JPH0937784A - Production of monoclonal antibody to be specifically reacted with pyrethroid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out a simply and readily treatable analysis high in accuracy in analyzing an ester of chrysanthemumic acid or 3-(2,2-dichlorovinyl-2,2- dimethylcyclopropanecarboxylate and pyrethrin alcohol, for example, in a food, in an environment such as soil, etc., or in an organism. SOLUTION: A mammal is immunized against a composite material obtained by directly bonding the carbonyl group of chrysanthemumic acid or 3-(2,2- dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate to a high-molecular weight carrier molecule and an immunological qualified B cell capable of forming an antibody against the composite material is developed from the mammal. The immunological qualified B cell is fused with a tumor cell capable of continuously subjecting the immunological qualified B cell to cell division to form a hybrid cell. The objective monoclonal antibody to be specifically reacted with an ester of chrysanthemumic acid or 3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropanecarboxylate and pyrethrin alcohol is liberated from the hybrid cell and collected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a monoclonal antibody which specifically reacts with an ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol.

[0002]

2. Description of the Prior Art Esters of chrysanthemic acid or dichlorovinyl chrysanthemic acid with pyrethroid alcohol have high insecticidal activity,
Further, since it has low toxicity to warm-blooded animals, it is used as a household insecticide, for example. Conventionally, a gas chromatography method or a high performance liquid chromatography method has been mainly adopted for the analysis of such pyrethroids in the environment such as crops, soils, foods, or biological samples. However, this analysis method has problems that it requires a considerable amount of labor and time to prepare a cleanup of a sample, and that a measurement apparatus, equipment, and the like require a high cost. In addition, analysis of residual pesticides requires a large number of analyzes, so simplicity and speed are important. Therefore, it has been desired to develop a simpler and faster method for analyzing residual pesticides as compared with conventional methods.

[0003]

Therefore, an attempt was made to establish a simple and rapid method for analyzing pyrethroids by an immunological detection method using an antibody. The most important factor in the immunological detection method is an antibody that specifically reacts with pyrethroid, but the ability of the produced antibody is determined by which method is used to produce an antibody that specifically reacts with pyrethroid. To do. Therefore, a better antibody production method is needed.

[0004]

Under these circumstances, the present inventors have sincerely studied a better method for producing an antibody, and as a result, have found that a certain compound has a high molecular weight at a specific functional group site of the compound. After immunizing a mammal with a complex directly bound to a carrier molecule as an antigen, immunocompetent B cells capable of forming an antibody against the complex are allowed to emerge from the mammal, and the immunocompetent B cells are continuously treated. A method of producing a hybrid cell by fusing with a tumor cell capable of cell division, and releasing and collecting a monoclonal antibody specifically reacting with an ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol from the hybrid cell. Found and completed the present invention. That is, the present invention provides chrysanthemic acid or dichlorovinyl chrysanthemic acid (3- (2,
2-dichlorovinyl) -2,2-dimethylcyclopropanecarboxyla
te), after immunizing a mammal against the complex in which the carbonyl group is directly bound to a high molecular weight carrier molecule, immunocompetent B cells capable of forming an antibody against the complex from the mammal are emerged, A monoclonal antibody that fuses the immunocompetent B cell with a tumor cell capable of continuous cell division to generate a hybrid cell, and specifically reacts with the ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol from the hybrid cell. The present invention provides a method for producing an antibody (hereinafter referred to as the method of the present invention), which comprises releasing and collecting the antibody.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol (hereinafter referred to as the pyrethroid) include compounds such as phenothrin, permethrin, cypermethrin, cyfluthrin, and pyrethrin. You can

The method of the present invention comprises the following steps. [Complete antigen (immunogen) formation step of chrysanthemic acid or dichlorovinyl chrysanthemic acid] The carbonyl group of chrysanthemic acid or dichlorovinyl chrysanthemic acid is directly bonded to a high molecular weight carrier molecule to obtain a complex. The chrysanthemic acid or dichlorovinyl chrysanthemic acid used is preferably highly pure. More preferably, it is about 99% or more.
Therefore, if necessary, it is purified in advance by a usual method such as high performance liquid chromatography. The high-molecular-weight carrier molecule used has a reactive group that can be freely used in the ligation reaction with the carbonyl group site of chrysanthemic acid or dichlorovinyl chrysanthemic acid (incomplete antigen), and is linked to the incomplete antigen. Any macromolecular compound that can impart immunogenicity to it or enhance their immunogenicity already present. In particular, macromolecular compounds containing freely available reactive amino groups are preferred. For example, a protein rich in lysine having a molecular weight of about 10,000 to about 150,000 can be mentioned. Specifically, bovine serum albumin (BS
A: molecular weight 66200), human serum albumin (HSA: molecular weight 58000), rabbit serum albumin (RSA: molecular weight
68000), goat serum albumin (GSA: molecular weight 68000)
, Ovalbumin (ovalbumin: molecular weight 45000)
Alternatively, keyhole limpet hemocyanin (KLH: molecular weight> 1000000) can be used. As long as other macromolecular compounds meet the above requirements, they can be used as carrier molecules, such compounds being, for example, porcine thyroglobulin, B2 microglobulin, hemocyanin, immunoglobulins. , Toxins (cholera toxin, tetanus toxin, diphtheria toxin, etc.), polysaccharides, lipopolysaccharides, natural or synthetic polyadenylic acid and polyuridylic acid, polyalanyl and polylysine polypeptides, or cell membrane components such as formalin- or glutaraldehyde-treated red blood cell membranes. You can The bond between the polymer carrier molecule and the carbonyl group site of chrysanthemic acid or dichlorovinyl chrysanthemic acid (incomplete antigen) is, for example,
Chem. Pharm. Bull. 31 ,, (11), 4001-4007 (1983), or the active ester method by H. Hosoda et al. Or J. Bio.
BFErla described in L. Chem., 234, 1090-1094 (1959).
It can be produced by the mixed acid anhydride method by Nger et al. [bonding chrysanthemic acid or dichlorovinyl chrysanthemic acid having a reactive carboxyl group to a reactive amino group of a high molecular weight carrier molecule]. Specifically, for example, carbodiimide, alkylchlorocarbonate, preferably 1-ethyl-3-
The carboxyl group of the incomplete antigen is attached to one of the reactive amino groups of the high molecular weight carrier molecule using (3-dimethylaminopropyl) carbodiimide hydrochloride, isobutylchlorocarbonate.

[Mammalian immunization step and present antibody acquisition step] To immunize mammals such as rats, rabbits and dogs with the thus obtained complex, for example, J. ASSOC. OFF. ANAL. CHEM. 70 ( 6) 1
It is carried out using a conventional immunization method such as WH Newsome described in 025-1027 (1987) etc., for example, by one or more administrations of the complex. For example, administration of 7 to 30 days, especially 2 to 3 times at intervals of 12 to 16 days is preferable. Injection, which may be intravenous, intraperitoneal or subcutaneous, is the preferred dosage form. Furthermore, the combination of subcutaneous injection and intraperitoneal injection is particularly preferable. In this case, the complex is an appropriate buffer solution, for example, a sodium phosphate buffer solution containing one of commonly used adjuvants such as complete Freund's adjuvant (Aracel A, Bayol F, a mixture of killed tuberculosis bacteria) and the like. It is used by dissolving in etc.
Here, an adjuvant means a substance that, when administered together with an antigen, nonspecifically enhances an immune response to the antigen. After leaving the above mammals untreated for 0.5 to 4 months, for example, 10 μg to 100
Another dose is given at a dose of 0 μg, in particular 25 μg to 500 μg of the complex. Immunocompetent B cells capable of forming an antibody against the complex are isolated from an immunized mammal 3 days to 2 months after the last administration by an ordinary method, and the immunocompetent B cells are continuously isolated. Hybrid cells that are fused with tumor cells capable of cell division, are isolated, and after selection, the hybrid cells that produce the desired antibody are cloned and the hybrid cells are used to produce monoclonal antibodies. By culturing in vitro or in vivo, it is possible to produce a monoclonal antibody (hereinafter referred to as the present antibody) that has a high degree of specificity and affinity and that specifically reacts with the present pyrethroid. .

The present antibody recognizes an ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol. For example, an IC ₅₀ (concentration of a test substance that inhibits the specific total binding rate by 50%) determined by a direct non-competitive inhibition method (ELISA method) described later is about 0.01 to 1.4 μg / m 2.
It has an affinity of about l. The antibody has a higher affinity for the above-mentioned pyrethroids having no cyano group at the alcohol site.

Examples of the use of the present antibody include analysis of the present pyrethroid by an indirect competitive inhibition method (ELISA method), a direct non-competitive inhibition method (ELISA method) and the like. The analysis is a simple and highly accurate analysis method that can be processed quickly.

The direct non-competitive inhibition method can be carried out, for example, by the following method. Basically, a certain amount of the first antibody bound to the solid support (that is, the present antibody) is reacted with the free pyrethroid contained in the sample, and then the unreacted first antibody is reacted. The antibody and the antigen of the antibody, which are labeled directly or through a spacer, are reacted with chrysanthemic acid or dichlorovinyl chrysanthemic acid and an ester of pyrethroid alcohol, and then the bound antigen is based on the label. By quantifying, the amount of the first antibody (that is, the present antibody) bound to the solid support, which reacted with the present pyrethroid in the free state contained in the sample, was calculated, and the amount contained in the sample from the calculated value. The amount of free pyrethroid in the free state is measured.

When indirectly linked via a spacer, the spacer used is a compound containing at least one or more reactive groups capable of forming a covalent bond with the freely available reactive groups of the high molecular weight carrier molecule. Is. For example, there may be mentioned compounds containing between 2 and 16 crosslinkable carbon atoms and having as a reactive group one or more reactive groups such as amino groups, carboxyl groups and the like. Specifically, the general formula H ₂ N (CH ₂ ) n
COOH (n is an integer from 2 to 16) is preferred. The spacer can be linked to chrysanthemic acid or dichlorovinyl chrysanthemic acid (incomplete antigen) by using 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. .

The indirect competitive inhibition method can be performed, for example, by the following method. Basically, competition between chrysanthemic acid or dichlorovinyl chrysanthemic acid, which is an antigen bound to a solid support, and the free pyrethroid contained in a sample, for example, chrysanthemum, which is an antigen bound to a solid support, is used. It is based on the competition between the acid or dichlorovinyl chrysanthemic acid and the present pyrethroid, which is the free antigen of the first antibody recognized by the labeled second antibody. In this context, the binding of the antigen chrysanthemic acid or dichlorovinyl chrysanthemic acid to the solid support is directly or via a spacer and / or a high molecular weight carrier molecule not recognized by the first antibody (ie the present antibody). It can happen indirectly. Here, the high molecular weight carrier molecule that is not recognized by the first antibody (that is, the present antibody) is a high molecular weight carrier molecule that can be used in the step of completely converting chrysanthemic acid or dichlorovinyl chrysanthemic acid (immunogen). Of these, high molecular weight carrier molecules that are not used in the production of primary antibodies. Also, when chrysanthemic acid or dichlorovinyl chrysanthemic acid, which is an antigen, is indirectly bound through a spacer and / or a high-molecular-weight carrier that is not recognized by the first antibody, these bonds include the above-mentioned chrysanthemic acid or dichlorovinyl chrysanthemic acid. A method similar to or similar to the step of converting an acid to a complete antigen (immunogen) can be used.

When indirectly linked via a spacer, the spacer used is a compound containing at least one or more reactive groups capable of forming a covalent bond with the freely available reactive groups of the high molecular weight carrier molecule. Is. For example, there may be mentioned compounds containing between 2 and 16 crosslinkable carbon atoms and having as a reactive group one or more reactive groups such as amino groups, carboxyl groups and the like. Specifically, the general formula H ₂ N (CH ₂ ) n
COOH (n is an integer from 2 to 16) is preferred. The spacer can be linked to chrysanthemic acid or dichlorovinyl chrysanthemic acid (incomplete antigen) by using 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. .

Solid supports used for direct or indirect binding of the antigen chrysanthemic acid or dichlorovinyl chrysanthemic acid include, for example, plastic surfaces of microtiter plates or test tubes, polystyrene, polypropylene, polyvinyl chloride, glass or plastic. And the like, surface of beads, filter paper, dextran, surface of strips of cellulose or nitrocellulose or other similar material. The antigen chrysanthemic acid or dichlorovinyl chrysanthemic acid is directly bound to these solid supports or indirectly through a spacer and / or a high molecular weight carrier molecule which is not recognized by the first antibody (the present antibody) (hereinafter, In the description of coating), for example, the solid support material is activated in advance by a usual method using glutaraldehyde, cyanogen bromide or the like. After the coating solution is added to the activated solid support material, the mixture is incubated to form a carrier with a conjugate of serum albumin of a mammal of the same species as the mammal producing the first antibody and chrysanthemic acid or dichlorovinyl chrysanthemic acid. Coat the surface. The coating liquid used here is, for example, 140
An example is a phosphate buffer (pH 7.4) of about 10 mM containing mM sodium chloride. As the coating conditions, for example, the concentration of chrysanthemic acid or dichlorovinyl chrysanthemic acid which is an antigen or chrysanthemic acid or dichlorovinyl chrysanthenic acid which is an antigen having a high molecular weight carrier molecule which is not recognized by the spacer and / or the first antibody is For example, chrysanthemic acid or dichlorovinyl chrysanthemic acid, which is an antigen, can be preferably about 0.05 μg / ml to about 1 μg / ml. When using a 96-well microplate, the amount used is preferably about 0.1 ml / well. The coating time is, for example, about 4 ° C., about 6 hours to 24 hours.
Time can be increased, preferably overnight. As a specific example of the above, for example, a 96-well microtiter plate made of polystyrene is used as a solid support, and a complex for producing the first antibody (chrysanthemic acid or dichlorovinyl chrysanthemic acid is used as a high molecular weight carrier molecule). When serum albumin of animal such as bovine or keyhole limpet hemocyanin is used as a high molecular weight carrier molecule used for (directly bound to), and when chrysanthemic acid or dichlorovinyl chrysanthemic acid is indirectly bound to a solid support material. As the high molecular weight carrier molecule used, it is possible to use serum albumin of another species of animal such as goat, which is a high molecular weight carrier molecule which is not recognized by the first antibody. In addition, after coating the carrier with a complex of serum albumin of a mammal of the same species as the mammal producing the first antibody and chrysanthemic acid or dichlorovinyl chrysanthemic acid,
Serum of a mammal of the same species as the mammal producing the first antibody, which is a protein other than serum albumin of the mammal of the same species as the mammal producing the first antibody, in order to prevent nonspecific binding of the antibody. It is desirable to block the part where albumin is not adsorbed. For this purpose, for example, a method of incubating a skim milk solution of about 3% and a carrier at about 20 ° C. for about 2 hours and the like can be mentioned as a simple method. The carrier thus obtained contains washing buffer (eg NaCl 0.8% (W / V), KCl 0.02% (W / V) and Tween 20).
A 10 mM phosphate buffer (pH 7.2) containing 0.2% (V / V) is preferred. ) After using, wash.

The chrysanthemic acid or dichlorovinyl chrysanthemate directly or indirectly bound to the solid support thus obtained is then the present pyrethroid and the first antibody (the present antibody) which are the antigens to be detected contained in the sample. Antibody) and the mixture is incubated. The pyrethroid, which is the antigen to be detected and is contained in the sample, may be present in this case in a free form or as a component in the environment such as water or crops, soil, foods, or the biological sample. After incubation for about 10 minutes to about 2 hours, the mixture is incubated with a second antibody labeled with an enzyme or the like that recognizes and binds to the first antibody (the present antibody). Examples of the secondary antibody labeled with this enzyme or the like include, for example, peroxidase, alkaline phosphatase, β-D
There may be mentioned an antibody against the first antibody (the present antibody) bound with an enzyme such as galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase. As a specific example, the first antibody (this antibody)
When a rabbit antiserum is used as the antibody, the second antibody is preferably anti-rabbit immunoglobulin (IgG) or goat immunoglobulin (IgG) bound to peroxidase. The rabbit IgG goat IgG
Is commercially available and is easily available. When labeled with peroxidase, it combines with hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent to give a brown or yellow color. In the case of labeling with glucose oxidase, for example, 2,2′-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) etc. is used as a substrate. The color developed by the enzymatic reaction between the enzyme and the substrate is measured as the absorbance using a device such as a multi-scanning spectrophotometer.

The present antibody is suitable for use under field conditions because it contains at least one kind of the present antibody as a reagent, and can detect the pyrethroid easily and accurately with high accuracy. In addition, means for immunological detection / analysis of the present pyrethroid in the form of a test kit may be included. The above-mentioned test kit may contain, for example, the following components: (1) A high molecular weight carrier which is not recognized by the antigen chrysanthemic acid or dichlorovinyl chrysanthemic acid directly or by the spacer and / or the first antibody (the present antibody) A solid support that is indirectly bound via a molecule,
(2) A reagent containing the antibody of the present invention, (3) a standardized solution of the present pyrethroid which is an antigen, (4) a second antibody labeled with an enzyme or the like that recognizes and binds to the first antibody (the present antibody). Reagents included, (5) buffer, (6) polypeptides that prevent non-specific adsorption and aggregate formation, additives such as surfactants, and (7) pipettes, reaction vessels, calculation curves, etc. The solid supports described above have a very wide range of designs and can have very different shapes depending on the particular purpose for which they are intended. Examples include plates, balls, plates, small rods, cells, small bottles, small tubes, fibers and nets. As a specific example, a microtiter plate made of a transparent plastic material such as polyvinyl chloride or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod, or the like can be used.

In the above-mentioned method, when a water system in the environment such as paddy water or tap water is used as a sample, the pyrethroid concentration in the sample should be measured in the range of about 0.01 μg / ml to about 10 μg / ml. You can Therefore, when the concentration of the present pyrethroid in the sample is higher than the above concentration, the sample is appropriately diluted before use. When the concentration is low, the sample is appropriately concentrated before use. When non-aqueous substances in the environment such as crops, soils, foods or biological samples are used as samples, after extracting the pyrethroid from the sample with a solvent such as methanol, the methanol extract is used, for example, the above-mentioned washing buffer. Use it after diluting it with the buffer solution of. The sample solution containing the present pyrethroid thus prepared is mixed with a buffer solution containing an excess amount of the first antibody (the present antibody), and the mixture is reacted by incubating at about 20 ° C. overnight. At this time, the first
It is desirable to dilute the antibody about 3,000 to about 5,000 times to react with the present pyrethroid in the sample, especially about 5,
A dilution of about 000 times can be more desirable.
That is, the first antibody diluted about 2,500 times is mixed with the sample solution containing the same amount of the present pyrethroid and mixed at about 20 ° C.
It is desirable to react overnight at. As the diluent, for example, one having the same composition as the above-mentioned washing buffer can be used. If necessary, 3% as a protective protein may be used to stabilize the first antibody when it is diluted too much.
It is desirable to add skim milk, etc.

The unreacted first antibody contained in the reaction solution of the sample and the first antibody prepared as described above is treated with serum albumin and chrysanthemic acid or dichloro of a mammal of the same species as the mammal producing the first antibody. The reaction is carried out by binding to a solid support whose surface is coated with a complex with vinyl chrysanthemic acid. Regarding this reaction condition, it is desirable to add a reaction solution of the sample and the first antibody to the solid support and to react at about 20 ° C. for about 1 hour. After the reaction, after washing the carrier with the above washing buffer, labeled with an enzyme or the like,
Further, it is subjected to reaction with an antibody (second antibody) against the first antibody derived from a mammal of a species different from the mammal producing the first antibody.

Hereinafter, the present invention will be described with reference to examples, but it goes without saying that the present invention is not limited to the following examples.

[0020]

【Example】

Production Example 1 Preparation of Complex (Linking of High Molecular Weight Carrier Molecule in Complete Antigen (Immunogen) Step and Linking of High Molecular Weight Carrier Molecule to Antigen Bound to Solid Support) Chrysanthemic Acid (hereinafter KCA) 50 mg of dichlorovinyl chrysanthemic acid (hereinafter referred to as DCPI) was dissolved in 4 ml of anhydrous dioxane, 100 μl of N-methylmorpholine was added to the solution, and the mixture was stirred at 10 ° C. for 20 minutes. Then, 40 μl of isobutyl chlorocarbonate was added little by little and stirred for 15 minutes.
After dissolving 80 mg of ovalbumin (manufactured by Wako Pure Chemical Industries, Ltd.) or 80 mg of bovine serum albumin (manufactured by Sigma) in 4.3 ml of distilled water,
2.6 ml of dioxane was added dropwise to the solution at 10 ° C. After dropping
The KCA or DCPI solution prepared above was gradually added dropwise to the solution adjusted to pH 9 with 1N NaOH while maintaining the pH at 9 with 1N NaOH. After dropping, the mixture was reacted at 10 ° C for 4 hours. After the reaction was completed, the obtained reaction product was placed in a dialysis tube, and 10 mM Tris-HCl (pH
It was dialyzed against 7.5) for 3 days at 4 ° C. During the dialysis, the buffer was changed twice. After dialysis, the reaction product was freeze-dried to obtain various complexes. Among the complexes thus obtained, the complex with ovalbumin was used as an immunogen. Also, the complex with bovine serum albumin is an antigen that is indirectly bound to the solid support (for ELISA).
Used as

Production Example 2 Immunization step of mammals and step of obtaining this antibody 100 μg of the complex with ovalbumin obtained in Production Example 1 was added to Dulbecco's phosphate buffer [KCl 200 mg / l,
KH ₂ PO ₄ 200 mg / l, NaCl 8 g / l, NaHPO ₄ 1.15 g / l, below, P
It is written as BS (-). ] Dissolved in 50 μl. An equal amount of complete Freund's adjuvant (manufactured by Yatron) was added to this aqueous solution,
Mixed well. 100 μl of the obtained mixture was added to mouse (BALB
/ cA, female, 6 weeks old, 20 g) was subcutaneously injected (first immunization). One month later, 1/4 of the initial immunization amount was mixed with an equal amount of incomplete Freund's adjuvant (manufactured by DIFCO LABORATORIES), and the mixture was additionally administered by subcutaneous injection (second immunization). Two months later,
Equivalent to 1/4 volume of incomplete Freund's adjuvant (DIFCO
(Manufactured by LABORATORIES) and then the mixture was additionally administered (final immunization) by intraperitoneal injection. 3 of the last dose
The spleen was removed from the immunized test mice after one day, and the removed spleen was removed from the Iscove's modified Dulbecco's medium [Iscove, N &
Melchers, F., J. Exp. Med., 147,923 (1978), hereinafter referred to as Iscove's medium. ], The cells were taken out from the spleen into Iscove's medium to obtain a cell suspension. Large solids were removed by filtering the cell suspension through a stainless mesh. The resulting spleen cells and mouse myeloma cells P3-X63-Ag8.653 [Kearney.JF, Radoruch,
A., Liesegang, B. & Rajewsky, K., J.Immumol., 123,1548 (1
979)] was washed three times with Iscove's medium, mixed at a cell number ratio of 5: 1 (spleen cells: myeloma cells), and centrifuged (1,200 rpm, 5 min, 25 ° C) to precipitate the cells. Got 1m of 50% polyethylene glycol (molecular weight 1,500) solution preheated to 37 ℃ in the obtained cell pellet
l was added slowly to fuse the cells. The cell fusion was stopped by adding 10 ml of Iscove's medium and 1 ml of fetal bovine serum (hereinafter referred to as FBS). The cells thus fused were treated with HAT medium (ISCOF / 10% F).
Hypoxanthine 100 μM and aminopterin were added to the BS medium.
0.4 μM, thymidine added at 1.6 μM) 2 × 10 ⁵ cells / ml
Was suspended in a 96-well microtiter plate and cultured at 37 ° C. in the presence of 5% carbon dioxide for 10 to 14 days. After culturing, the culture solution of the hybrid cells was screened by the following method for the presence or absence of antibody production against the complex with bovine serum albumin obtained in Example 1. 1. (Screening of antibody-producing cells by ELISA) 4 μl of the complex with bovine serum albumin obtained in Example 1 was used.
It was diluted with PBS (-) at a concentration of g / ml, the diluted solution was dispensed into a 96-well microtiter plate at 50 µl / well, and left at 4 ° C overnight to immobilize. Next, after removing the coating solution, a microtiter plate coated with a complex with bovine serum albumin was borate buffer (85 mM borate buffer (pH 8.0), 62 mM NaCl,
Hereinafter referred to as BBS. ], Wash once by immersing in
The water in the plate was removed by tapping on a paper towel with the upper surface of the microtiter plate facing down at 4 ° C. Bovine serum albumin was added to this (1%)
After adding 250 μl / well of the prepared BBS, the mixture was allowed to stand at room temperature for 1 hour (blocking). This 96-well microtiter plate was washed three times with BBS, and then 50 μl / well of a culture solution (supernatant) of hybrid cells was added to it and reacted at room temperature for 1 hour. This 96-well microtiter plate was washed again three times with BBS and then diluted 1,000 times with an antibody diluent (0.3% bovine serum albumin / BBS) to obtain commercially available peroxidase-labeled anti-mouse immunoglobulin (IgG) goat immunoglobulin (IgG). ) [Organon Technica] was added at 100 μl / well and reacted for 1 hour. After the reaction, the plate was washed 3 times with BBS, and then the substrate solution of peroxidase [100 mM Sodium-acetate buffer (pH5.5), 100 μg /
ml3,3 ', 5,5'-Tetramethylbenzidine, 0.006 % H 2 O ]
Was added and developed by incubating for 10 minutes at room temperature. Then, the enzymatic reaction was stopped by adding an equal amount of 1N sulfuric acid, and the color development in the microtiter plate was measured by measuring the absorbance at 450 nm using a multi-scanning spectrophotometer (manufactured by Titer Tech). In this way, antibody producing wells were identified. 2. (Cloning of antibody-producing cells) The antibody-producing wells identified as described above were subjected to cell cloning by the limiting dilution method. As a result of cell cloning, cloned cells producing an antibody against the complex with bovine serum albumin were obtained. The obtained cloned cells were cultured in Iscove's medium containing 10% FBS at 5% CO _{2 and} 37 ° C.
The cells were cultured under the conditions, and the culture solution was used as an antibody solution. Further, the antibody solution was purified by affinity chromatography using Avid AL gel (manufactured by Cypress), and the obtained protein fraction was dialyzed with PBS (-) to obtain the present antibody. The results are shown in Table 1.

[0023]

[Table 1] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Incomplete antigen This antibody subclass ━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Chrysanthemic acid (KCA) KCA226 IgG2a KCA190 IgG2b ━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Dichlorovinyl DCPI4 IgG2b Chrysanthemic acid DCPI4-5 IgG2a (DCPI) DCPI4-3 IgG2a DCPI4-17 IgG2b DCPI6 IgG1 ━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ The cloned cells obtained in this way are currently the Institute of Biotechnology, Institute of Industrial Science and Technology. Has been deposited with. KCA2
In the case of the 26 antibody-producing cells, the identification for the identification is "Hybridoma KCA226", and the deposit number is FERM P-15051 (deposit date: July 20, 1995).
Day).

Test Example 1 Analysis by Indirect Competitive Inhibition Method Cattle obtained in Example 1 by the same method as the indirect competitive inhibition method (ELISA method) shown in Example 2 (screening of antibody-producing cells by ELISA method). A 96-well microtiter plate on which a complex with serum albumin was immobilized was prepared, and borate buffer [85 mM Borate-buffer (pH 8.
0), 150 mM NaCl], and a solution of the present pyrethroid of the following (Table 2) at a predetermined concentration was dispensed. Further, an equal amount of the antibody solution [KCA226] obtained in Example 2 was added and immediately mixed, and reacted at room temperature for 1 hour. After washing this 96-well microtiter plate three times with BBS, add 100 μl / well of peroxidase-conjugated anti-mouse IgG antibody diluted 1,000-fold with antibody diluent (0.3% bovine serum albumin / BBS) for 1 hour. Reacted After the reaction, the plate was washed 3 times with BBS, and then the substrate solution of peroxidase [100 mM Sodium
-acetate buffer (pH5.5), 100 μg / ml 3,3 ', 5,5'-Tetr
amethylbenzidine, 0.006% H ₂ O] was added to develop the color. After 10 minutes, an equal amount of 1N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured. The combinations (incomplete antigen, high molecular weight carrier molecule, present pyrethroid) are shown in Table 2.

[0025]

[Table 2] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Incomplete antigen High molecular weight carrier molecule This pyrethroid ━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Chrysanthemic acid (KCA) Bovine serum albumin pyrethrin ━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Dichlorovinyl bovine serum albumin permethrin chrysanthemate (DCPI) ━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━

As a result, in the indirect competitive inhibition method, the present antibody [KCA226] recognized the present pyrethroid well.

Test Example 2 Analysis by Direct Noncompetitive Inhibition Method KCA was labeled with peroxidase by the active ester method. KCA 0.625 μmol (255 μg) in DMF 25 μl and 5
2.5 mg of a 6.5 mg / ml Dicyclocarbodiimide / DMF solution was added, and the mixture was reacted at room temperature for 1 hour. After the reaction, 20 μl of 1M NaHCO ₃ solution and 125 μl of 20 mg / ml peroxidase solution were added, and the mixture was further reacted at room temperature for 3 hours. Next, in advance
The KCA-peroxidase conjugate was purified using a PD-10 column (Pharmacia) equilibrated with BBS.
The present antibody [KCA 226] obtained in Example 2 was dissolved in a 50 mM NaHCO ₃ solution at a concentration of 8 μg / ml, the solution was dispensed into a 96-well microtiter plate at 50 μl / well, and the mixture was diluted at 4 ° C. It was solidified by allowing it to stand overnight. Next, after removing the coating solution, the microtiter plate coated with the antibody was immersed in BBS to
The microtiter plate was washed twice, and the water in the plate was removed by gently striking it on a paper towel with the upper surface of the microtiter plate facing down at 4 ° C. After adding BBS containing bovine serum albumin (1%) at 250 μl / well,
It was left at 25 ° C for 1 hour (blocking). After washing this 96-well microtiter plate 3 times with BBS,
100 μl of a solution of pyrethrin diluted to the specified concentration with BBS
/ Well was added and reacted at room temperature for 1 hour. After washing this 96-well microtiter plate 5 times with BBS, the purified conjugate of KCA and peroxidase (0.4 μg / ml) was added.
100 μl / well was added, and the mixture was reacted at room temperature for 1 hour. The 96-well microtiter plate was washed again with BBS 5 times, and then the substrate solution of peroxidase [100 mM Sodium-a
cetate buffer (pH5.5), 100 μg / ml 3,3 ', 5,5'-Tetram
ethylbenzidine, 0.006% H ₂ O] was added, and the mixture was added at room temperature for 10
Color was developed by incubating for minutes. Then, by adding an equal amount of 1N sulfuric acid, the enzymatic reaction was stopped, and the color development in the microtiter plate was measured using a multi-scanning spectrophotometer (Titer Tech).
Was used to measure the absorbance at 450 nm. as a result,
This antibody [KCA 226] contains pyrethrin at about 0.01-1.0 μg / ml.
It was recognized in the concentration range of (Fig. 2). Then, it was confirmed that simple and rapid processing with pyrethrin enables highly accurate analysis.

Test Example 3 Reactivity of this antibody with various pyrethroids Using the direct non-competitive inhibition method shown in Test Example 2, the reactivity of this antibody [KCA 226] with various pyrethroids was examined. The tested pyrethroid was made into a solution state by dissolving it in BBS. The result shows the absorbance when using BBS without pyrethroid as a sample.
The concentration of the substance to be tested (pyrethroid) that inhibits 50% was defined as IC ₅₀ (μg / ml). Table 3 shows the results.

[0029]

[Table 3] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Test Pyrethroid IC ₅₀ (μg / ml) Remark ━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Phenothrin 0.17 Pyrethroid permethrin 1.0 Cyfluthrin 1.4 Pyrethrin 0. 11 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━ 40.0 Other pyrethroid fluvalinates> 40. 0 Cycloprotoline> 40.0 Etofenprox> 40.0 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

Test Example 4 Methyl alcohol resistance of the present antibody Using the direct non-competitive inhibition method shown in Test Example 2, the methyl alcohol resistance of the present antibody [KCA 226] was examined.
Methyl alcohol final concentration 0%, 10%, 20
Pyrethrin dissolved in BBS containing 10%, 30% and 40% was used as a sample. The results are shown in Fig. 3. Analysis using this antibody [KCA 226]
It was sufficiently possible when the concentration of methyl alcohol added was in the range of 0% to 40%.

[0031]

INDUSTRIAL APPLICABILITY The method of the present invention enables the production of a monoclonal antibody that specifically reacts with an ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol.
The antibody enables, for example, simple and rapid high-accuracy analysis in the analysis of an ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol in an environment such as crops, soil, foods or a biological sample. did.

[Brief description of drawings]

FIG. 1 is a diagram showing a synthetic pathway of a complex of chrysanthemic acid or dichlorovinyl chrysanthemic acid and ovalbumin, or a complex of chrysanthemic acid or dichlorovinyl chrysanthemic acid and bovine serum albumin.

FIG. 2 is a diagram showing an analysis by a direct non-competitive inhibition method (ELISA method).

FIG. 3 is a diagram showing an analysis by a direct non-competitive inhibition method (ELISA method) for examining methyl alcohol resistance of the present antibody. The final concentration of methyl alcohol is 0%, 10%, 20%, 30%, 4 in the order of white square symbol, black circle symbol, white circle symbol, white triangle symbol, and black square symbol.
Represents 0%.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:91) (72) Inventor Hideo Kaneko 8-12-6 Mikagehonmachi, Higashinada-ku, Kobe-shi, Hyogo

Claims (2)

[Claims] 1. A chrysanthemic acid or a dichlorovinyl chrysanthemic acid (3- (2,2-dichlorovinyl) -2,2-dimethylcyclo).
immunizing a mammal against a complex in which the carbonyl group of (propanecarboxylate) is directly bound to a high molecular weight carrier molecule, and then allowing the mammal to develop immunocompetent B cells capable of forming an antibody against the complex, A monoclonal antibody that fuses the immunocompetent B cell with a tumor cell capable of continuous cell division to generate a hybrid cell, and specifically reacts with the ester of chrysanthemic acid or dichlorovinyl chrysanthemic acid and pyrethroid alcohol from the hybrid cell. Of releasing antibody and collecting the antibody 2. A method for producing an antibody, wherein the pyrethroid according to claim 1 is a compound having no cyano group at its alcohol site.