JP2000111552A - IMMUNOLOGIC MEASURING METHOD FOR beta-hCG - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which β-hCG is immunologically measured by a new chemiluminescence system by a method wherein a peroxidase enzyme is used as a marker substance. SOLUTION: This immunologic measuring method by a chemiluminescence method for β-hCG uses a peroxidase enzyme as a marker substance. In the method, 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, the β-hCG is immunologically measured with high accuracy by the chemiluminescence method 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]

TECHNICAL FIELD [0001] The present invention relates to a human chorionic gonadotropin β-subunit (hereinafter referred to as a human chorionic gonadotropin β-subunit) widely used for diagnosis of ectopic human chorionic gonadotropin-producing tumors and the like.
It is called “β-hCG”. The present invention relates to a method for immunologically measuring the concentration in serum of the present invention using a chemiluminescence method. More specifically, the present invention relates to the use of a peroxidase enzyme as a labeling substance, and the use of a specific novel chemiluminescent reagent to increase the serum concentration of β-hCG. The present invention relates to an immunological measurement method for measuring a medium concentration with high sensitivity.

[0002]

2. Description of the Related Art β-hCG is a gonadotropin secreted from the placenta, a glycoprotein having a molecular weight of about 38,000, comprising an α-subunit and a β subunit,
-The amino acid sequence of the subunit is determined by luteinizing hormone (LH) secreted from the anterior pituitary gland, follicle stimulating hormone (F
SH), similar to that of TSH, the amino acid sequence of the β-subunit is considerably different from that of FSH and TSH, but is known to have similarity to the β-subunit of LH. Each of the subunits has no biological activity by itself, and most of the subunits exist in a combined form in vivo.

[0003] Although hCG is secreted in large quantities during pregnancy, it is widely used for pregnancy diagnosis and the like. However, it has been found that the β-subunit of hCG is produced as ectopic hCG in many malignant tumors. It has been used as a tumor marker. As a method for measuring β-hCG, radioimmunoassay and enzyme immunoassay are performed, but the measurement sensitivity is not always sufficient, and β-hCG leads to early detection of ectopic hCG-producing malignant tumor.
Development of a highly sensitive method for measuring hCG has been desired.

[0004]

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

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of intensive studies on a method for measuring β-hCG with higher sensitivity than in the past in order to solve the above-mentioned problem, β-hCG was immunized with a novel chemiluminescent reagent capable of ultra-sensitive measurement of peroxidase enzyme activity. The present inventors have found that blood β-hCG can be measured on the order of several picograms by performing a biological measurement, and have arrived at the present invention based on these findings.

That is, the present invention relates to a method wherein an anti-β-hCG antibody labeled with a peroxidase enzyme is brought into contact with β-hCG in a sample to form an immune complex comprising a peroxidase enzyme-labeled-antigen-antibody complex by an antigen-antibody reaction. The immobilized anti-β-hC immobilized on an insoluble carrier
After reacting with the G antibody to capture on the insoluble carrier and separating, the N, N′-disubstituted-9,9′-bisacridinium salt is added to the N, N-disubstituted carboxylic acid amide compound. When reacting by light irradiation in the presence, a chemiluminescent reagent containing a chemiluminescent substance obtained by adding an amino alcohol compound during the reaction and / or after the reaction is added, and in the presence of a hydrogen acceptor, Chemiluminescence is performed, and the emission intensity is measured.
Β-hC characterized by quantifying the content of hCG
The present invention relates to a highly sensitive immunoassay for G by chemiluminescence.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The immunoassay of β-hCG of the present invention by chemiluminescence method comprises an immunoreaction step of capturing β-hCG as an immune complex with an anti-β-hCG antibody including a peroxidase enzyme-labeled antibody by an antigen-antibody reaction, A chemiluminescence reaction step of measuring the produced immune complex by a chemiluminescence method using a labeling enzyme present in the molecule.

[0008] The method of the antigen-antibody reaction constituting the immune reaction step is arbitrary, 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 a β-hCG antibody to be measured in a sample is captured by an anti-β-hCG antibody bound to an insoluble carrier, and then reacted with a peroxidase enzyme-labeled anti-β-hCG antibody, Anti-β bound to
Using an anti-β-hCG antibody derived from an animal species different from that of the hCG antibody, using a second anti-β-hC antibody labeled with the anti-β-hCG antibody against the sandwich complex formed.
A two-antibody method in which a G antibody is reacted, and a β-hCG to be measured in a sample is added to a peroxidase enzyme-labeled anti-β-hCG antibody in an anti-β-hCG antibody bound to an insoluble carrier.
a competitive method in which the reaction is carried out in the presence of an hCG antibody; a labeled anti-β-hCG antibody which reacts specifically with the β-hCG antibody is allowed to act on a sample containing β-hCG to be aggregated and precipitated; An agglutination precipitation method for detecting a labeling substance in the separated immune complex, and a biotin-avidin method for reacting a biotin-labeled anti-β-hCG antibody and a peroxidase enzyme-labeled avidin can be used without limitation.

The insoluble carrier used in the immunoassay by β-hCG chemiluminescence method of the present invention includes, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide and the like. Examples include molecular compounds, glass, metals, magnetic particles, and combinations thereof. Further, as the shape of the insoluble carrier, for example, tray-like, spherical, fibrous, rod-like, disk-like, container-like,
It can be used in various shapes such as a cell, a microplate, and a test tube. 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, or the like can be used.

The antibodies used in the immunoassay of β-hCG by the chemiluminescence method of the present invention can be either monoclonal antibodies or polyclonal antibodies. (A
b ′) ₂ , Fab and other fragments can be used. Although the origin of the antibody is arbitrary, mouse, rat, rabbit,
Antibodies derived from sheep, goats, chickens and the like are preferably used.

Further, the chemiluminescence reaction step, which constitutes the latter stage of the immunoassay for β-hCG by the chemiluminescence method of the present invention, comprises N, N′-disubstituted-9,
When a 9'-bisacridinium salt is reacted by light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound, a product obtained by adding an amino alcohol compound during and / or after the reaction And, if desired, the activity of a peroxidase enzyme, which is a labeling substance captured on an insoluble carrier by acting a hydrogen acceptor in the presence of a luminescence enhancer, and the measuring method is optional. In general, a measuring reagent containing a chemiluminescent substance and a luminescence enhancer is used as a peroxidase enzyme-labeled anti-β-h
A method of adding a CG antibody to an insoluble carrier that has been immunologically captured, adding an aqueous solution of hydrogen peroxide in a specific basic pH region to cause a chemiluminescence reaction, and measuring the amount of chemiluminescence with a luminescence measuring device is performed. ing.

Next, the chemiluminescent reagent used in the immunoassay of β-hCG of the present invention by the chemiluminescence method will be described. The chemiluminescent reagent is an N, N'-disubstituted-
When reacting a 9,9'-bisacridinium salt by light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound, a specific amino alcohol compound is added during and / or after the reaction. Is a reaction mixture containing a chemiluminescent substance obtained by The amino alcohol compound may be added to the mixture of the N, N'-disubstituted-9,9'-bisacridinium salt and the N, N-disubstituted carboxylic acid amide compound before the light irradiation. May be added at the time of reaction and / or after the reaction, that is, either at the time of light irradiation or after the light irradiation, or both at the time of the light irradiation and after the light irradiation. 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. The alkyl group has 1 to 14 carbon atoms.
Straight or branched ones, in particular,
Those having 1 to 10 carbon atoms are preferred, and specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, an isopentyl group, a hexyl group, and an isohexyl group. it can. 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 from each other. 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 has 6 carbon atoms.
-20, and the aryl group may be a group having an alkyl group having 1-14 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 6 carbon atoms.
It has up to 20 aryl groups. 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; 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. , the aryl group is an alkyl group, a nitro group, a hydroxyl group may be substituted by an amino group and a halogen atom, R ₂ is selected from the group consisting of methyl and ethyl, R ₃ is C 1 -C An alkyl group of 10 to 10,
It is selected from the group consisting of an alkenyl group having 2 to 10 carbon atoms and an aryl group having 6 to 20 carbon atoms, and the aryl group is at least one selected from the group consisting of an alkyl group, a nitro group, a hydroxyl group, an amino group, and a halogen atom. And 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 is a divalent aliphatic hydrocarbon having 1 to 5 carbon atoms, and m is an integer of 1 to 3. Specific examples include monoethanolamine, diethanolamine, triethanolamine,
Monoisopropanolamine, diisopropanolamine, triisopropanolamine and the like can be mentioned without limitation.

The addition of the amino alcohol compound necessary for the production of the chemiluminescent reagent used in the immunoassay of β-hCG by the chemiluminescence method of the present invention has the effect of reducing the blank value and the effect of peroxidase during the luminescence reaction. Has the effect of increasing the luminescence intensity in the high concentration region, and has the effect of improving the storage stability of the chemiluminescent reagent during storage, preventing the degradation of the luminescence performance, and the sensitivity and the like of the chemiluminescent reagent. It greatly contributes to the improvement of performance such as stability.

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 substance because it shows strength and, when this reaction is performed in the absence of light, a chemiluminescent reagent having a peroxidase concentration dependence 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.

The N, N'-disubstituted-9,9'-bisacridinium salts, the N, N-disubstituted carboxylic acid amide compound and the amino alcohol compound are mixed at a molar ratio of:
N, N-disubstituted -9,9'-bisacridinium salts can be combined with N, N-disubstituted carboxylic acid amide compound by 1 to
The amino alcohol compound can be used in a molar amount of 10,000 to 10,000 times, and the same kind and / or
Alternatively, a different N, N-disubstituted carboxylic acid amide compound and / or another solvent can be used as a reaction solvent.

The reaction temperature of the light irradiation reaction varies depending on the presence or absence and type of the solvent used, but is generally -10 to -10.
+ 150 ° C, preferably 0-120 ° C, particularly preferably 20-90 ° C. The reaction time is 1
The reaction can be carried out for a period ranging from minutes to one day, preferably from 10 minutes to 15 hours, particularly preferably from 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, preferably in the range of 50 to 300 μl. Further, the luminescence promoter is used in an amount of 0.01 to 1 with respect to the chemiluminescence reagent.
It is used in the range of 00 mole, preferably 0.1 to 10 mole, and its concentration is 10 ^{-6 to 1} M, preferably 10 ^-4 to 10 M.
^It is desirable to use in the range of -2M.

The hydrogen acceptor used for the chemiluminescence reaction in the immunoassay of β-hCG 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. No,
Organic peroxides, inorganic peroxides and the like are optionally used. In particular, 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 is in the range of 1000 times mol. In addition, in the case of quantifying β-hCG by labeling an anti-β-hCG antibody, a nucleic acid or the like using peroxidase as a labeling substance, horseradish peroxidase (HRP) is preferably used as the peroxidase, although it is not particularly limited. Can be

As the basic buffer used in 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 desirably in the range of 1 mM to 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 in the immunoassay for β-hCG by the chemiluminescence method of the present invention 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 at which the light emission amount is stable and the concentration of the light emission amount is high. 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]

The present invention will be specifically described below with reference to examples together with reference examples. However, the present invention is not limited by the examples and the like. In addition,% 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] A rabbit anti-β-hCG polyclonal antibody immobilized on an insoluble carrier
A solution of anti-beta-hCG polyclonal antibody 10mM phosphate buffered saline (PBS) (pH7.4) to a concentration of 10 [mu] g / ml derived from animal such as a rabbit with specific reactivity to the production beta-hCG 0.1 ml to each well of a white 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. The mixture was left at 37 ° C. for 1 hour to carry out a post-coating treatment to obtain a rabbit anti-β-hCG polyclonal antibody-immobilized white microplate.

[Reference Example 3] Peroxidase enzyme-labeled mouse anti-β-hCG monochrome
Mouse anti-beta-hCG having a specific reactivity to the production beta-hCG for 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 0.1% was added and reacted at a temperature of 25 ° C. for 30 minutes.
Gel filtration was performed with a 1 M phosphate buffer (pH 6.0) to separate maleimide-conjugated mouse anti-β-hCG monoclonal antibody from unreacted MBS.

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 1M hydroxylamine containing 5 mM ethylenediaminetetraacetic acid (EDTA).
Add 0.8 ml of 1M phosphate buffer (pH 6.0) and add 30 ml
After deacetylation of the S-acetylmercapto group introduced into the HRP molecule by stirring for 4 minutes at a temperature of 5 mM, 5 mM EDTA was applied using a column packed with Sephadex G-25.
Gel filtration was performed using a 0.1 M phosphate buffer (pH 6.0) to obtain a fraction containing thiolated HRP.

Next, a maleimidated mouse anti-β-hCG monoclonal antibody and a thiolated HRP were mixed, concentrated using a collodion bag under ice cooling to a protein concentration of 4 mg / ml, and left overnight at 4 ° C. The mixture was subjected to gel filtration using a column packed with Ultrogel AcA44 (SEPRACOR) to obtain a mouse anti-β-hCG monoclonal antibody labeled with a peroxidase enzyme.

Example 1 Determination of β-hCG by Simultaneous Sandwich Method CLEIA Purified β-hCG (standard substance) was added to a white microplate on which a rabbit anti-β-hCG polyclonal antibody was immobilized, at 0, 0.01 and 0.1, respectively. 50% of 2% BSA-containing PBS solution (pH 7.2) containing 1, 10, 100 ng / ml and 2% BSA containing peroxidase enzyme-labeled mouse anti-β-hCG monoclonal antibody at a concentration of about 2 μg / ml 100 μl of a PBS solution (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 wells was removed by suction, the wells were washed with physiological saline, and then 75 mM Tris-HCl buffer (pH 7.8) was added to each well.
0 μl was added thereto, and 100 μl of the chemiluminescence reagent solution prepared in Reference Example 1 and 0.0017% hydrogen peroxide were added thereto.
After sequentially injecting 50 μl of 5 mM Tris-HCl buffer (pH 7.8) to emit light, the amount of the emitted light was measured by integrating for 1 to 5 seconds using a luminometer (Diatron Co., Ltd., Luminous CT-9000D). Was plotted against the concentration of the standard substance to obtain a calibration curve with good concentration dependency shown in FIG. Using this calibration curve, β-hC
G could be measured to a concentration of 0.01 ng / ml.

[Reference Example 4] Mouse anti-β-hCG monoclonal antibody immobilized on an insoluble carrier
Mouse anti-beta-hCG having a specific reactivity to the production beta-hCG body
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, thereby obtaining a mouse anti-β-hCG monoclonal antibody-immobilized white microplate.

Example 2 Measurement of β-hCG by Simultaneous Sandwich Method CLEIA Purified β-hCG (standard substance) was placed on a white microplate on which a mouse anti-β-hCG monoclonal antibody was immobilized, at 0, 0.01 and 0.1, respectively. 50% of 2% BSA-containing PBS solution (pH 7.2) containing 1, 10, 100 ng / ml
And 2% B containing peroxidase enzyme-labeled mouse anti-β-hCG monoclonal antibody at a concentration of about 2 μg / ml.
100 μl of SA-containing PBS solution (pH 7.2) was added, and 37
Incubated for 1 hour at a temperature of ° C. Next, after aspirating and removing the solution in the well, the inside of the well is washed with physiological saline, and then a 75 mM Tris-HCl buffer (pH 7.
8) 100 μl was added, and 100 μl of the chemiluminescent reagent solution prepared in Reference Example 1 and 50 μl of 75 mM Tris-HCl buffer (pH 7.8) containing 0.0017% hydrogen peroxide were sequentially injected to emit light. The amount was measured by integrating for 1 to 5 seconds using a luminometer (Luminous CT-9000D manufactured by Diatron), and this value was plotted against the concentration of the standard substance to obtain a calibration with good concentration dependency shown in FIG. Got a line. Using this calibration curve, β-h
It was possible to measure CG up to a concentration of 0.01 ng / ml.

[0041]

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

[Brief description of the drawings]

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

FIG. 2 is a monoclonal antibody calibration curve for β-hCG measurement prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Example 2 as a function of the concentration of β-hCG (standard substance). It is.

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[Procedure amendment]

[Submission date] May 21, 1999 (1999.5.2
1)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction contents]

Example 1 Determination of β-hCG by Simultaneous Sandwich Method CLEIA Purified β-hCG (standard substance) was applied to a white microplate on which a rabbit anti-β-hCG polyclonal antibody was immobilized, at 0, 0.01 and 0.1, respectively. 2, containing 1, 10, 100 mIU / ml
PB containing 2% BSA containing 50 μl of a PBS solution (pH 7.2) containing 2% BSA and a mouse anti-β-hCG monoclonal antibody labeled with a peroxidase enzyme at a concentration of about 2 μg / ml.
100 μl of S solution (pH 7.2) and add 1
Incubated for hours. 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.
And the chemiluminescent reagent solution 1 prepared in Reference Example 1 was added thereto.
75 mM with 00 μl and 0.0017% hydrogen peroxide
After 50 μl of Tris-HCl buffer (pH 7.8) was sequentially injected to emit light, the amount of the emitted light was integrated and measured by a luminometer (Diatron Co., Ltd., Luminous CT-9000D) for 1 to 5 seconds, and this value was measured. By plotting against the standard substance concentration, a calibration curve having good concentration dependency shown in FIG. 1 was obtained. Using this calibration curve, it was possible to measure β-hCG in a serum sample up to a concentration of 0.01 mIU / ml.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0040

[Correction method] Change

[Correction contents]

Example 2 Measurement of β-hCG by Simultaneous Sandwich Method CLEIA Purified β-hCG (standard substance) was placed on a white microplate on which a mouse anti-β-hCG monoclonal antibody was immobilized, at 0, 0.01 and 0.1, respectively. 50 μl of a PBS solution (pH 7.2) containing 2% BSA at a concentration of 1, 10, 100 mIU / ml and 2% BSA containing a peroxidase enzyme-labeled mouse anti-β-hCG monoclonal antibody at a concentration of about 2 μg / ml
100 μl of a PBS solution (pH 7.2) 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 inside of the well was washed with physiological saline, and then a 75 mM Tris-HCl buffer (pH 7.8) was added to each well.
Then, 100 μl of the chemiluminescent reagent solution prepared in Reference Example 1 and 75 μm containing 0.0017% hydrogen peroxide were added thereto.
After 50 μl of M Tris-HCl buffer (pH 7.8) was sequentially injected to emit light, the amount of the emitted light was integrated and measured for 1 to 5 seconds using a luminometer (Diatron, Luminous CT-9000D). Was 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 β-hCG in a serum sample up to a concentration of 0.01 mIU / ml.

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

[Procedure amendment 4]

[Document name to be amended] Drawing

[Correction target item name] Figure 2

[Correction method] Change

[Correction contents]

FIG. 2

 ──────────────────────────────────────────────────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-β-hCG antibody labeled with a peroxidase enzyme is brought into contact with β-hCG to be measured in a sample, and a peroxidase enzyme-labeled antibody is reacted by an antigen-antibody reaction.
After forming an immune complex comprising an antigen-antibody complex, the complex is separated and the N, N'-disubstituted-9,9'-bisacridinium salt is added to an N, N-disubstituted carboxylic acid amide compound. When the reaction is carried out under light irradiation, a chemiluminescent reagent containing a chemiluminescent substance obtained by adding an amino alcohol compound at the time of the reaction and / or after the reaction is added, and the reaction is carried out in the presence of a hydrogen acceptor. A method for immunological measurement of β-hCG by chemiluminescence, characterized in that the content of β-hCG in a sample is quantified by emitting light and measuring the emission intensity. 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. The immunoassay of β-hCG 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 immunoassay of β-hCG 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 group, an amino group, a halogen atom and the like, and R ₁ and R ₃ are bonded to each other to form a carbon atom of a carbonyl group to which each is bonded. And a ring 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:
-Immunological measurement method by chemiluminescence of hCG. 5. The method according to claim 1, wherein the antigen-antibody reaction is carried out in the presence of an anti-antibody immobilized on an insoluble carrier to form a labeled immune complex on the insoluble carrier. The immunological measurement method of β-hCG by the chemiluminescence method described in 1. 6. The method according to claim 1, wherein the N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide or N, N
The immunoassay of β-hCG by chemiluminescence according to any one of claims 1 to 5, wherein the immunoassay is dimethylacetamide. 7. The amino alcohol compound has the following general formula (3): (Wherein, in the general formula (3), R is a divalent aliphatic hydrocarbon having 1 to 5 carbon atoms, and m is an integer of 1 to 3). 7. The immunoassay of β-hCG according to any one of the above items 6 to 6 by a chemiluminescence method. 8. The β-hCG according to any one of claims 1 to 7, wherein the amino alcohol compound is at least one compound selected from the group consisting of monoethanolamine, diethanolamine and triethanolamine. Immunoassay by chemiluminescence method. 9. The immunoassay according to claim 1, wherein the hydrogen acceptor is hydrogen peroxide or a hydrogen peroxide source. 10. The immunoassay of β-hCG according to any one of claims 1 to 9, further comprising a luminescence enhancer when performing the chemiluminescence reaction. Method.