JP2000191699A - Monoclonal antibody to be specifically reacted with coplanar pcb substantially not reducing antigen recognition characteristic even in 50 (v/v)% aqueous solution of organic solvent, and its measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a monoclonal antibody capable of omitting the pretreatment of extraction with an organic solvent and analyzing a coplanar PCB simply quickly in high accuracy by making the antigen recognition characteristics not lowered even in an aqueous solution of an organic solvent having a fixed concentration. SOLUTION: This monoclonal antibody recognizes a coplanar PCB such as 3,3,3'4'-tetrachlorobiphenyl (PCB-#77), 3,4,5,3',4'-pentachlorobiphenyl (PCB-#126) or 3,4,5,3'4',5'-hexachlorobiphenyl (PCB-#169) and will not be substantially lowered in antigen recognition characteristics even in 50 (v/v)% aqueous solution of an organic solvent. A coplanar PCB existing in an aqueous solution of an organic solvent or a buffer solution not containing an organic solvent is brought into contact with the monoclonal antibody to form an antigen-antibody complex and the amount of the antigen-antibody complex formed is measured to measure the coplanar PCB.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a coplanar PCB.
TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically reacts with, and a method for measuring coplanar PCB in an oily sample using the same.

[0002]

2. Description of the Related Art Due to its toxicity, PCBs have caused harm to humans, such as the case of Kanemi Yusho, and have been designated as Class 1 Specified Chemical Substances in the Law Concerning the Examination and Production of Chemical Substances, and their manufacture and use have been prohibited. I have. However, PCB is unintentionally generated by incineration with dioxins, and is hardly decomposable and accumulates in living organisms.
Dangerous to humans and wildlife. Furthermore, in recent years, PCB has been attracting attention as a chemical substance which is highly suspect as a so-called environmental hormone (endocrine disrupting substance). Under such circumstances, it is important to measure a coplanar PCB having high toxicity to living organisms among PCBs having various homologs and isomers.
Conventionally, gas chromatography or high-performance liquid chromatography is mainly used for analysis of such PCB-containing oils (especially chemically treated oils), soil, water, food, and other environmental or biological samples. Have been. However, in the analysis method, considerable labor and time are required for preparation of a sample cleanup and the like, and high costs are required for measuring devices and equipment, and the technician also requires education and training. There was a problem. In addition, the analysis of PCBs requires a large number of analysis cases, so that simplicity and speed are important, especially at the measurement site (on-site).
The development of analytical methods in has been awaited. Thus, an enzyme immunoassay was developed as a simple analysis method. However, the reported monoclonal antibody used in the conventional enzyme immunoassay cannot retain antigen recognition properties in the presence of an organic solvent such as the antibody described in the present invention. Quantification by enzyme immunoassay is not always satisfactory, and monoclonal antibodies resistant to organic solvents have been expected.

[0003]

Therefore, an attempt was made to establish a simple and rapid method for analyzing PCBs by an immunological detection method using an antibody. In the immunological detection method, the most important factor is an antibody that specifically reacts with the coplanar PCB, and the measurement target of the coplanar PCB is extracted from environmental water, soil, a biological sample, and the like with an organic solvent. In some cases, the coplanar PCB in an organic solvent or oil may be used, so it is necessary that the PCB can be measured in the presence of an organic solvent in addition to the aqueous solvent. Even with such a measurement system, it was necessary to obtain an anti-coplanar PCB monoclonal antibody having high antigen recognition characteristics.

[0004] The organic solvent resistance of the monoclonal antibody is necessary in the step of recognizing the antigen of the antibody. In the enzymatic reaction step, which is the next step of the enzyme immunoassay, the reaction is carried out with a buffer solution (aqueous solution). The above-mentioned monoclonal antibodies do not require organic solvent resistance. However, it goes without saying that the presence of the organic solvent does not affect the enzyme activity of the enzyme-labeled antibody.

[0005] By labeling the antibody with a fluorescent dye instead of labeling the enzyme, the target substance can be measured in the same manner as in the case of labeling the enzyme.

[0006]

SUMMARY OF THE INVENTION The present invention provides (1) a monoclonal antibody that recognizes coplanar PCB,
(2) Coplanar PC, a monoclonal antibody that does not substantially reduce antigen recognition properties even in a (v / v)% organic solvent aqueous solution
B is 3,4,3 ', 4'-tetrachlorobiphenyl (P
CB- # 77), 3,4,5,3 ', 4'-pentachlorobiphenyl (PCB- # 126) or 3,4,5,
3 ', 4', 5'-hexachlorobiphenyl (PCB-
# 169) the monoclonal antibody according to the above (1) or (3) the coplanar PCB present in an aqueous solution of an organic solvent or a buffer solution (including an aqueous solution) containing no organic solvent, using the monoclonal antibody according to the above (1) or (2). A method for measuring coplanar PCB, comprising: forming an antigen-antibody complex by contacting with an antibody; and measuring the amount of the complex formed.
(4) An oily sample containing a coplanar PCB is brought into contact with an organic solvent to extract the coplanar PCB in the oily sample into the organic solvent, and the coplanar PCB in the organic solvent is measured using a monoclonal antibody. A method for measuring coplanar PCB in an oily sample, wherein the method is converted into coplanar PCB in the sample, and (5) the monoclonal antibody is any one of the monoclonal antibodies described in (1) or (2) above. (4) The method for measuring coplanar PCB in an oily sample according to (4).

The monoclonal antibody of the present invention is obtained by immunizing a mammal with a complex obtained by binding a coplanar PCB to a high molecular weight carrier via a linker, and then producing an antibody against the complex from the mammal. Emerges immunocompetent B cells that can form, fuses the immunocompetent cells with tumor cells capable of continuous cell division to produce hybrid cells, and converts the hybrid cells to coplanar PCB substantially in the presence of an organic solvent in the presence of an organic solvent. It can be obtained by isolating a hybridoma that produces a monoclonal antibody that specifically reacts with the above, and purifying and isolating the monoclonal antibody produced by the hybridoma.

The method for measuring coplanar PCB using the monoclonal antibody of the present invention can be constituted by adding an improvement based on the characteristics of the monoclonal antibody of the present invention to a conventionally known immunoassay method.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Among coplanar PCBs, those having particularly high toxicity include, for example, PCB- # 12.
6, PCB- # 77, PCB- # 169 and the like. Therefore, a method for producing monoclonal antibodies against the above three compounds will be described as a representative.
Anti-PCB- # 77 antibody obtained by the present invention, anti-PC
The isotypes of the B- # 126 antibody and the anti-PCB- # 169 antibody were both IgG. When coplanar PCB is measured using the obtained anti-coplanar PCB monoclonal antibody, IgG or a fragment thereof, for example, F (a'b) _{2 is} used.

A) [Step of Converting Coplanar PCB to Complete Antigen (Immunogen)] By binding the target coplanar PCB to a high molecular weight carrier molecule via a linker, a complex having antigenicity can be obtained. Coplanar PCB used
Is preferably high in purity. The high-molecular-weight carrier molecule used has a reactive group that can be freely used for a ligation reaction with a compound in which a linker has been introduced into coplanar PCB (incomplete antigen), and is linked to the incomplete antigen. Any macromolecular compound that can confer immunogenicity or enhance their existing immunogenicity can be used. In particular, macromolecular compounds containing freely available reactive amino groups are preferred. For example, lysine-rich proteins having a molecular weight of about 10,000 to 150,000 can be mentioned. Specifically, bovine serum albumin (B
SA: molecular weight 66200), human serum albumin (HS)
A: molecular weight 58000), rabbit serum albumin (RS
A: molecular weight 68000), goat serum albumin (GSA:
Molecular weight 68000), ovalbumin (ovalbumin: molecular weight 45,000), or keyhole limpet hemocyanin (KLH: molecular weight 1,000,000). If other macromolecular compounds meet the above requirements, they can be used as carrier molecules, such as butathyroglobulin, B2 microglobulin, hemociamine, immunoglobulins, toxins ( Cholera toxin, tetanus toxin, diphtheria toxin and others),
Polysaccharides, lipopolysaccharides, natural or synthetic polyadenylic and polyuridylic acids, polyalanyl and polylysine polypeptides, or cell membrane components such as formalin or glutaraldehyde treated red blood cell membranes and the like. Specifically, for example, carbodiimide, alkyl chlorocarbonate, preferably 1-ethyl-3-
The carboxyl group of the incomplete antigen is bound to one of the reactive amino groups of the high molecular weight carrier using (3-dimethylaminopropyl) carbodiimide hydrochloride, isobutyl chlorocarbonate.

B) [Mammalian Immunization Step and the Present Antibody Obtaining Step] To immunize mammals such as mice, rats, rabbits, dogs, etc. with the complex thus obtained, It is performed using known immunization methods, for example, by one or more administrations of the conjugate. For example, 7
2 to 3 days at intervals of 30 days, especially 12 to 16 days
Single administration and the like are preferred. Injection, which can be performed intravenously, intraperitoneally or subcutaneously, is a preferred form of administration. Furthermore, a combination of subcutaneous injection and intraperitoneal cavity is particularly preferred. In this case, the complex is dissolved in an appropriate buffer, for example, a sodium phosphate buffer containing one of commonly used adjuvants such as Freund's complete adjuvant (a mixture of killed tuberculosis). Used. here,
An adjuvant refers to a substance that, when administered with an antigen, nonspecifically enhances the immune response to that antigen. Then, after leaving the mammal without treatment for 0.5 to 4 months, for example, 10 μg to 1000 μg, particularly
Another dose is given at a dose of 25 μg to 500 μg of the conjugate. Three to two months after the last administration, immunocompetent B cells capable of producing an antibody against the complex are isolated from the immunized mammal by a conventional method, and the immunocompetent B cells are continuously isolated. The resulting hybrid cells are fused with tumor cells that are capable of cell division. Selecting a hybrid cell producing the desired antibody,
By culturing the cloned cells in a test tube (in vitro) or in vivo (in vivo), the present coplanar PCB having a high degree of specificity and affinity can be obtained.
(Hereinafter referred to as the present antibody) can be produced. This antibody recognizes the difference in the chlorine bond position and the chlorine bond number of each homologue / isomer of coplanar PCB.

C) [Measuring Method Using Monoclonal Antibody] As a measuring method using the present antibody, immunoassay of the present coplanar PCB by an indirect competitive inhibition method (ELISA method), a direct non-competitive inhibition method (ELISA method), or the like. Can be given. The analysis is a highly accurate analysis method that can be performed easily and quickly.

The direct non-competitive inhibition method can be performed, for example, by the following method. Basically, a certain amount of the first antibody (ie, the present antibody) bound to the solid support is allowed to react with the free coplanar PCB contained in the sample, and then the unreacted first coplanar PCB is reacted. After binding the labeled coplanar PCB (directly or via a linker), which serves as an antigen of the antibody, the bound antigen is quantified based on the label to be included in the sample. Primary antibody bound to a solid support (ie, the present antibody) that has reacted with the present coplanar PCB in a free state
Is calculated, and the amount of the present coplanar PCB contained in the sample in a free state is measured from the calculated value.

When indirectly linked via a linker,
The linker used is a compound containing at least one or more reactive groups capable of forming a covalent bond of freely available reactive groups of the high molecular weight carrier molecule. For example, compounds containing between 2 and 16 crosslinkable carbon molecules and having one or more reactive groups such as amino groups, carboxyl groups, etc. as reactive groups can be mentioned. Specifically, the general formula H ₂ (CH ₂ ) _n COOH (n
Is an integer of 2 to 16). A method similar to the method of binding the reactive group of the incomplete antigen to one of the reactive groups of the high molecular weight carrier molecule can be used.

The indirect competitive inhibition method can be performed, for example, by the following method. Basically, the competition between the antigen bound to the solid support and the free coplanar PCB contained in the sample, for example, the coplanar PCB that is the antigen bound to the solid support is labeled with the coplanar PCB. Based on competition of the first antibody recognized by the two antibodies with coplanar PCB, the free antigen. In this context, attachment of the antigen, coplanar PCB, to the solid support can occur directly or indirectly via a linker and / or a high molecular weight carrier that is not recognized by the first antibody (ie, the present antibody). Here, the high molecular weight carrier that is not recognized by the first antibody (that is, the present antibody) is a high molecular weight carrier that can be used in the above-mentioned complete antigen (immunogen) conversion step, and is used in the production of the first antibody. It is a high molecular weight carrier that cannot be obtained. When the coplanar PCB as an antigen is indirectly bound via a linker and / or a high molecular weight carrier that is not recognized by the first antibody,
For such binding, a method similar to or a method analogous to the above-described complete antigen (immunogen) conversion step can be used.

When indirectly linked via a linker,
The linker used is a compound containing at least one or more reactive groups capable of forming a covalent bond in a freely available reactor of a high molecular weight carrier. For example, compounds containing between 2 and 16 crosslinkable carbon molecules and having one or more reactive groups such as amino groups, carboxyl groups, etc. as reactive groups can be mentioned.
Specifically, the general formula H ₂ (CH ₂ ) _n COOH (n is an integer of 2 to 16) is preferable.

The solid support used for the direct or indirect binding of the coplanar PCB as an antigen includes, for example, plastic surfaces of microtiter plates or test tubes, beads made of polystyrene, polypropylene, polyvinyl chloride, glass or plastic, etc. surface,
Examples include filter paper, dextran, the surface of a strip of cellulose or nitrocellulose or other similar material, and the like.

The coplanar PCB, which is an antigen, is directly or indirectly bound to these solid supports through a spacer and / or a high molecular weight carrier molecule which is not recognized by the first antibody (the present antibody) (hereinafter referred to as coating). ), The solid support is activated in advance by an ordinary method using glutaraldehyde or cyanogen bromide, for example. After a coating solution is added to the coating material on the activated solid support material, the surface is coated with a conjugate with serum albumin of a mammal of the same species as the mammal producing the first antibody by incubating. Examples of the coating liquid used here include about 10 mM phosphate buffer (pH 7.4) containing 140 mM sodium chloride. Coating conditions include, for example, coplanar P which is an antigen.
The concentration of the CB or the antigen having a high molecular weight carrier not recognized by the linker and / or the first antibody in the coating solution may be, for example, about 0.05 μg / ml to about 1
μg / ml and the like can be mentioned as preferable conditions.
When a 96-well microtest plate is used, for example, about 0.1 ml / well can be used as a preferable condition. The coating conditions include about 4 ° C. and about 6 to 24 hours, preferably overnight. As a specific example of the above, for example, a solid support material made of polystyrene
A 96-well microtiter plate is used as a high molecular weight carrier molecule for a complex (coplanar PCB bound to a high molecular weight carrier via a linker) for producing the first antibody. Use serum albumin or keyhole limpet hemocyanin,
As the high molecular weight carrier molecule used when indirectly binding to the solid support material, use can be made of serum albumin of another animal such as goat, which is a high molecular weight carrier molecule that is not recognized by the first antibody. In addition, serum albumin of a mammal of the same species as the mammal producing the first antibody and coplanar PCB bound to a high molecular weight carrier via a linker, but after coating the complex on the carrier, the non-specific In order to prevent binding, serum albumin of a mammal of the same species as the mammal producing the first antibody is adsorbed by a protein other than serum albumin of the same species of mammal producing the first antibody. It is desirable to block missing parts. For this purpose, for example, a simple method of incubating a carrier containing about 3% skim milk solution and a carrier at about 20 ° C. for about 2 hours can be mentioned. The carrier thus obtained can be used as a washing buffer (for example,
10 mM containing 0.15 M sodium chloride containing 0.05% tween 20
Used after washing with phosphate buffer (pH 7.4).

The coplanar PCB directly or indirectly bound to the solid support thus obtained then contains the coplanar PCB to be detected contained in the sample and the first antibody (the present antibody). The test solution is mixed and the mixture is incubated. At this time, the coplanar PCB which is the antigen to be detected contained in the sample may be present in a free form or as a component in water or an environment such as a crop, soil, food or a biological sample.
After incubation for about 10 minutes to about 2 hours, the mixture is incubated with a second antibody that recognizes the first antibody and is labeled with a binding enzyme or the like. Examples of the second antibody labeled with this enzyme include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and glucose-6-
An antibody against the first antibody (the present antibody) to which an enzyme such as phosphate dehydrogenase or urease has been bound can be mentioned. As a specific example, the first antibody (the present antibody)
When a rabbit antiserum is used as the above, a suitable example of the second antibody is a peroxidase-conjugated anti-rabbit immunoglobulin (IgG) or goat immunoglobulin (IgG). The rabbit IgG goat IgG is commercially available and easily available. When labeled with peroxidase, it produces a brown or yellow color in combination with hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent. In the case of labeling with glucose oxidase, for example, 2,2′-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or the like is used as a substrate, and the label bound to the second antibody is used. The color developed by the enzymatic reaction between the enzyme and the substrate is measured as absorbance using a device such as a multi-scanning spectrophotometer.

In addition, a fluorescent dye is previously bound to the second antibody in addition to the above enzyme, and the second antibody is subjected to an antigen-antibody reaction.
By measuring the amount of bound antibody not by enzyme activity but by fluorescence intensity, a more sensitive measurement method can be obtained.

The present antibody may be used by containing at least one of the present antibodies as a reagent and using the present coplanar PC.
Means for immunological detection and analysis of the present coplanar PCB in the form of a test kit suitable for use under field conditions for simple and rapid processing and accurate detection and analysis of B Can also be raised. The test kit described above can contain, for example, the following components. : (1)
A solid support material that directly binds the coplanar PCB as an antigen or indirectly via a linker and / or a high molecular weight carrier molecule that is not recognized by the first antibody (the present antibody);
(2) Reagent containing the antibody of the present invention (3) Standard solution of the present coplanar PCB as an antigen (4) Second reagent labeled with an enzyme or a fluorescent dye which recognizes and binds to the first antibody (the present antibody) Reagent containing antibody, (5) buffer, (6)
Additives such as polypeptides and surfactants for preventing non-specific adsorption and formation of aggregates, and (7) pipettes, reaction vessels, calculation curves and the like. The solid supports described above have a very wide range of designs and can have very different shapes depending on the particular purpose intended in use. For example, dishes, balls, plates, small rods, cells,
Small bottles, small tubes, fibers, nets and the like can be mentioned. As a specific example, a transparent plastic material, for example, a microtiter plate made of polyvinyl chloride or polystyrene, a small ball made of polystyrene, a tube or a rod, or the like can be used.

In the above method, when using a water system in the environment such as paddy water or tap water as a sample, the coplanar PCB in the sample can be measured. Therefore, when the concentration of the coplanar PCB in the sample is higher than the above, the sample is appropriately diluted before use. When sampling non-aqueous substances in environmental or biological samples such as oil (chemically treated oil, etc.) crops, soil, food, etc., extract the coplanar PCB from a solvent sample such as methanol, or use an appropriate solid After being adsorbed on the phase support, the coplanar PCB is used from the solid support by an appropriate method and from the support using an appropriate organic solvent or the like. The buffer solution (or aqueous solution) containing the organic solvent containing the present coplanar PCB thus prepared is mixed, and for example, about 2
React by incubating overnight at around 0 ° C. At this time, the first antibody (the present antibody) is about 3,000 to about 5,0
It is desirable to dilute it by a factor of 00 and react it with the present coplanar PCB in the sample. Particularly, a concentration of about 5,000 times can be mentioned as a desirable condition. That is, about 2,500
Co-planar PC with the same dose of primary antibody diluted about 1-fold
It is desirable to mix with a sample solution containing B and react at about 20 ° C. overnight. As the diluent, for example, one having the same composition as the above-mentioned washing buffer can be used.
If necessary, it is desirable to add skim milk or the like as a protective protein for stabilization when the first antibody is excessively diluted.

The surface of the unreacted first antibody contained in the reaction solution of the sample and the first antibody prepared as described above was coated with a complex of serum albumin of the same species as the mammal producing the first antibody. A reaction is performed to bind to the solid support. Regarding the reaction conditions, it is desirable to add a reaction solution of the sample and the first antibody to a solid support material and to react at, for example, about 20 ° C. for about 1 hour. After the reaction, after washing the carrier with the above-mentioned washing buffer, an antibody against the first antibody (second antibody) labeled with an enzyme or the like and derived from a mammal of a different species from the mammal producing the first antibody is used. For the reaction.

[0024]

The present invention will be described below with reference to examples. It goes without saying that the present invention is not limited only to the following examples.

Example 1 Preparation of Immunization Antigen Preparation Example 1 Preparation of Complex (Linking of High Molecular Weight Carrier Molecule in Complete Antigen (Immunogen) Step and High Molecular Weight Carrier to Antigen Bound to Solid Support Material) [Preparation of complex for preparing anti-PCB- # 77 antibody] 4
After protecting the hydroxyl group of -bromo-2-chlorophenol with methyl ether, Grignard-cross coupling reaction with 3,4-dichloroiodobenzene gave
By synthesizing 3 ', 4-trichloro-4'methoxybiphenyl and deprotecting the methyl ether group to a hydroxyl group with boron tribromide, 3,3', 4-trichloro-4 '
Hydroxybiphenyl was obtained. This is reacted with 6-bromohexanoic acid and ethyl ester, and the ethyl ester is hydrolyzed to carboxylic acid to give 6- (3,3).
3 ', 4-Trichlorobiphenyl-4'-oxy) hexanoic acid was synthesized in 5 steps with a total yield of 44%. For binding to the carrier protein, the carboxylic acid was converted into an active ester form with hydroxysuccinimide, and then an amide bond was formed with the amino group to prepare a conjugate in which PCB- # 77 was bound. 4-bromo-2-phenol ↓ 4-bromo-2-chloroanisole ↓ 3,4-dichloroiodobenzene ↓ 3,3 ', 4-trichloro-4'methoxybiphenyl ↓ 3,3', 4-trichloro-4 ' Hydroxybiphenyl ↓ 6-ethyl- (3,3 ', 4-trichlorobiphenyl-
4'-oxy) hexanoate ↓ 6- (3,3 ', 4-trichlorobiphenyl-4'-oxy) hexanoic acid ↓ 6-N-hydroxysuccinimide- (3,3', 4-
Trichlorobiphenyl-4'-oxy) hexanoate

[Preparation of Complex for Preparing Anti-PCB- # 126 Antibody] After protecting a hydroxyl group with a methyl ether group in the same manner as in the synthesis of conjugate # PCB-77,
3,3 ', 4,5-Tetrachloro-4'methoxybiphenyl is synthesized by a Grignard-cross coupling reaction with 5-trichloroiodobenzene, and the methyl ether group is deprotected to a hydroxyl group by boron tribromide. Thus, 3,3 ', 4,5-tetrachloro-4'hydroxybiphenyl was obtained. This is reacted with 6-bromohexanoic acid and ethyl ester, and the ethyl ester is hydrolyzed to carboxylic acid to give 6- (3,3 ', 4,5-
Tetrachlorobiphenyl-4'-oxy) hexanoic acid was synthesized in 5 steps with a total yield of 35%. For binding to the carrier protein, the carboxylic acid was converted into an active ester form with hydroxysuccinimide, and then an amide bond was formed with an amino group to prepare a conjugate in which PCB- # 126 was bound. 3,4,5-trichloroiodobenzene ↓ 3,3 ', 4,5-tetrachloro-4'methoxybiphenyl ↓ 3,3', 4,5-tetrachloro-4'hydroxybiphenyl ↓ 6-ethyl- (3 , 3 ', 4,5-tetrachlorobiphenyl-4'-oxy) hexanoate ↓ 6- (3,3', 4,5-tetrachlorobiphenyl-
4′-oxy) hexanoic acid ↓ 6-N-hydroxysuccinimide- (3,3 ′, 4,
5-tetrachlorobiphenyl-4'-oxy) hexanoate

[Preparation of conjugate for preparing anti-PCB- # 169 antibody] By reacting 2,6-dichlorophenol with an equal amount of a dioxane dibromide complex in dioxane, 4-bromo-2,6- After synthesizing dichlorophenol and protecting the hydroxyl group with a methyl ether group in the same manner as in the synthesis of conjugate # PCB-77, 3,3 ', 4,3 was performed by Grignard-cross coupling reaction.
5,5'-Pentachloro-4'hydroxybiphenyl was obtained. This is reacted with 6-bromohexanoic acid and ethyl ester, and the ethyl ester is hydrolyzed to carboxylic acid to give 6- (3,3 ', 4,5,5'-pentachlorobiphenyl-4'-oxyl. ) Hexanoic acid was synthesized in 6 steps with a total yield of 23%. For binding to the carrier protein, the carboxylic acid was converted into an active ester form with hydroxysuccinimide, and then an amide bond was formed with an amino group to prepare a conjugate in which PCB- # 169 was bound. 4-bromo-2,6-dichlorophenol ↓ 4-bromo-2,6-dichloroanisole ↓ 3,3 ', 4,5,5'-pentachloro-4'methoxybiphenyl ↓ 3,3', 4,5 5'-pentachloro-4'hydroxybiphenyl ↓ 6-ethyl- (3,3 ', 4,5,5'-pentachlorobiphenyl-4'-oxy) hexanoate ↓ 6- (3,3', 4,5, 5'-pentachlorobiphenyl-4'-oxy) hexanoic acid ↓ 6-N-hydroxysuccinimide- (3,3 ', 4,
5,5'-pentachlorobiphenyl-4'-oxy) hexanoate

Example 2 Immunization Production Example 2 Immunization step of mammal and step of obtaining the present antibody Bovine serum albumin is dissolved in distilled water and equilibrated with phosphate buffer (pH 8.0). The resultant was applied to a PD-10 column, and developed and eluted with a phosphate buffer (pH 8.0).
By this operation, bovine serum albumin in a phosphate buffer (pH 8.0) was obtained. 16.7mg bovine serum albumin / 0.5m
l To the phosphate buffer (pH 8.0), 3.88 mg (10 μmol, 1:40 equivalent) of the conjugate obtained in Production Example 1 was added.
A solution prepared by adding and dissolving 0.5 ml of dimethylformamide (DMSO) was added, and the mixture was stirred at room temperature for 30 seconds and left at 4 ° C. overnight. Each reaction solution was applied to a PD-10 column equilibrated with a phosphate buffered saline, and developed and eluted with a phosphate buffered saline. PCB determined from absorbance measurement
The number of derivatives introduced into the conjugate was 10 to 16 molecules per bovine serum albumin molecule, and about 30 to 50% of the available amino groups in the bovine serum albumin molecule were used for binding. First, 100 μg / mouse of each immunogen using Balb / C mice (6 weeks old, female) per group, 50 μl or less
Up to 8-9 immunizations were performed with g / mouse. The antigen was emulsified with an adjuvant, and the route of administration was intraperitoneal and in the footpad of mice.

Example 3 Confirmation of Anti-PCB Serum Antibody Production and Preparation of Hybridoma Cells Mice in which production of a specific antibody against hapten was confirmed,
The antigen conjugate was administered into the tail vein, and three days later, the spleen was excised and spleen cells were prepared. Myeloma cells (P3-
X63 / Ag. 8) and spleen cells were mixed at a ratio of 1: 5, and cell fusion was performed by the polyethylene glycol (PEG) method. RPs containing 10% fetal bovine serum with HAT
The cells are suspended in MI1640 medium, seeded at a density of 2 to 5 × 10 ⁵ cells / well on a 96-well culture microtest plate, and cultured under conditions of 5% carbon dioxide and 37 ° C. From 4 to 5 days from the start of the culture, the medium was exchanged, and screening was performed from 1 week to 10 days, up to the first month, using the culture supernatant of the wells in which the clones were only propagated.

Example 4 Evaluation of Monoclonal Antibody; Measurement of PCB by Competition Inhibition ELISA (Screening of antibody-producing cells by ELISA method) Hydroxysuccinimide ester of PCB derivative 0.02
300 μl of DM in 0.024 mmol of biotin-hydrazide and 0.024 mmol of
After adding SO and stirring, the mixture is reacted at room temperature for 16 hours. After completion of the reaction, reverse phase HPLC (ODS-80T column: Tosoh, solvent: PCB- # 77: 72% acetonitrile line)
ar, PCB- # 126: 80% acetonitrile linear,
PCB- # 169: 90% acetonitrile linear, flow rate: 2 ml / min, 1 cycle: 40 minutes) to prepare a purified biotin-labeled PCB. 10 μl with PBS (-) [phosphate buffered saline without calcium and magnesium]
g / ml of anti-mouse IgG antibody (50 μl)
After incubating for 1 hour at ℃, PB with 0.05% tween20
Each well of the 96-well microtiter plate was washed three times with S (-) (hereinafter abbreviated as "washing solution"). To this PBS
Gelatin (manufactured by BIO-RAD) 300% prepared by (-)
μl was added and left at room temperature for 2 hours (blocking). After washing the 96-well microtiter plate three times with the above washing solution, a culture solution (supernatant) of the hybrid cells was added thereto at 50 μl / well, and reacted at room temperature for 1 hour. After washing the 96-well microtiter plate again three times, the biotin-labeled PCB adjusted to 0.8 μM with 50% DMSO and 0.01 μM, 0.1 μM with 50% DMSO were used.
M, 1 μM unlabeled PCB (PCB- # 77,
PCB- # 126, PCB- # 169) were added in an amount of 25 μl each, followed by reaction at room temperature for 1 hour. Wash wells with the above washing solution
After washing once, 50 μl of commercially available peroxidase-labeled streptavidin (manufactured by Amasham) diluted 1,000-fold with 0.1% gelatin-PBS (−) was added, and after reaction at room temperature for 1 hour, each well was washed three times with the above washing solution. . Substrate solution (3,3 ',
5,5'-tetramethylbenzidine substrate system: K
PL Co.] was added, and the color was developed by incubating at room temperature for 15 minutes. The enzyme reaction was stopped by adding an equal volume of 1 M phosphoric acid, and the color of each well of the microtiter plate was measured using a multi-scanning spectrophotometer (Titertec Co., Ltd.).
Absorbance at 450 nm (control wavelength: 655 nm). In the absence of unlabeled PCB (50% D
The absorbance (in the presence of MSO) was set to 100%, and a well having a high degree of inhibiting color development by an enzyme reaction in the presence of an unlabeled PCB was defined as a PCB-specific antibody production well.

2. (Cloning of antibody-producing cells) The antibody-producing wells identified as described above were subjected to cell cloning by limiting dilution. As a result of cell cloning, cloned cells producing antibodies were obtained.
The obtained cloned cells were cultured in a medium containing 10% fetal bovine serum under conditions of 5% carbon dioxide and 37 ° C., and the resulting culture was used as an antibody solution. Further, the antibody solution was purified by affinity chromatography, and the obtained protein fraction was dialyzed with PBS (-) to obtain the present antibody. The reactivity of the monoclonal antibody derived from the cloned hybridoma was examined by the method described in Example 4 using the antibody obtained by purifying the culture supernatant. Also,
In this process, the anti-mouse I
Effect of gG (Fc recognition) antibody,
Influence of Tween20 (selection of optimal concentration), appropriate combination of anti-PCB antibody, label and competitor (type and concentration of label)
It was determined. Under the resulting test conditions, suitable antibodies were selected. As a measurement system for PCB- # 77, antibody 8-5B-12B (biotin-labeled PCB- # 7
7: 1.6 × 10 −7 M), the antibody 4-6B-19F (biotin-labeled P
CB- # 77: Used at 8 × 10-7M), PCB-16
As the 9 measurement systems, antibody 7-3C-4C (used with biotin-labeled PCB- # 169: 1.3 × 10 −7 M) was selected. Antibody 8-5B-12B was hybridoma PCB
77-B (FE deposited with the National Institute of Advanced Industrial Science and Technology)
RMP-17098). Antibody 4-6B-19F was hybridoma PC
B77-A (Funded by the National Institute of Advanced Industrial Science and Technology
ERM P-17097). Antibody 7-3C-4C was hybridoma PCB
169-E (Funded by the National Institute of Advanced Industrial Science and Technology
ERM P-17099).

FIGS. 1 to 3 show standard curves of coplanar PCB. When 20% inhibition is defined as the detection limit of antigen concentration,
The detection limit value of each measurement system under this condition is PCB- #
In the 77 measurement system, 8.08 × 10 −8 M (corresponding to 23.0 ppb, detection limit value per assay system: 0.59 ng), PCB
-For # 126 measurement system, 2.10 × 10 −8 M (6.85 ppb
, Detection limit per assay system: 0.17 ng), P
In the CB- # 169 measurement system, 1.22 × 10 −8 M (4.40
ppb, the limit of detection per assay system: 0.11 ng).

[Example 5] Reactivity between a PCB other than the object to be measured and the antibody of the present invention (confirmation of specificity of the present antibody) In a real sample (waste oil, environmental sample, etc.) whose object is PCB Since it is presumed that isomers other than the target PCB coexist, evaluation of antibody specificity involves not only comparison of cross-reactivity of individual isomers but also PCB
The antibody specificity was evaluated in a manner that reflects the situation where measurement of the target PCB in the presence of isomers was required.

The anti-PCB- # 77 antibody (8-5B-1
2B) Specificity evaluation For anti-PCB- # 77 antibody (8-5B-12B),
As shown in FIG. 4, a preliminary test was performed in advance to determine the cross-reactivity with a mixture of PCB isomers (Table 1) where cross-reactivity is expected.

[0035]

[Table 1]

From these results, it was determined that the antibody was specific to PCB- # 77 although cross-reactivity with PCB- # 126 was observed. For this reason, PCB- # 1
26, an anti-PCB- # 77 antibody (8-5B
-12B) and the effect of PCB- # 77 on the antigen-antibody reaction were examined. As a result, as shown in FIG.
If the mixed amount of # 126 is 10 times or less, it is considered that the influence on the measurement result is small.

The anti-PCB- # 126 antibody (4-6B-
Evaluation of Specificity of 19F) For the anti-PCB- # 126 antibody (4-6B-19F), as shown in FIG. 6, a preliminary test was performed to determine the cross-reactivity with the PCB isomer. PCB- # 1
The highest reactivity with 26 was observed, but cross-reactivity was also observed with PCB- # 77 and group 11 (a mixture of PCB- # 78 and PCB- # 81). Therefore, PCB- # 77,
Adjust the mixture of PCB- # 78, PCB- # 81, PCB- # 169 to an initial concentration of 1 × 10 −6 M, 1 × 10 −7 M,
Equivalent amount of PCB- # 126 is 2 × 10 −7 M to 1 × 10 −5
M was mixed in the concentration range of M (initial concentration) and measured.
The effect of antibody 126 (4-6B-19F) on the antigen-antibody reaction of PCB- # 126 was examined. The result is shown in FIG.
Shown in In the measurement system of PCB- # 126, it was considered that there was almost no influence of the mixing if the mixing amount was 5 times or less.

The anti-PCB- # 169 antibody (7-3C-
4C) Specificity evaluation For anti-PCB- # 169 antibody (7-3C-4C),
As shown in FIG. 8, a preliminary test was conducted to determine the cross-reactivity with the PCB isomer in order to determine the cross-reactivity with the PCB isomer. Although the reactivity with PCB- # 169 is highest, PCB- # 126 and group 11 (P
Mixture of CB- # 78 and PCB- # 81), group 1
Cross-reactivity with 2 (PCB- # 127) was observed. Therefore, PCB- # 77, PCB- # 126, group 1
1 (equal concentration mixture of PCB- # 78, PCB- # 81),
Group 12 (PCB- # 127) mixture at 1 × 10 −8
M was measured in the presence of 5 × 10 −8 M in the presence of M. As a result, as shown in FIG. 9, in the PCB- # 169 measurement system, if the mixed amount of the PCB that could interfere with the presence of the ppb level PCB- # 169 is twice or less, the measurement result The influence on the measured value is small, but when the amount is more than 10 times, a positive error is given to the measured value.

Example 6 Solvent Resistance of Antibody (Dimethyl Sulfoxide and Methanol) Using each antibody selected in Example 4, dimethyl sulfoxide (DMSO) was obtained by the competitive ELISA method shown in the same method.
Resistance was examined. DMSO was added so that the final concentrations contained in the solution during the competition reaction were 10% to 50%, respectively. FIG. 10 shows the effect of DMSO on the PCB- # 77 antibody, and FIG. 11 shows the effect of DMSO on the PCB- # 126 antibody.
FIG. In the case of the PCB- # 77 antibody shown in FIG.
A decrease in the reaction value was observed when the 77 concentration was on the high concentration side. However, as shown in FIGS.
By decreasing the concentration of O, the measurement range and the detection sensitivity tended to increase. Although not shown in the figure, D
When the concentration of MSO was 70% or more, almost no reactivity was observed. From the above results, this monoclonal antibody
When the final concentration of DMSO was 50%, it was found that each PCB could be measured although the measurement sensitivity was reduced.

Similarly, the resistance of this antibody to methanol (methyl alcohol) was determined by dissolving the coplanar PCB biotin-labeled product in a solution state by dissolving it in a buffer containing 50%, 70%, and 90% of the final concentration of methanol. Was used as a sample. The analysis using each antibody showed that the anti-PCB- # 126 antibody (4-
6B-19F) and anti-PCB- # 77 antibody (8-5B-1)
In 2B), inhibition of the reaction was observed, but at 50%, the reaction was observed. Therefore, these antigen-antibody reactions were sufficiently possible in the methanol concentration range of 0 to 50%. Furthermore, 70% of anti-PCB- # 169 antibody (7-3C-4C)
Since the reaction was observed even with methanol, the effect on the antigen-antibody reaction was minimal in the range of methanol concentration of 0 to 70%, and the measurement was sufficiently possible.

[0041]

According to the method of the present invention, coplanar PCB,
Particularly high toxicity PCB- # 77, PC
It has become possible to provide a monoclonal antibody that specifically reacts with B- # 126 and PCB- # 169 and retains its properties even in an organic solvent (methanol, dimethyl sulfoxide). The antibody is useful for analysis of the compound in an environment such as oil (chemically treated oil or the like), soil, water, food, or a biological sample, particularly in a sample that requires pretreatment for organic solvent extraction. And high-precision analysis that can be processed quickly.

[Brief description of the drawings]

FIG. 1 is a standard curve of PCB- # 77.

FIG. 2 is a standard curve of PCB- # 126.

FIG. 3 is a standard curve of PCB- # 169.

FIG. 4 shows cross-reactivity / single product of anti-PCB- # 169 antibody.

FIG. 5 shows the measurement of PCB- # 77 in the presence of contaminated PCB.

FIG. 6 shows the cross-reactivity / single product of anti-PCB- # 77 antibody.

FIG. 7 shows the effect of mixing other components on the antigen-antibody reaction of the anti-PCB- # 126 antibody.

FIG. 8 shows the cross-reactivity / single product of anti-PCB- # 169 antibody.

FIG. 9 shows measurement of PCB- # 169 in the presence of contaminated PCB.

FIG. 10 shows the solvent resistance of anti-PCB- # 126 antibody.

FIG. 11 shows the solvent resistance of anti-PCB- # 77 antibody.

FIG. 12 shows the solvent resistance of anti-PCB- # 169 antibody.

[Procedure amendment]

[Submission date] June 30, 1999 (1999.6.3)
0)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/08 C12N 15/00 C (C12N 5/10 C12R 1:91) (C12N 15/02 C12R 1: 91) (72) Inventor Masumi Watanabe 103-43 Kitanoyama, Okubo-cho, Uji-city, Kyoto F-term (reference) 4B024 AA11 BA41 GA05 HA15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AB05 BA08 CA25 CA46 4H045 AA11 AA30 DA76 EA50 FA72 GA10

Claims (5)

[Claims] 1. A monoclonal antibody recognizing coplanar PCB, wherein the antigen recognition property does not substantially decrease even in a 50% (v / v) aqueous organic solvent solution. 2. The coplanar PCB having 3, 4, 3 ', 4'
-Tetrachlorobiphenyl (PCB- # 77), 3,
4,5,3 ', 4'-pentachlorobiphenyl (PCB
-# 126) or 3,4,5,3 ', 4', 5'-hexachlorobiphenyl (PCB- # 169). 3. An antigen-antibody complex is formed by contacting a coplanar PCB present in an aqueous solution of an organic solvent or a buffer solution containing no organic solvent (including an aqueous solution) with the monoclonal antibody according to claim 1 or 2; A method for measuring coplanar PCB, comprising measuring the amount of body formation. 4. An oily specimen containing a coplanar PCB is brought into contact with an organic solvent to obtain a coplanar PCB in the oily specimen.
In an organic solvent, and coplanar PC in the organic solvent.
A method for measuring coplanar PCB in an oily sample, wherein B is measured using a monoclonal antibody and converted to coplanar PCB in an oily sample. 5. The method for measuring coplanar PCB in an oily sample according to claim 4, wherein the monoclonal antibody is any one of the monoclonal antibodies according to claim 1.