JPH11221097A - Measurement of peroxidase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure peroxidase activities in the presence of a hydrogen receptor in high sensitivity by using a chemical fluorescent reagent obtained by bringing N,N'-disubstituted-9,9'-bisacridinium salt into contact with N,N- disubstituted-carboxylic acid amide. SOLUTION: The objective peroxidase activities are measured in high sensitivity by measuring the peroxidase enzyme activities by using a chemical fluorescent reagent prepared by bringing N,N'-disubstituted-9,9'-bisacridinium salts (e.g. lucigenin) of the formula [R<1> and R<2> are each an alkyl or a (halogenated) aryl; R<3> to R<6> are each H, an alkyl, an aryl, an alkoxy, an aryloxy or a halogen; X is a (n)-valent anion; (n) is 1 or 2] into contact with N,N-disubstituted- carboxylic acid amide (e.g. N,N-dimethylacetamide), in the presence of a hydrogen acceptor (e.g. hydrogen peroxide). Antigens and antibodies are measured by an enzyme immunity measurement, and nucleic acids are measured by a hybridization assay, by using the peroxidase as a marker.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring peroxidase activity using a chemiluminescence method.

[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 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, an analysis method using peroxidase as a labeling substance includes an immunological measurement method using an antigen-antibody reaction, a nucleic acid hybridization assay method using nucleic acid complementation, and avidin-biotin, a hormone-hormone receptor. , Sugar-
It is used for an analysis system utilizing specific affinity such as lectin.

In order to measure such peroxidase activity, a colorimetric method is used in which a hydrogen donor is oxidized using hydrogen peroxide as a hydrogen acceptor to form a dye, and the amount of coloring 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. 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 it is possible to quantify peroxidase activity with high sensitivity. It is possible.

[0004]

However, when the measurement of peroxidase enzyme activity is applied to an enzyme immunoassay, it is often necessary to further increase the quantification of the enzyme in a low concentration region. It has been desired to further increase the sensitivity of activity measurement. In view of the above circumstances, 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.

[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 reagent, N, N'-disubstituted-9,9'-bisacridy. Chemiluminescent system using a hydrogen salt-N, N-disubstituted carboxylic acid amide compound complex, hydrogen peroxide as a hydrogen acceptor and a specific phenolic compound as a luminescence enhancer, and luminol as a chemiluminescent substance The inventors have found that it is possible to quantify peroxidase activity with higher sensitivity than the system used, and have reached the present invention based on these findings.

Accordingly, the first aspect of the present invention is to provide the following general formula (1)

Embedded image (In the 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;
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 n is 1 or 2
It is. ), N, N'-disubstitution-9,9'-
Using a chemiluminescent reagent prepared by contacting a bisacridinium salt with an N, N-disubstituted carboxylic acid amide compound,
The present invention relates to a method for measuring peroxidase activity by a chemiluminescence method, which comprises measuring peroxidase enzyme activity in the presence of a hydrogen acceptor.

A second aspect of the present invention is the following general formula (1):

Embedded image (In the 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;
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 n is 1 or 2
It is. ), N, N'-disubstitution-9,9'-
Using a chemiluminescent reagent prepared by contacting a bisacridinium salt with an N, N-disubstituted carboxylic acid amide compound,
The present invention relates to a method for measuring peroxidase activity by a chemiluminescence method, wherein a phenolic compound is used as a luminescence enhancer when measuring peroxidase activity in the presence of a hydrogen acceptor.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The method for measuring peroxidase activity of the present invention comprises:
A chemiluminescent reagent prepared by contacting an N, N'-disubstituted-9,9'-bisacridinium salt with an N, N'-disubstituted carboxylic acid amide compound in the presence of a hydrogen acceptor; In some cases, it is to measure the peroxidase enzyme activity in the presence of a luminescence enhancer, the method of measurement is optional, but generally, a chemiluminescence reagent, a luminescence enhancer and a measurement sample containing a peroxidase enzyme is mixed, A method of adding a hydrogen acceptor solution in a specific basic pH region to cause a chemiluminescence reaction, and measuring the amount of chemiluminescence with a luminescence measuring device has been performed. Next, the chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention will be described. N, N′-disubstitution—a component of the chemiluminescent reagent
The 9,9′-bisacridinium salt is represented by the following general formula (1)

[0009]

Embedded image In general formula (1), R ¹ and R ²
Is 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.

The alkyl, aryl and halogenated aryl groups have 1 to 20 carbon atoms, and the preferred alkyl groups have 1 to 10 carbon atoms. Examples include linear alkyl groups such as methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, and decyl group, and their branched alkyl groups. . The aryl group preferably has 6 to 20 carbon atoms, and includes a phenyl group, a tolyl group, a xylyl group, and the like, and may be further substituted with an alkyl group. As the aryl group, a phenyl group is particularly preferred. Examples of the halogenated aryl group include a halogenated phenyl group, a halogenated tolyl group, and a halogenated xylyl group, and a chlorophenyl group is particularly preferable.

In the general formula (I), 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. Examples of the number of carbon atoms of the hydrocarbon group include 1 to 20, preferably 1 to 14.
In the general formula (1), X is an n-valent anion, and n is 1 or 2. Specific examples of the anion include a nitrate ion, a halogen ion, a phosphate ion, a sulfate ion, and a sulfonate ion. Of these, nitrate ions are particularly preferred.

Specific examples of the N, N'-disubstituted-9,9'-bisacridinium salts include N, N-dimethyl-9,9'-bisacridinium salt and N, N-diethyl. -9,9'-bisacridinium salt, N, N-diphenyl-9,9'-bisacridinium salt, N, N'-di-
Examples thereof include m-chlorophenyl-9,9′-bisacridinium salt. As the counter ion, the above-mentioned anion is preferable, and a nitrate ion is particularly preferable. N,
N'-disubstituted-9,9'-bisacridinium salts include, but are not limited to, N, N'-
Dimethyl-9,9'-bisacridinium dinitrate (lucigenin) is preferred.

N, N- used in the production of a chemiluminescent reagent
Examples of the disubstituted carboxylic acid amide compound include N, N-dimethylformamide, N, N-dimethylacetamide,
N, N-dimethylacrylamide, N, N-dimethylpropionamide, N, N-dimethylbenzamide, N-
Specific examples include methyl-2-pyrrolidone and the like,
Particularly, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone and the like are preferable.

Among these N, N-disubstituted carboxylic acid amide compounds, those which are liquid at room temperature can be used in large excess also as a solvent, or other suitable solvents may be used. A solid at room temperature may be heated and melted, or an appropriate solvent may be used. Examples of such a solvent include dimethyl sulfoxide, hexamethylphosphamide and the like.

In the production of a chemiluminescent reagent, N, N '
Di-substituted-9,9'-bisacridinium salts and N, N
By bringing into contact with the di-substituted carboxylic acid amide compound, the luminescence intensity is increased by the contact time, and a phenomenon that a peak is formed and attenuated is observed.
N-disubstituted carboxylic acid amide compound reacts with N, N'-disubstituted-9,9'-bisacridinium salts to form an activated state, and then changes over time to become inactivated it is conceivable that.

Therefore, the above-mentioned chemiluminescent reagent is basically produced by a contact treatment between an N, N'-disubstituted-9,9'-bisacridinium salt and an N, N-disubstituted carboxylic acid amide compound. It is carried out by mixing and maintaining both under the reaction conditions described below. Specifically, when performing chemiluminescence analysis using the chemiluminescent reagent of the present invention, a method is employed in which each component of the chemiluminescent reagent is prepared and reacted for a predetermined time before use.

As the above reaction conditions, the reaction temperature varies depending on the type of the reagent and the solvent to be used.
The temperature ranges from 10 to + 200 ° C, preferably from 0 to 120 ° C, particularly preferably from 20 to 60 ° C. Reaction time is 1
A time in the range of minutes to day and night, preferably 5 minutes to 12 hours, particularly preferably 10 minutes to 5 hours can be employed. The ratio of each component of the chemiluminescent reagent is N, N'-
It may be in the range of equimolar to a large excess of N, N-disubstituted carboxylic acid amide compound per mol of disubstituted-9,9'-bisacridinium salt.

The chemiluminescent reagent obtained as described above emits light under basic conditions of pH 7.5 to 13 in an amount depending on the concentration of peroxidase in the presence of excess hydrogen acceptor. It is recognized that this luminescence is enhanced by a luminescence enhancer such as a phenolic compound. Such phenolic compounds include p-hydroxycinnamic acid, p-
-Phenylphenol, p- (4-chlorophenyl) phenol, p- (4-bromophenyl) phenol, p
-(4-Iodophenyl) phenol, p-iodophenol, p-bromophenol, p-chlorophenol, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like can be mentioned without limitation. The hydrogen acceptor is not particularly limited as long as it can serve as a substrate for peroxidase.Organic peroxides, inorganic peroxides and the like can be arbitrarily used. Is preferred.

The concentration of the chemiluminescent reagent used in the method for measuring peroxidase enzyme activity of the present invention is 10 ^-6.
１1M, preferably 10 ^-4 to 10 ^-2 M, and the amount used is 10 to 500 μl, preferably 50 to 300 μl.
A range of μl is desirable. The amount of the luminescence enhancer used is 0.01 to 100 times the molar amount of the chemiluminescent reagent, preferably 0.1 to 100 times.
It is in the range of 10 times mol. 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 organic peroxides and inorganic peroxides are optionally used.
In particular, hydrogen peroxide is preferred. It is necessary to use the hydrogen acceptor in a sufficient excess amount with respect to the chemiluminescent substance.
Preferably, it is in the range of 10 to 1000 times mol.

Examples of the basic buffer used in the above chemiluminescence reaction include Tris buffer, phosphate buffer, borate buffer, carbonate buffer, glycine-sodium hydroxide buffer and the like. Buffer concentration should be between 1 mM and
A range of 1M is desirable. In addition, additives such as a surfactant and a chelating agent can be optionally used during the reaction.

[0021]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples together with Reference Examples and Comparative Examples, but the present invention is not limited to the following.

Example 1 1 mg of lucigenin was placed in a test tube, 2 ml of N, N-dimethylformamide was added and dissolved in the test tube.
lucigenin and N, N'-
A chemiluminescent reagent comprising a complex with dimethylformamide was obtained.

[Measurement of peroxidase activity] The lucigenin-N, N'-dimethylformamide complex was prepared at 4.0 ×
0.1 M Tris-HCl buffer (pH 8.4) containing 10 ^-5 M and p-iodophenol at a concentration of 10 ^-3 M
To 100 μl, 100 μl of an aqueous solution of horseradish peroxidase (HRP) at various concentration levels were mixed.
50 μl of a 0034% by weight aqueous hydrogen peroxide solution was added, and a luminometer (Luminous CT-900 manufactured by Diatron Co., Ltd.) was added.
As a result of integrating the light emission amounts in 0D) for 0 to 5 seconds, it was possible to measure HRP up to 1 × 10 ⁻¹⁸ mol / assay as shown in Table 1.

[0024]

[Table 1] [Example 2] 1 mg of lucigenin was placed in a test tube, 2 ml of N, N-dimethylacetamide was added and dissolved therein, and the mixture was allowed to stand at room temperature for 90 minutes, and 2 ml of pure water was added and mixed. And a N, N′-dimethylacetamide complex are obtained. Next, a 0.1 M Tris-HCl buffer (pH 8.0) containing this lucigenin-N, N'-dimethylformamide complex at a concentration of 4.0 × 10 ⁻⁵ M and p-iodophenol at a concentration of 10 ⁻³ M is used. 4) 100 μl of 100 μl of an aqueous solution of horseradish peroxidase (HRP) at various concentrations was mixed with 100 μl of this solution, and the mixture was added to a 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 2, HRP was 1 × 10 ⁻¹⁸ mol / a.
It was possible to measure up to ssay.

[0025]

[Table 2] [Example 3] 1 mg of lucigenin was collected in a test tube, 2 ml of N-methyl-2-pyrrolidone was added and dissolved therein, and the mixture was allowed to stand at room temperature for 90 minutes, and then mixed with 2 ml of pure water. As a result, a chemiluminescent reagent comprising a complex of lucigenin and N, N'-dimethylacetamide was obtained. Next, a 0.1 M Tris-HCl buffer solution containing the complex of lucigenin and N-methyl-2-pyrrolidone at a concentration of 4.0 × 10 ⁻⁵ M and p-iodophenol at a concentration of 10 ⁻³ M ( pH 8.4) was mixed with 100 μl of horseradish peroxidase (HRP) aqueous solution of various concentration levels and mixed with a 0.0034 wt% aqueous hydrogen peroxide solution.
μl was added, and the luminescence amount was integrated for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron). As a result, as shown in Table 3, HRP was 5 × 10 ⁻¹⁸ mol / a.
It was possible to measure up to ssay.

[0026]

[Table 3] Comparative Example 1 Luminol was added at 5.6 × 10 ^-5 M and p-
0.1 M containing iodophenol at a concentration of 10 ^-3 M
After 100 μl of Tris-HCl buffer (pH 8.4) was mixed with 100 μl of HRP aqueous solution of various concentration levels, 50 μl of 0.0034 wt% hydrogen peroxide aqueous solution was added thereto,
Luminometer (Luminous CT-9 manufactured by Diatron)
As a result of integrating the light emission amounts for 0 to 5 seconds at 000 D), it was confirmed that HRP as shown in Table 4 could be measured up to 10 ⁻¹⁷ mol / assay.

[0027]

[Table 4]

[0028]

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 and widely used assay system using luminol. Antigens and antibodies can be detected or quantified more sensitively by enzyme immunoassay and nucleic acids by hybridization assay, respectively.

 ──────────────────────────────────────────────────続 き Continuing on 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 9-4 Dainichi Seika Kogyo Co., Ltd. Technology Research Center

Claims (5)

[Claims] [Claim 1] The following general formula (1) (In the 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;
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 n is 1 or 2
It is. ), N, N'-disubstitution-9,9'-
Using a chemiluminescent reagent prepared by contacting a bisacridinium salt with an N, N-disubstituted carboxylic acid amide compound,
A method for measuring peroxidase activity by a chemiluminescence method, comprising measuring peroxidase enzyme activity in the presence of a hydrogen acceptor. 2. The following general formula (1): (In the 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;
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 n is 1 or 2
It is. ), N, N'-disubstitution-9,9'-
Using a chemiluminescent reagent prepared by contacting a bisacridinium salt with an N, N-disubstituted carboxylic acid amide compound,
A method for measuring peroxidase activity by a chemiluminescence method, wherein a phenolic compound is used as a luminescence enhancer when measuring peroxidase enzyme activity in the presence of a hydrogen acceptor. 3. The N, N-disubstituted-9,9′-bisacridinium salt is lucigenin, and the N, N′-
The method according to claim 1 or 2, wherein the disubstituted carboxylic acid amide compound is N, N'-dimethylformamide or N, N'-dimethylacetamide. 4. The method according to claim 1, wherein the hydrogen acceptor is hydrogen peroxide. 5. The method according to claim 2, wherein the luminescence enhancer is at least one phenolic compound selected from p-iodophenol, p-phenylphenol, and 6-hydroxybenzothiazole.