JPH10295399A - Measurement of peroxidase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring peroxidase enzyme activity in high sensitivity by making a reduction product of an N,N'-di-substituted-9,9'- bisacridinium salt generate a chemiluminescence in the presence of a hydrogen acceptor through the action of peroxidase. SOLUTION: This method comprises measuring peroxidase activity, by use of chemiluminescece, useful for analytical methods using peroxidase as a labeling substance, such as enzyme immunoassays or nucleic acid hybridization assays, by using, as a chemiluminescent substance, a reaction product including a compound expressed by formula II (R<11> to R<16> are the same as R<1> to R<6> , respectively), obtained by reduction treatment of an N,N'-di-substituted-9,9'-bisacridinium salt expressed by formula I [R<1> and R<2> are each an alkyl, or (halogenated)aryl; R<3> to R<6> are each H, an alkyl, aryl, alkoxy, aryloxy or a halogen; X<n-> is an n-valent anion; (n) is 1 or 2], and measuring peroxidase enzyme activity in the presence of a hydrogen acceptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring peroxidase activity using a chemiluminescence method (chemiluminescence). More specifically, the present invention uses a reduction product of a specific acridinium salt as a chemiluminescent substance. The present invention relates to a method for measuring peroxidase activity.

[0002]

2. Description of the Related Art Peroxidase is an enzyme that catalyzes the oxidation reaction of a hydrogen donor in the presence of a hydrogen acceptor. Such catalytic activity is also observed in non-enzymatic substances such as hemoglobin, for example, and is collectively referred to as peroxidase activity. Since the catalytic activity of peroxidase is easily detected with high sensitivity even in a trace amount, it is used in a wide range of fields as an analytical reagent which is an indicator of various substances to be detected, as well as detecting peroxidase enzyme activity. As described above, as an analysis method using peroxidase as a labeling substance, an immunological assay using an antigen-antibody reaction, a nucleic acid hybridization assay using nucleic acid complementation, other avidin-biotin, a hormone-hormone receptor, An analysis method utilizing specific affinity such as sugar-lectin and the like can be mentioned.

[0003] In addition, such a peroxidase activity is measured by a colorimetric method in which hydrogen peroxide is used as a hydrogen acceptor to oxidize a hydrogen donor to form a dye, and the color development is measured by a spectrophotometer or the like. Alternatively, a fluorescence method of generating a fluorescent substance and measuring its fluorescence with a fluorescence spectrophotometer has been performed. However, when a colorimetric method is used to measure peroxidase activity, there is a problem that the quantitativeness of peroxidase in a low concentration region is lacking, and a fluorescence method has been developed to solve this problem. The quantification in the area is not yet sufficient. For the purpose of solving this problem, a method for measuring peroxidase activity by a chemiluminescence method has been developed. The chemiluminescence method is a method of quantifying peroxidase enzyme activity by measuring the amount of luminescence emitted when the chemiluminescent substance is put into an excited state via an intermediate by the catalytic activity of the peroxidase enzyme and returning from this state to the ground state. Conventionally, a chemiluminescence system using luminol as a chemiluminescent substance, hydrogen peroxide as a hydrogen acceptor, and p-iodophenol as a luminescence enhancer has been developed, and the peroxidase activity is quantified with high sensitivity. It has become possible.

[0004]

However, when the measurement of peroxidase enzyme activity is applied to an enzyme immunoassay, it is often necessary to further improve the quantification of the enzyme in a low concentration region. It has been desired to further increase the sensitivity of activity measurement. Accordingly, an object of the present invention is to provide a novel measurement system based on a chemiluminescence method capable of measuring peroxidase activity with higher sensitivity in view of the development circumstances as described above.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, as a chemiluminescent substance, N, N'-disubstituted-9,9'-bisacrylic acid is used. A chemiluminescence system that uses a reduction reaction product of dinium salts, hydrogen peroxide as a hydrogen acceptor, and more preferably a specific phenolic compound as a luminescence enhancer, and a chemical luminescence system that uses luminol as a chemiluminescent substance. The inventors have found that the peroxidase activity can be quantified with higher sensitivity than the luminescence system, and have completed the present invention.

That is, a first aspect of the present invention is the following general formula (1):

[0007]

Embedded image (In the above general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ And R ⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X ^n- is an n-valent anion; n is 1 or 2.)
Using a reaction product obtained by reduction treatment of N'-disubstituted-9,9'-bisacridinium salts as a chemiluminescent substance, measuring peroxidase enzyme activity in the presence of a hydrogen acceptor. And a method for measuring peroxidase activity.

A second aspect of the present invention is a compound represented by the following general formula (1):

[0009]

Embedded image (In the above general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ And R ⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X ^n- is an n-valent anion; n is 1 or 2.)
The reaction product obtained by reduction treatment of N'-disubstituted-9,9'-bisacridinium salts is used as a chemiluminescent substance to enhance luminescence when measuring peroxidase enzyme activity in the presence of a hydrogen acceptor. The present invention relates to a method for measuring peroxidase activity, characterized by using an agent.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail.

The method for measuring peroxidase activity according to the present invention uses N, N'-disubstituted-9,9 'as a chemiluminescent substance.
Using a reaction product obtained by a reduction treatment of bisacridinium salts, and measuring peroxidase enzyme activity by a chemiluminescence reaction in the presence of a hydrogen acceptor; more preferably, the peroxidase enzyme activity is The present invention relates to a method for measuring by a chemiluminescence reaction using a luminescent substance in the presence of a hydrogen acceptor and a luminescence enhancer. The measurement method is arbitrary, and generally, a measurement sample containing a chemiluminescent substance, a luminescence enhancer and a peroxidase enzyme is mixed, and a hydrogen acceptor is added in a specific basic pH region to cause a chemiluminescence reaction. A method of measuring the amount of luminescence with a luminescence measuring device or the like can be employed.

The starting material of the chemiluminescent substance used in the method for measuring peroxidase activity of the present invention is N, N'-
Disubstituted-9,9′-bisacridinium salts represented by the general formula (1)

[0013]

Embedded image It is a compound represented by these. In the general formula (1), R
¹ and R ² are each an alkyl group, an aryl group or a halogenated aryl group, which may be the same or different from each other. The alkyl group has 1 to 2 carbon atoms.
0, preferably any of 1 to 14 linear or branched, and the aryl group and the halogenated aryl group each have 6 to 20 carbon atoms, and each has 1 to 14 carbon atoms. Although it may be substituted with an alkyl group, a phenyl group and a halogenated phenyl group are particularly preferred. In the formula, R ³ , R ⁴ , R ⁵ and R ⁶ are each a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group or a halogen atom, and may be the same or different. The alkyl group and the alkoxy group may be linear or branched having 1 to 20 carbon atoms, preferably 1 to 14 carbon atoms, and the aryl group and the aryloxy group are each having 6 to 20 carbon atoms. It may have a group.

In the general formula (1), X ^n- is an n-valent anion, and n is 1 or 2. The anion is not particularly limited, but may be a nitrate ion (N
O ₃ ⁻ ), halide ions (for example, chloride ions,
Fluoride ion and the like). Among these anions, nitrate ions are particularly preferred.

Specific examples of N, N'-disubstituted-9,9'-bisacridinium salts include: N, N'-dimethyl-9,9'-bisacridinium salts, N, N '-Diethyl-9,9'-bisacridinium salt, N, N'-dipropyl-9,9'-bisacridinium salt, N, N '
-Dibutyl-9,9'-bisacdinium salt, N, N '
-Dipentyl-9,9'-bisacridinium salt, N,
N′-diphenyl-9,9′-bisacridinium salt,
N, N'-di-m-chlorophenyl-9,9'-bisacridinium salt and the like can be mentioned. In particular, N, N '
-Dimethyl-9,9'-bisacridinium dinitrate is preferred.

The product of the reduction of N, N'-disubstituted-9,9'-bisacridinium salts is N, N'-disubstituted-
It may contain a 9,9'-bisdihydroacridine derivative, which is represented by the following general formula (2)

[0017]

Embedded image Can be represented by

In the general formula (2), R ¹¹ and R ²²
Are selected from the group consisting of an alkyl group, an aryl group and an aryl halide group, and may be the same or different from each other. The alkyl group has 1 to 20 carbon atoms,
Preferably, any of 1 to 14 linear or branched groups can be used. The aryl group and the halogenated aryl group each have 6 to 20 carbon atoms and are each substituted with an alkyl group. Good, but particularly preferred are a phenyl group and a halogenated phenyl group. R ³³ ,
R ⁴⁴ , R ⁵⁵ and R ⁶⁶ are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different. The alkyl group and the alkoxy group may be linear or branched having 1 to 20 carbon atoms, preferably 1 to 14 carbon atoms, and the aryl group and the aryloxy group are each having 6 to 20 carbon atoms and are substituted with an alkyl group. It may be done.

These chemiluminescent substances have a pH of 7.5 to 7.5.
Under 13 basic conditions, it emits light in the presence of an excess of hydrogen acceptor, for example hydrogen peroxide, in an amount dependent on the concentration of peroxidase.

It has been recognized that this luminescence is enhanced by a luminescence enhancer such as a phenolic compound. As such a phenolic compound, p-iodophenol, p-phenylphenol, and 6-hydroxybenzothiazole are particularly preferable, but p-hydroxycinnamic acid, p- (4-chlorophenyl) phenol, and p-iodophenol are also preferable.
Examples thereof include (4-bromophenyl) phenol, p- (4-iodophenyl) phenol, p-bromophenol, p-chlorophenol, 2-naphthol, firefly luciferin and the like.

The concentration of the chemiluminescent substance used in the method for measuring peroxidase activity of the present invention is based on the concentration of N, N'-disubstituted-9,9'-bisacridinium salts which are starting materials for the reduction treatment. In conversion, it is in the range of 10 ^{-6 to 1} M, preferably 10 ^-4 to 10 ^-2 M, and the amount used is 10 to 500 μl, preferably 50 to 300 μl.
1 range. Further, the amount of the luminescence enhancer is 0.01 to 100 times mol of the chemiluminescent substance, preferably 0.1 mol.
The concentration is in the range of 10 to 10 times, and the concentration is preferably in the range of 10 ^{-6 to 1} M, particularly 10 ^-4 to 10 ^-2 M.

The hydrogen acceptor used in the chemiluminescence reaction is not particularly limited as long as it can serve as a substrate for a peroxidase enzyme, and an organic peroxide, an inorganic peroxide or the like is optionally used. However, hydrogen peroxide is particularly preferred. It is necessary to use the hydrogen acceptor in a sufficient excess amount with respect to the chemiluminescent substance. It is preferable to use it in a range of 1000 times mol.

As the basic buffer used for the chemiluminescence reaction, a Tris-HCl buffer, a phosphate buffer, a borate buffer, a carbonate buffer, a glycine-sodium hydroxide buffer and the like can be optionally used. The concentration of these buffers is preferably 1 mM to 1M. During the reaction, additives such as a surfactant and a chelating agent can be optionally used.

The reaction product obtained by the reduction treatment of the N, N'-disubstituted-9,9'-bisacridinium salt is oxidized by nascent oxygen generated by the decomposition reaction of hydrogen peroxide by peroxidase. N, N'-disubstitution-9,
It becomes a 9'-bisacridinium compound, further reacts with excess hydrogen peroxide to form a dioxetane structure in an excited state, and then decomposes with alkali to produce acridone and emit light when returning to a ground state. It is presumed that.

The luminescence of the chemiluminescence reaction of the present invention is measured using a luminescence photometer. The starting point of the measurement and the integration time are arbitrary, but the luminescence is stable and the concentration of the luminescence is measured. It is preferable to select a time with high dependence. For example, the measurement start point is 0 to 1 hour, preferably 0 to 30 minutes, particularly preferably 0 to 15 minutes after mixing the reagent, and the integration time of the measurement is 1 second to 1 minute, preferably 1 to 30 seconds, particularly Preferably, it is 1 to 10 seconds.

The method for producing the reduction reaction product of N, N'-disubstituted-9,9'-bisacridinium salts used in the method for measuring peroxidase activity of the present invention is optional, but N, N '-Disubstituted-9,9'-bisacridinium salts can be easily produced by reduction treatment with a reducing agent. Examples of the reducing agent to be used include lithium aluminum hydride, lithium boron hydride, sodium boron hydride and the like, and lithium aluminum hydride is particularly preferable. In the case of the reduction treatment, an N, N-disubstituted carboxylic acid amide compound such as N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylbenzamide and the like are used. It can also be added.

The reduction reaction is preferably performed in a reaction solvent. The reaction solvent is not limited as long as it is inert to the reaction reagent and has solubility in the reaction reagent, and particularly, cyclic ethers such as tetrahydrofuran and dioxane are preferably used. . The reaction temperature varies depending on the type of the reducing agent and the reaction solvent used, but is generally in the range of -10 to + 150 ° C, preferably 0 to 120 ° C, particularly preferably 20 to 90 ° C. The reaction time is in the range of 1 minute to 24 hours, preferably 10 minutes to 12 hours, particularly preferably 30 minutes to 5 hours.

Further, according to the present invention, the N, N'-disubstituted-9,
A chemiluminescence measuring reagent comprising a reduction reaction product of 9'-bisdihydroacridine salts can be provided;
To provide a reagent kit for measuring peroxidase activity, comprising a combination of an N'-disubstituted-9,9'-histacridium salt reduction reaction product, hydrogen peroxide as a hydrogen acceptor, a phenolic compound as a luminescence enhancer, and the like. it can. This reagent kit for measuring chemiluminescence is extremely useful for efficient chemiluminescence measurement.

[0029]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples along with Reference Examples and Comparative Examples, but the present invention is not limited to these Examples. In addition,% in the examples
Means% by weight.

[Reference Example 1] Preparation of chemiluminescent reagent 1 mg of lucigenin (N, N'-dimethyl-9,9'-bisacridinium dinitrate) was placed in a test tube, and stirred with 1,4-dioxane. Lucigenin was finely dispersed in 1,4-dioxane in addition to 1 ml. Next, 150 mg of lithium aluminum hydride powder was added thereto, and the mixture was subjected to a reduction treatment by stirring at 80 ° C. for 2 hours, and the reaction product was poured into deionized water little by little under ice-cooling to add an excess amount of hydrogen. Lithium aluminum chloride was decomposed. Next, the formed precipitate such as aluminum hydroxide was filtered off, and the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a chemiluminescent reagent containing a lucigenin reduction reaction product.

Example 1 Measurement of Peroxidase Activity The reduction reaction product of the chemiluminescent reagent lucigenin prepared in Reference Example 1 was treated with 0.1 M Tris-HCl buffer (pH 8.4).
10 μl of a 0-fold diluted solution and 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing p-iodophenol at a concentration of 10 ⁻³ M are mixed with 50 μl of an aqueous solution of horseradish peroxidase (HRP) at various concentration levels. Is mixed with 50% of a 0.0034% by weight aqueous hydrogen peroxide solution.
μl was added, and the luminescence amount was integrated for 0 to 5 seconds using a luminometer (Luminous CT-9000D manufactured by Diatron). As a result, as shown in Table 1, HRP was 5 × 10 ⁻¹⁹ mol / a.
It was possible to measure up to ssay.

[0032]

Example 2 Measurement of Peroxidase Activity The reduction reaction product of the chemiluminescent reagent lucigenin prepared in Reference Example 1 and 6-hydroxybenzothiazole were used at 10 ^−3.
0.1 M Tris-HCl buffer (pH
8.4) 50 μl of HRP aqueous solution of various concentration levels were mixed with 100 μl, and 50 μl of 0.0034 wt% aqueous hydrogen peroxide solution was added thereto, and 0 to 5 seconds using a luminometer (Luminas CT-9000D manufactured by Diamondtron). As a result of integrating the light emission amounts, as shown in Table 2, HRP was 10 ⁻¹⁸ m.
It was possible to measure up to ol / assay.

[0034]

Comparative Example 1 Luminol was added at 5.6 × 10 ^−5.
100 μM of 0.1 M Tris-HCl buffer (pH 8.4) containing M and p-iodophenol at a concentration of 10 ⁻³ M
After mixing with 50 μl of HRP aqueous solution of various concentration levels, the mixture was mixed with 50% aqueous solution of 0.0034 wt% hydrogen peroxide.
μl was added, and the luminescence amount was integrated for 0 to 5 seconds using a luminometer (Luminous CT-9000D manufactured by Diatron). As a result, as shown in Table 3, HRP was 10 ^-17 mol / as.
It was recognized that measurement could be performed up to the day of the day.

[0036]

[0037]

According to the method for measuring peroxidase activity of the present invention, it becomes possible to measure peroxidase enzyme activity with higher sensitivity than the conventional measurement system using luminol, and peroxidase can be used as a labeling substance. Antigens and antibodies can be detected with higher sensitivity by the enzyme immunoassay used, and nucleic acids can be detected with higher sensitivity by the hybridization assay, and can be quantified.

Continuation of the front page (72) Inventor Toshifumi Katsuragi 1-9-4 Horinouchi, Adachi-ku, Tokyo Dainichi Seika Industry Co., Ltd. Technology Research Center (72) Inventor Mio Sato 1-chome, Horinouchi, Adachi-ku, Tokyo No. Dainichi Seika Kogyo Co., Ltd. Technology Research Center

Claims (7)

[Claims] [Claim 1] The following general formula (1) (In the above general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ And R ⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X ^n- is an n-valent anion; n is 1 or 2.)
Using a reaction product obtained by reduction treatment of N'-disubstituted-9,9'-bisacridinium salts as a chemiluminescent substance, measuring peroxidase enzyme activity in the presence of a hydrogen acceptor. A method for measuring peroxidase activity by a chemiluminescence method. 2. The following general formula (1): (In the above general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ And R ⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other, and X ^n- is an n-valent anion; n is 1 or 2.)
The reaction product obtained by the reduction treatment of N'-disubstituted-9,9'-bisacridinium salts is used as a chemiluminescent substance and emits light when measuring the peroxidase enzyme activity in the presence of a hydrogen acceptor. A method for measuring peroxidase activity by a chemiluminescence method, comprising using an enhancer. 3. The reaction product is represented by the following general formula (2). (In the above general formula (2), R ¹¹ and R ²² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³³ , R ⁴⁴ , and R ⁵⁵ And R ⁶⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different from each other.) Item 3. The method for measuring peroxidase activity according to Item 1 or 2. 4. The N, N′-disubstituted-9,9′-
Bisacridinium salts are N, N'-dimethyl-9,
The method for measuring peroxidase activity according to claim 1 or 2, which is 9'-bisacridinium dinitrate. 5. The method for measuring peroxidase activity according to claim 1, wherein the hydrogen acceptor is hydrogen peroxide. 6. The method for measuring peroxidase activity according to claim 2, wherein the luminescence enhancer is a phenolic compound. 7. The method according to claim 6, wherein the phenolic compound is at least one compound selected from the group consisting of p-iodophenol, p-phenylphenol and 6-hydroxybenzothiazole. .