JP4286357B2 - Chemiluminescent enzyme immunoassay method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a chemiluminescent enzyme immunoassay method, and more particularly to a method for immunologically measuring an antigen or antibody by a chemiluminescent system using a peroxidase enzyme as a labeling substance and a chemiluminescent reagent. .
[0002]
[Prior art]
The enzyme immunoassay method has been developed as an immunoassay method that can maintain a good measurement environment because it does not use a radioisotope as a labeling substance and has a low risk to the human body. It's being used. As an enzyme used in this enzyme immunoassay method, various enzymes such as peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase are used. As a method for detecting the enzyme activity of an enzyme-labeled antibody or antigen using these enzymes, an enzyme substrate such as hydrogen peroxide / o-phenylenediamine, 4-nitrophenyl-phosphate, 2-nitrophenyl-β-galactoside is used. A colorimetric method is generally used in which the amount of color produced by a degradation reaction by an enzyme is measured to quantify the enzyme activity, and the amount of antibody or antigen having a correlation with the enzyme activity is quantified. However, in clinical chemistry analysis, the measurement target is a biological sample (mainly serum, urine, etc.), and the measured value is often used for diagnosis of disease state or follow-up of the disease. Although accurate measurement is required, it is difficult to completely satisfy this requirement by the colorimetric method. Therefore, a fluorescence method has been proposed for the purpose of satisfying this requirement. The fluorescence method is a method of decomposing fluorescent substrates such as 4-hydroxyphenylacetic acid, 4-methylumbelliferyl-β-galactoside, 4-methylumbelliferyl-phosphate by the catalytic activity of the enzyme used for labeling. After the generation, the fluorescence intensity is measured to quantify the enzyme activity, and the amount of antibody or antigen having a correlation with the enzyme activity is quantified. However, in the fluorescence method, it is difficult to satisfy the above requirement because of the scattering of excitation light. In addition, the colorimetric method and the fluorescence method require a light source such as a xenon lamp, and the background level increases due to the stray phenomenon derived from the light from the light source and the Raman light derived from the solvent. In principle, it is difficult to increase the sensitivity of the measurement by the colorimetric method and the fluorescence method. In recent years, a chemiluminescent enzyme immunoassay method (CLEIA) has been developed and attracts attention as a highly sensitive enzyme immunoassay method exceeding the colorimetric method and the fluorescence method.
[0003]
CLEIA determines the enzyme activity by measuring the amount of luminescence emitted when the chemiluminescent substance enters an excited state through an intermediate due to the catalytic activity of the enzyme and returns to the ground state from this state, and correlates with this enzyme activity. It is a method for quantifying the amount of antibodies or antigens that have a high molecular weight.A chemiluminescent substance is caused to emit light by a chemical reaction, so no light source is required, and there is no increase in the background caused by the light source, so the measurement can be highly sensitive. is there. Examples of enzymes used in CLEIA include peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, and dehydrogenase. A chemiluminescence system using luminol and p-iodophenol as a luminescence enhancer has been developed, and various substances can be quantified immunologically with high sensitivity by a chemiluminescence method.
[0004]
[Problems to be solved by the invention]
However, in CLEIA using a peroxidase enzyme as a labeling substance, it is often necessary to further improve the quantitativeness of a measurement target substance in a low concentration region, and the sensitivity of CLEIA using a peroxidase enzyme as a labeling substance is further increased. It was desired. In view of the above circumstances, an object of the present invention is to provide a chemiluminescent enzyme immunoassay method using a novel immunoassay system based on a chemiluminescence method capable of measuring a measurement target substance with higher sensitivity.
[0005]
[Means for Solving the Problems]
Therefore, as a result of intensive studies to solve the above problems, the present inventors have determined that N, N′-disubstituted-9,9′-bisacridinium salts are used as chemiluminescent substances. A chemiluminescent system using a chemiluminescent reagent produced by contacting with a reducing agent in the presence of a substituted carboxylic acid amide compound and formic acid, and further using a specific phenolic compound as a luminescence enhancer, luminol is added to the chemiluminescent substance. It has been found that the substance to be measured can be immunologically quantified with higher sensitivity than the chemiluminescent system used, and the present invention has been achieved based on these findings.
[0006]
Therefore, the present invention mixes a peroxidase enzyme-labeled antibody or antigen with the antigen or antibody to be measured in the sample or an aggregate thereof, and forms an immune complex comprising a peroxidase enzyme label-antigen antibody complex by an antigen-antibody reaction. If necessary, the immune complex is reacted with an antibody or antigen immobilized on an insoluble carrier and captured on the insoluble carrier, and then the following general formula (1)
[0007]
[Chemical formula 2]
(In the general formula (1), R¹ And R² Are each selected from the group consisting of an alkyl group, an aryl group, and a halogenated aryl group, and may be the same or different from each other;^Three , R^Four , R^Five And R⁶ 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, X is an n-valent anion, and n is 1 or 2 It is. )
A chemiluminescent reagent obtained by contacting an N, N′-disubstituted-9,9′-bisacridinium salt represented by formula (I) with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid A chemiluminescent enzyme immunoassay method characterized by measuring the content of an antigen or antibody in a sample by chemiluminescence in the presence of a hydrogen acceptor and measuring the amount of chemiluminescence is there.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
The chemiluminescent enzyme immunoassay method of the present invention comprises an immune reaction stage in which an antigen or antibody to be measured by an antigen-antibody reaction is captured as a peroxidase enzyme-labeled immune complex, and the generated immune complex is present in the molecule. It consists of a chemiluminescence reaction step measured by a chemiluminescence method using a labeling enzyme. The method of antigen-antibody reaction that constitutes the immune reaction stage is arbitrary, and N, N′-disubstituted-9,9′-bisacridinium salts in the presence of N, N-disubstituted carboxylic acid amide compound and formic acid In the method, any method can be adopted as long as the chemiluminescent reagent obtained by contacting with a reducing agent using an organic solvent can be used.
[0009]
For example,
(1) Sandwich method in which an antibody bound to an insoluble carrier is captured with an antigen to be measured and then reacted with a peroxidase enzyme-labeled antibody.
(2) A two-antibody method in which an antibody derived from a different animal species than an antibody bound to an insoluble carrier is used in the sandwich method, and the resulting sandwich complex is further reacted with a labeled second antibody against this antibody.
(3) A competitive method in which an antibody to be measured is reacted with an antibody bound to an insoluble carrier in the presence of a peroxidase enzyme-labeled antigen.
(4) The labeled substance in the immune complex separated by centrifugation after the labeled antibody or antigen that reacts specifically with the sample to be measured is allowed to act on the sample containing the antigen or antibody to be measured. Coagulation sedimentation method to detect,
(5) An antibody detection method in which a peroxidase enzyme-labeled anti-human gamma globulin antibody is allowed to act on an antibody to be measured in an antigen bound to an insoluble carrier,
(6) Biotin-avidin method in which peroxidase enzyme-labeled avidin is reacted with a biotin-labeled antibody, etc.
Can be used without limitation.
[0010]
Examples of the insoluble carrier used in the chemiluminescent enzyme immunoassay method of the present invention include, for example, high molecular compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, glass, metal, and magnetic particles. And combinations thereof. In addition, as the shape of the insoluble carrier, for example, a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, and various shapes such as a cell, a microplate, and a test tube can be used. Furthermore, the immobilization method of the antigen or antibody on these insoluble carriers is arbitrary, and any of physical adsorption method, covalent bond method, ion bond method and the like can be used.
[0011]
The antibody used in the chemiluminescent enzyme immunoassay method of the present invention can be either a monoclonal antibody or a polyclonal antibody, and the form is F (ab ′)₂ Fragments such as Fab 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.
[0012]
Furthermore, the chemiluminescent reaction that constitutes the latter stage of the chemiluminescent enzyme immunoassay method of the present invention comprises N, N′-disubstituted-9,9′-bisacridinium salts as chemiluminescent reagents. Using a reaction product obtained by contacting with a reducing agent in an organic solvent in the presence of a carboxylic acid amide compound and formic acid, and trapping on an insoluble carrier by acting a hydrogen acceptor in the presence of a luminescence enhancer The activity of the peroxidase enzyme, which is a labeled substance, is measured, and the measurement operation is optional. Generally, however, a measurement reagent containing a chemiluminescent substance and a luminescence enhancer is immunized with a peroxidase enzyme-labeled antibody or antigen. In addition to a chemically captured insoluble carrier, a hydrogen acceptor aqueous solution is added in a specific basic pH region to cause a chemiluminescence reaction, and the amount of chemiluminescence is measured by a luminescence measuring device. In a method in which measurements are being made.
[0013]
The chemiluminescent reagent used in the chemiluminescent enzyme immunoassay method of the present invention comprises N, N′-disubstituted-9,9′-bisacridinium salts in the presence of N, N-disubstituted carboxylic acid amide compounds and formic acid. It contains the reaction product obtained by contacting with a reducing agent below.
N, N'-disubstituted-9,9'-bisacridinium salts are represented by the following general formula (1)
[0014]
[Chemical 3]
Where R is¹ And R² Are each selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same as or different from each other. The alkyl group, aryl group and halogenated aryl group have 1 to 20 carbon atoms, and preferred alkyl groups are those having 1 to 10 carbon atoms. Examples thereof include linear or branched alkyl groups such as methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, and decyl group. The aryl group preferably has 6 to 14 carbon atoms, and includes a phenyl group, a tolyl group, a xylyl group, and the like, and may be further substituted with an alkyl group. As the aryl group, a phenyl group is particularly preferable. Examples of the halogenated aryl group include a halogenated phenyl group, a halogenated tolyl group, and a halogenated xylyl group, and a chlorophenyl group is particularly preferable.
[0015]
In the general formula (1), R^Three , R^Four , R^Five And R⁶ 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 as or different from each other. These hydrocarbon groups preferably have 1 to 20 carbon atoms, particularly 1 to 10 carbon atoms.
[0016]
In the general formula (1), X is an n-valent anion, and n is 1 or 2. Specific examples of the anion include nitrate ion, halogen ion, phosphate ion, sulfate ion, sulfonate ion and the like. Of these anions, nitrate ions are particularly preferred.
[0017]
Specific examples of N, N′-disubstituted-9,9′-bisacridinium salts include N, N-dimethyl-9,9′-bisacridinium salts, N, N′-diethyl-9, 9'-bisacridinium salt, N, N'-diphenyl-9,9'-bisacridinium salt, N, N'-di-m-chlorophenyl-9,9'-bisacridinium salt, etc. N, N′-dimethyl-9,9′-bisacridinium dinitrate (lucigenin) is particularly preferable.
[0018]
Examples of the N, N-disubstituted carboxylic acid amide compound used in the production of the chemiluminescent reagent include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N- Nonlimiting examples include dimethylpropionamide, N, N-dimethylbenzamide, N-methyl-2-pyrrolidone and the like. These N, N-disubstituted carboxylic acid amide compounds are essential for the production of the chemiluminescent reagent of the present invention, and N, N′-disubstituted-9,9′-bisacridinium salts and complexes are formed. After the formation, it is considered that the compound is converted into a compound having a higher luminescence yield with the reduction reaction of the acridinium salt structure, and is involved as a reaction component in the reaction in the production of the chemiluminescent reagent of the present invention. It is thought that it contributes to imparting water solubility to the product. Therefore, the amount used is 1 to 1000 equivalents, preferably 1.5 to 500 equivalents, particularly preferably 2 to 300 equivalents, based on the N, N′-disubstituted-9,9′-bisacridinium salts. Is enough.
[0019]
Furthermore, the formic acid used in the production of the chemiluminescent reagent has an effect of promoting the reaction and significantly increasing the reaction yield of the chemiluminescent reagent. The amount used is N, N′-disubstituted-9. , 9'-bisacridinium salts in a proportion of 0.1 to 5000 equivalents, preferably 0.5 to 1000 equivalents, particularly preferably 1 to 500 equivalents.
[0020]
Examples of the reducing agent used in the production of the chemiluminescent reagent include lithium aluminum hydride, lithium boron hydride, sodium boron hydride, and the like. Among these, lithium aluminum hydride is particularly preferably used.
[0021]
The reaction solvent used in carrying out the reduction reaction is not particularly limited as long as it is inert to the reaction reagent and has solubility in the reaction reagent, such as tetrahydrofuran and dioxanes. Cyclic ethers and the like are preferably used.
[0022]
Further, as the reduction reaction conditions, the reaction temperature varies depending on the type of reducing agent and solvent used, but is generally in the range of −10 to + 150 ° C., preferably 0 to 120 ° C., particularly preferably 20 to 90 ° C., The reaction time is 1 minute to 1 day, preferably 10 minutes to 12 hours, particularly preferably 30 minutes to 5 hours.
[0023]
In the chemiluminescent enzyme immunoassay method of the present invention, the chemiluminescent reagent emits light in an amount depending on the concentration of peroxidase in the presence of excess hydrogen acceptor under basic conditions of pH 7.5-13. It is recognized that this luminescence is enhanced by a luminescence enhancer such as a phenolic compound. Such phenolic compounds include p-hydroxycinnamic acid, p-phenylphenol, p- (4-chlorophenyl) phenol, p- (4-bromophenyl) phenol, p- (4-iodophenyl) phenol, p -Iodophenol, p-bromophenol, p-chlorophenol, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like.
[0024]
In the chemiluminescent enzyme immunoassay method of the present invention, the concentration of the chemiluminescent reagent is 10^-6~ 1M, preferably 10^-Four-10^-2The range is M, and the amount used is 10 to 500 μl, preferably 50 to 300 μl. The amount of the luminescence enhancer used is in the range of 0.01 to 100 times mol, preferably 0.1 to 10 times mol of the chemiluminescence reagent, and its concentration is 10 times.^-6~ 1M, preferably 10^-Four-10^-2The range of M.
[0025]
The hydrogen acceptor used in the chemiluminescence reaction is not particularly limited as long as it can serve as a substrate for the peroxidase enzyme, and organic peroxides, inorganic peroxides, and the like are arbitrarily used. Of these, hydrogen peroxide is particularly preferable. It is necessary to use the hydrogen acceptor in an amount that is sufficiently excessive with respect to the chemiluminescent substance, and the use amount is 3 to 10,000 times mol, preferably 10 to 1000 mol, with respect to the chemiluminescent substance. The range is double moles. Further, when various substances are quantified by labeling antibodies, nucleic acids and the like using peroxidase as a labeling substance, horseradish peroxidase (HRP) is preferably used as the peroxidase.
[0026]
As a basic buffer solution used for the chemiluminescence reaction, a Tris buffer solution, a phosphate buffer solution, a borate buffer solution, a carbonate buffer solution, a glycine-sodium hydroxide buffer solution, or the like can be arbitrarily used. The concentration of these buffers is preferably in the range of 1 mM to 1M. Moreover, additives, such as surfactant and a chelating agent, can be arbitrarily used at the time of reaction.
[0027]
In the chemiluminescent enzyme immunoassay method of the present invention, the amount of luminescence of the chemiluminescent reaction is measured using a luminescence photometer. The starting point and integration time of the measurement are arbitrary, but it is preferable to select a time in which the light emission amount is stable and the light emission amount is highly concentration dependent. For example, the measurement starting point is 0 to 1 hour, preferably 0 to 30 minutes, particularly preferably 0 to 15 minutes after mixing the reagents, and the measurement integration time is 1 second to 1 minute, preferably 1 to 30 seconds, particularly Preferably, it is 1 to 10 seconds.
[0028]
【Example】
EXAMPLES Hereinafter, although an Example and a comparative example are shown with a reference example, this invention is demonstrated concretely, This invention is not limited by an Example etc.
In addition,% in a reference example, an Example, etc. means weight%.
[0029]
[Reference Example 1]
Preparation of chemiluminescent reagents
Take 1 mg of lucigenin in a test tube, add 150 μl of N, N-dimethylformamide to dissolve it, add to 1 ml of 1,4-dioxane with stirring, and finely disperse lucigenin in 1,4-dioxane. It was. Next, 20 μl of formic acid was added and stirred well, 200 μg of lithium aluminum hydride powder was added, and the mixture was stirred at 60 ° C. for 3 hours for reduction reaction, and then the reaction mixture was removed under ice cooling. An excessive amount of lithium aluminum hydride was decomposed by injecting it little by little into 1 ml of ionic water. Next, the produced precipitate such as aluminum hydroxide was separated by filtration, and then the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a chemiluminescent reagent.
[0030]
[Reference Example 2]
Preparation of insoluble carrier-immobilized polyclonal antibody
A solution prepared by dissolving a polyclonal antibody derived from an animal such as a spider having specific reactivity with an antigen in 10 mM phosphate buffered saline (pH 7.4) (PBS) at a concentration of 10 mg / ml is prepared using a white microplate (laboratory). 0.1 ml to each well of System Co.), left at 37 ° C. for 1 hour, washed with PBS, then added with 0.3 ml of 1% bovine serum albumin (BSA) aqueous solution at 37 ° C. A post-coating treatment was performed by allowing to stand at temperature for 1 hour to obtain a polyclonal antibody-immobilized white microplate.
[0031]
[Reference Example 3]
Preparation of peroxidase enzyme-labeled monoclonal antibody
In a 1 ml solution of a monoclonal antibody derived from a mouse having specific reactivity with an antigen or the like dissolved in 10 mM phosphate buffered saline (PBS) (pH 7.4) at a concentration of 1.0 mg / ml, N- (m- 0.1 ml of a 10 mg / ml dimethylformamide solution of maleimidobenzoic acid) -N-succinimide ester (MBS) was added and reacted at a temperature of 25 ° C. for 30 minutes. Next, this reaction mixture was subjected to gel filtration with a 0.1 M phosphate buffer (pH 6.0) using a column packed with Sephadex G-25 to separate the maleimidated monoclonal antibody and unreacted MBS.
On the other hand, to a 1.0 mg / ml PBS solution of horseradish peroxidase (HRP) as a peroxidase enzyme, an ethanol solution of N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) at a concentration of 10 mg / ml was added, The reaction was performed at a temperature of 25 ° C. for 30 minutes. Next, this reaction mixture was purified by gel filtration using a column packed with Sephadex G-25 and filtered with 10 mM acetate buffer (pH 4.5), and a fraction containing pyridyl disulfide HRP was collected. In the collodion bag, the solution was concentrated about 10 times under ice cooling. Next, 1 ml of 0.1 M acetate buffered saline (pH 4.5) containing 0.1 M dithiothreitol was added thereto, and the mixture was stirred at a temperature of 25 ° C. for 30 minutes to introduce pyridyl into the HRP molecule. After reducing the disulfide group, 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 thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice-cooling using a collodion bag, and allowed to stand overnight at 4 ° C., and then Ultrogel AcA44 (SEPRACOR) was used. Gel filtration was performed using a packed column to obtain a peroxidase enzyme-labeled monoclonal antibody.
[0032]
[Example 1]
Measurement of α-fetoprotein (AFP) by simultaneous sandwich method CLEIA
(2) 50 μl of 2% BSA-containing PBS solution (pH 7.4) containing purified human AFP (standard substance) in the range of 0 to 800 ng / ml on a white microplate on which anti-human AFP polyclonal antibody is immobilized and peroxidase enzyme labeling 100% of a 2% BSA-containing PBS solution (pH 7.4) containing a mouse anti-human AFP monoclonal antibody at a concentration of about 3 μg / ml was added and incubated at a concentration of 37 ° C. for 1 hour. Next, the solution in the well is removed by suction, and then the well is washed with physiological saline, and then 10 parts of p-iodophenol is added to each well.^-3100 μl of 0.1 M Tris-HCl buffer (pH 8.4) contained at a concentration of M is added, and 100 μl of a 50-fold diluted chemiluminescent reagent prepared in Reference Example 1 and 0 containing 0.0034% hydrogen peroxide are added thereto. Inject 50 μl of 1 M Tris-HCl buffer (pH 8.4) to emit light, and measure the amount of luminescence by integrating for 0 to 5 seconds with a luminometer (Diatron Luminous CT-9000D). By plotting with respect to the substance concentration, a calibration curve having good concentration dependency shown in FIG. 1 was obtained. Using this calibration curve, it was possible to measure human AFP in serum samples up to a concentration of 0.05 ng / ml.
[0033]
Example 2
Measurement of prolactin (PRL) by simultaneous sandwich method CLEIA
白色 50 μl of 2% BSA-containing PBS solution (pH 7.4) containing purified human PRL (standard substance) in the range of 0 to 200 ng / ml on a white microplate immobilized with anti-human PRL polyclonal antibody and peroxidase enzyme labeling 100% of 2% BSA-containing PBS solution (pH 7.4) containing mouse anti-human PRL monoclonal antibody at a concentration of about 2 μg / ml was added and incubated at a temperature of 37 ° C. for 1 hour. Next, the solution in the well is removed by suction, the inside of the well is washed with physiological saline, and p-iodophenol is then added to each well.^-3100 μl of 0.1 M Tris-HCl buffer (pH 8.4) contained at a concentration of M is added, and 100 μl of a 50-fold diluted chemiluminescent reagent prepared in Reference Example 1 and 0 containing 0.0034% hydrogen peroxide are added thereto. Inject 50 μl of 1 M Tris-HCl buffer (pH 8.4) to emit light, and measure the amount of luminescence by integrating for 0 to 5 seconds with a luminometer (Diatron Luminous CT-9000D). By plotting against the substance concentration, a calibration curve having a good concentration dependency shown in FIG. 2 was obtained. Using this calibration curve, it was possible to measure human prolactin in serum samples up to a concentration of 0.1 ng / ml.
[0034]
[Example 3]
Measurement of human chorionic gonadotropin β chain (βhCG) by simultaneous sandwich method CLEIA
A white microplate on which an anti-human hCG polyclonal antibody is immobilized, 50 μl of 2% BSA-containing PBS solution (pH 7.4) containing purified βhCG (standard substance) in the range of 0 to 200 mIU / ml and a peroxidase enzyme-labeled mouse 100% of 2% BSA-containing PBS solution (pH 7.4) containing anti-βhCG monoclonal antibody at a concentration of about 2 μg / ml was added and incubated at a temperature of 37 ° C. for 1 hour.
Next, the solution in the well is removed by suction, and then the well is washed with physiological saline, and then 10 parts of p-iodophenol is added to each well.^-3100 μl of 0.1 M Tris-HCl buffer (pH 8.4) contained at a concentration of M is added, and this well contains 100 μl of a 50-fold dilution of the chemiluminescent reagent prepared in Reference Example 1 and 0.0034% hydrogen peroxide. Inject 50 μl of 0.1 M Tris-HCl buffer (pH 8.4) to emit light, and measure the amount of luminescence by integrating it with a luminometer (Diatron Luminous CT-9000D) for 0 to 5 seconds. By plotting against the standard substance concentration, a calibration curve with good concentration dependency shown in FIG. 3 was obtained. Using this calibration curve, it was possible to measure βhCG in serum samples up to a concentration of 0.1 mIU / ml.
[0035]
[Comparative Example 1]
Determination of α-fetoprotein (AFP) by simultaneous sandwich method CLEIA using luminol
(2) 50 μl of 2% BSA-containing PBS solution (pH 7.4) containing purified human AFP (standard substance) in the range of 0 to 800 ng / ml on a white microplate on which anti-human AFP polyclonal antibody is immobilized and peroxidase enzyme labeling 100% of a 2% BSA-containing PBS solution (pH 7.4) containing a mouse anti-human AFP monoclonal antibody at a concentration of about 3 μg / ml was added and incubated at a concentration of 37 ° C. for 1 hour. Next, the solution in the well is removed by suction and washed with physiological saline, and then 10 ml of p-iodophenol is added to each well.^-3100 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing M at a concentration of M was added, and luminol was added to the wells at 5.6 × 10 6.^-Five100 μl of 0.1 M Tris-HCl buffer (pH 8.4) contained at a concentration of M and 50 μl of 0.1M Tris-HCl buffer (pH 8.4) of 0.0034% hydrogen peroxide were injected to emit light. The amount of luminescence is measured by integrating for 0 to 5 seconds with a luminometer (Diatron Luminous CT-9000D), and this value is plotted against the standard substance concentration, thereby having the concentration dependency shown in FIG. A calibration curve was obtained. Using this calibration curve, it was only possible to measure human AFP in serum samples to a concentration of 2.0 ng / ml.
[0036]
【The invention's effect】
The chemiluminescent enzyme immunoassay method of the present invention uses a peroxidase enzyme that is easily available and relatively easy to handle as a labeling substance, and is a novel material that can be easily manufactured using lucigenin, which is inexpensive and easily available, as a starting material. Various antigens or antibodies that are measurement target substances can be measured immunologically with high sensitivity by a chemiluminescence method using a chemiluminescent reagent.
[Brief description of the drawings]
FIG. 1 is a 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 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 is a calibration curve for human AFP measurement prepared by plotting the amount of chemiluminescence chemiluminescent using the reaction system described in Comparative Example 1 as a function of the concentration of human αAFP (standard substance).

Claims (8)

After peroxidase enzyme-labeled antibody or antigen is mixed with the antigen or antibody to be measured in the sample or an aggregate thereof, after forming an immune complex consisting of a peroxidase enzyme label-antigen-antibody complex by antigen-antibody reaction,
The following general formula (1)
(In the general formula (1), R ¹ and R ² are each selected from the group consisting of an alkyl group having 1 to 2 carbon atoms , a phenyl group or a chlorophenyl group, and may be the same or different from each other. R ³ , R ⁴ , R ⁵ and R ⁶ are each a hydrogen atom, X is a nitrate ion, and n is 2.)
N, N′-disubstituted-9,9′-bisacridinium salts represented by the formula: N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide or N, N-dimethylpropion A chemiluminescent reagent obtained by contacting with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound consisting of an amide and formic acid is added to cause chemiluminescence in the presence of a hydrogen acceptor. A chemiluminescent enzyme immunoassay method for measuring the amount of antigen or antibody in a sample by measuring    The chemiluminescence according to claim 1, wherein the N, N'-disubstituted-9,9'-bisacridinium salt is N, N'-disubstituted-9,9'-bisacridinium dinitrate. Enzyme immunoassay method.    The chemiluminescent enzyme immunity according to claim 1 or 2, wherein the N, N-disubstituted carboxylic acid amide compound is at least one compound selected from the group consisting of N, N-dimethylformamide and N, N-dimethylacetamide. Measuring method.    The chemiluminescent enzyme immunoassay method according to any one of claims 1 to 3, wherein the reducing agent is lithium aluminum hydride.    The chemiluminescent enzyme immunoassay method according to any one of claims 1 to 4, wherein the hydrogen acceptor is hydrogen peroxide.    The chemiluminescent enzyme immunoassay method according to any one of claims 1 to 5, further comprising a luminescence enhancer when the chemiluminescent reaction is performed.    The chemiluminescent enzyme immunoassay method according to claim 6, wherein the luminescence enhancer is a phenolic compound.    The chemiluminescent enzyme immunoassay method according to claim 7, wherein the phenolic compound is at least one compound selected from the group consisting of p-iodophenol, p-phenylphenol and 6-hydroxybenzothiazole.