JPH11225792A - Chemiluminescent enzyme immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for assaying an antigen or an antibody with a high sensitivity by a chemiluminescent system using a peroxidase enzyme as a labeling substance. SOLUTION: This method is an enzyme immunoassay using a peroxidase enzyme as a labeling substance and comprises immunologically assaying the amount of an antigen or an antibody in a sample by a chemiluminescent system using a chemiluminescent reagent containing a complex comprising N,N'- disubstituted-9,9'-bisacridinium salts represented by the general formula [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 mutually be same or different; R<3> , R<4> , R<5> and R<6> are each selected from the group consisting of hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom and may mutually be same or different; X is an (n)-valent anion; (n) is 1 or 2] and an N,N-disubstituted carboxamide compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for immunoassay of a chemiluminescent enzyme, and more particularly, to a method for immunologically measuring an antigen or an antibody by a chemiluminescence system using a peroxidase enzyme as a labeling substance. Things.

[0002]

2. Description of the Related Art Enzyme immunoassays have been developed as immunoassays which do not use a radioisotope as a labeling substance, can maintain a good measurement environment, and have little danger to the human body. Used in substance measurement systems.
Enzymes used in this enzyme immunoassay include peroxidase, alkaline phosphatase, β-
Various enzymes such as galactosidase and glucose oxidase have been used. As a method for detecting the enzyme activity of an enzyme-labeled antibody or an antigen using these enzymes, a method for detecting an enzyme substrate such as hydrogen peroxide / o-phenylenediamine, 4-nitrophenyl-phosphate, 2-nitrophenyl-β-galactoside, etc. A colorimetric method is generally used, in which the amount of a color-forming substance produced by a decomposition reaction by an enzyme is measured to determine the enzyme activity, and the amount of an antibody or antigen having a correlation with the enzyme activity is determined. However, in clinical chemistry analysis, the measurement target is a biological sample (mainly, serum, urine, etc.), and the measured value is often used for diagnosing a disease state or observing its follow-up. Accurate measurement 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. The fluorescence method is based on the catalytic activity of the enzyme used for labeling.
After decomposing a fluorescent substrate such as β-galactoside or 4-methylumbelliferyl-phosphate to generate fluorescence,
In this method, the fluorescence intensity is measured to quantify the enzyme activity, and the amount of the antibody or antigen correlated with the enzyme activity is determined. However, it is difficult to say that the fluorescence method is sufficient to satisfy the above requirements because of the scattering of excitation light. In addition, the colorimetric method and the fluorescent method require a light source such as a xenon lamp. 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 immunoassay (CLEIA) has been developed and attracted attention as a more sensitive enzyme immunoassay than colorimetry and fluorescence.

[0003] In CLEIA, a chemiluminescent substance is put into an excited state via an intermediate due to the catalytic activity of an enzyme, and the amount of luminescence released when returning from this state to a ground state is measured to quantify the enzymatic activity. This is a method for quantifying the amount of an antibody or an antigen having a correlation with the light source.The light source is unnecessary because the chemiluminescent substance emits light by a chemical reaction, and the background is not increased due to the light source. Is possible. Enzymes used for CLEIA include peroxidase, alkaline phosphatase, β-
Galactosidase, glucose oxidase, dehydrogenase, etc. are mentioned, but peroxidase is preferably used in terms of ease of handling, availability, etc., luminol is used as the chemiluminescent substance, and p-iodophenol is used as the luminescence enhancer. A luminescence system has been developed, and various substances can be quantified with high sensitivity immunologically by a chemiluminescence method.

[0004]

However, in the case of CLEIA using a peroxidase enzyme as a labeling substance, it is often necessary to further improve the quantification of the substance to be measured in a low concentration region, and the peroxidase enzyme is used as a labeling substance. It has been desired to further increase the sensitivity of CLEIA. In view of the above circumstances, an object of the present invention is to provide a chemiluminescent enzyme immunoassay using a novel immunoassay system based on a chemiluminescence method capable of measuring a substance to be measured with higher sensitivity.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, as a chemiluminescent substance, N, N'-disubstituted-9,9'-bisacridinium was used. A chemiluminescent system using a complex composed of salts and an N, N-disubstituted carboxylic acid amide compound and using a specific phenolic compound as a luminescence enhancer measures with higher sensitivity than a system using luminol as a chemiluminescent substance The present inventors have found that a target substance can be quantitatively determined immunologically, 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. After forming an antibody and, if necessary, reacting the immune complex with an antibody or antigen immobilized on an insoluble carrier and capturing it on the insoluble carrier, the compound is represented by the following general formula (1) as a chemiluminescent substance:

[0007]

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'-
A complex comprising a bisacridinium salt and an N, N-disubstituted carboxylic acid amide compound is added, chemiluminescence is caused in the presence of a hydrogen acceptor, and the amount of antigen in the sample is determined by measuring the amount of chemiluminescence. Alternatively, the present invention relates to a chemiluminescent enzyme immunoassay method comprising measuring the amount of 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 of capturing an antigen or an antibody to be measured by an antigen-antibody reaction as a peroxidase enzyme-labeled immune complex, and the produced immunocomplex is present in the molecule. A chemiluminescence reaction step measured by a chemiluminescence method using a labeling enzyme. The method of the antigen-antibody reaction constituting the immune reaction step is optional, and a complex of an N, N'-disubstituted-9,9'-bisacridinium salt and an N, N-disubstituted carboxylic acid amide compound is prepared. Any method can be adopted as long as the contained chemiluminescent reagent can be used.

[0009] For example, a sandwich method in which an antigen to be measured in a sample is captured by an antibody bound to an insoluble carrier, and then a peroxidase enzyme-labeled antibody is reacted. In the sandwich method, the antibody is derived from an animal species different from the antibody bound to the insoluble carrier. A two-antibody method using an antibody to be reacted, and further reacting a labeled second antibody against the generated sandwich complex with the antibody. A competition method in which an antigen to be measured in a sample is reacted with an antibody bound to an insoluble carrier in the presence of a peroxidase enzyme-labeled antigen, a labeled antibody which specifically reacts with a sample containing the antigen or antibody to be measured or An agglutination sedimentation method is used to detect the labeling substance in the immune complex separated by centrifugation after the antigen is allowed to act for coagulation and sedimentation. An antibody detection method in which a human gamma globulin antibody is acted on, and a bition-avidin method in which a peroxidase enzyme-labeled avidin is reacted with a bition-labeled antibody can be used without limitation.

The insoluble carrier used in the chemiluminescent enzyme immunoassay of the present invention includes, for example, polymer compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, glass, and metal. , Magnetic particles and combinations thereof. As the shape of the insoluble carrier, for example, 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 antigen or antibody on these insoluble carriers is arbitrary, and any of a physical adsorption method, a covalent bonding method, and an ionic bonding method can be used.

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

Further, the chemiluminescent reaction constituting the latter stage of the chemiluminescent enzyme immunoassay of the present invention is carried out using N, N'-disubstituted-9,9'-bisacridinium salts-N, N as a chemiluminescent reagent. Using a di-substituted carboxylic acid amide compound conjugate, and measuring the activity of a peroxidase enzyme that is a labeling substance captured on an insoluble carrier by acting a hydrogen acceptor in the presence of a luminescence enhancer; The measurement method 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 hydrogen is added at a specific basic pH range. A method of adding a receptor (aqueous hydrogen peroxide solution) to cause a chemiluminescence reaction, and measuring the amount of chemiluminescence with a luminescence measuring device has been used.

The chemiluminescent reagent N, N'-disubstituted-9,9 'used in the chemiluminescent enzyme immunoassay of the present invention.
A complex comprising a bisacridinium salt and an N, N-disubstituted carboxylic acid amide compound is represented by the following general formula (1)

[0014]

Embedded image Can be easily produced by bringing an N, N'-disubstituted-9,9'-bisacridinium salt represented by the following formula into contact with an N, N-disubstituted carboxylic acid amide compound.

In the above 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 group, the aryl group and the aryl halide group have 1 to 20 carbon atoms, and the preferred alkyl group has 1 to 10 carbon atoms. For example,
Examples include a linear 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, or a branched alkyl group thereof. The aryl group preferably has 6 to 20 carbon atoms, for example, a phenyl group,
Examples thereof include a treyl group and a xylyl group, and those further substituted with an alkyl group may be used. 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 (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, and may be the same or different from each other. Among these, examples of the carbon number of the hydrocarbon group include those having 1 to 20, preferably 1 to 14 carbon atoms.

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. Among these anions, nitrate ion is particularly preferred.

As the reaction conditions for the N, N'-disubstituted-9,9'-bisacridinium salt and the N, N-disubstituted carboxylic acid amide compound, the reaction temperature varies depending on the type of reagent and solvent used. But generally -10 to +200
° C, preferably 0 to 120 ° C, particularly preferably 20 ° C.
To 60 ° C, and the reaction time may be 1 minute to 24 hours, preferably 5 minutes to 12 hours, particularly preferably 10 minutes to 5 hours. Also, N,
The N, N-disubstituted carboxylic acid amide compound can be used in an equimolar amount to a large excess with respect to 1 mol of the N'-disubstituted-9,9'-bisacridinium salt.

Specific examples of N, N'-disubstituted-9,9'-bisacridinium salts include N, N-dimethyl-
9,9′-bisacridinium salt, N, N′-diethyl-9,9′-bisacridinium salt, N, N′-diphenyl-9,9′-bisacridinium salt, N, N And '-di-m-chlorophenyl-9,9'-bisacridinium salt. In particular, N, N'-dimethyl-9,
9'-bisacridinium dinitrate (lucigenin) can be suitably used, but is not limited to these.

The N, N-disubstituted carboxylic acid amide compounds used for the production of chemiluminescent reagents include N, N
Non-limiting examples include N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropionamide, N, N-dimethylbenzamide, N-methyl-2-pyrrolidone, and the like. Especially, N, N-dimethylformamide, N, N
-Dimethylacetamide, N-methyl-2-pyrrolidone and the like are preferred.

Of 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, and an appropriate solvent can be used if necessary. In addition, those which are solid at room temperature may be heated and melted, and an appropriate solvent may be used. Examples of such a solvent include dimethyl sulfoxide, hexamethylphosphamide and the like.

The above chemiluminescent reagent has a pH of 7.5 to 13.
Under basic conditions, in the presence of excess hydrogen acceptor
It emits light in an amount depending on the concentration of peroxidase. Examples of the hydrogen acceptor include hydrogen peroxide, perboric acid, and organic hydroperoxide, and hydrogen peroxide is preferably used. It is also 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- (4-iodophenyl) phenol -Iodophenol, p-bromophenol, p-chlorophenol, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like are non-limiting examples.

In the chemiluminescent enzyme immunoassay of the present invention, the chemiluminescent reagent is 10 ^{-6 to 1} M, preferably 10 ^-4.
It is used at a concentration in the range of 10 ^-2 M and its amount is 10
The range is preferably from 500 to 500 µl, particularly preferably from 50 to 300 µl. In addition, the amount of the luminescence enhancer used is 0.1% of the chemiluminescence reagent.
It is in the range of 0.1 to 100-fold molar, preferably 0.1 to 10-fold molar, and its concentration is 10 ^{-6 to} 1 M, preferably 1 to 10-fold.
The range of 0 ^-4 to 10 ^-2 M is preferred. Further, the amount of hydrogen peroxide used in the chemiluminescence reaction needs to be used in a sufficiently excessive amount with respect to the chemiluminescent reagent, and the amount of use is preferably 30,000 to 10,000 times, preferably mol, based on the chemiluminescent reagent. Is 10
It is in the range of 1000 times mol. In addition, when peroxidase is used as a labeling substance to label antibodies, nucleic acids, and the like and various substances are quantified, it is not particularly limited, and horseradish peroxidase (HRP) may be used as the peroxidase. preferable.

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 used arbitrarily. The concentration of these buffers is between 1 mM and
A range of 1M is preferred. In addition, additives such as a surfactant and a chelating agent can be optionally used during the reaction.

In the chemiluminescent enzyme immunoassay of the present invention, the amount of luminescence of the chemiluminescence reaction is measured by using a luminescence photometer. It is preferable to select a time during which the light emission amount is stable and the emission amount 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, and the integration time of the measurement is 1 second to
1 minute, preferably 1 to 30 seconds, particularly preferably 1 to 30 seconds
10 seconds.

[0026]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples along with Reference Examples, but the present invention is not limited to Examples and the like. The percentages in Reference Examples and Examples are% by weight.
Means

Reference Example 1 N, N'-disubstituted-9,9'-bisacridinium salts
Of a complex of the compound with an N, N-disubstituted carboxylic acid amide compound
Take 1 mg of the produced lucigenin into a test tube, dissolve by adding 2 ml of N, N-dimethylacetamide, leave the mixture at room temperature for 90 minutes, add 2 ml of pure water and mix to obtain lucigenin-N, N. -A chemiluminescent reagent comprising a dimethylacetamide complex was obtained.

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 by adding 10 mM phosphate buffered saline (pH
7.4) 0.1 ml of a solution dissolved in (PBS) 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 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 A monoclonal antibody derived from a mouse or the like having specific reactivity with an antigen was prepared from 10 mM phosphate buffered saline (PBS).
A solution of N- (m-maleimidobenzoic acid) -N-succinimide ester (MBS) in dimethylformamide having 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 25
The reaction was carried out at a temperature of ° C for 30 minutes. Then, using a column packed with Sephadex G-25,
Gel filtration was performed with a 0.1 M phosphate buffer (pH 6.0) to separate the maleimidated monoclonal antibody from unreacted MBS.

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. Then, the reaction mixture was applied to a column packed with Sephadex G-25 using 1
Purification by gel filtration with 0 mM acetate buffer (pH 4.5), collection of a fraction containing pyridyl disulfide-modified HRP,
This was concentrated about 10 times under ice cooling in a collodion bag. Next, 1 m of 0.1 M acetate buffered saline (pH 4.5) containing 0.1 M dithiothreitol was added thereto.
and stirred at a temperature of 25 ° C. for 30 minutes to add HRP
After reducing the pyridyl disulfide group introduced into the molecule, the reaction mixture was subjected to gel filtration using a column packed with Sephadex G-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 is obtained.

[Example 1] α-fetoprotein by simultaneous sandwich method CLEIA
(AFP) Measurement A 2% BSA-containing PBS solution (pH 7.0) containing purified human AFP (standard substance) in the range of 0 to 800 ng / ml is immobilized on a white microplate on which a rabbit anti-human AFP polyclonal antibody is immobilized. 4) 50 μl of peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody was added to about 3 μl.
100 μl of a PBS solution (pH 7.4) containing 2% BSA containing a concentration of g / ml was added, and the mixture was incubated at a concentration of 37 ° C. for 1 hour. Next, after the solution in the well is removed by suction, the well is washed with a physiological saline, and then the lucigenin-N, N'-dimethylacetamide complex is added to each well.
0.1 M Tris-HCl buffer (pH 8.4) containing 0 × 10 ^-5 M and p-iodophenol at a concentration of 10 ^-3 M 25
Add 0 μl and add 0.0034% hydrogen peroxide in 0.1 ml.
50 μl of 1 M Tris-HCl buffer (pH 8.4) was injected to cause luminescence, and the luminescence was integrated and measured for 0 to 5 seconds with a luminometer (Diatron, Luminous CT-9000D). By plotting against the concentration, a calibration curve with good concentration dependency shown in FIG. 1 was obtained. Using this calibration curve, it was possible to measure human AFP in a serum sample up to a concentration of 0.5 ng / ml.

[Example 2]Prolactin (P) by simultaneous sandwich method CLEIA
RL) measurement White rabbit immobilized with rabbit anti-human PRL polyclonal antibody
In a microplate, purified human PRL (standard substance)
Contains 2% BSA in the range of 0 to 200 ng / ml
50 μl of PBS solution (pH 7.4) and peroxidase enzyme
Approximately 2 μl of an unlabeled mouse anti-human PRL monoclonal antibody
g / ml PBS solution containing 2% BSA
(PH 7.4) 100 μl and 1 hour at 37 ° C.
For a while. Then, aspirate the solution in the well
Remove and wash with saline before adding Lucige to each well.
The nin-N, N-dimethylacetamide complex was 4.0 ×
10 ^-FiveM, p-iodophenol in 10^-3Included at M concentration
0.1 μM Tris-HCl buffer (pH 8.4) 250μ
1 and then 0.1M of 0.0034% hydrogen peroxide
Inject 50 μl of Tris-HCl buffer (pH 8.4) and emit light
And the luminescence is measured using a luminometer (Dyatron
Luminous CT-9000D) for 0-5 seconds
And plot this value against the standard concentration.
As a result, a calibration curve with good concentration dependency shown in FIG. 2 was obtained.
Was. Using this calibration curve, human prolactin in serum samples
Can be measured to a concentration of 1.0 ng / ml.
Was.

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 about 200 μIU / ml and about 2 μg of a peroxidase enzyme-labeled mouse anti-βhCG monoclonal antibody
2% BSA-containing PBS solution (p / p
H7.4) and incubated at 37 ° C. for 1 hour.

Next, after the solution in the well is removed by suction,
After washing with saline, lucigenin- was added to each well.
4.0 × 10 ^{−5 of} N, N-dimethylacetamide complex
250 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing M, p-iodophenol at a concentration of 10 ⁻³ M was added thereto, and to this was added 0.0034% hydrogen peroxide in 0.1 M Tris-HCl buffer ( pH 8.4) Inject 50 μl to emit light,
This luminescence amount was measured by integrating for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron),
By plotting this value against the concentration of the standard substance, a calibration curve with good concentration dependency shown in FIG. 3 was obtained. Using this calibration curve, βhCG in the serum sample was determined to be 1.0 mIU.
/ Ml could be measured.

Comparative Example 1 Simultaneous sandwich method using luminol CLEIA
Measurement of α-fetoprotein (AFP) 2% BSA-containing PBS solution containing purified human AFP (standard substance) in a range of 0 to 800 ng / ml on a white microplate on which a rabbit anti-human AFP polyclonal antibody is immobilized. (PH 7.4) 50 μl and peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody
100 μl of a PBS solution (pH 7.4) containing 2% BSA containing a concentration of g / ml was added, and the mixture was incubated at a concentration of 37 ° C. for 1 hour. Next, after the solution in the wells was removed by suction, the wells were washed with physiological saline, and 5.6 × 10 ⁻⁵ M luminol and 1 p-iodophenol were added to each well.
0 ^-3 0.1 M Tris-HCl buffer containing a concentration of M (p
H8.4) of 100 μl, and 50 μl of 0.0034% hydrogen peroxide in 0.1 M Tris-HCl buffer (pH 8.4).
1 was injected to emit light, and the amount of emitted light was measured with a luminometer (Luminous CT-9000D manufactured by Diatron Co., Ltd.).
Measurement was performed by integrating for 〜5 seconds, and this value was plotted against the standard substance concentration to obtain a calibration curve having a concentration dependency shown in FIG. It was only possible to measure human AFP in serum samples to a concentration of 2.0 ng / ml using this calibration curve.

[0037]

The method for immunoassay of a chemiluminescent enzyme according to the present invention comprises:
A peroxidase enzyme, which is easily available and relatively easy to handle, is used as a labeling substance, and as a chemiluminescent reagent, N, N'-disubstituted-9, which is extremely easy to produce, is used.
Various antigens or antibodies to be measured are immunologically sensitive by a chemiluminescence method using a novel chemiluminescent reagent comprising a 9'bisacridinium salt-N, N-disubstituted carboxylic acid amide compound complex. Can be measured.

[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 the amount of chemiluminescence obtained by chemiluminescence using the reaction system (using luminol) described in Comparative Example 1 and human αAF.
It is a calibration curve for human αAFP measurement prepared by plotting as a function of the concentration of P (standard substance).

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C07D 219/04 C07D 219/04 C09K 11/07 C09K 11/07 (72) Inventor Toshifumi Katsuragi 1-chome Horinouchi, Adachi-ku, Tokyo 9-4 Dainichi Seika Kogyo Co., Ltd. Technology Research Center (72) Inventor Mio Sato 1-9-4 Horinouchi, Adachi-ku, Tokyo Dainichi Seika Kogyo Co., Ltd. Technical Research Center

Claims (7)

[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. After that, the following general formula (1) is used as a chemiluminescent reagent: (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'-
A complex comprising a bisacridinium salt and an N, N-disubstituted carboxylic acid amide compound is added, chemiluminescence is caused in the presence of a hydrogen acceptor, and the amount of antigen in the sample is determined by measuring the amount of chemiluminescence. Alternatively, a chemiluminescent enzyme immunoassay method comprising measuring the amount of an antibody. 2. The N, N′-disubstituted-9,9′-
Bisacridinium salts are N, N'-disubstituted-9,9 '
The chemiluminescent enzyme immunoassay according to claim 1, which is -bisacridinium dinitrate. 3. 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 method for immunoassay of a chemiluminescent enzyme according to claim 1. 4. The method according to claim 1, wherein the hydrogen acceptor is hydrogen peroxide. 5. The chemiluminescent enzyme immunoassay according to claim 1, further comprising a luminescence enhancer when performing the chemiluminescence reaction. 6. The method according to claim 5, wherein the luminescence enhancer is a phenolic compound. 7. The method according to claim 6, wherein the phenolic compound is at least one compound selected from the group consisting of p-iodophenol, p-phenylphenol and 6-hydroxybenzothiazole. .