JP2000111554A - IMMUNOLOGIC MEASURING METHOD FOR alpha-FETOPROTEIN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which α-fetoprotein is immunologically mea sured with high sensitivity by a new chemiluminescence system by a method wherein a peroxidase enzyme is used as a marker substance. SOLUTION: In this immunologic measuring method by a chemiluminescence method which uses a peroxidase enzyme as a marker substance, N,N'- disubstituted-9,9'-bisacridinium salts are reacted while light is irradiated under the existence of an N,N-disubstituted carboxylic acid amide compound. At this time, α-fetoprotein is immunologically measured with high sensitivity by a chemiluminescence reaction which uses a chemiluminescence reagent obtained by adding a specific amino alcohol compound in their reaction and/or after their reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring the concentration of α-fetoprotein (hereinafter abbreviated as “AFP” as necessary) in serum, which is widely used in the diagnosis of malignant tumors such as liver cancer. The present invention relates to a method for immunological measurement using a luminescence method. More specifically, the present invention relates to an immunological method for measuring the serum concentration of AFP with high sensitivity using a specific novel chemiluminescent reagent using a peroxidase enzyme as a labeling substance. It relates to a method of measuring statically.

[0002]

2. Description of the Related Art AFP is a fetal protein which migrates at the position of α-globulin and has a molecular weight of about 70,00.
0, the isoelectric point is 4.7, showing very similar physicochemical properties to albumin, and the amino acid sequences are both known to have high similarity. However, AFP, unlike albumin, is a glycoprotein containing about 4% sugar,
Although its function is not clear, it is a major serum protein in the early embryo, which decreases as the development progresses, and increases in the amount of albumin in parallel with the regulation of blood osmotic pressure. Is considered to be involved in the action of suppressing fetuses from maternal immunity. This fetal protein appears in the serum of patients with malignant tumors such as hepatocellular carcinoma and Yorksack tumor, patients with liver diseases such as hepatitis and cirrhosis, and in pregnant women, and particularly in serum of patients with malignant tumors. It is widely used as a tumor marker because it appears in the rate. For the measurement of AFP in blood, radioimmunoassay, enzyme immunoassay, one-way immunodiffusion method, lectin binding analysis for analyzing the difference in sugar chains of AFP, and the like are performed.
The method of quantification in the low concentration range of not more than / ml is not always sufficient. In healthy adults, what concentration actually exists in the blood and low A concentration in the early stage of malignant tumor
Sufficient knowledge has not yet been obtained on the issue of whether early detection of cancer is possible by detecting FP. Therefore, there has been a demand for the development of an ultra-sensitive method for measuring AFP, which is necessary for examining the possibility of serodiagnosis capable of early detection of malignant tumors such as hepatocellular carcinoma and observation of subtle changes in pathological conditions.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a highly sensitive method for immunological measurement of AFP in view of the above circumstances.

[0004]

Means for Solving the Problems Accordingly, the present inventors have:
In order to solve the above-mentioned problems, the present inventors conducted intensive studies on a method for measuring AFP with higher sensitivity than before, and as a result, used a chemiluminescence method for AFP using a new chemiluminescent reagent capable of ultra-sensitive measurement of peroxidase enzyme activity. The present inventors have found that AFP in blood can be measured on the order of several picograms by immunological measurement, and have completed the present invention based on these findings.

That is, the present invention comprises contacting an anti-α-fetoprotein antibody labeled with a peroxidase enzyme with α-fetoprotein in a sample to form an immune complex comprising a peroxidase enzyme-labeled-antigen-antibody complex by an antigen-antibody reaction. Reacts the immune complex with an anti-α-fetoprotein antibody immobilized on an insoluble carrier, captures the immunocomplex on the insoluble carrier, separates it, and adds it to the N, N′-disubstituted-9,9′-bis When an acridinium salt is reacted by light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound, a chemiluminescent reagent obtained by adding an amino alcohol compound during and / or after the reaction is added. Then, chemiluminescence was performed in the presence of a hydrogen acceptor, and the emission intensity was measured to determine the amount of α-fetoprotein in the sample. The present invention relates to a method for highly sensitive immunological measurement of α-fetoprotein by a chemiluminescence method, which comprises quantifying the content.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The immunoassay method for AFP by the chemiluminescence method of the present invention comprises an immunoreaction step of capturing AFP to be measured by an antigen-antibody reaction as an immune complex with an anti-AFP antibody containing a peroxidase enzyme-labeled antibody. A chemiluminescence reaction step in which the immune complex is measured by a chemiluminescence method using a labeling enzyme present in the molecule.

[0007] The method of the antigen-antibody reaction constituting the immune reaction step is optional, and includes N, N'-disubstituted-9,9'-bisacridinium salts, N, N-disubstituted carboxylic acid amide compounds and amino acids. Any method can be adopted as long as a chemiluminescent reagent obtained from an alcohol compound can be used.

For example, in a sandwich method in which an AFP to be measured in a sample is captured by an anti-AFP antibody bound to an insoluble carrier, and then reacted with an anti-AFP antibody labeled with a peroxidase enzyme. In the sandwich method, anti-AFP bound to an insoluble carrier is used.
Using an anti-AFP antibody derived from an animal species different from the FP antibody, a two-antibody method in which the generated sandwich complex is further reacted with a labeled second anti-AFP antibody against the anti-AFP antibody, the anti-AFP antibody bound to an insoluble carrier is used. A competition method in which an AFP to be measured in a sample is reacted with an AFP antibody in the presence of a peroxidase enzyme-labeled anti-AFP antibody. A sample containing the AFP to be measured is reacted with a labeled anti-AFP antibody that specifically reacts with these. Non-limiting examples include a coagulation sedimentation method for detecting a labeling substance in an immune complex separated by centrifugation after coagulation and sedimentation, and a biotin-avidin method for reacting a biotin-labeled anti-AFP antibody and a peroxidase enzyme-labeled avidin. Can be used.

Examples of the insoluble carrier used in the immunoassay of AFP of the present invention by a chemiluminescence method include, for example,
Examples thereof include polymer compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, and polysaccharide, 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 antigen or antibody 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 immunoassay for AFP of the present invention by the chemiluminescence method may be either monoclonal antibodies or polyclonal antibodies, and may be in the form of whole antibody or F (ab). ')
₂ , fragments such as Fab can be used. The origin of the antibody is arbitrary, but antibodies derived from mice, rats, rabbits, sheep, goats, chickens and the like are preferably used.

Further, the chemiluminescence reaction step, which constitutes the latter stage of the method for immunological measurement of AFP by the chemiluminescence method of the present invention, comprises the step of converting the N, N'-disubstituted-9,9'-bisacridinium salt into N , N-disubstituted carboxylic acid amide compound, when the reaction is performed by light irradiation, a chemiluminescent reagent obtained by adding an amino alcohol at the time of the reaction and / or after the reaction is used, and if necessary, a luminescence enhancer is used. The activity of a peroxidase enzyme, which is a labeling substance captured on an insoluble carrier by the action of a hydrogen receptor in the presence of the enzyme, is measured.The measuring method is optional, but generally, a chemiluminescent substance and luminescence enhancement are used. The chemiluminescent reagent containing the agent is added to an insoluble carrier immunologically captured with a peroxidase enzyme-labeled anti-AFP antibody, and an aqueous solution of hydrogen peroxide is added in a specific basic pH range. Pressurizing and by chemiluminescent reaction method and the like are performed to measure the amount of chemiluminescence in a luminometer.

Next, the chemiluminescent reagent used in the immunoassay of AFP of the present invention by the chemiluminescence method will be described. The chemiluminescent reagent comprises N, N′-disubstituted-9,
When reacting 9'-bisacridinium salts by light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound, it is obtained by adding a specific amino alcohol compound during and / or after the reaction. Reaction mixture containing the resulting chemiluminescent substance.

The amino alcohol compound is N, N '
Di-substituted-9,9'-bisacridinium salts and N, N
May be added to the mixture with the di-substituted carboxylic acid amide compound before light irradiation, but during the reaction and / or after the reaction, that is, either at the time of light irradiation or after light irradiation, or at the time of light irradiation and light irradiation You may add in both later. In particular, from the viewpoint of establishing the stability of light emission performance, it is important that the compound be present after light irradiation, that is, after the reaction. The N, N'-disubstituted-9,9'-bisacridinium salts are represented by the following general formula (1)

[0014]

Embedded image Is represented by

In the general formula (1), R ¹ and R ²
Are 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. Examples of the alkyl group include linear or branched ones having 1 to 14 carbon atoms, particularly those having 1 to 10 carbon atoms, and specifically, a methyl group, an ethyl group, a propyl group, Isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl,
Examples include an isohexyl group. The aryl group has 6 to 20 carbon atoms, and the aryl group may have an alkyl group having 1 to 14 carbon atoms bonded thereto. Specific examples include a phenyl group, a tolyl group, and a xylyl group. 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. The alkyl group is a linear or branched one having 1 to 14 carbon atoms, and particularly preferably one having 1 to 10 carbon atoms, specifically, a methyl group, an ethyl group, a propyl group, an isopropyl group, and a butyl group. Group, isobutyl group, pentyl group, isopentyl group and the like. The aryl group may have 6 to 20 carbon atoms, and the aryl group may have an alkyl group having 1 to 14 carbon atoms bonded thereto. Specific examples include a phenyl group, a tolyl group, and a xylyl group. The alkoxy group has a linear or branched alkyl group having 1 to 14 carbon atoms, and the aryloxy group has an aryl group having 6 to 20 carbon atoms. In the above formula (1), X is an n-valent anion, and n is 1 or 2. Specific examples of the anion (counter ion) include a nitrate ion, a halogen ion, a phosphate ion, a sulfate ion, and a sulfonate ion.

Specific examples of the N, N'-disubstituted-9,9'-bisacridinium salts include N, N'-dimethyl-9,9'-bisacridinium salt and N, N '-Diethyl-9,9'-bisacridinium salt, N, N'-diphenyl-9,9'-bisacridinium salt, N, N '
-Di-m-chlorophenyl-9,9'-bisacridinium salt and the like, in particular, N, N'-dimethyl-9,
9'-bisacridinium dinitrate (lucigenin) is preferred. The N, N-disubstituted carboxylic acid amide compound has the following general formula (2)

[0017]

Embedded image Is represented by

In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms. , An aryl group is an alkyl group, a nitro group,
May be substituted with a hydroxyl group, an amino group, a halogen atom, or the like; R ₂ is selected from the group consisting of a methyl group and an ethyl group; R ₃ is an alkyl group having 1 to 10 carbon atoms; It is a selected from alkenyl groups and the group consisting of an aryl group having 6 to 20 carbon atoms, an aryl group an alkyl group, a nitro group, a hydroxyl group, may be substituted by an amino group, and a halogen group, etc., also, R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group to which they are bonded and the nitrogen atom of the amide group.

Specific examples of the N, N-disubstituted carboxylic acid amide compound include N, N-dimethylformamide,
N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropionamide,
Non-limiting examples include N-dimethylbenzamide, N-methyl-2-pyrrolidone, and the like. The amino alcohol compound has the following general formula (3)

[0020]

Embedded image Is represented by

In the general formula (3), R represents a divalent aliphatic hydrocarbon group having 1 to 5 carbon atoms, and m represents 1 to 5
Represents an integer of 3. Specific examples thereof include, but are not limited to, monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, diisopropanolamine, and triisopropanolamine.

The addition of the amino alcohol compound necessary for the production of the chemiluminescent reagent has the effect of reducing the blank value and the effect of increasing the luminescence intensity in the high concentration region of peroxidase during the luminescence reaction. During storage of the reagent, it has an effect of improving storage stability for preventing a decrease in luminescence performance, and greatly contributes to improvement of performance such as sensitivity and stability of the chemiluminescent reagent.

The light beam for light irradiation used for producing the chemiluminescent reagent has a wavelength range of about 290 to 800 nm.
Is used, and in particular, about 400 to 80
Visible light in the range of 0 nm is desirable. As these light sources, high-pressure mercury lamps, low-pressure mercury lamps, germicidal lamps, fluorescent lamps, incandescent lamps and the like can be used without limitation, and incandescent lamps are particularly preferably used. The chemiluminescent reagent obtained by this light irradiation has a very high luminescence in a short time when manufactured at the same raw material lucigenin concentration and used in the same amount, compared to a chemiluminescent reagent manufactured under natural light without using a light source. Light irradiation plays an important role in the production of a chemiluminescent compound because it shows strength and, when this reaction is performed under light blocking, a chemiluminescent reagent having a peroxidase concentration-dependent property cannot be obtained. Is recognized.

The N, N'-disubstituted-9,9'-bisacridinium salts do not cause a remarkable luminescence reaction with hydrogen peroxide at around pH 8 or with hydrogen peroxide in the presence of peroxidase. N, N'-disubstitution-9,9'-
This is probably because the bisacridinium cation forms a stable salt with the counter ion, particularly the nitrate ion. However, by performing light irradiation in the presence of a highly polar compound such as an N, N-disubstituted carboxylic acid amide compound, N, N
The charge transfer from the counter anion to the N'-disubstituted-9,9'-bisacridinium cation is promoted, and the salt is changed from a highly ionic salt to a radical charge transfer complex. The N-disubstituted carboxylic acid amide compound is stabilized by coordination or solvation, and the stabilized biradical compound reacts with active oxygen generated by enzymatic decomposition of hydrogen peroxide to form an excited state dioxetane structure. It is considered that since the light is emitted after passing through, the light emission intensity depending on the peroxidase concentration can be obtained. In the reaction by light irradiation, the addition of the amino alcohol compound promotes the reaction by participating in charge transfer to the N, N'-disubstituted-9,9'-bisacridinium cation, and promotes the reaction during the luminescence reaction. Suppress the generation of by-products that increase the blank value,
Further, it is considered to have an action of further stabilizing the generated biradical.

Mixing ratio (molar ratio) of the N, N'-disubstituted-9,9'-bisacridinium salts, N, N-disubstituted carboxylic acid amide compound and amino alcohol compound
Is N, N-disubstituted-9,9'-bisacridinium salts, N, N-disubstituted carboxylic acid amide compound is 10,000 to 10,000 times mole, amino alcohol compound is 10,000 to 10,000 times. Each of them can be used in a molar amount, and further, the same and / or different N, N-disubstituted carboxylic acid amide compounds and / or other solvents can be used as the reaction solvent.

The reaction temperature of the light irradiation reaction varies depending on the presence or absence and the type of the solvent used.
The temperature is in the range of -150C, preferably 0-120C, particularly preferably 20-90C. The reaction time is in the range of 1 minute to one day, preferably 10 minutes to 15 hours, particularly preferably 30 minutes to 10 hours.

The chemiluminescent reagent emits light under basic conditions of pH 7.5 to 13 in the presence of excess hydrogen peroxide in an amount depending on the concentration of peroxidase. It is recognized that this luminescence is enhanced by a luminescence promoter 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, 2,4-dichlorophenol, p-coumaric acid, 6-hydroxybenzothiazole , 2-naphthol, firefly luciferin and the like.

The concentration of the chemiluminescent reagent is from 10 ^-8 to 1
M, preferably 10 ^{-6 to} 1 M, more preferably 10 ^-4 to M
It is in the range of 10 ^-2 M, and the amount of use is
1, especially in the range of 50 to 300 μl. The luminescence promoter is used in an amount of 0.01 to 100 times, preferably 0.1 to 10 times, the molar amount of the chemiluminescent reagent, and the concentration thereof is 10 ^{-6 to 1} M, particularly 10 ^-4 to 10. ^It is desirable to use in the range of -2M.

The hydrogen acceptor used in the chemiluminescence reaction in the immunoassay for AFP of the present invention by the chemiluminescence method is not particularly limited as long as it can serve as a substrate for a peroxidase enzyme. A peroxide, an inorganic peroxide or the like is optionally used, and hydrogen peroxide is particularly preferred. It is necessary to use the hydrogen acceptor in a sufficient excess amount with respect to the chemiluminescent reagent.
It is desirable to use it in a range of 0 mole. In addition, antibodies, nucleic acids, and the like are labeled using peroxidase as a labeling substance and AF
When quantifying P, although not particularly limited, horseradish peroxidase (HRP) is preferably used as the peroxidase.

The basic buffer used for the chemiluminescence reaction includes 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.

The amount of light emitted by the chemiluminescence reaction can be measured using a luminescence photometer. At this time, the start point and the integration time of the light emission amount measurement are arbitrary, but it is desirable to select a time in which the light emission amount is stable and the light emission amount has high concentration dependency. 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 reagent, and the integration time of the measurement is 1 second to 1 minute, preferably 1 to 1 minute.
It is 30 seconds, particularly preferably 1 to 10 seconds.

[0032]

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 the following. Incidentally,% in Reference Examples and Examples means% by weight.

[Reference Example 1] Preparation of chemiluminescent reagent 1.5 mg of lucigenin was placed in a test tube, and N, N-
After adding and dissolving 1 ml of dimethylacetamide, 3
After irradiation with a 250 W copy lamp for 7 hours in a water bath at a temperature of 0 ° C., 0.5 ml of triethanolamine was added and mixed to obtain a chemiluminescent reagent. Next, this chemiluminescent reagent was added to 8 × 10 ⁻⁴ M p-iodophenol 7
A chemiluminescent reagent solution was prepared by diluting 500-fold with a 5 mM Tris-HCl buffer (pH 8.0) solution.

[Reference Example 2] Rabbit anti-human AFP polyclonal antibody immobilized on an insoluble carrier
A solution of a concentration of 10 [mu] g / ml anti-human AFP polyclonal antibody 10mM phosphate buffered saline (PBS) (pH7.4) from an animal such as a rabbit with specific reactivity to manufacture human AFP, white 0.1 ml was added to each well of a microplate (Lab System), left at 37 ° C. for 1 hour, washed with PBS, and then 0.4 ml of 1% bovine serum albumin (BSA) aqueous solution was added. And left at 37 ° C. for 1 hour to perform post-coating treatment to obtain a rabbit anti-human AFP polyclonal antibody-immobilized white microplate.

[Reference Example 3] Peroxidase enzyme-labeled mouse anti-human AFP monochrome
Mouse anti-human AFP having a specific reactivity to manufacture human AFP in Naru antibody
N- (m-maleimidobenzoic acid) -N was added to 1 ml of a solution obtained by dissolving the monoclonal antibody at a concentration of 1.0 mg / ml in 10 mM phosphate buffered saline (PBS) (pH 7.4).
-10 mg / ml of succinimide ester (MBS)
0.1 ml of a dimethylformamide solution having a concentration of 5% was added and reacted at a temperature of 25 ° C. for 30 minutes. Next, the reaction mixture was subjected to gel filtration using a column filled with Sephadex G-25 using a 0.1 M phosphate buffer (pH 6.0), and a maleimidated mouse anti-human AFP monoclonal antibody was combined with unreacted MBS. Was isolated.

On the other hand, as a peroxidase enzyme, 5.0 m of horseradish peroxidase (HRP) was used.
g / ml of 0.1 M phosphate buffer (pH 6.5) solution 1 ml
In addition, S-acetylmercaptosuccinic anhydride (SAM)
50 μl of a 35 mg / ml dimethylformamide solution of S) was added, and the mixture was reacted at a temperature of 25 ° C. for 2 hours. The reaction mixture was then added to 0.1 M hydroxylamine in 0.1 mM containing 5 mM ethyldiaminetetraacetic acid (EDTA).
0.8 ml of M phosphate buffer (pH 6.0) was added, and the mixture was stirred at a temperature of 30 ° C. for 4 minutes to deacetylate the S-acetylmercapto group introduced into the HRP molecule. Then, Sephadex G-25 was added. Was purified by gel filtration using a 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA to obtain a fraction containing thiolated HRP.

Next, a maleimidated mouse anti-human AFP monoclonal antibody and thiolated HRP were mixed, concentrated using a collodion bag under ice cooling to a protein concentration of 4 mg / ml, and allowed to stand at 4 ° C. for 24 hours. Gel filtration was performed using a column packed with Ultrogel AcA44 (SEPRACOR) to obtain a peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody.

[Example 1] α-fetoprotein by simultaneous sandwich method CLEIA
Measurement of in (AFP) A 2% BSA-containing PBS solution (pH 7.2) containing purified human AFP (standard substance) in the 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 and about 2 μg / mouse anti-human AFP monoclonal antibody labeled with peroxidase enzyme
2% BSA-containing PBS solution (pH 7.
2) 100 μl was added, and the mixture was incubated at a temperature of 37 ° C. for 1 hour. Next, after aspirating and removing the solution in the well, the well was washed with physiological saline, and then 100 μl of 75 mM Tris-HCl buffer (pH 7.8) was added to each well,
100 µl of the chemiluminescent reagent solution prepared in Reference Example 1
50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide and 0.0017% hydrogen peroxide were sequentially injected to emit light, and the amount of emitted light was measured using a luminometer (Diatron, Luminous CT-9000D). The measurement was performed by integrating for 5 seconds, and this value was plotted against the concentration of the standard substance to obtain a calibration curve having good concentration dependency shown in FIG.
Using this calibration curve, human AFP in serum samples was
It was possible to measure up to a concentration of ng / ml.

[Reference Example 4] Mouse anti-human AFP monoclonal antibody immobilized on an insoluble carrier
Mouse anti-human AFP having a specific reactivity to the body of the production human AFP
10 μl of monoclonal antibody in 10 mM PBS (pH 7.2)
g / ml solution was added to each well of a white microplate (Lab System) in an amount of 0.1 ml, left at 37 ° C. for 1 hour, washed with PBS, and then washed with 1% bovine. 0.4 ml of an aqueous solution of serum albumin (BSA) 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 mouse anti-human AFP monoclonal antibody-immobilized white microplate.

Example 2 α-fetoprotein by simultaneous sandwich method CLEIA
Measurement of in (AFP) 2% BS containing purified human AFP (standard substance) in the range of 0 to 800 ng / ml was placed on a white microplate on which a mouse anti-human AFP monoclonal antibody was immobilized.
A containing 50 μl of a PBS solution (pH 7.2) and peroxidase enzyme-labeled mouse anti-human AFP monoclonal antibody
PBS solution containing 2% BSA at a concentration of μg / ml
(pH 7.2) was added, and the mixture was incubated at a temperature of 37 ° C. for 1 hour. Next, after the solution in the well was removed by suction, the well was washed with physiological saline, and then 100 μl of 75 mM Tris-HCl buffer (pH 7.8) was added to each well. Luminescent reagent solution 100
μl and 50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide were sequentially injected to emit light. Measurement was performed by accumulating for 、 5 seconds, and this value was plotted against the standard substance concentration to obtain a calibration curve with good concentration dependency shown in FIG. Using this calibration curve, human AFP in serum samples was determined to be 0.
It was possible to measure up to a concentration of 01 ng / ml.

[0041]

The immunoassay of the present invention utilizing the α-fetoprotein chemiluminescence method uses a peroxidase enzyme which is easily available and relatively easy to handle as a labeling substance, and is inexpensive and easily available. The α-fetoprotein in a sample such as serum can be measured immunologically with high sensitivity by a chemiluminescence method using a certain lucigenin or the like as a starting material and a novel chemiluminescent reagent which can be easily produced.

[Brief description of the drawings]

FIG. 1 is a polyclonal antibody-based calibration curve for human AFP measurement prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 1 as a function of the concentration of human AFP (standard substance). .

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

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Genichiro Aratani 1-9-4 Horinouchi, Adachi-ku, Tokyo Dainichi Seika Industry Co., Ltd. Technology Research Center (72) Inventor Toshifumi Katsuragi One-Horiuchi, Adachi-ku, Tokyo No. 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 (10)

[Claims] 1. An anti-α labeled with a peroxidase enzyme.
A fetoprotein antibody is brought into contact with α-fetoprotein in the sample to form an immune complex consisting of a peroxidase enzyme-labeled-antigen-antibody complex by an antigen-antibody reaction, followed by separation, and N, N′-disubstitution-9 , 9′-Bisacridinium salts are obtained by adding an amino alcohol compound during and / or after the reaction of the N, N-disubstituted carboxylic acid amide compound by light irradiation. A chemiluminescence method for α-fetoprotein, which comprises adding a chemiluminescence reagent, causing chemiluminescence in the presence of a hydrogen acceptor, and measuring the luminescence intensity to determine the content of α-fetoprotein in the sample. Method for immunological measurement. 2. The N, N′-disubstituted-9,9′-
Bisacridinium salts are represented by the following general formula (1): (In the general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group, and a halogenated aryl group, and may be the same or different from each other; 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. 2. The immunoassay method for α-fetoprotein according to claim 1, which is a compound represented by the formula: 3. The N, N′-disubstituted-9,9′-
When bisacridinium salts are N, N'-dimethyl-9,
The immunological assay method for α-fetoprotein by chemiluminescence according to claim 1 or 2, which is 9′-bisacridinium dinitrate (lucigenin). 4. The N, N-disubstituted carboxylic acid amide compound represented by the following general formula (2): (In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms. An aryl group is an alkyl group, a nitro group, a hydroxyl group,
May be substituted with an amino group and a halogen atom,
R ₂ is a methyl group or an ethyl group; R ₃ is selected from the group consisting of an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms; The aryl group may be substituted with an alkyl group, a nitro group, a hydroxyl amino group, a halogen atom, or the like, and R ₁ and R ₃ are bonded to each other to form a carbon atom and a carbon atom of a carbonyl group to which each is bonded. A ring may be formed together with the nitrogen atom of the amide group. The compound according to any one of claims 1 to 3, which is a compound represented by the formula:
-Immunoassay of fetoprotein by chemiluminescence method. 5. The method according to claim 1, wherein the N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide or N, N
The immunoassay method for α-fetoprotein according to any one of claims 1 to 4, wherein the α-fetoprotein is -dimethylacetamide. 6. The amino alcohol compound represented by the following general formula (3): (Wherein, in the general formula (3), R is a divalent aliphatic hydrocarbon group having 1 to 5 carbon atoms, and m is an integer of 1 to 3). An immunological method using chemiluminescence of α-fetoprotein according to any one of the above. 7. The α-fetoprotein according to claim 1, wherein the amino alcohol compound is at least one compound selected from the group consisting of monoethanolamine, diethanolamine and triethanolamine. Immunoassay by chemiluminescence method. 8. The method according to claim 1, wherein the antigen-antibody reaction is performed in the presence of an antibody immobilized on an insoluble carrier to form a labeled immune complex on the insoluble carrier. An immunoassay method for the above-mentioned α-fetoprotein by a chemiluminescence method. 9. The immunoassay method for α-fetoprotein according to claim 1, wherein the hydrogen acceptor is hydrogen peroxide or a hydrogen peroxide source. 10. The immunological measurement of α-fetoprotein according to claim 1, further comprising a luminescence enhancer when performing the chemiluminescence reaction. Method.