JPH10295398A - Chemiluminescent enzyme immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay for measuring an antigen or antibody in a sample with high sensitivity, by reaction of a peroxidase-labeled antibody or antigen with the sample and then subjecting the reaction product to chemiluminescence with a reduction product of an N,N'-di-substituted-9,9'- bisacridinium salt. SOLUTION: This immunoassay measures an antigen or antibody in a sample by mixing a peroxidase enzyme-labeled antibody or antigen with the antigen or antibody, etc., to be measured in the sample to conduct an antigen-antibody reaction so as to produce an immune complex composed of a peroxidase enzyme-labeled-antigen antibody complex, then, supplementing the complex with, 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), i.e., a reduction product from an N,N'-di-substituted-9,9'-bisacridinium salts 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], followed by conducting a chemiluminescence in the presence of a hydrogen acceptor, and measuring the quantity of the emission.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an enzyme immunoassay using a chemiluminescence method, and more particularly to a method using a peroxidase enzyme as a labeling substance.
The present invention relates to a method for immunologically measuring an antigen or an antibody using a reduction reaction product of a specific acridinium salt as a chemiluminescent substance.

[0002]

2. Description of the Related Art Enzyme-linked immunosorbent assays have been developed as immunoassays that do not use radioactive isotopes as labeling substances and thus can maintain a favorable assay environment and are less dangerous to the human body. It is used in the measurement system for substances. In this enzyme immunoassay, various enzymes such as peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase are used. Methods for detecting the enzyme activity of an enzyme-labeled antibody or antigen using these enzymes include hydrogen peroxide / o-
Phenylenediamine, 4-nitrophenyl-phospho-
And an enzyme or an antigen having a correlation with the enzyme activity by measuring the amount of coloring of a chromogenic substance produced by an enzymatic decomposition reaction of an enzyme substrate such as 2-nitrophenyl-β-galactoside. A colorimetric method for quantifying the amount of is generally used. However, in clinical chemistry analysis, the measurement target is a biological sample (mainly, serum, urine, etc.), and the measured value is often used for diagnosis or follow-up of a disease state. Is required, but it is difficult to completely satisfy this requirement by a colorimetric method. Therefore, a fluorescence method has been proposed for the purpose of satisfying this requirement. Fluorescence is a method of decomposing a fluorescent substrate such as 4-hydroxyphenylacetic acid, 4-methylumbelliferyl-β-galactoside, or 4-methylumbelliferyl-phosphate to generate fluorescence by the catalytic activity of the enzyme used for labeling. After the generation, the fluorescence intensity is measured to quantify the enzyme activity, and to quantify the amount of the antibody or antigen correlated with the enzyme activity. However, in the fluorescence method, the excitation light is scattered, so that it is hardly sufficient to satisfy the above-mentioned requirements. In addition, in the colorimetric method and the fluorescent method, a light source such as a xenon lamp is required, and a stray phenomenon derived from light from the light source and Raman light derived from a solvent cause a background level to increase. In principle, it is difficult to increase the sensitivity of the measurement by the colorimetric method and the fluorescent method. In recent years, a chemiluminescent enzyme-linked immunosorbent assay (CLEIA) has been developed and attracted attention as a highly sensitive enzyme-linked immunosorbent assay that exceeds colorimetric and fluorescent methods.

[0003] In the chemiluminescent enzyme immunoassay, a chemiluminescent substance is put into an excited state via an intermediate by the catalytic activity of an enzyme, and the amount of light emitted when returning from this state to a ground state is measured to measure the enzyme activity. This is a method for quantifying the amount of an antibody or an antigen having a correlation with this enzyme activity.A light source is unnecessary because a chemiluminescent substance emits light by a chemical reaction, and an increase in the background caused by the light source is prevented. Because there is no measurement, it is possible to increase the sensitivity of the measurement. Enzymes used in the chemiluminescent enzyme immunoassay include peroxidase, alkaline phosphatase, β-galactosidase,
Glucose oxidase, dehydrogenase, etc. are mentioned, but peroxidase is preferably used in terms of ease of handling and availability, using luminol as a chemiluminescent substance, and using p-iodophenol as a luminescence enhancer. A chemiluminescence method has been developed, and it has become possible to quantify various substances with high sensitivity immunologically by the chemiluminescence method.

[0004]

However, in the chemiluminescent enzyme immunoassay (CLEIA) using a peroxidase enzyme as a labeling substance, it may be necessary to further increase the quantitativeness of the substance to be measured in a low concentration region. In many cases, it has been desired to further increase the sensitivity of a chemiluminescent enzyme immunoassay using a peroxidase enzyme as a labeling substance.
In view of the above circumstances, an object of the present invention is to provide a novel enzyme immunoassay using a chemiluminescence method capable of measuring a substance to be measured with higher sensitivity.

[0005]

Accordingly, the present inventors have:
As a result of intensive studies to achieve the above object, as a chemiluminescent substance, a reaction product obtained by reduction treatment of N, N'-disubstituted-9,9'-bisacridinium salts was used, In the presence of a hydrogen acceptor, a chemiluminescence system using a specific phenolic compound as a luminescence enhancer particularly preferably immunologically converts a substance to be measured with higher sensitivity than a chemiluminescence system using luminol as a chemiluminescence substance. They have found that they can be quantified, and have arrived at the present invention based on these findings.

Accordingly, the present invention provides an immunoconjugate comprising a peroxidase enzyme-labeled-antigen-antibody complex by mixing an antibody or an antigen labeled with a peroxidase enzyme with an antigen or an antibody to be measured in a sample or an aggregate thereof. And then, preferably, reacting the immune complex with an antibody or antigen immobilized on an insoluble carrier and capturing the complex on the insoluble carrier.

[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 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 ^n- is an n-valent anion; Is 1 or 2.)
The reaction product obtained by the reduction treatment of the di-substituted-9,9'-bisacridinium salts is added, chemiluminescence is caused in the presence of a hydrogen acceptor, and the amount of chemiluminescence is measured. The present invention relates to a chemiluminescent enzyme immunoassay method for measuring an antigen or an antibody.

[0008]

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

The chemiluminescent enzyme immunoassay of the present invention comprises an immunoreaction step in which an antigen or antibody to be measured by an antigen-antibody reaction is captured as an immune complex labeled with a peroxidase enzyme; And a chemiluminescence reaction step which is measured by a chemiluminescence method using a labeling enzyme present in the above.

The method of the immunoreaction constituting the immunoreaction step is arbitrary, and the detection and quantification of the enzymatic activity in the chemiluminescence reaction step is carried out for the chemiluminescent substance N, N'-disubstitution-9,9'-.
Any method can be adopted as long as a reaction product obtained by a reduction treatment of a bisacridinenium salt can be used.

[0011] For example, a sandwich method in which an antibody bound to an insoluble carrier is reacted with a peroxidase enzyme-labeled antibody simultaneously with or after the antigen to be measured in the sample is captured by the antibody bound to the insoluble carrier. Using an antibody derived from a different animal species, the generated sandwich complex is further reacted with a labeled second antibody against the antibody, a two-antibody method, and the antigen to be measured in the sample is added to the antibody bound to the insoluble carrier. A competitive method in which the reaction is performed in the presence of a peroxidase enzyme-labeled antigen, a sample containing the antigen or antibody to be measured is reacted with a labeled antibody or antigen that specifically reacts with the sample, and aggregated and precipitated. The coagulation sedimentation method to detect the labeling substance in the separated immune complex or the antigen bound to an insoluble carrier A method such as an antibody detection method in which a peroxidase enzyme-labeled anti-human gamma globulin antibody is acted upon after capturing an antibody to be measured therein, and a method using a biotin-labeled antibody and a peroxidase-labeled avidin are used without limitation. be able to.

The insoluble carrier used in the immunoreaction step of the chemiluminescent enzyme immunoassay of the present invention includes, for example, high molecular compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, and polysaccharide. , Others, paper, glass, metal, magnetic particles and combinations thereof. As the shape of the insoluble carrier, various shapes such as a tray, a sphere, a fiber, a bar, a disk, a container, a cell, a microplate, and a test tube can be used. Further, the method of immobilizing the antigens and antibodies on these insoluble carriers is arbitrary, but a physical adsorption method, a covalent bonding method, an ionic bonding method and the like can be used.

The antibodies used in the chemiluminescent enzyme immunoassay of the present invention may be either monoclonal antibodies or polyclonal antibodies, and the form may be a fragment such as F (ab ') ₂ or Fab even if it is a whole antibody. You can also. The origin of the antibody is arbitrary, but antibodies derived from mice, rats, rabbits, sheep, goats, chickens and the like are preferably used.

The chemiluminescence reaction constituting the latter stage of the chemiluminescence enzyme immunoassay of the chemiluminescent enzyme immunoassay according to the present invention comprises N, N'-disubstituted-9,9'-bisacridinium salts as chemiluminescent substances. There is specificity in using the reaction product obtained by the reduction treatment of Further, it is preferable that a luminescence enhancer is present in the reaction system, and the activity of a peroxidase enzyme as a labeling substance can be measured by allowing a hydrogen receptor to act in the presence of the luminescence enhancer. The measurement technique is optional, but generally, a measurement reagent containing a chemiluminescent substance and a luminescence enhancer is added to an insoluble carrier immunologically capturing a peroxidase enzyme-labeled antibody or antigen, and a specific basic pH region is used. For example, a method of adding a hydrogen acceptor to cause a chemiluminescence reaction and measuring the amount of chemiluminescence by a luminescence measuring device can be used.

The chemiluminescent substance N, N'-disubstituted-9,
The reaction product obtained by the reduction treatment of 9′-bisacridinium salt is a reaction product obtained by performing the reduction treatment of the compound represented by the general formula (1) in a solvent using a reducing agent. Yes, a single compound or a mixture. 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 from each other. The alkyl group has 1 carbon atom.
-20, preferably 1-14 linear or branched, and the aryl group and the halogenated aryl group each have 6-20 carbon atoms, and have 1-14 carbon atoms. May be substituted with an alkyl group, but 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 selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different. Alkyl group,
The alkoxy group has 1 to 20 carbon atoms, preferably 1 to 14 carbon atoms.
The aryl group and the aryloxy group may have 6 to 20 carbon atoms and may be substituted with an alkyl group.

In the above 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), phosphate ion (PO ₃ ⁻ ) and the like. Among these anions, nitrate ions are particularly preferred.

As a specific example of N, N'-disubstituted-9,9'-bisacridinium salts, N, N'-dimethyl-9,9'-bisacridinium salts are preferably used. But also N, N'-diethyl-9,9'-bisacridinium salt, N, N'-dipropyl-9,9'-
Bisacridinium salt, N, N'-dibutyl-9,9 '
Bisacridinium salts, 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 without limitation. Among these, N, N'-dimethyl-9,9'-bisacridinium dinitrate is particularly preferred.

The reduction reaction product may contain an N, N'-disubstituted-9,9'-bisdihydroacridine derivative, which may be represented by, for example, the following general formula (2). it can.

[0019]

Embedded image In the 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. The alkyl group has 1 to 20 carbon atoms, preferably,
The aryl group and the halogenated aryl group each have 6 to 20 carbon atoms, each of which may be substituted with an alkyl group having 1 to 14 carbon atoms. Although a phenyl group and a halogenated phenyl group are particularly preferable. R ³³ ,
R ⁴⁴ , 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. The alkyl group and the alkoxy group may have 1 to 20 carbon atoms, preferably 1 to 14 linear or branched groups, and the aryl group and the aryloxy group may have 6 carbon atoms.
To 20 and may be substituted with an alkyl group.

These chemiluminescent substances have a pH of 7.5 to 7.5.
Under 13 basic conditions, light is emitted in the presence of an excess of hydrogen acceptor 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 preferred.
-(4-chlorophenyl) phenol, p- (4-bromophenyl) phenol, p- (4-iodophenyl)
Examples include, but are not limited to, phenol, p-bromophenol, p-chlorophenol, 2-naphthol, firefly luciferin and the like.

The chemiluminescent substance N, N'-di-substituted used in the chemiluminescent enzyme immunoassay of the present invention.
The concentration of the reduction reaction product of the 9,9′-bisacridinium salt is 10 ^{−8 to 1} M, preferably, in terms of the N, N′-substituted-9,9′-bisacridinium salt of the starting material. 1
The range is from 0 ^-5 to 10 ^-2 M.
The range is preferably from 500 to 500 µl, particularly 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 chemiluminescent substance composed of the reduction reaction product, and the concentration is 10 ^-6 to 1.
M, preferably in the range of 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 preferably used. It is necessary to use the hydrogen acceptor in a sufficient excess amount with respect to the chemiluminescent substance, and the use amount is 30,000 to 10,000 times, preferably 10 to 10 times, the mole of the chemiluminescent substance. It can be used in a range of 1000 times mol.

As the basic buffer used for the chemiluminescence reaction, a Tris buffer, a phosphate buffer, a borate buffer, a carbonate buffer, a glycine-sodium hydroxide buffer and the like can be arbitrarily used. The concentration of these buffers is between 1 mM and
It is preferable to use in the range of 1M. In addition, additives such as a surfactant and a chelating agent can be optionally used during the reaction.

The amount of luminescence of the chemiluminescence reaction of the present invention can be measured using a luminescence photometer, and the starting point of the measurement and the integration time are arbitrary, but the luminescence is stable and the luminescence is small. It is desirable to select a time that is highly concentration dependent. 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 reagents,
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 chemiluminescent enzyme immunoassay of the present invention is optional, It can be easily produced by subjecting the compound represented by (1) to a reduction treatment using 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 preferably used. The reduction reaction is preferably performed in a reaction solvent, but the reaction solvent is not particularly limited as long as it is inert to the reaction reagent and has solubility in the reaction reagent, and tetrahydrofuran, Cyclic ethers such as 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, a reagent for measuring chemiluminescence comprising a reduction reaction product of N, N'-disubstituted-9,9'-bisacridinium salts can be provided, and a peroxidase enzyme can be provided. It is possible to provide a reagent kit for enzyme immunoassay using a labeling substance, a reagent for measuring chemiluminescence, hydrogen peroxide in a hydrogen acceptor solution, a phenolic compound as a luminescence enhancer, and the like. This measurement reagent or these measurement reagent kits are extremely useful for performing an efficient chemiluminescence enzyme immunoassay.

[0027]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited to these Examples. In addition,% in a reference example, an example, etc. means 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.

Reference Example 2 Production of Polyclonal Antibody Immobilized on Insoluble Carrier A polyclonal antibody derived from an animal such as a rabbit having specific reactivity to an antigen was prepared from 10 mM phosphate buffered saline (pH 7.0).
4) 0.1 ml of a solution of (PBS) dissolved at a concentration of 10 mg / ml was added to each well of a white microplate (Lab System), left at 37 ° C. for 1 hour, and then washed with PBS. Thereafter, 0.3 ml of a 1% bovine serum albumin (BSA) aqueous solution was added thereto, and the mixture was allowed to stand at a temperature of 37 ° C. for 1 hour to perform a post-coating treatment to obtain a polyclonal antibody-immobilized white microplate.

Reference Example 3 Production of Peroxidase Enzyme-Labeled Monoclonal Antibody Monoclonal antibody derived from a mouse or the like having specific reactivity to an antigen was prepared using 10 mM phosphate buffered saline (PBS).
A solution of N- (m-maleimidobenzoic acid) -N-succinimide ester (MBS) in dimethylformamide at a concentration of 10 mg / ml was dissolved in 1 ml of a solution of 1.0 mg / ml in 1.0 (pH 7.4). Add 1 ml and add 2
The reaction was performed at a temperature of 5 ° C. for 30 minutes. Next, the reaction mixture was subjected to gel filtration using a column packed with Sephadex G-25 using a 0.1 M phosphate buffer (pH 6.0), and a maleimide-conjugated monoclonal antibody and unreacted MB were reacted.
S was separated.

On the other hand, horseradish peroxidase (HRP) was used as a peroxidase enzyme.
g / ml of PBS solution in N-succinimidyl-3-
(2-pyridylthio) propionate (SPDP) 1
0 mg / ml ethanol solution was added,
At 30 ° C. for 30 minutes.

Next, the reaction mixture was purified by gel filtration using a column packed with Sephadex G-25 using a 10 mM acetate buffer (pH 4.5), and a fraction containing pyridyl disulfide-modified HRP was collected. This was concentrated about 10-fold in a collodion bag under ice-cooling. Next, it contains 0.1 M dithiothreitol.
After adding 1 ml of 1 M acetate buffered saline (pH 4.5) and stirring at a temperature of 25 ° C. for 30 minutes to reduce the pyridyl disulfide group introduced into the HRP molecule, the reaction mixture was separated with Sephadex G- Gel filtration was performed using a column packed with 25 to obtain a fraction containing thiolated HRP.

Next, the maleimidated monoclonal antibody and the thiolated HRP were mixed, concentrated using a collodion bag under ice cooling to a protein concentration of 4 mg / ml,
And left overnight for Ultrogel AcA44 (SEP
Gel filtration using a column packed with (RACOR),
A peroxidase enzyme-labeled monoclonal antibody was obtained.

Example 1 α-fetoprotein by simultaneous sandwich method CLEIA
Measurement of in (AFP) A 2% BSA-containing PBS solution (pH 7.4) containing purified human AFP (standard substance) in a range of 0 to 800 ng / ml is placed on a white microplate on which a rabbit anti-human AFP polyclonal antibody is immobilized. ) 50 μl of peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody
100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing a concentration of μg / ml was added thereto.
Incubated for hours. Next, the solution in the well is removed by suction, and the well is washed with physiological saline. Then, the chemiluminescent reagent prepared in Reference Example 1 is added to each well 10 times with 0.1 M Tris-HCl buffer (pH 8.4). Was added and 240 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing p-iodophenol at a concentration of 10 ⁻³ M were added thereto, and 0.134 M of 0.0034% hydrogen peroxide was added thereto. 50 μl of a hydrochloric acid buffer (pH 8.4) was injected to cause luminescence, and the luminescence was integrated and measured with a luminometer (Luminas CT-9000D manufactured by Diatron) for 0 to 5 seconds. By plotting, a calibration curve having good concentration dependency shown in FIG. 1 was obtained.
Using this calibration curve, human AFP in serum sample was
It was possible to measure up to a concentration of ng / ml.

Example 2 Prolactin (P) by simultaneous sandwich method CLEIA
(RL) Measurement 50 μl of a 2% BSA-containing PBS solution (pH 7.4) containing purified human PRL (standard substance) in the range of 0 to 200 ng / ml was placed on a white microplate on which a rabbit anti-human PRL polyclonal antibody was immobilized. And peroxidase enzyme-labeled mouse anti-human PRL monoclonal antibody
100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing a concentration of μg / ml was added thereto.
Incubated for hours. Next, the solution in the well was removed by suction, and the well was washed with physiological saline. Then, the reduction reaction of the N, N′-dimethyl-9,9′-bisacridinium salt prepared in Reference Example 2 was formed in each well. The solution was diluted 10-fold with 0.1 M Tris-HCl buffer (pH 8.4) 10 μl
240 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing p-iodophenol at a concentration of 10 ⁻³ M
Was added thereto, and 50 μl of 0.0034% hydrogen peroxide in 0.1 M Tris-HCl buffer (pH 8.4) was injected to cause luminescence. The luminescence was measured using a luminometer (Diatron, Luminous CT-9000D). The measurement was performed by integrating for 0 to 5 seconds, and this value was plotted against the standard substance concentration to obtain a calibration curve having good concentration dependency shown in FIG. Using this calibration curve, it was possible to measure human prolactin in serum samples to a concentration of 0.1 ng / ml.

Example 3 Human Chorionic Gonado by Simultaneous Sandwich Method CLEIA
Measurement of tropin β chain (βhCG) Purified βhCG (standard substance) was added to a white microplate on which a rabbit anti-human hCG polyclonal antibody was immobilized.
50 μl of a 2% BSA-containing PBS solution (pH 7.4) containing 200 μlU / ml and about 2 μl of a peroxidase enzyme-labeled mouse anti-βhCG monoclonal antibody
100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing g / ml at a concentration of 1 g / ml.
Incubated for hours. Next, after the solution in the wells is removed by suction, the wells are washed with physiological saline, and a chemiluminescent reagent is added to each well with a 0.1 M Tris-HCl buffer (pH 8.4).
Then, 10 μl of the solution diluted 10-fold with, and 240 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing p-iodophenol at a concentration of 10 ⁻³ M were added thereto, and the mixture was added to 0.1 μl.
0.1 M Tris-HCl buffer of 0034% hydrogen peroxide (p
H8.4) 50 μl was injected to emit light, and the amount of this light was measured using a luminometer (Diatron, Luminous CT-9).
000D) for 0 to 5 seconds, and the measured values were plotted against the concentration of the standard substance to obtain a calibration curve with good concentration dependency shown in FIG. Using this calibration curve, it was possible to measure βhCG in serum samples to a concentration of 0.1 mIU / ml.

Comparative Example 1 Simultaneous sandwich method CLEIA using luminol
Measurement of α-fetoprotein (AFP) Purified human AFP (standard substance) containing 2% BSA containing PSA in a range of 0 to 800 ng / m was placed on a white microplate on which a rabbit anti-human AFP polyclonal antibody was immobilized.
50 μl of a BS solution (pH 7.4) and about 3 μl of peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody
100 μl of a 2% BSA-containing PBS solution (pH 7.4) containing g / ml at a concentration of 1 g / ml.
Incubated for hours. Next, after the solution in the well is removed by suction, the well is washed with physiological saline, and each well contains luminol at a concentration of 5.6 × 10 ⁻⁵ M and p-iodophenol at a concentration of 10 ⁻³ M. 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) was added, and 0.0034
% Hydrogen peroxide in 0.1 M Tris-HCl buffer (pH 8.
4) Inject 50 μl to emit light, and measure the amount of emitted light by using a luminometer (Luminous CT-9000 manufactured by Diatron).
In D), the measurement was performed by integrating for 0 to 5 seconds, and this value was plotted against the concentration of the standard substance to obtain a calibration curve having a concentration dependency shown in FIG. Using this calibration curve, it was possible to measure human AFP in serum samples up to a concentration of 2.0 ng / ml.

[0038]

The enzyme immunoassay of the present invention uses a peroxidase enzyme which is easily available and relatively easy to handle as a labeling substance, and N, N as a chemiluminescent substance.
It is possible to measure various antigens or antibodies to be measured with high sensitivity immunologically by a chemiluminescence method using a reduction reaction product of N′-disubstituted-9,9′-bisacridinium salts. it can.

Further, using an enzyme-labeled product obtained by labeling an antigen, an antibody, a nucleic acid or the like using peroxidase as a labeling substance,
Antibodies, antigens, etc., by enzyme immunoassay, proteins by Western blotting, and DNA by enzyme-labeled nucleic acid probes by Southern and Northern blotting.
And nucleic acids such as RNA can be measured specifically and with high sensitivity.

[Brief description of the drawings]

FIG. 1 is a calibration curve for measuring human αAFP prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 1 as a function of the concentration of human αAFP (standard substance).

FIG. 2 is a calibration curve for measuring human prolactin prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 2 as a function of the concentration of human prolactin (standard substance).

FIG. 3 is a calibration curve for βhCG measurement prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 3 as a function of the concentration of βhCG (standard substance).

FIG. 4 shows a human αA prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system according to the conventional method described in Comparative Example 1 as a function of the concentration of human αAFP (standard substance).
It is a calibration curve for FP measurement.

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 (8)

[Claims] 1. An antibody or an antigen labeled with a peroxidase enzyme is mixed with an antigen or an antibody to be measured in a sample or an aggregate thereof to form an immune complex comprising a peroxidase enzyme-labeled-antigen-antibody complex by an antigen-antibody reaction. The immunoconjugate as a chemiluminescent substance is 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, and X ^n- is an n-valent anion; n is 1 or 2.)
A reaction product obtained by reduction treatment of N'-disubstituted-9,9'-bisacridinium salts is added, chemiluminescence is caused in the presence of a hydrogen acceptor, and the amount of chemiluminescence is measured to obtain a sample. A chemiluminescent enzyme immunoassay, comprising measuring the content of an antigen or an antibody therein. 2. The reaction product according to 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 7. The chemiluminescent enzyme immunoassay according to Item 1. 3. The method according to claim 1, wherein the immune complex reacts with an antibody or antigen immobilized on an insoluble carrier and is captured on the insoluble carrier. 4. The method according to claim 1, wherein the N, N′-disubstituted-9,9′-bisacridinium salt is N, N′-dimethyl-9,9′-.
2. The method according to claim 1, wherein the method is bisacridinium dinitrate. 5. The method according to claim 1, wherein the hydrogen acceptor is hydrogen peroxide. 6. The method of any one of claims 1 to 5, further comprising a luminescence enhancer. 7. The method according to claim 6, wherein the luminescence enhancer is a phenolic compound. 8. The at least one phenolic compound selected from the group consisting of p-iodophenol, p-phenylphenol, and 6-hydroxybenzothiazole.
The method according to claim 7, which is a kind of phenolic compound.