JPH11279549A - Chemiluminescent reagent and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel chemiluminescent reagent which enables the measurement of the activity of an peroxidase enzyme in the presence of hydrogen peroxide or the like and can be utilized in the detection, microanalysis, or the like of various substances. SOLUTION: The chemiluminescent reagent is obtained by bringing an N,N'- di-substituted 9,9'-bisacridinium salt into contact with a reducing agent under irradiation with ultraviolet rays in the presence of an N,N-di-substituted carboxamide compound, desirably, formic acid in an organic solvent and enables the measurement of the activity of a peroxidaze enzyme in the presence of hydrogen peroxide or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel chemiluminescent reagent which can be used for detecting and quantifying various substances by chemiluminescence analysis as a chemiluminescent reagent capable of controlled chemiluminescence, and a method for producing the same. is there.

[0002]

2. Description of the Related Art Lucigenin (N, N'-dimethyl-9,9'-bisacridinium dinitrate), which has been used for a long time as a chemiluminescent reagent, exhibits a faint light in an alkaline aqueous solution. It is known that light emission is strong when hydrogen oxide is added, and it is used for various trace analysis. However, since lucigenin emits light quantitatively due to the presence of hydrogen peroxide and alkali, it can be used for the quantitative determination of hydrogen peroxide. The luminescent enzyme immunoassay (CLEIA) has a problem that it is difficult to use. As a means to solve this problem, chemiluminescence depending on the concentration of the substance to be measured is achieved by using glucose oxidase as a labeling enzyme and causing hydrogen peroxide generated by the oxidation reaction of glucose to act on lucigenin in the presence of an alkali. Methods for measuring the amount and the like have been developed.

However, chemiluminescence analysis using glucose oxidase as a labeling enzyme requires complicated preparation of reagents and operation of a luminescence system, and development of an inexpensive chemiluminescence reagent for chemiluminescence analysis using peroxidase as a labeling enzyme. Was desired.

[0004]

Accordingly, an object of the present invention is to provide a novel chemiluminescent reagent which can use peroxidase using hydrogen peroxide as a substrate, and a method for producing the same.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
In view of the state of development of conventional chemiluminescent reagents, as a result of intensive studies to achieve the above object, N, N'-disubstituted-9,9'-bisdiacridinium salts such as lucigenin were converted to N When contacting with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound, the chemiluminescent reagent obtained by irradiating ultraviolet rays does not react with hydrogen peroxide under a specific alkaline pH condition. The present inventors have found that in the presence of hydrogen peroxide and peroxidase, chemiluminescence is detected depending on the amount of peroxidase, and based on these findings, the present invention has been achieved.

Accordingly, a first aspect of the present invention is to contact an N, N'-disubstituted-9,9'-bisacridinium salt with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound. The present invention relates to a chemiluminescent reagent characterized by containing a reaction product obtained by irradiating an ultraviolet ray during the treatment.

A second aspect of the present invention is to contact-treat an N, N'-disubstituted-9,9'-bisacridinium salt with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound. The present invention relates to a method for producing a chemiluminescent reagent, which comprises irradiating an ultraviolet ray at the time.

[0008]

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

[0009] The chemiluminescent reagent of the present invention is used for reducing N, N'-disubstituted-9,9'-bisacridinium salts in the presence of an N, N-disubstituted carboxylic acid amide compound. It contains a reaction product obtained by irradiation with ultraviolet rays.

The method for producing a chemiluminescent reagent of the present invention comprises the steps of: preparing an N, N'-disubstituted-9,9'-bisacridinium salt in the presence of an N, N-disubstituted carboxylic acid amide compound; Preferably, the method comprises irradiating an ultraviolet ray when the contact treatment with the reducing agent is performed in an organic solvent.

[0011] N, used in the chemiluminescent reagent of the present invention
N′-disubstituted-9,9′-bisacridinium salts are
The following general formula (1)

[0012]

Embedded image Can be represented by

In the general formula (1), R ¹ and R ²
Are each selected from the group consisting of an alkyl group, an aryl group and an aryl halide group, and may be the same or different. Alkyl, aryl and halogenated aryl groups have 1 to 14 carbon atoms, and preferred alkyl groups have 1 to 10 carbon atoms. For example, a linear or branched alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group can be exemplified. The aryl group has 6 to 20 carbon atoms.
Are preferable, and examples thereof include 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 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. These hydrocarbon groups include those having 1 carbon atom.
To 20, preferably 1 to 10. For example, a linear or branched alkyl group having 1 to 20 carbon atoms, an alkoxy group, an aryl group having 6 to 20 carbon atoms,
An aryloxy group may be mentioned, and the aryl group and the aryloxy group may be substituted with an alkyl group.

Specific examples of the N, N'-disubstituted-9,9'-bisacridinium salts include N, N'-dimethyl-9,9'-bisacridinium salts and N, N '-Diethyl-9,9'-bisacridinium salt, N, N'-dipropyl-9,9'-bisacridinium salt, N, N '
-Diisopropyl-9,9'-bisacridinium salt,
N, N'-dibutyl-9,9'-bisacridinium salt, N, N'-diisobutyl-9,9'-bisacridinium salt, N, N-diphenyl-9,9'-bisacridinium salt Ammonium salt, N, N'-di-m-chlorophenyl-9,
9'-bisacridinium dinitrate (lucigenin) and the like can be mentioned. In particular, N, N'-dimethyl-9,9'-bisacridinium dinitrate (lucigenin) is preferred.

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

The N, N-disubstituted carboxylic acid amide compound used for producing the chemiluminescent reagent of the present invention has the following general formula (2)

[0018]

Embedded image Can be represented by In the general formula (2), R ₁ is
Hydrogen atom, alkyl group having 1 to 10 carbon atoms, 2 to 1 carbon atoms
0 is an alkenyl group or an aryl group having 6 to 20 carbon atoms, and the aryl group may be substituted with an alkyl group, a halogen atom, a nitro group, a hydroxyl group, an amino group, or the like. R ₂
Is a methyl group or an ethyl group, and R ₃ has 1 to 1 carbon atoms.
It is an alkyl group having 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms, and the aryl group may be substituted with an alkyl group, a halogen atom, a nitro group, a hydroxyl group, an amino group, or the like. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which each is bonded.
Specific examples of the N, N-disubstituted carboxylic acid amide compound include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide,
N, N-dimethylpropionamide, N, N-dimethylbenzamide and the like can be mentioned. Further, N-substituted-pyrrolidone, for example, N-alkyl-2-pyrrolidone can be mentioned, and specifically, N-methyl-2-pyrrolidone is used.
Examples include pyrrolidone, N-propyl-2-pyrrolidone, N-butyl-2-pyrrolidone, and N-pentyl-2-pyrrolidone.

The N, N-disubstituted carboxylic acid amide compound is essential for producing the chemiluminescent reagent of the present invention,
After forming a complex with the N, N'-disubstituted-9,9'-bisacridinium salt, it is considered that the compound changes to a compound having a higher luminescence yield with the reduction reaction of the acridinium salt structure. It is considered that it participates as a reaction component in the production reaction of the chemiluminescent reagent of the present invention and contributes to the water solubility of the reaction product.

The formic acid used for the production of the chemiluminescent reagent of the present invention is used in an amount of 0 to 5000 equivalents, preferably 0.1 equivalent, based on the N, N'-disubstituted-9,9'-bisacridinium salt. 5 to 1000 equivalents, particularly preferably 1 to 500
N, N'-disubstituted-9,9'-
It has the effect of promoting the reduction reaction of bisacridinium salts and significantly increasing the reaction yield of the chemiluminescent reagent.

Examples of the reducing agent used for producing the chemiluminescent reagent of the present invention include lithium aluminum hydride, lithium boron hydride, sodium boron hydride, and the like, with lithium aluminum hydride being particularly preferred.

The organic solvent used in the reduction treatment is not particularly limited as long as it is inert to the reaction reagent and has solubility, and examples thereof include tetrahydrofuran and dioxane. Cyclic ethers and the like are preferably used.

As the ultraviolet light source used for producing the chemiluminescent reagent of the present invention, a high-pressure mercury lamp, a low-pressure mercury lamp, a germicidal lamp and the like can be used without limitation, but a high-pressure mercury lamp is preferably used. The chemiluminescent reagent obtained by this ultraviolet irradiation shows extremely high luminescence intensity when used at the same concentration as compared to a chemiluminescent reagent manufactured without irradiation with ultraviolet light. It is considered that the compound has an action of accelerating the formation reaction of the compound.

The reaction temperature varies depending on the type of the reducing agent and the reaction solvent used, but is generally -10 to +15.
0 ° C, preferably 0 to 120 ° C, particularly preferably 20 to
Temperatures in the range of 90 ° C. Reaction time is one minute to one day and night,
Preferably from 10 minutes to 12 hours, particularly preferably from 30 minutes to
Times in the range of 5 hours can be employed.

The chemiluminescent reagent of the present invention reacts in the presence of hydrogen peroxide and peroxidase under basic conditions to emit light, and this reaction can be used for detecting hydrogen peroxide and quantifying peroxidase. By using peroxidase as a labeling substance, it becomes possible to further use it for quantification of various substances.

The chemiluminescent reagent of the present invention has a pH of 7.5 to 1
Under basic conditions of 3, light is emitted in the presence of excess hydrogen peroxide in an amount dependent on the concentration of peroxidase.
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-
Non-limiting examples include bromophenol, p-chlorophenol, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like.

When performing chemiluminescence analysis using the novel chemiluminescent reagent of the present invention, 10 ^{-8 to} 1M, preferably 1 to ⁸ M
It is used at a concentration ranging from 0 ^-6 to 10 ^-2 M, and the amount used may range from 10 to 500 μl, preferably from 50 to 300 μl. The amount of the luminescence enhancer used is in the range of 0.01 to 100 times, preferably 0.1 to 10 times the mol of the chemiluminescence reagent, and the concentration is 10 ^{-6 to 1} M, preferably 10 ^-4. It is preferable to use in the range of from 10 to ² M. Also,
The amount of hydrogen peroxide used in the chemiluminescent reaction needs to be used in a sufficient excess amount with respect to the chemiluminescent reagent, and is used in an amount of 30,000 to 10,000 times, preferably 10 to 10 times, the molar amount of the chemiluminescent reagent. It is desirable to use it in the range of up to 1000 times mol.

In the case where various substances are quantified by labeling an antibody, nucleic acid or the like using peroxidase as a labeling substance, horseradish peroxidase (HRP) is preferably used as the peroxidase, although it is not particularly limited. . As the basic buffer used for the chemiluminescence reaction, Tris buffer, phosphate buffer, borate buffer,
Carbonate buffer, glycine-sodium hydroxide buffer and the like can be optionally used. The concentration of these buffers was 1 m
It is desirable to use in the range of M to 1M. In addition, additives such as a surfactant and a chelating agent can be optionally used during the reaction.

[0029]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 1 mg of lucigenin was placed in a test tube, and 150 μl of N, N-dimethylacetamide was added thereto.
And then dissolved in 1,4-dioxane 1 with stirring.
and lucigenin was finely dispersed in 1,4-dioxane. Next, formic acid (20 μl) was added thereto, and the mixture was stirred well. Then, lithium aluminum hydride powder (5 mg) was added, and the mixture was irradiated with a 1 KW high-pressure mercury lamp at 25 ° C. for 3 hours.
After a reduction reaction by stirring for 0 minutes, the reaction mixture was poured into 1 ml of deionized water little by little under ice cooling to decompose an excessive amount of lithium aluminum hydride. Next, the precipitate such as aluminum hydroxide formed is filtered off, and the p
A new chemiluminescent reagent was obtained by adjusting H to 7.0 with 1N hydrochloric acid. [Measurement of Peroxidase Activity] After this chemiluminescent reagent was diluted 100-fold with 0.1 M Tris-HCl buffer (pH 8.4), 20 μl of each was filled into a plurality of wells for chemiluminescence measurement, and horseradish peroxidase (HRP) was used. Is added to a mixture containing 200 μl of a 0.1 M Tris-HCl buffer (pH 8.4) solution at various concentration levels and 20 μl of a 10 mM p-iodophenol 10 mM Tris-HCl buffer (pH 8.4) solution,
Next, a 0.0034% by weight aqueous hydrogen peroxide solution (50 μm) was added thereto.
1 and adding the luminescence amount for 0 to 5 seconds with a luminometer (Luminas CT-9000D manufactured by Diatron), the luminescence intensity as shown in Table 1 is obtained, and the peroxidase activity is measured with good concentration dependency. It was recognized that it was possible.

[0031]

[Table 1]

Example 2 1 mg of lucigenin was placed in a test tube, and 150 μl of N, N-dimethylformamide was added thereto.
And then dissolved in 1,4-dioxane 1 with stirring.
and lucigenin was finely dispersed in 1,4-dioxane. Next, formic acid (20 μl) was added thereto, and the mixture was stirred well. Then, lithium aluminum hydride powder (5 mg) was added, and the mixture was irradiated with a 1 KW high-pressure mercury lamp at 25 ° C. for 3 hours.
After a reduction reaction by stirring for 0 minutes, the reaction mixture was poured into 1 ml of deionized water little by little under ice cooling to decompose an excessive amount of lithium aluminum hydride. Next, the precipitate such as aluminum hydroxide formed is filtered off, and the p
A new chemiluminescent reagent was obtained by adjusting H to 7.0 with 1N hydrochloric acid. [Measurement of Peroxidase Activity] After this chemiluminescent reagent was diluted 100-fold with 0.1 M Tris-HCl buffer (pH 8.4), 20 μl of each was filled into a plurality of wells for chemiluminescence measurement, and horseradish peroxidase (HRP) was used. ) Of a 0.1 M Tris-HCl buffer (pH 8.4) solution at various concentrations and 20 μl of a 10 mM p-iodophenol 10 mM Tris-HCl buffer (pH 8.4) solution. 50 μl of a 0034% by weight aqueous hydrogen peroxide solution was added, and a luminometer (Luminous CT manufactured by Diatron Co., Ltd.) was added.
As a result of integrating the luminescence amount for 0 to 5 seconds at −9000 D), it was confirmed that the luminescence intensity as shown in Table 2 was obtained, and that the peroxidase activity could be measured with good concentration dependency.

[0033]

[Table 2]

Example 3 1 mg of lucigenin was placed in a test tube, and 150 μl of N-methyl-2-pyrrolidone was added and dissolved therein. Then, the mixture was added to 1 ml with stirring to add lucigenin to 1,4-dioxane. Finely disperse in it. next,
After adding 20 μl of formic acid and stirring well, 5 mg of lithium aluminum hydride powder was added, and 1 KW
The mixture was stirred at 25 ° C. for 30 minutes under a high pressure mercury lamp to effect a reduction reaction, and the reaction mixture was cooled with ice to 1 m of deionized water.
An excess amount of lithium aluminum hydride was decomposed by adding a small amount into the 1L. Next, the resulting precipitate of aluminum hydroxide and the like was separated by filtration, and the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a novel chemiluminescent reagent. [Measurement of Peroxidase Activity] After this chemiluminescent reagent was diluted 100-fold with 0.1 M Tris-HCl buffer (pH 8.4), 20 μl of each was filled into a plurality of wells for chemiluminescence measurement, and horseradish peroxidase (HRP) was used. ) Of a 0.1 M Tris-HCl buffer (pH 8.4) solution at various concentrations and 20 μl of a 10 mM p-iodophenol 10 mM Tris-HCl buffer (pH 8.4) solution. 50 μl of a 0034% by weight aqueous hydrogen peroxide solution was added, and a luminometer (Luminous CT manufactured by Diatron Co., Ltd.) was added.
As a result of integrating the luminescence amount for 0 to 5 seconds at −9000 D), it was confirmed that the luminescence intensity as shown in Table 2 was obtained, and that the peroxidase activity could be measured with good concentration dependency.

[0035]

[Table 3]

Comparative Example 1 1 mg of lucigenin was placed in a test tube, and 150 μl of N, N-dimethylformamide was added thereto.
And then dissolved in 1,4-dioxane 1 with stirring.
and lucigenin is finely dispersed in 1,4-dioxane. Next, formic acid (20 μl) was added thereto, and the mixture was stirred well, and then lithium aluminum hydride powder (200 μl) was added.
g, and the mixture was stirred at 60 ° C. for 3 hours to effect a reduction reaction. The reaction mixture was poured into 1 ml of deionized water little by little under ice cooling to decompose excess lithium aluminum hydride. . Next, the precipitate of the formed aluminum hydroxide and the like was separated by filtration, and the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a chemiluminescent reagent. [Measurement of Peroxidase Activity] After this chemiluminescent reagent was diluted 100-fold with 0.1 M Tris-HCl buffer (pH 8.4), 20 μl of each was filled into a plurality of wells for chemiluminescence measurement, and horseradish peroxidase (HRP) was used. ) Of a 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations and 20 μl of a 10 mM p-iodophenol solution in 10 mM Tris-HCl buffer solution (pH 8.4). 50 μl of a 0034% by weight aqueous hydrogen peroxide solution was added, and a luminometer (Luminous CT manufactured by Diatron Co., Ltd.) was added.
As a result of integrating the luminescence amount for 0 to 5 seconds at −9000 D), the luminescence intensity as shown in Table 4 was obtained, and it was recognized that the peroxidase activity could not be measured accurately at this chemiluminescent reagent concentration.

[0037]

[Table 4]

[0038]

The novel chemiluminescent reagent provided by the present invention can be easily produced in a relatively short period of time using inexpensive raw materials, and is capable of producing peroxidase in the presence of hydrogen peroxide and peroxidase. By utilizing the property of chemiluminescence depending on the concentration, a peroxidase enzyme can be detected with high sensitivity. Furthermore, using an enzyme labeled product obtained by labeling an antigen, an antibody, a nucleic acid, or the like with peroxidase as a labeling substance, an antibody, an antigen, or the like is subjected to a protein by Western blotting using an enzyme immunoassay, and a DNA or RNA is subjected to Southern or Northern blotting. Each of the nucleic acids can be specifically and highly sensitively measured using an enzyme-labeled nucleic acid probe.

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[Procedure amendment]

[Submission date] July 13, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction contents]

[0018]

Embedded image Can be represented by In the general formula (2), R ₁ is
Hydrogen atom, alkyl group having 1 to 10 carbon atoms, 2 to 1 carbon atoms
0 is an alkenyl group or an aryl group having 6 to 20 carbon atoms, and the aryl group may be substituted with an alkyl group, a halogen atom, a nitro group, a hydroxyl group, an amino group, or the like. R ₂
Is a methyl group or an ethyl group, and R ₃ has 1 to 1 carbon atoms.
It is an alkyl group having 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms, and the aryl group may be substituted with an alkyl group, a halogen atom, a nitro group, a hydroxyl group, an amino group, or the like. Further, R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which each is bonded.
Specific examples of the N, N-disubstituted carboxylic acid amide compound include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide,
N, N-dimethylpropionamide, N, N-dimethylbenzamide , N-methyl-2-pyrrolidone and N-E
Tyl-2-pyrrolidone and the like can be mentioned.

 ──────────────────────────────────────────────────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 (10)

[Claims] 1. When an N, N′-disubstituted-9,9′-bisacridinium salt is contact-treated with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound, an ultraviolet ray is irradiated. A chemiluminescent reagent comprising a reaction product obtained by irradiation. 2. The N, N′-disubstituted-9,9′-
Bisacridinium salts are represented by 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; X is an n-valent anion;
Is 1 or 2. The chemiluminescent reagent according to claim 1, which is a compound represented by the formula: 3. The N, N′-disubstituted-9,9′-
Bisacridinium salts are N, N'-dimethyl-9,
The chemiluminescent reagent according to claim 1 or 2, which is 9'-bisacridinium dinitrate. 4. The N, N-disubstituted carboxylic acid amide compound is at least one compound selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone. The chemiluminescent reagent according to any one of claims 1 to 3. 5. The N, N′-disubstituted-9,9′-
The formic acid is further added when the bisacridinium salt is contact-treated with the reducing agent in the presence of the N, N-disubstituted carboxylic acid amide compound.
The chemiluminescent reagent according to claim 4. 6. An ultraviolet ray when contacting an N, N′-disubstituted-9,9′-bisacridinium salt with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound. A method for producing a chemiluminescent reagent, comprising irradiating. 7. The N, N′-disubstituted-9,9′-
Bisacridinium salts are represented by 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; X is an n-valent anion;
Is 1 or 2. 7. The method for producing a chemiluminescent reagent according to claim 6, which is a compound represented by the formula: 8. The method for producing a chemiluminescent reagent according to claim 6, further comprising adding formic acid during the contact treatment with the reducing agent. 9. The N, N′-disubstituted-9,9′-
The method for producing a chemiluminescent reagent according to claim 8, wherein the N, N-disubstituted carboxylic acid amide compound and the formic acid are each at least equimolar to 1 mol of the bisacridinium salt. 10. The chemiluminescent reagent according to claim 6, wherein the reducing agent is lithium aluminum hydride.