JP4286356B2 - Method for measuring peroxidase activity - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring peroxidase activity using a chemiluminescent method, and more specifically, measures peroxidase activity in the presence of a hydrogen acceptor by using a chemiluminescent method using a specific chemiluminescent reagent. It is about the method.
[0002]
[Prior 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 highly sensitive and easy to detect, it is used not only for detection of peroxidase enzyme activity but also as a reagent for analysis that serves as an indicator of various substances to be detected in a wide range of fields. As described above, analysis methods using peroxidase as a labeling substance include immunoassays using antigen-antibody reactions, nucleic acid hybridization assays using nucleic acid complementation, other avidin-biotin, hormone-hormone receptors, sugars. -Used for analysis systems utilizing specific affinity such as lectins.
[0003]
In addition, for the measurement of such peroxidase activity, a colorimetric method in which hydrogen peroxide is used as a hydrogen acceptor, a hydrogen donor is oxidized, a dye is formed, and the amount of color development is measured with a spectrophotometer or the like, or a fluorescent substance The fluorescence method etc. which produce this and measure the fluorescence with a fluorescence spectrophotometer etc. are performed. However, when a colorimetric method is used for measuring peroxidase activity, there is a problem that the quantitative property in the low concentration region of peroxidase is lacking, and a fluorescence method has been developed to solve this problem. It is not yet sufficient for quantitativeness in the area. In order to solve this problem, a method for measuring peroxidase activity by a chemiluminescence method has been developed. The chemiluminescence method is a method for quantifying peroxidase enzyme activity by measuring the amount of luminescence emitted when the chemiluminescent substance is excited through an intermediate due to the catalytic activity of the peroxidase enzyme and returns to the ground state from this state. A chemiluminescence system has been developed that uses luminol as the chemiluminescent substance, hydrogen peroxide as the hydrogen acceptor, and p-iodophenol as the luminescence enhancer, and can quantify peroxidase activity with high sensitivity. It has become.
[0004]
[Problems to be solved by the invention]
However, when measuring peroxidase enzyme activity in enzyme immunoassay, there are many cases in which it is necessary to further improve the quantitativeness of the enzyme in a low concentration region, and the sensitivity of peroxidase enzyme activity measurement is further increased. It was desired. The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel measurement method by a chemiluminescence method capable of measuring peroxidase activity with higher sensitivity.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have determined that N, N′-disubstituted-9,9′-bisacridinium salts are N, N-disubstituted as chemiluminescent reagents. Chemiluminescence using a reaction product obtained by contact with a reducing agent in the presence of a carboxylic acid amide compound and formic acid, using hydrogen peroxide as the hydrogen acceptor and preferably a specific phenolic compound as the luminescence enhancer It has been found that the system can quantify peroxidase activity with higher sensitivity than the chemiluminescent system using luminol as a chemiluminescent substance, and the present invention has been achieved based on these findings.
Therefore, the first of the present invention is the following general formula (1)
[0006]
[Chemical 3]
(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 a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ and R ⁶ is 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, and n is 1 Or 2.
A reaction product obtained by contacting a N, N′-disubstituted-9,9′-bisacridinium salt represented by formula (I) with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid The present invention relates to a method for measuring peroxidase activity by a chemiluminescence method, wherein the peroxidase enzyme activity is measured in the presence of a hydrogen acceptor using a chemiluminescent reagent comprising:
The second of the present invention is
The following general formula (1)
[0007]
[Formula 4]
(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 a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ and R ⁶ is 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, and n is 1 Or 2.
A reaction product obtained by contacting a N, N′-disubstituted-9,9′-bisacridinium salt represented by formula (I) with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid 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 using a chemiluminescent reagent comprising .
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
In the method for measuring peroxidase activity of the present invention, N, N′-disubstituted-9,9′-bisacridinium salts are brought into contact with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid. The peroxidase enzyme activity is measured in the presence of a hydrogen acceptor, more preferably in the presence of a luminescence enhancer, using a chemiluminescent reagent comprising the reaction product obtained by In general, a measurement sample containing a chemiluminescent substance, a luminescence enhancer and a peroxidase enzyme is prepared, and a hydrogen acceptor solution is added to the chemiluminescent reaction in a specific basic pH region. A method of measuring the amount with a luminescence measuring device or the like can be employed.
[0009]
The chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention comprises N, N′-disubstituted-9,9′-bisacridinium salts, N, N′-disubstituted carboxylic acid amide compounds, and presence of formic acid. It contains the reaction product obtained by contacting with a reducing agent in an organic solvent.
N, N′-disubstituted-9,9′-bisacridinium salts are
The following general formula (1)
[0010]
[Chemical formula 5]
Wherein R ¹ and R ² are each 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 group, aryl group and halogenated aryl group have 1 to 20 carbon atoms, and preferred alkyl groups are those having 1 to 10 carbon atoms. Examples thereof include linear or branched 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. The aryl group preferably has 6 to 14 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 preferable. 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.
[0011]
In the general formula (1), 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, Or it may be different. These hydrocarbon groups preferably have 1 to 20 carbon atoms, particularly 1 to 10 carbon atoms.
[0012]
In the general formula (1), X is an n-valent anion, and n is 1 or 2. Specific examples of the anion include nitrate ion, halogen ion, phosphate ion, sulfate ion, sulfonate ion and the like. Of these anions, nitrate ions are particularly preferred.
[0013]
Specific examples of 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′-diphenyl-9,9′-bisacridinium salt, N, N′-di-m-chlorophenyl-9,9′-bisacridinium salt, etc. Is mentioned. Of course, although not limited thereto, N, N′-dimethyl-9,9′-bisacridinium dinitrate (lucigenin) is particularly preferable.
[0014]
Examples of the N, N′-disubstituted carboxylic acid amide compound used for the preparation of the chemiluminescent reagent include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, and N, N-dimethyl. Nonlimiting examples include propionamide, N, N-dimethylbenzamide, N-methyl-2-pyrrolidone, and the like. This N, N-disubstituted carboxylic acid amide compound is essential for the preparation of the chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention, and N, N'-disubstituted-9,9'-bisacrylic acid is used. After forming a complex with dinium salts, it is estimated that the compound will have a higher emission yield due to the reduction reaction of the acridinium salt structure. It is presumed that it contributes to imparting water solubility to the product. Therefore, the amount used is 1 to 1000 equivalents, preferably 1.5 to 500 equivalents, particularly preferably 2 to 300 equivalents, based on the N, N′-disubstituted-9,9′-bisacridinium salts. It is.
[0015]
Formic acid used in the preparation of the chemiluminescent reagent together with the N, N′-disubstituted-9,9′-bisacridinium salts and the N, N′-disubstituted carboxylic acid amide compounds promotes the reduction reaction, It has the effect of significantly increasing the reaction yield of the chemiluminescent reagent, and the amount used is 0.1 to 5000 equivalents, preferably 0, based on the N, N′-disubstituted-9,9′-bisacridinium salts. It is a ratio of 0.5 to 1000 equivalents, particularly preferably 1 to 500 equivalents.
[0016]
Examples of the reducing agent used in the preparation of the chemiluminescent reagent include lithium aluminum hydride, lithium boron hydride, and sodium boron hydride. Among these, lithium aluminum hydride is particularly preferable.
A reaction solvent is preferably used when performing the reduction reaction, and the reaction solvent is not particularly limited as long as it is inert to the reaction reagent and has solubility in the reaction reagent. An organic solvent is used. In particular, cyclic ethers such as tetrahydrofuran and dioxane are preferable.
Moreover, although reaction temperature changes with kinds of reducing agent and solvent to be used, generally it is -10- + 150 degreeC, Preferably it is 0-120 degreeC, Especially preferably, it is employ | adopted in the range of 20-90 degreeC. A reaction time in the range of 1 minute to 1 day, preferably 10 minutes to 12 hours, particularly preferably 30 minutes to 5 hours is sufficient.
[0017]
The chemiluminescent reagent prepared as described above reacts in the presence of a hydrogen acceptor and peroxidase under basic conditions, and emits light. This reaction is used for hydrogen peroxide detection and peroxidase quantification. By using peroxidase as a labeling substance, it can be further used for quantification of various substances.
[0018]
The chemiluminescent reagent emits light in an amount depending on the concentration of peroxidase in the presence of an excess of hydrogen acceptor under basic conditions of pH 7.5-13. 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.
[0019]
In the method for measuring peroxidase activity of the present invention, the chemiluminescent reagent is used at a concentration in the range of 10 ^{−6 to 1} M, preferably 10 ⁻⁴ to 10 ⁻² M. The amount used is 10 to 500 μl, preferably 50 to The range is 300 μl. The amount of the luminescence enhancer used is in the range of 0.01 to 100 times mol, preferably 0.1 to 10 times mol of the amount of the chemiluminescent reagent, and its concentration is 10 ^{−6 to 1} M, preferably 10 ^−. It is in the range of ⁴ to 10 ⁻² M.
[0020]
The hydrogen acceptor used in the chemiluminescence reaction is not particularly limited as long as it can serve as a substrate for the peroxidase enzyme, but organic peroxides, inorganic peroxides and the like are arbitrarily used. Hydrogen oxide is preferably used. The amount of the hydrogen acceptor used must be sufficiently excessive with respect to the chemiluminescent reagent, and the amount used is 3 to 10,000 times mol, preferably 10 to 1000 times the amount of the chemiluminescent reagent. The range of moles.
[0021]
Further, when peroxidase is used as a labeling substance and various substances are quantified by labeling antibodies, nucleic acids, etc., horseradish peroxidase (HRP) is preferably used as the peroxidase. .
[0022]
Examples of the basic buffer used in the chemiluminescence reaction include Tris buffer, phosphate buffer, borate buffer, carbonate buffer, glycine-sodium hydroxide buffer, and the like. These buffers are preferably used at a concentration in the range of 1 mM to 1M. Further, during the reaction, additives such as a surfactant and a chelating agent can be arbitrarily used.
[0023]
【Example】
EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated concretely, this invention is not limited to the following Example etc.
[0024]
[Example 1]
[Preparation of chemiluminescent reagent]
1 mg of lucigenin (N, N′-dimethyl-9,9′-bisacridinium dinitrate) is put in a test tube, and 150 μl of N, N-dimethylformamide is added and dissolved therein. , 4-Dioxane was added to 1 ml, and lucigenin was finely dispersed in 1,4-dioxane. Next, 20 μl of formic acid was added and stirred well, 200 μg of lithium aluminum hydride powder was added, and the mixture was stirred at 60 ° C. for 3 hours for reduction reaction, and then the reaction mixture was removed under ice cooling. An excessive amount of lithium aluminum hydride was decomposed by injecting it little by little into 1 ml of ionic water. Next, the produced 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 novel chemiluminescent reagent.
[Measurement of peroxidase activity]
200 μl of 0.1 M Tris-HCl buffer (pH 8.4) solution with various concentrations of horseradish peroxidase (HRP) and p-iodophenol 10 mM Tris-HCl buffer (pH 8.4) in various chemiluminescence assay wells After 20 μl was added, 20 μl of the chemiluminescent reagent diluted 10-fold and 50 μl of 0.0034 wt% aqueous hydrogen peroxide were sequentially added thereto, and 0-5 with a luminometer (Diatron Luminous CT-9000D). As a result of integrating the luminescence amount per second, the luminescence intensity as shown in Table 1 was obtained, and it was possible to measure HRP up to 5 × 10 ⁻¹⁹ mol / assay.
[0025]
[Table 1]
[0026]
[Example 2]
[Preparation of chemiluminescent reagent]
Take 1 mg of lucigenin in a test tube, add 150 μl of N, N-dimethylacetamide to dissolve it, add to 1 ml of 1,4-dioxane with stirring, and finely disperse lucigenin in 1,4-dioxane. . Next, 20 μl of formic acid was added and stirred well, 200 μg of lithium aluminum hydride powder was added, and the mixture was stirred at 60 ° C. for 3 hours for reduction reaction, and then the reaction mixture was removed under ice cooling. An excessive amount of lithium aluminum hydride was decomposed by injecting it little by little into 1 ml of ionic water. Next, the produced 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 novel chemiluminescent reagent.
[Measurement of peroxidase activity]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations of horseradish peroxidase (HRP) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) were added to a plurality of chemiluminescence wells. After the addition, 20 μl of the chemiluminescent reagent diluted 10 times and 50 μl of 0.0034% by weight aqueous hydrogen peroxide solution were sequentially added thereto, and luminescence was emitted for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron). As a result of integrating the amounts, emission intensity as shown in Table 2 was obtained, and HRP could be measured up to 5 × 10 ⁻¹⁹ mol / assay.
[0027]
[Table 2]
[0028]
[Example 3]
[Preparation of chemiluminescent reagent]
Take 1 mg of lucigenin in a test tube, add 150 μl of N-methyl-2-pyrrolidone to dissolve it, add to 1 ml of 1,4-dioxane with stirring, and finely disperse lucigenin in 1,4-dioxane. Let Next, 20 μl of formic acid was added and stirred well, 200 μg of lithium aluminum hydride powder was added, and the mixture was stirred at 60 ° C. for 3 hours for reduction reaction, and then the reaction mixture was removed under ice cooling. An excessive amount of lithium aluminum hydride was decomposed by injecting it little by little into 1 ml of ionic water. Next, the produced 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 novel chemiluminescent reagent.
[Measurement of peroxidase activity]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations of horseradish peroxidase (HRP) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) were added to a plurality of chemiluminescence wells. After the addition, 20 μl of the chemiluminescent reagent diluted 10 times and 50 μl of 0.0034% by weight aqueous hydrogen peroxide solution were sequentially added thereto, and luminescence was emitted for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron). As a result of integrating the amounts, emission intensity as shown in Table 3 was obtained, and HRP could be measured up to 5 × 10 ⁻¹⁹ mol / assay.
[0029]
[Table 3]
[0030]
[Comparative Example 1]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations of horseradish peroxidase (HRP) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) were added to a plurality of chemiluminescence wells. After the addition, 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing luminol at a concentration of 5.6 × 10 ⁻⁵ M and 50 μl of 0.0034 wt% aqueous hydrogen peroxide solution were sequentially added to the luminometer. As a result of integrating the amount of luminescence for 0-5 seconds with (Diatron Luminous CT-9000D), the luminescence intensity shown in Table 4 is obtained, and HRP can be measured up to 5 × 10 ^-17 mol / assay. there were.
[0031]
[Table 4]
[0032]
【The invention's effect】
The method for measuring peroxidase activity of the present invention makes it possible to measure peroxidase enzyme activity with higher sensitivity than the measurement system using luminol, which has been widely used in the past, and the enzyme immunoassay method using peroxidase as a labeling substance. Antigens and antibodies, and nucleic acids can be detected or quantified with higher sensitivity, respectively, by hybridization assays.

Claims (6)

The following general formula (1)
(In the general formula (1), R ¹ and R ² are each selected from the group consisting of an alkyl group having 1 to 2 carbon atoms , a phenyl group, and a chlorophenyl group, and may be the same or different from each other. R ³ , R ⁴ , R ⁵ and R ⁶ are each a hydrogen atom, X is a nitrate ion , and n is 2.)
N, N′-disubstituted-9,9′-bisacridinium salts represented by the formula: N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropion Chemistry consisting of an N, N-disubstituted carboxylic acid amide compound consisting of amide, N, N-dimethylbenzamide or N-methyl-2-pyrrolidone and a reaction product obtained by contacting with a reducing agent in the presence of formic acid A method for measuring peroxidase activity by a chemiluminescence method, wherein a peroxidase enzyme activity is measured in the presence of a hydrogen acceptor using a luminescent reagent. The following general formula (1)
(In the general formula (1), R ¹ and R ² are each selected from the group consisting of an alkyl group having 1 to 2 carbon atoms , a phenyl group, and a chlorophenyl group, and may be the same or different from each other. R ³ , R ⁴ , R ⁵ and R ⁶ are each a hydrogen atom, X is a nitrate ion, and n is 2.)
N, N′-disubstituted-9,9′-bisacridinium salts represented by the formula: N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropion Chemistry consisting of an N, N-disubstituted carboxylic acid amide compound consisting of amide, N, N-dimethylbenzamide or N-methyl-2-pyrrolidone and a reaction product obtained by contacting with a reducing agent in the presence of formic acid A method for measuring peroxidase activity by a chemiluminescence method, wherein a phenolic compound is used as a luminescence enhancer when a peroxidase enzyme activity is measured in the presence of a hydrogen acceptor using a luminescent reagent.    The N, N′-disubstituted-9,9′-bisacridinium salt is lucigenin, and the N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide, N, N-dimethylacetamide, and The method for measuring peroxidase activity according to claim 1 or 2, which is at least one compound selected from the group consisting of N-methyl-2-pyrrolidone.    The method for measuring peroxidase activity according to any one of claims 1 to 3, wherein the reducing agent is lithium aluminum hydride.    The method for measuring peroxidase activity according to any one of claims 1 to 4, wherein the hydrogen acceptor is hydrogen peroxide.    The peroxidase activity according to any one of claims 2 to 5, wherein the phenolic compound is at least one compound selected from the group consisting of p-iodophenol, p-phenylphenol and 6-hydroxybenzothiazole. Measuring method.