JP2001275698A - Carrier for holding luminescent reagent and method for assaying atp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a solid phase carrier for holding luminescent reagents, and to provide a convenient and high sensitive method for assaying ATP by using the same carrier. SOLUTION: This carrier features holding luciferin, luciferase and an ATP regenerating enzyme (e.g. pyruvate orthophosphate dikinase) as luminescent reagents. The method for assaying ATP in a specimen features contacting the above carrier with the specimen and then measuring generated light emission.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a carrier holding a luminescent reagent and a method for measuring ATP.

[0002]

2. Description of the Related Art Hitherto, it has been known that luminescence occurs when adenosine-3-phosphate (hereinafter referred to as "ATP") reacts with insect luciferin and luciferase in the presence of magnesium ions. This is called a bioluminescence reaction, and utilizes this principle to make ATP in a sample
And a method for measuring the number of cells by measuring intracellular ATP have been developed. In addition, many reagents involved in bioluminescence (hereinafter referred to as “luminescent reagents”) such as luciferin and luciferase have been developed.

The amount of ATP in a sample and the amount of AT contained in cells
When the amount of P is small, a sufficient measurement result may not be obtained with the conventional luminescent reagent or method. A using luminescent reagent
As a method for improving the measurement sensitivity of the method for measuring TP, there is known a method in which pyruvate orthophosphate dikinase (hereinafter referred to as "PPDK"; EC 2.7.9.1) is added to the reaction system (Japanese Patent Application Laid-Open No. 9-1991). No. 234099, JP-A-11-
No. 69994). When ATP, luciferin and luciferase react, ATP is consumed, and the luminescence rapidly attenuates. Adenosine monophosphate (AMP) and pyrophosphate are generated with the bioluminescence reaction. Since PPDK catalyzes a reaction that generates ATP by acting on these products in the presence of magnesium ions (ATP regeneration reaction), luminescence is not attenuated in the bioluminescence reaction, and light can be emitted for a long time. This makes it possible to increase the sensitivity of the measurement method.

It is known that a luminescent reagent, luciferin and luciferase, is held or immobilized on a carrier such as a sheet of paper or a polysaccharide membrane (US Pat.
US5188965, US5736351). A carrier on which a luminescent reagent is immobilized is useful because the operability and stability of the reagent are improved. There is also a demand for a carrier holding a luminescent reagent to have even higher sensitivity.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a solid phase carrier for holding a luminescent reagent and a simple and highly sensitive method for measuring ATP using the same.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on the above-mentioned problems, and as a result, by combining a carrier holding luciferin and luciferase with an ATP regeneration reaction system using PPDK, Was found to be easily and highly sensitive, and the present invention was completed.

That is, the present invention relates to the following carriers and methods. 1. A carrier which holds luciferin, luciferase and ATP regenerating enzyme as a luminescent reagent. 2. A carrier characterized by holding luciferin, luciferase and at least one reagent selected from the following (1) to (3) as a luminescent reagent. (1) PPDK (2) phosphoenolpyruvate (3) pyrophosphate The above-mentioned 2. which further holds magnesium ions. Carrier.

[0008] 4. A method for measuring ATP in a sample including the following three steps. First step: a step of bringing a carrier holding luciferin, luciferase and one or more reagents selected from the following (1) to (3) into contact with a sample. (1) PPDK (2) Phosphoenolpyruvate (3) Pyrophosphate Second step: Carrier after completion of the first step and the above (1) to
And (3) a step of bringing the reagent into contact with a reagent that is not held by the carrier. Third step: a step of measuring the generated luminescence.

[0009] 5. The above 1. 2. A method for measuring ATP in a sample, comprising measuring the emitted light after contacting the carrier according to 2. or 3. with the sample.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION "Carrier holding luminescent reagent" The carrier of the present invention is characterized by holding luciferin, luciferase and ATP-regenerating enzyme as luminescent reagents. The ATP regenerating enzyme means an enzyme that catalyzes a reaction for generating ATP from AMP generated in a bioluminescence reaction. By using the enzyme, the regenerated A
Since TP is used again in the bioluminescence reaction, the luminescence is maintained for a long time. As the ATP regenerating enzyme, for example, phosphoenolpyruvate synthetase (EC 2.7.9.
2), PPDK.

[0011] Phosphoenolpyruvate synthetase is derived from AMP + phosphoenolpyruvate + phosphate.
It can catalyze the reaction that produces ATP + pyruvic acid + water. When the enzyme is used as an ATP-regenerating enzyme, it is preferable that phosphoenolpyruvate and phosphoric acid be further retained on a carrier.

[0012] In another aspect, the carrier of the present invention is characterized by holding luciferin, luciferase and one or more reagents selected from the following (1) to (3) as a luminescent reagent. (1) PPDK (2) phosphoenolpyruvate (3) pyrophosphate

The carrier of the present invention is used as a luminescent reagent as a reagent (luciferin, luciferase,
In addition to ze), a reagent involved in the ATP regeneration reaction (PPD
K, one or more selected from phosphoenolpyruvate or pyrophosphate). The carrier is suitable as a carrier used in the ATP measurement method described later. When one or two reagents are selected from the reagents involved in the ATP regeneration reaction, when the ATP is measured using the carrier of the present invention, a reagent not supported on the carrier is added in an appropriate step. Thus, the bioluminescence reaction and the ATP regeneration reaction need to occur.

Since the bioluminescence reaction and the ATP regeneration reaction require magnesium ions, it is preferable that the carrier of the present invention further retains magnesium ions. From the viewpoint of simplification of the operation in the ATP measurement, the carrier of the present invention is a reagent necessary for the bioluminescence reaction and the ATP regeneration reaction, that is, luciferin, luciferase, PP
It is preferable that DK, phosphoenolpyruvate, pyrophosphate and magnesium ions are all retained.

As the PPDK, any enzyme can be used as long as it acts on AMP and pyrophosphate to generate ATP. As plant-derived PPDK, for example, corn leaf-derived [Biochemistry 12, 2862-2867 (19
73)] and from sugarcane leaves [The Biochemical Journal 1
14, 117-125 (1969)], and those derived from microorganisms include, for example, Propionibacterium shermanii [Biochemistry 10, 721].
-729 (1971)], Acetobacter xylinum (Acetobacte
r xylinum) [Journal of Bacteriology (1970)], Bacteroides symbiosus (Meth
ods in Enzymology 42, 199-212 (1975)] and the genus Microbispora [for example, Microbispora thermomorosa (Mic
robispora thermorosea) IFO 14047] and the like.

As the luciferin and luciferase, those derived from, for example, insects (genji firefly, Heike firefly, firefly from North America, click beetle, glowworm) can be used. Luciferase is a natural luciferase purified from the luminescent tissue of the above organism, a natural luciferase prepared by genetic engineering techniques, and addition, deletion, substitution, etc. to one or more amino acids in the amino acid sequence of the natural luciferase. Mutant luciferase into which the above-mentioned mutation has been introduced can be used. Commercial products can be used for other luminescent reagents such as phosphoenolpyruvate, pyrophosphate and magnesium ion.

The material and shape of the carrier are not limited. Examples of the carrier material include paper such as filter paper, synthetic resins, glass filters, biopolymers such as polysaccharides and proteins, and materials used as filtration membranes. Examples of the shape of the carrier include a microtiter plate shape, a flat plate shape, a film shape, a sheet shape, a disk shape, a tubular shape, a rod shape and the like. The carrier may have a multilayer structure or a porous structure. The method for retaining the luminescent reagent on the carrier is not limited, but the reagent is preferably sufficiently immobilized on the carrier, and a known method for immobilizing enzymes and other reagents on the carrier can be employed.

As a method for immobilizing a reagent on a carrier, a method in which a luminescent reagent is dissolved in a suitable solvent, then dropped or sprayed on the carrier, and then the solvent is volatilized by freeze-drying can be used. In addition, a method of drying the luminescent reagent solution in a sheet-shaped mold, a method of impregnating the luminescent reagent into a carrier such as filter paper, superabsorbent resin, cellulose powder, and the like, and a method of drying the luminescent reagent solution on a nitrocellulose membrane or the like. A method of adsorbing, a method of attaching a luminescent reagent to a carrier by printing or the like can be used.

When the carrier is in the form of a film or sheet, the carrier of the present invention can be mass-produced at low cost by preparing a large film or sheet in advance and cutting it into the final target size. There are advantages. Note that the carrier of the present invention may hold other reagents, for example, a pH adjuster, a chelating agent, an antioxidant, a preservative, etc., for the purpose of improving the storage stability and stability of the luminescent reagent. .

"Measurement method of ATP in sample" The measurement method of the present invention includes the following three steps. This method is characterized by causing a bioluminescence reaction and an ATP regeneration reaction to occur on the carrier of the present invention. Since the bioluminescence reaction and the ATP regeneration reaction require magnesium ions, the carrier of the present invention desirably holds magnesium ions. Magnesium ion is first or second
It may be added to the reaction system when performing the step, or may be added to the sample when preparing the sample.

In the first step, a carrier holding luciferin, luciferase and one or more reagents selected from the following (1) to (3) is brought into contact with a sample. (1) PPDK (2) phosphoenolpyruvate (3) pyrophosphate

Examples of the sample include foods and drinks, pharmaceuticals, cosmetics, seawater, river water, industrial water, sewage, soil, urine, feces, blood, sputum, pus, and cell cultures.
Further, the above sample may be diluted with an appropriate solvent (for example, distilled water, physiological saline, phosphate buffer, Tris buffer, sodium acetate buffer, etc.).

The method of bringing the carrier into contact with the sample is not limited. For example, the sample solution may be dropped or sprayed on the portion of the carrier where the luminescent reagent is held. Further, the carrier may be immersed in the sample solution.

In order to measure the number of cells, intracellular A
When TP is measured, an ATP extractant such as a surfactant, trichloroacetic acid or the like is added to the sample, and AT
Extract P. AT can also be achieved by heating the sample.
P extraction is possible. In the second step, the carrier after the completion of the first step is brought into contact with a reagent not held by the carrier among the reagents of (1) to (3). Thereby, in the presence of magnesium ions, bioluminescence reaction (luminescence by reaction of luciferin, luciferase, ATP in a sample) and ATP regeneration reaction (regeneration of ATP by reaction of PPDK, phosphoenolpyruvate, pyrophosphate, AMP) ) Happens.

The first step and the second step are preferably carried out simultaneously, in which case a carrier holding luciferin, luciferase, PPDK, phosphoenolpyruvate, pyrophosphate, magnesium ion is used, What is necessary is just to make the said carrier and sample contact.

In the third step, the generated light emission is measured. In this case, a method in which the carrier after the second step is set in a luminometer and the amount of luminescence is measured can be used. As the luminometer, a luminometer using a photomultiplier or a photodiode can be used. For example, a luminescence reader BLR-201 manufactured by Aloka, a luminescence tester C-100 manufactured by Kikkoman, or the like can be used. When the carrier is in the form of a film, the amount of luminescence can be reduced by imaging the luminescence on the film using a bioluminescence image analysis system device, for example, ARGUS-50 / CL (with a tapered fiber: manufactured by Hamamatsu Photonic Co., Ltd.). Measurement becomes possible. By comparing the measurement result of the luminescence amount with the calibration curve prepared in advance, the A
The amount of TP and the number of cells can be calculated.

In carrying out the measurement method of the present invention, the above-mentioned carrier of the present invention, that is, luciferin, luciferase and a reagent relating to ATP regeneration reaction (selectable from PPDK, phosphoenolpyruvate, pyrophosphate) as a luminescent reagent. It is preferable to use a carrier characterized by holding at least one of the above, or a carrier holding magnesium. When using these carriers,
After contacting the carrier with the sample, the resulting luminescence is measured.
If the carrier does not hold all the luminescent reagents necessary for the bioluminescence reaction and the ATP regeneration reaction, the luminescent reagent that is lacking in an appropriate step is added.

[0028]

The present invention will be described below with reference to examples. In Examples, a plastic sheet-like carrier holding a luminescent reagent was prepared as the carrier of the present invention.

1. Preparation of carrier of the present invention (1) Luminescent reagent solution: A solution containing the following luminescent reagent and protective agent was prepared. 1.5 mM luciferin (manufactured by Sigma) 2.05 mg / ml Genji firefly luciferase 0.13 mg / ml PPDK (from Microbispora thermoserum) 0.2 mM PPi 2Na 10H ₂ O (sodium pyrophosphate decahydrate) 4.2 mM PEP 3Na 7.5H ₂ O (phosphoenolpyruvate 3
Sodium 7.5 hydrate) 15 mM Mg Ac 4H ₂ O (magnesium acetate tetrahydrate) Protectant (0.2% BSA, 2% sucrose, 20 mM HEPE
S pH7.8, 1mM EDTA 2Na, 1mM DTT)

(2) Immobilization of a luminescent reagent on a carrier 10 ml of the luminescent reagent solution was freeze-dried, and the freeze-dried product was pulverized. The pulverized product is mixed with 1 ml of n-hexyl alcohol.
Suspended in a transparent plastic sheet (vertical)
It was applied uniformly on one side (10 cm × 10 cm × 0.2 mm thick) and dried at 80 ° C. for 10 minutes. As described above, the luminescent reagent was immobilized on the carrier.

2. Preparation of Carrier of Comparative Example 1 As the luminescent reagent, the following 1. The product obtained by removing PPDK from (1) was used. When this luminescent reagent is used, A in the sample
Bioluminescence reaction occurs by contact with TP, but PP
Since no DK is included, no ATP regeneration reaction occurs. The luminescent reagent was immobilized on a carrier in the same manner as described above to obtain a carrier of Comparative Example.

3. Preparation of Carrier of Comparative Example 2 A luminescent reagent solution having the following composition was prepared. When this luminescent reagent is used, a bioluminescent reaction occurs when it comes into contact with ATP in a sample, but since a reagent involved in the ATP regeneration reaction is not included, the ATP regeneration reaction does not occur. In the same manner as described above, the luminescent reagent was immobilized on a carrier to obtain a carrier of Comparative Example 2. Composition of luminescence reagent solution: 1.5 mM luciferin (manufactured by Sigma) 2.05 mg / ml Genji firefly luciferase 15 mM Mg Ac 4H ₂ O (magnesium acetate tetrahydrate) Protecting agent (0.2% BSA, 2% sucrose, 20 mM HEPE
S pH7.8, 1mM EDTA 2Na, 1mM DTT)

4. A using a carrier holding a luminescent reagent
TP measurement 5 mm × 5 mm plastic sheet with luminescent reagent attached
And fixed in a white plastic rod recess with a 6 mm x 6 mm recess. 25 μl of a 1 × 10 ⁻⁹ M ATP solution was applied to the carrier, and the luminescence amount was measured by setting it on a horizontal Lumitester C100 (manufactured by Kikkoman Corporation). The light emission amount when the carrier of the present invention was used was 55,080 RLU. On the other hand, the light emission amount when the carrier of Comparative Example 1 was used was 100,500 RLU, and the light emission amount when the carrier of Comparative Example 2 was used was 120,153 RLU.

In the carriers of Comparative Examples 1 and 2, only the bioluminescence reaction occurs upon contact with ATP, and the luminescence is rapidly attenuated. On the other hand, in the carrier of the present invention, since the ATP regeneration reaction occurs, the bioluminescence reaction is continued. For this reason, when the carrier of the present invention was used, a higher luminescence amount could be obtained than when the carrier of the comparative example was used.

[0035]

Effects of the Invention According to the present invention, a solid phase carrier holding a luminescent reagent and a simple and highly sensitive method for measuring ATP using the same are provided. The present invention can be suitably used for measuring the amount of ATP and the number of cells in a sample.

Claims (5)

[Claims] 1. A carrier which holds luciferin, luciferase and ATP regenerating enzyme as luminescent reagents. 2. A carrier comprising luciferin, luciferase and one or more reagents selected from the following (1) to (3) as luminescent reagents: (1) pyruvate orthophosphate dikinase (2) phosphoenolpyruvate (3) pyrophosphate 3. The carrier according to claim 2, further comprising magnesium ions. 4. A method for measuring ATP in a sample, comprising the following three steps. First step: a step of bringing a carrier holding luciferin, luciferase and one or more reagents selected from the following (1) to (3) into contact with a sample. (1) Pyruvate orthophosphate dikinase (2) Phosphoenolpyruvate (3) Pyrophosphate Second step: The carrier after completion of the first step, and the above (1) to
And (3) a step of bringing the reagent into contact with a reagent that is not held by the carrier. Third step: a step of measuring the generated luminescence. 5. A method for measuring ATP in a sample, comprising: contacting the carrier according to claim 1, 2 or 3 with the sample, and measuring the generated luminescence.