder 16. Further, the apparatus has a support 8 for holding a test tube or the like 3 which is generating bioluminescence, and a shutter cover 9 for shielding light. The support may have a structure capable of holding a plurality of test tubes or the like. After exposure, the instant film 10 is removed from the outlet 11. (2) is a perspective view of the device. As shown in (3), if a cover 12 for shielding light is installed, it can be used in places other than dark rooms.

Fig. 3 shows an example of the device when the photosensitive substrate is a CCD. This device consists of a dark box-shaped main body 2 containing a CCD 13 and an entrance 5 (with a lid 4) for inserting a test tube 3 which is a bioluminescence reaction container. The CCD reads the bioluminescence light information and converts it into electrical signals. The electrical signal is sent to the It is output to a peripheral device such as a printer and reproduced as an image. Other devices necessary for collecting, digitizing, outputting, and reproducing optical information will be installed as appropriate. Other devices include, for example, microlenses, A / D converters, and CDS (core 1 at ion double sampling).

As shown in FIG. 4 (1), the apparatus for carrying out the invention is provided with a condenser lens 16 to collect the bioluminescent light 15 on the photosensitive substrate 1 and improve the measurement sensitivity. Is also good. When a bioluminescence reaction is caused in a test tube or cell, a structure 17 in which a part of the test tube or cell functions as a condenser lens as shown in (2) may be used.

In addition, it is preferable that the surface other than the film contact surface of the test tube or the cell used in the present invention has been subjected to processing for preventing light scattering. The distance between a photosensitive substrate such as a photographic film and a test tube or the like in which bioluminescence is generated is preferably set to be as short as possible, and both may be in contact with each other.

Example

Hereinafter, the present invention will be described with reference to examples.

1. Preparation of reagents

(1) Bioluminescent reagent containing ATP regenerating enzyme

In order to carry out the present invention, a luminescent reagent containing PPDK as an ATP regenerating enzyme (HPPDK luminescent reagent) was prepared. PPM luminescent reagents are bioluminescent reagents such as Heike firefly luciferase, luciferin, magnesium ion, and ATP regenerating enzyme.Microbispolar 'PPDK derived from thermolasa. It contains benzalkonium chloride and a protective agent as reagents necessary for ATP extraction from microorganisms. Its composition is as follows:

Composition of PPDK luminescence reagent:

0.5 mM luciferin (Sigma)

0.27 mg / ml mutant firefly luciferase (Kikko Iman)

0.13 mg / ml PPDK (from Microbispora. Thermolosa)

0.2 mM PPi K (potassium pyrophosphate) 1.4 mM PEP K (potassium phosphoenorubirbate)

6.67 mM Mg Ac 4¾0 (magnesium acetate .4hydrate)

0.01 Benzalconium chloride

Protecting agent (0.06% BSA, 20mM Tricine (ρΗ7.8), 1mM EDTA 2Na, 0.023mM DTT) (2) Bioluminescence reagent of comparative example

The following two types were used as comparative bioluminescent reagents.

As a normal sensitivity reagent: Lucifer 250 Plus (manufactured by Kikkoman) As a high sensitivity reagent: Lucifer HS Plus (manufactured by Kikkoman)

2. Implementation of bioluminescence reaction

(1) PPM luminescence reagent

To 0.15 ml of the luminescence reagent was added 0.01 ml of ATP solution (2X10- 10-fold dilution series from ⁶ to 2X10- ¹³ M), were mixed with the reaction solution by pipetting to produce bioluminescence.

(2) Bioluminescent reagent for comparison

It was added ATP solution (4XL (ATP extraction reagent of 0.05 ml in 10-fold dilution series) from T ⁶ to 4X10- ¹² Μ of 0.05 ml, giving rise to a bioluminescence was further added light emission reagent of 0.05 ml.

3. Measurement of bioluminescence

(1) Measurement with a luminometer

A test tube (trade name: Lumitube, manufactured by Kikkoman Co., Ltd.) was used as a reaction vessel for each reagent, and the amount of luminescence was measured using a Lumitester K-100 (manufactured by Kikkoman).

(2) Measurement using photographic film

The reaction was carried out by using a Nunc immo module / Module plate C8 (manufactured by Nunc) as a reaction vessel and dropping each reagent in one row. In addition, the distance between the wells used for the reaction was adjusted appropriately according to the intensity of the light emission to prevent interference of the light emission.

An instant B & W film FP-3000B (manufactured by Fuji Photo Film Co., Ltd.) was mounted on a film holder (manufactured by Boraroid), and the shutter cover was opened in a dark room. The bioluminescent well was then exposed on a film. Exposure time is 5 Minutes or 15 minutes.

4. Measurement results

(1) Measurement results using a luminometer

Table 1 shows the measurement results of the amount of luminescence by the PPDK luminescence reagent and the bioluminescence reagent (Lucifer 250 Plus and HS Plus). From this, it was found that the PPDK luminescent reagent showed a luminescence amount that was about 50 times higher than Lucifer 250 plus and about 2.5 times higher than Lucifer HS plus.

However, PPDK luminescence reagent has a high blank value, and the detection limit is calculated from twice the blank value.The lower sensitivity of PPM luminescence reagent and Lucifer 250 Plus is 2X ΚΓ ¹⁵ M with the same sensitivity, and It was of the highest sensitivity 2X10- ¹⁶ M.

table 1

ATP light intensity (RLU)

(mol / assay) PPM luminescence reagent Lucifer 2507 ° ras Lucifer HS. Lath

2X10 " ¹² Scale over 50,475 Scale over

2X10— ⁱ³ 265,312 5,245 107,542

2X10— ¹⁴ 27, 564 517 11,546

2X10 " ¹⁵ 3,678 65 1,125

2X10 " ¹⁶ 1,385 12 106

2X10- ¹⁷ 1,235 7 28

2X10 " ¹⁸ 1,197 8 20

Blank 1,240 7 17

(2) Measurement results using photographic film

Fig. 5 shows the result of 5 minutes of exposure time, and Fig. 6 shows the result of 15 minutes.

The 5 minute exposure time, light emission image of PPDK luminescent reagent can be bright et crab measured up 2X10- ¹³ mol / assay, the sensitivity drop from the case of using a luminometer was only 100 times. On the other hand, the detection limit of Rushifuesore 250 plus 2X10- H mol / assa y, the lower limit of detection Rushifue Ichiru HS Plus is 2X10- ¹² mol / assay, luminometer one The decrease in sensitivity from the case of using a filter was 10,000 times. That is, when luminescence measurement was performed using a photographic film, it was shown that the PPDK luminescence reagent can measure 100 times more sensitively than Lucifer 250 plus and 10 times more sensitively Lucifer HS brasserie.

This is because when the bioluminescent reagent of Comparative Example was used, the amount of luminescence attenuated over time, but when the PPDK luminescent reagent was used, luminescence continued due to the ATP regeneration reaction, and the film was sufficiently exposed.

For reference, Table 2 shows the residual luminescence (%) after a certain period of time (5, 15 minutes) when the bioluminescent reagent or PPDK luminescent reagent of Comparative Example was used. The amount of residual luminescence was calculated by the following equation.

100 X (the amount of light emitted per unit time after a certain period of time) / (the amount of light emitted when the amount of light emitted per unit time immediately after the start of the luminescence reaction is maximized)

Table 2

Residual light emission ^)

PPDK Luminescent Reagent Lucifer 250F. Ras Lucif I-Les HS7 ° Lath

After 5 minutes 99.7 11.6 5.4

After 15 minutes 97.9 1.5 0.2 On the other hand, further extending the exposure time to 15 minutes, the lower detection limit of PPDK emission reagent as compared to the case 2X ¹ 0- 1 ⁴ mol / assay, and the 5-minute A 10-fold increase in sensitivity, that is, a 10-fold decrease in sensitivity from using a luminometer. On the other hand, the lower detection limits of Lucifer 250 brass and Lucifer HS Plus were not different from those of 5 minutes, and the sensitivity reduction from using the luminometer was 10,000 times. That is, the PPDK luminescent reagent was able to measure 1000 times more sensitively than Lucifer 250 plus and 100 times more sensitive than Lucifer HS plus.

Table 3 summarizes the lower detection limits (mol / assay.) Of ATP obtained by the methods 2. (1) and (2) above. Table 3

From the above results, in the method of measuring bioluminescence using a photographic film instead of a luminometer, the PPDK luminescence reagent has less decrease in sensitivity than when a bioluminescence reagent is used, and as a result, the highest sensitivity is obtained. It clearly shows that the measurement is possible. In particular, by prolonging the exposure time, the characteristics of the PPDK luminescent reagent and photographic film could be maximized. It has been clarified that the method of the present invention, that is, the method of contacting a sample, a bioluminescent reagent and an ATP-regenerating enzyme and exposing the resulting bioluminescence to a photosensitive substrate is excellent as a method for measuring bioluminescence. Industrial applicability

According to the present invention, a method for simply and highly sensitively measuring bioluminescence is provided. According to the present invention, luminescence can be recorded as an image on a photosensitive substrate such as a photographic film without using an expensive luminometer. When a photographic film or photographic paper is used as the photosensitive substrate, the device for practicing the present invention does not require an electric device. Therefore, it is possible to design a device that is simplified and has excellent portability.

INDUSTRIAL APPLICABILITY The present invention can be used in the fields of medical treatment, hygiene inspection, and test research as a method for measuring a specific substance in a sample using a bioluminescent reagent and an ATP regenerating enzyme. The present invention is particularly useful as a method for measuring ATP in a sample or intracellular ATP, or a method for counting the number of living cells captured on a filtration membrane.

The present description is based on the specification of Japanese Patent Application No. 2000-2012 which is the basis of the priority of the present application. The contents described in this document are included. All publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Claims

The scope of the claims

(2) The method for measuring bioluminescence according to claim 1, wherein the bioluminescent reagent contains luciferin, luciferase, and magnesium ion.

(3) The bioluminescence measurement according to claim 1, wherein the ATP-regenerating enzyme is pyruvate orthophosphate dikinase or phosphonorubyruvate synthetase, or a mixture of adenylate kinase and pyruvate kinase. Law.

(4) The method for measuring bioluminescence according to claim 1, wherein the photosensitive substrate is a photographic film, an instant film, or a photographic paper.

(5) A method for measuring bioluminescence including the following steps.

Second step: The second step in which the bioluminescence generated in the first step is exposed to a photosensitive substrate.

(6) The method for measuring bioluminescence according to claim 5, wherein the photosensitive substrate is a photographic film, an instant film, or a photographic paper.