JP2001089484A - Hapten compound of tricyclazole, antibody and assay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hapten compound of a tricyclazole (5-methyl-1,2,4- triazolo[3,4-b]benzothiazole) analog compound and an antibody and to provide a method of immunochemical assay using the antibody. SOLUTION: This hapten compound has a structure in which a spacer arm and a functional group for bonding are bonded by a covalent bond to the tricyclazole analog compound or its part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to 5-methyl-1,
The present invention relates to a hapten compound of 2,4-triazolo [3,4-b] benzothiazole (hereinafter, referred to as “tricyclazole”), an antigen, an antibody, and a fragment thereof.

[0002] The present invention further relates to an immunochemical assay using the antigen, antibody and fragment thereof.

[0003]

2. Description of the Related Art Tricyclazole is represented by the following formula (3):

[0004]

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Is a compound having a structure represented by the following formula: More specifically, tricyclazole is a rice blast control agent that inhibits melanin biosynthesis in the blast fungus appressorium,
By reducing the physical strength of the appressorium wall, it prevents the invasion of bacteria by rice.

[0006] The tricyclazole agent has a systemic property to penetrate the rice body, is absorbed from all parts of rice and is distributed in the body, prevents the invasion of blast fungus, and shows a stable preventive effect. There is aftereffect. It also has the effect of suppressing spore formation of the blast fungus, reducing the pathogenicity of spores, and preventing secondary infection. Tricyclazoles are effective when applied prophylactically. For leaf blast, spray it when it is expected to occur or immediately after the first occurrence. For ear blast, it is sprayed at the late stage of booting, but when it is expected to occur frequently, it is further sprayed after heading (Agrochemical Handbook pages 246-249 and 666).
1998 Edition, Japan Plant Protection Association.

[0007] In recent years, there has been great social interest in pesticide residues in the environment such as soil, water, and the atmosphere, and in post-harvest pesticides in imported agricultural products, which have recently increased in particular. For tricyclazole, the standard for pesticide residues in the Food Sanitation Law is 3 ppm in rice. Regarding water quality, the registration suspension standard value for water pollution is 1 mg.
/ L (Food Hygiene Magazine 1999, v
ol. 40, no. 1, J-50; revised 3rd edition Agricultural Chemical Registration Reservation Standard Handbook pages 533-536, 1
Published by the Chemical Daily on September 25, 998). Therefore, in order to ensure the safety of the environment and food, it is necessary to measure the amount of tricyclazole contained therein quickly and accurately.

Conventionally, for example, tricyclazole in agricultural crops has been extracted from rice and the like, purified, and then analyzed by gas chromatography (GC). That is, for example, a method is employed in which a sample is extracted with acetone, treated with a coagulating solution, purified by silica gel column chromatography using hexane-ethyl acetate as a solvent, and then measured by GC ("Latest Latest"). Pesticide Residue Analysis Method ”, pp. 651-652, edited by the Pesticide Residue Analysis Research Group, published by Chuo Hokoku Shuppan '95 .4.1). These methods have problems in that the preparation of the sample is complicated, requires a large amount of procedures and time, requires a high level of skill in the analysis, and requires high costs for measuring devices and equipment. The measurement of tricyclazole requires the analysis of a large number of samples in a short time,
There has been a demand for a new measurement method that is not only accurate but also simple, quick and economical.

[0009] The immunochemical assay is a method for detecting an antigen or an antibody based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen.
In recent years, it has attracted attention due to its economic efficiency. In immunochemical assay methods, a wide variety of labels have been applied as detection methods, for example, enzymes, radiotracers, chemiluminescent or fluorescent substances, metal atoms, sols, latexes and bacteriophages.

[0010] Among the immunochemical assay methods, the enzyme immunoassay (EIA) using an enzyme has been widely used as being particularly excellent in terms of economy and convenience. An excellent review of enzyme immunoassays can be found in Tijssen
P, "Practice and theory of
Enzyme immunoassays ”inL
laboratory technologies in b
iochemistry and molecular
biology, Elsevier Amster
dam New York, Oxford ISBN
0-7204-4200-1 (1990).

Generally, for a molecule having a large molecular weight,
By inoculating animals without further modification,
An appropriate immune response can be elicited to produce an antibody that recognizes the antigen. However, small molecule compounds such as tricyclazole usually fail to elicit an immune response when inoculated into animals. These molecules only act as a group of epitopes by binding to immunogenic macromolecules (proteins, polysaccharides, etc.).
An immune response is elicited in the presence of a cell receptor, resulting in the production of antibodies by a panel of B lymphocytes. Molecules that produce immunogenicity only when bound to a polymer compound in this way are collectively referred to as “haptens”.

However, even when a low molecular compound is combined with a high molecular compound as an antigen, the resulting antibody often does not recognize the desired molecule or has only a very low affinity. Therefore, it is generally necessary to use not a low molecular compound itself but a hapten into which a spacer arm (bond) is introduced together with a functional group available for bonding. However, in such a case, a preferable antibody cannot be obtained unless one having been appropriately introduced in consideration of all problems such as the arrangement of the bond / functional group and the size of the bond is used. Proper introduction must be devised for each molecule.

[0013] Despite the great need for tricyclazole, no hapten for producing such antibodies as well as a suitable antibody had been obtained before the present invention.

[0014]

DISCLOSURE OF THE INVENTION The present invention provides a novel antibody or a fragment thereof which reacts with tricyclazole and an analog thereof (hereinafter referred to as "tricyclazole analog" or simply "tricyclazole" depending on the context), and a method for producing the same. The purpose is to provide. As used herein, the term “fragment” of an antibody means a part of an antibody capable of binding to an antigen, for example, a _Fab fragment or the like.

[0015] In one aspect, the present invention provides a monoclonal antibody reactive with tricyclazole analogs. Another object of the present invention is to provide a hapten compound (tricyclazole hapten) which constitutes an antigen for producing a novel antibody reactive with a tricyclazole analog.

Another object of the present invention is to provide a conjugate of a tricyclazole hapten and a polymer compound. Another object of the present invention is to provide a hybridoma that produces the antibody or a fragment thereof.

The present invention further provides a method for immunochemically measuring a tricyclazole-related compound, which comprises using a conjugate of the above-mentioned antibody or fragment thereof and / or the above-mentioned tricyclazole hapten with a polymer compound or a labeling substance. The purpose is to:

[0018]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a tricyclazole derivative in which a functional group usable for bonding to a spacer arm and a polymer compound is introduced into a tricyclazole analog or a portion thereof. By using as a hapten, an antibody reactive with the compound was successfully obtained, and the present invention was completed.

The tricyclazole analogs which are the object of the present invention are represented by the following formula (2):

[0020]

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In the formula (2), R is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms.

According to one aspect of the present invention, there is provided, in particular, the following formula (3):

[0023]

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The target is tricyclazole having a structure represented by the formula: The antibody of the present invention can be obtained, for example, by using, as an antigen, a derivative obtained by introducing a spacer arm and a functional group available for binding into a portion of a tricyclazole analog compound and binding it to a suitable polymer compound as a hapten. . For example, the following equation (1):

[0025]

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In the formula (1), R is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms; and n is an integer of 1 to 10. The compound having the structure is used as a hapten for producing an antibody.

In the formula (1), preferably, R is a methyl group, and the oxygen atom bonded to the methylene group is bonded to the 7-position of the condensed ring. Preferably, n is 5. The present invention relates to the hapten compound, a conjugate of the hapten compound and a polymer compound, an antibody that reacts with a tricyclazole analog compound, a method for producing the same, and an immunochemical measurement method of the hapten compound or a tricyclazole analog compound using the antibody. . Production of tricyclazole hapten The tricyclazole hapten represented by the formula (1) can be produced according to a known method. Although not limited, for example, the following method can be used.

First, the following equation (Z1):

[0029]

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In the formula (Z1), P ¹ is a protecting group for a hydroxyl group; and R is as defined above. ,
The following formula (Z2):

[0031]

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A thiocyan compound having a structure represented by the formula (Z2), wherein L ¹ is an atom or a molecule selected from sodium, potassium, calcium and ammonium is reacted. Then, by reacting a halogenating agent such as sulfuryl chloride, chlorine and bromine, the following formula (Z3):

[0033]

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A compound having a structure represented by the formula (Z3), wherein P ¹ and R are as defined above, is synthesized. The protecting group P ^{1 for the} hydroxyl group may be a known protecting group, for example, an acetyl group, a methyl group, an ethyl group, a tert-butyl group, a benzyl group, a p-methoxybenzyl group, a 3,4
-Dimethoxybenzyl group, trichloroethyl group and the like.

Chlorobenzene, nitrobenzene, carbon tetrachloride, acetic acid and the like can be used as an organic solvent for synthesizing the compound of the formula (Z3).
Hydrochloric acid, nitric acid, hydrobromic acid and the like can be used. The reaction is carried out at a temperature of from 0 ° C. to the boiling point of the solvent, preferably at 50 ° C. to 150 ° C., for 5 minutes to 10 hours, preferably for 1 hour to 5 hours before adding the halogenating agent. The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably from room temperature to 70 ° C., for 5 minutes to 10 hours, preferably 1 hour to 3 hours.

Next, the compound of the formula (Z3) is reacted with hydrazine hydrate in the presence of an acid such as hydrochloric acid, nitric acid, sulfuric acid or the like to give the following formula (Z4):

[0037]

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A compound having a structure represented by the formula (Z4), wherein P ¹ and R are as defined above, is obtained. By the reaction, the amino group of the compound of the formula (Z3) is converted to a hydrazino group.

The reaction is carried out using ethylene glycol, xylene,
The reaction is carried out in an organic solvent such as toluene or ethanol at a temperature from 0 ° C. to the boiling point of the solvent, preferably 100 ° C. to 150 ° C., for 5 minutes to 10 hours, preferably 1 hour to 4 hours.

Next, the compound of the formula (Z4) is added to the compound of the following formula (Z5):

[0041]

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[In the formula (Z5), R ′ is a group having 1 to 3 carbon atoms.
The cyclization reaction is carried out by reacting a trialkyl orthoformate derivative or formic acid having a structure represented by the following formula (Z6):

[0043]

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A compound having the structure represented by the formula (Z6), wherein P ¹ and R are as defined above, is synthesized. When a cyclization reaction is carried out using a trialkyl orthoformate derivative, the reaction is carried out in an organic solvent such as xylene, toluene, ethanol, chloroform or carbon tetrachloride at a temperature from 0 ° C to the boiling point of the solvent, preferably from 50 ° C to 150 ° C. ° C
For 1 to 50 hours, preferably 5 to 25 hours. However, when the trialkyl orthoformate derivative is used in a large excess, the use of the organic solvent can be omitted.

When a cyclization reaction is carried out using formic acid,
The reaction is carried out at a temperature between 0 ° C. and the boiling point of the solvent, preferably between 50 ° C. and 150 ° C., optionally with the addition of
It is carried out for a period of from minutes to 10 hours, preferably from 30 minutes to 2 hours.

Next, the protecting group for the hydroxyl group is removed from the compound of the formula (Z6) to give the following formula (Z7):

[0047]

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A compound having a structure represented by the formula (Z7), wherein P ¹ and R are as defined above, is obtained. The removal of the hydroxyl-protecting group P ¹ can be carried out by a known method such as acid hydrolysis or alkali hydrolysis.

That is, in the case of acid hydrolysis, the formula (Z
The compound of 6) is preferably dissolved in an organic solvent such as acetic acid, formic acid, benzene, dichloromethane, 1,2-dichloroethane and the like, and then hydrochloric acid, sulfuric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid and the like are dissolved. In addition, the compound of the formula (Z7) can be obtained by stirring and reacting at a temperature of 0 ° C. to the boiling point of the solvent, preferably room temperature to 150 ° C., for 5 minutes to 10 hours, preferably 1 hour to 5 hours.

In the case of alkali hydrolysis, the formula (Z
Preferably, the compound of 6) is methanol, ethanol,
Dissolve in an organic solvent such as tetrahydrofuran and ethylene glycol, and then add an aqueous solution such as sodium hydrogen carbonate, sodium carbonate, potassium carbonate, lithium hydroxide, sodium hydroxide or potassium hydroxide, and the temperature from 0 ° C. to the boiling point of the solvent, Preferably at 0 ° C. to room temperature for 5 minutes to 1
The compound of formula (Z7) can be obtained by stirring and reacting for 0 hour, preferably 1 hour to 2 hours.

Further, when P ¹ is a benzyl group, the removal can be carried out by hydrogenolysis with hydrogen.
When P ¹ is a methyl group, boron tribromide, boron trichloride,
The compound of formula (Z7) can also be obtained by demethylation with a halide such as aluminum chloride.

Next, the compound of the formula (Z7) is added to the compound of the formula (Z7) in an organic solvent in the presence of a base:

[0053]

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[In the formula (Z8), L ² is a halogen atom selected from Cl, Br and I; P ² is a protecting group for a carboxyl group; and n is as defined above. And reacting the compound having the structure represented by the following formula (Z9):

[0055]

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A compound represented by the formula (Z9), wherein R, P ² and n are as defined above, is synthesized.
The protecting group for the carboxyl group of P ² may be a known one, and specific examples include, for example, a methyl group, an ethyl group, and a tert- group.
Butyl group, benzyl group, p-methoxybenzyl group, 3,
4-dimethoxybenzyl group, trichloroethyl group, trimethylsilyl group, tert-butyldimethylsilyl group,
Examples thereof include a tert-butyldiphenylsilyl group, a triethylsilyl group, a triisopropylsilyl group, and a trimethylsilylethyl group.

The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably at room temperature to 120 ° C., for 5 minutes to 10 hours, preferably 30 minutes to 3 hours. As a solvent for synthesizing the compound of the formula (Z9), for example, methanol, ethanol, benzene, toluene, xylene, dichloromethane,
Chloroform, carbon tetrachloride, diethyl ether, tetrahydrofuran, dioxane, acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, N, N-dimethylformamide, dimethylsulfoxide, water and the like can be used. Examples of the base include sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate, sodium hydride and the like.

Finally, the compound of the formula (1) can be obtained by removing the protecting group P ² for the carboxyl group from the compound of the formula (Z9). Carboxyl protecting group P
² removal can be performed in the same manner as the removal of the hydroxyl-protecting group P ¹ as described for the synthesis of compounds of formula (Z2), alkaline hydrolysis in a known manner an acid hydrolysis and the like.

By subjecting the compound obtained by the above-described production method to silica gel chromatography or recrystallization, if necessary, a highly purified product can be obtained.

Hereinafter, the preparation of the antigen and antibody of the present invention and the immunochemical assay will be described. In addition, these preparations can be carried out according to a known method, for example, a method described in Seikagaku Experimental Lecture, Immunobiochemical Research Method (edited by the Japanese Biochemical Society), or the like. Of conjugates of tricyclazole hapten and polymer
Preparation The tricyclazole hapten synthesized as described above is bound to an appropriate polymer compound and then used as an antigen for immunization or an antigen for immobilization.

Examples of preferred polymer compounds include keyhole limpet hemocyanin (hereinafter referred to as “KLH”), ovalbumin (hereinafter referred to as “OVA”), bovine serum albumin (hereinafter referred to as “BSA”), Rabbit serum albumin (hereinafter referred to as “RSA”) and the like. KLH and BSA are preferred.

The binding between the tricyclazole hapten and the polymer compound can be performed, for example, by the activated ester method (AEKA).
RU et al. : J. Agric. Food Ch
em. 42 301-309 (1994)), or the mixed acid anhydride method (BF Erlanger et a).
l. : J. Biol. Chem. 234 1090-1
094 (1954)).

The activated ester method can be generally carried out as follows. First, a hapten compound is dissolved in an organic solvent and reacted with N-hydroxysuccinimide in the presence of a coupling agent to form an N-hydroxysuccinimide activated ester.

As the coupling agent, a usual coupling agent commonly used for a condensation reaction can be used.
Dicyclohexylcarbodiimide, carbonyldiimidazole, water-soluble carbodiimide and the like. Examples of the organic solvent include dimethyl sulfoxide (hereinafter referred to as “D
MSO), N, N-dimethylformamide (hereinafter, referred to as "DMF"), dioxane, and the like.
The molar ratio of the hapten compound and N-hydroxysuccinimide used in the reaction is preferably 1:10 to 10: 1,
More preferably from 1: 1 to 1:10, most preferably 1: 1. The reaction temperature is 0 ° C to 100 ° C, preferably 5 ° C to 50 ° C, more preferably 22 ° C to 27 ° C.
The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours, more preferably 1 hour to 2 hours.

After the coupling reaction, the reaction solution is added to a solution in which the polymer compound is dissolved, and the reaction is performed. For example, when the polymer compound has a free amino group, an acid is added between the amino group and the carboxyl group of the hapten compound. An amide bond is formed. The reaction temperature is from 0 ° C to 60 ° C, preferably 5 ° C.
C. to 40.degree. C., more preferably 22.degree. C. to 27.degree. C., and the reaction time is 5 minutes to 24 hours, preferably 1 hour to 16 hours.
Hours, more preferably from 1 hour to 2 hours. The reaction product can be purified by dialysis, a desalting column, or the like to obtain a conjugate of a tricyclazole hapten and a polymer compound.

On the other hand, the acid anhydride used in the mixed acid anhydride method is obtained by a usual Schotten-Baumann reaction, and this is reacted with a polymer to produce the desired hapten-polymer compound conjugate. Is done. The Schotten-Baumann reaction is performed in the presence of a basic compound. As the basic compound, a compound commonly used in the Schotten-Baumann reaction can be used, for example, tributylamine, triethylamine, trimethylamine,
Organic bases such as N-methylmorpholine, pyridine, N, N-dimethylaniline, DBN, DBU, and DABCO; and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogencarbonate, and sodium hydrogencarbonate. The reaction is usually carried out at a temperature between -20 ° C and 150 ° C, preferably at 0 ° C.
To 100 ° C., and the reaction time is 5 minutes to 10
Time, preferably from 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually carried out at minus 20.
C. to 100.degree. C., preferably 0.degree. C. to 50.degree. C., and the reaction time is 5 minutes to 10 hours, preferably 5 minutes to 5 hours. The mixed anhydride method is generally performed in a solvent. As the solvent, any solvent commonly used in the mixed acid anhydride method can be used, and specifically, ethers such as dioxane, diethyl ether, tetrahydrofuran, and dimethoxyethane, and halogenated solvents such as dichloromethane, chloroform, and dichloroethane. Hydrocarbons, aromatic hydrocarbons such as benzene, toluene and xylene, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, etc. Is mentioned. Examples of the haloformate used in the mixed acid anhydride method include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, and isobutyl chloroformate. The proportions of the hapten, the haloformate and the polymer compound used in the method can be appropriately selected from a wide range.

In a similar manner as described above, a product obtained by binding a labeling substance such as an enzyme to a tricyclazole hapten can be used in an immunochemical assay. The labeling substance, (hereinafter referred to "HRP") horseradish peroxidase, an enzyme such as alkaline phosphatase, fluorescein isothiocyanate, a fluorescent substance such as rhodamine, radioactive substances such as ³² P, ¹²⁵ I, and the like chemiluminescent substance. Preparation of polyclonal antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of a tricyclazole hapten and a polymer compound. For example, a conjugate of a tricyclazole hapten and KLH is dissolved in a sodium phosphate buffer (hereinafter, referred to as “PBS”) and mixed with an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or alum. It can be obtained by immunizing an animal as an antigen for use. As the animal to be immunized, any of those commonly used in the art can be used, and examples thereof include mice, rats, rabbits, goats, and horses. However, humans are not included.

The method of administration upon immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred. Immunization can be performed once or at appropriate intervals, preferably multiple times at intervals of one to five weeks.

Blood is collected from the immunized animal, and serum separated therefrom can be used to evaluate the presence of a polyclonal antibody that reacts with tricyclazole. Preparation of Monoclonal Antibody The monoclonal antibody of the present invention can be prepared by a known method using a conjugate of a tricyclazole hapten and a polymer compound.

In producing a monoclonal antibody,
At least the following work steps are required. (A) Preparation of a conjugate of a tricyclazole hapten used as an antigen for immunization and a polymer compound (b) Immunization of an animal (c) Collection of blood, assay, and preparation of antibody-producing cells (d) Preparation of myeloma cells (E) Cell fusion between antibody-producing cells and myeloma cells and selective culture of hybridomas (f) Screening and cell cloning of hybridomas producing the desired antibody (g) Monoclones by culturing hybridomas or transplanting hybridomas into animals Preparation of Antibodies (h) Measurement of Reactivity of Prepared Monoclonal Antibodies Conventional methods for producing hybridomas that produce monoclonal antibodies include, for example, Hybridoma Technologies, Cold Spring Harbor Laboratories (Co.)
ld Spring Harbor Laborato
ry, 1980 edition), and Cell Histochemistry (Shuji Yamashita et al., edited by the Japanese Society for Tissue and Cellular Chemistry; Interdisciplinary Planning, 1986).

Hereinafter, a method for preparing a monoclonal antibody against the tricyclazole analog of the present invention will be described, but it will be apparent to those skilled in the art that the present invention is not limited thereto.

The steps (a) and (b) can be performed by a method substantially similar to the method described for the polyclonal antibody. The antibody-producing cells in step (c) are lymphocytes, which can generally be obtained from the spleen, thymus, lymph nodes, peripheral blood or a combination thereof, but splenocytes are most commonly used. Therefore, after the final immunization, a site where antibody-producing cells are present, for example, a spleen is excised from a mouse in which antibody production has been confirmed, and splenocytes are prepared.

The myeloma cells which can be used in the step (d) include, for example, myeloma cell line P3 / X63-Ag8 (X63) (Nat) derived from Balb / c mouse.
ure, 256, 495-497 (1975)), P3
/ X63-Ag8. U1 (P3U1) (Current
Topics. in Microbiology
d Immunology, 81, 1-7 (198
7)), P3 / NSI-1-Ag 4-1 (NS-1)
(Eur. J. Immunol., 6, 511-519).
(1976)), Sp2 / 0-Ag14 (Sp2 / 0)
(Nature, 276, 269-270 (197
8)), FO (J. Immuno. Meth., 35,
1-21 (1980)), MPC-11, X63.6.
Myeloma cell lines such as S.53 and S194, or rat-derived 210. RCY3. Ag 1.2.3. (Y3)
(Nature, 277, 131-133, (197
9)) etc. can be used.

The above-mentioned myeloma cells are subcultured in Dulbecco's modified Eagle's medium (DMEM) or Iscove's modified Dulbecco's medium (IMDM) containing fetal bovine serum, and a cell number of about 3 × 10 ³ or more is secured on the day of fusion.

Cell fusion in the step (e) can be performed by a known method, for example, the method of Milstein et al.
things in Enzymology, 73, 3
(1981)). Currently, the most commonly used is polyethylene glycol (P
EG). The PEG method is described in, for example, Cell Histochemistry, Shuji Yamashita et al. (Supra). As another fusion method, a method by electric treatment (electric fusion) can be adopted (Etsuko Okochi et al., Experimental Medicine 5.1315-19, 1987). Other methods can be appropriately adopted. In addition, the ratio of cells used may be the same as in a known method. For example, spleen cells may be used about 3 to 10 times as much as myeloma cells.

The selection of a hybridoma group in which spleen cells and myeloma cells are fused to obtain antibody secretion ability and proliferation ability is performed, for example, when a hypoxanthine guanine phosphoribosyltransferase-deficient strain is used as a myeloma cell line, for example, as described above. It can be performed by using a HAT medium prepared by adding hypoxanthine / aminopterin / thymidine to DMEM or IMDM.

In the step (f), a part of the culture supernatant containing the selected hybridoma group is taken and, for example, E
The antibody activity against tricyclazole is measured by the LISA method.

Further, cell cloning of a hybridoma which has been found by measurement to produce an antibody reactive with tricyclazole is performed. As the cell cloning method, a method of limiting the dilution so that one hybridoma is contained in one well by limiting dilution “Limiting dilution method”; a method of collecting a colony spread on a soft agar medium; and a method of separating one cell by a micromanipulator Extraction method: a "sorter clone method" in which one cell is separated by a cell sorter, and the like. The limiting dilution method is simple and often used.

For wells in which an antibody titer has been recognized, cloning is repeated 1-4 times, for example, by the limiting dilution method, and a stably obtained antibody titer is selected as an anti-tricyclazole monoclonal antibody-producing hybridoma strain. As a medium for culturing a hybridoma, for example,
DMEM or IMDM containing fetal calf serum (FCS) is used. The hybridoma is preferably cultured, for example, at a carbon dioxide concentration of about 5 to 7% and at 37 ° C. (in a thermostat at 100% humidity).

The mass culture for preparing the antibody in the step (g) is performed by a follow-fiber type culture device or the like. Alternatively, mice of the same strain (for example, Bal
b / c) or hybridomas can be grown in the peritoneal cavity of Nu / Nu mice, and antibodies can be prepared from ascites fluid.

The culture supernatant or ascites fluid obtained as described above can be used as an anti-tricyclazole monoclonal antibody. Further, dialysis, salting out with ammonium sulfate, gel filtration, freeze-drying, etc. are performed to collect and purify the antibody fraction. By doing so, an anti-tricyclazole monoclonal antibody can be obtained. Further, when purification is necessary, it can be carried out by combining commonly used methods such as ion exchange column chromatography, affinity chromatography, and high performance liquid chromatography (HPLC).

The anti-tricyclazole monoclonal antibody obtained as described above can be determined for its subclass, antibody titer and the like by using a known method such as ELISA. Measurement of Tricyclazole Analogues Using Antibodies As a method for measuring tricyclazole analogs using an antibody used in the present invention, radioisotope immunoassay (RI
A method), ELISA method (Engval, E., Met)
hods in Enzymol. , 70, 419-4
39 (1980)), fluorescent antibody method, plaque method, spot method, agglutination method, Ouchterlon (Ouchterlon)
y) and other commonly used methods for detecting antigens ("Hybridoma method and monoclonal antibody", R & D Planning, pp. 30-53, Showa 5)
March 5, 7). The ELISA method is widely used from the viewpoints of sensitivity, simplicity, and the like.

The measurement of the tricyclazole analog compound can be performed by the following procedure using, for example, an indirect competitive ELISA method among various ELISA methods. (A) First, a conjugate of a tricyclazole hapten, which is an antigen for immobilization, and a polymer compound is immobilized on a carrier.

(B) The surface of the solid phase on which the antigen for immobilization is not adsorbed is blocked by a substance unrelated to the antigen, for example, a protein. (C) A sample and an antibody containing various concentrations of tricyclazole are added thereto, and the antibody is allowed to competitively react with the immobilized antigen and tricyclazole to form an immobilized antigen-antibody complex and a tricyclazole-antibody complex. Is generated.

(D) By measuring the amount of the immobilized antigen-antibody complex, the amount of tricyclazole in the sample can be determined from a previously prepared calibration curve. In the step (a), the carrier for immobilizing the antigen for immobilization is not particularly limited, and any carrier commonly used in the ELISA method can be used. For example,
Examples include a 96-well microtiter plate made of polystyrene.

To immobilize the solid phase antigen on a carrier,
For example, a buffer containing an antigen for immobilization may be placed on a carrier and incubated. As the buffer, a known buffer can be used, and examples thereof include a phosphate buffer. The concentration of the antigen in the buffer solution can be selected from a wide range, but is usually about 0.01 μg / ml to 100 μg / ml, preferably 0.05 μg / ml to 5 μg / ml. When a 96-well microtiter plate is used as a carrier, it is desirable that the concentration be about 300 μl / well or less and about 20 μl / well to about 150 μl / well. Furthermore, the conditions for incubation are not particularly limited, but usually incubation at about 4 ° C. overnight is suitable.

The antigen to be immobilized on the carrier includes:
Not only the conjugate of the tricyclazole hapten for which the antibody was prepared and the polymer compound itself, but also the conjugate of the other hapten represented by the formula (1) and the polymer compound can be used as the immobilized antigen. is there. For example, a compound in which R or n in formula (1) is different from that used for antibody production can be used as the immobilized antigen. Furthermore, other tricyclazole-like compounds not included in the formula (1) can be used as the immobilized antigen.

In the blocking in the step (b), in the carrier on which the antigen (combination of the tricyclazole hapten and the polymer compound) is immobilized, there is a portion other than the tricyclazole hapten portion to which the antibody to be added later can be adsorbed. May be done solely to prevent it. As a blocking agent, for example, BSA or skim milk solution can be used. Alternatively, block ace ("Bloc
k-Ace ", manufactured by Dainippon Pharmaceutical Co., Ltd., code No. UK-2
A commercially available blocking agent such as 5B) can also be used. Specifically, for example, an appropriate amount of a buffer containing a blocking agent (for example, a PBS solution containing 1% BSA) is added to a portion where the antigen is immobilized, and the mixture is incubated at about 4 ° C. overnight. After that, cleaning is performed by a cleaning solution. Although the washing solution is not particularly limited, for example, 10 mM PBS (p
H7.2), 0.8% (w / v) NaCl, 0.02%
(W / v) KCl, 0.02% (v / v) Tween2
A composition having a composition of 0 can be used.

Next, in step (c), the sample containing tricyclazole and the antibody are brought into contact with the immobilized antigen, and the antibody is reacted with the immobilized antigen and tricyclazole, whereby the immobilized antigen-antibody complex and tricyclazole- An antibody complex is formed.

At this time, as the antibody, an antibody against the tricyclazole of the present invention is added as a first antibody, and an antibody against the first antibody to which a labeling enzyme is bound is sequentially added as a second antibody.

The first antibody is dissolved in a buffer and added. Although not limited, the reaction is carried out at 10 to 40 ° C,
Preferably, the heat treatment may be performed at about 25 ° C. to about 37 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the first antibody that has not bound to the immobilized antigen. As a cleaning solution,
For example, 10 mM PBS (pH 7.2), 0.8%
A composition of (w / v) NaCl and 0.02% (w / v) KCl can be used.

Next, a second antibody is added. For example, when a mouse monoclonal antibody is used as the first antibody, it is appropriate to use an antibody against a mouse antibody to which an enzyme (eg, peroxidase or alkaline phosphatase) is bound. The first antibody bound to the carrier has a final absorbance of about 500 to 10,000 times, preferably 0.5 to 3.5 times.
It is desirable to react the second antibody diluted so that Buffer is used for dilution. Although not limited, the reaction is carried out at 37 ° C. for about 1 hour, and after the reaction, it is washed with a buffer. By the above reaction, the second antibody binds to the first antibody. Alternatively, a labeled first antibody may be used, in which case the second antibody is not required.

Next, in step (d), a chromogenic substrate solution that reacts with the labeling substance of the second antibody bound to the carrier is added, and the amount of tricyclazole can be calculated from the calibration curve by measuring the absorbance.

When peroxidase is used as an enzyme that binds to the second antibody, for example, hydrogen peroxide and 3,3 ′, 5,5′-tetramethylbenzidine or o
-A chromogenic substrate solution containing phenylenediamine (hereinafter referred to as "OPD") can be used. Although not limited, a chromogenic substrate solution is added and reacted at about 25 ° C. for about 20 minutes, and then the enzyme reaction is stopped by adding 2N sulfuric acid. When 3,3 ', 5,5'-tetramethylbenzidine is used, the absorbance at 450 nm is measured. If OPD is used, measure the absorbance at 492 nm. On the other hand, when alkaline phosphatase is used as the enzyme that binds to the second antibody, the color is developed using, for example, p-nitrophenyl phosphate as a substrate, the enzyme reaction is stopped by adding a 2N NaOH solution, and the absorbance at 415 nm is measured. The method is suitable.

With respect to the absorbance of the reaction solution to which tricyclazole was not added, the rate of decrease in the absorbance of the solution in which they were added and reacted with the antibody was calculated as the inhibition rate. The concentration of tricyclazole in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which tricyclazole at a known concentration has been added.

Alternatively, the measurement of tricyclazole can be carried out, for example, by a direct competitive ELISA method using the monoclonal antibody of the present invention as described below. (A) First, the monoclonal antibody of the present invention is immobilized on a carrier.

(B) The surface of the carrier on which the antibody is not immobilized is blocked with a substance unrelated to the antigen, for example, a protein. (C) Separately from the above steps, a mixture is prepared by adding an enzyme-bound hapten obtained by binding a tricyclazole hapten and an enzyme to a sample containing various concentrations of tricyclazole.

(D) The mixture is reacted with the antibody-immobilized carrier. (E) By measuring the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of tricyclazole in the sample is determined from a previously prepared calibration curve.

The carrier on which the monoclonal antibody is immobilized in the step (a) is not particularly limited and may be an ELISA.
Conventional methods can be used, and examples include a 96-well microtiter plate.
The immobilization of the monoclonal antibody can be carried out, for example, by placing a buffer containing the monoclonal antibody on a carrier and incubating. The composition and concentration of the buffer may be the same as those in the indirect competitive ELISA method described above.

In the blocking in the step (b), the carrier on which the antibody is immobilized may have a portion where tricyclazole and enzyme-bound hapten in a sample to be added later are adsorbed independently of the antigen-antibody reaction. So do it to prevent it. As the blocking agent and the method therefor, those similar to the indirect competitive ELISA method described above can be used.

The preparation of the enzyme-linked hapten used in the step (c) is not particularly limited, and may be carried out by any method as long as the method binds the tricyclazole hapten to the enzyme. For example, the activated ester method described above can be employed. The prepared enzyme-linked hapten is mixed with a sample containing tricyclazole.

The hapten to be bound to a labeling substance such as an enzyme is not limited to the tricyclazole hapten itself used for the production of the antibody, but can be represented by the formula (1) as in the case of the immobilized antigen in the indirect competitive ELISA method. It is also possible to use a conjugate of another hapten and a polymer compound as an antigen for labeling. For example, in the formula (1), R
Alternatively, a compound in which n is different from that for antibody production can also be used as a labeling antigen. Further, other tricyclazole-like compounds not included in the formula (1) can be used as the labeling antigen.

In the step (d), a sample containing tricyclazole and an enzyme-bound hapten are brought into contact with an antibody-immobilized carrier, and a complex between the tricyclazole and the enzyme-bound hapten is formed by a competitive inhibition reaction between the tricyclazole and the enzyme-bound hapten. I do. The sample containing tricyclazole is used after being diluted with an appropriate buffer. Without limitation, the reaction is carried out, for example, at room temperature for approximately one hour. After the completion of the reaction, the carrier is washed with a buffer to remove the enzyme-bound hapten that has not bound to the immobilized antibody. As the washing solution, for example, PBS can be used.

Further, in step (e), the chromogenic substrate solution that reacts with the enzyme of the enzyme-bound hapten is combined with the indirect competitive E
The amount of tricyclazole can be calculated from the calibration curve by measuring the absorbance in addition to the LISA method.

The monoclonal antibody TCZ1-1 of the present invention
2 is about 0.01 ng / in the direct competitive ELISA method.
ml to about 100 ng / ml, preferably about 0.1 ng
Reacts with tricyclazole in a concentration range from about 10 ng / ml to about 10 ng / ml (Example 8, FIG. 1).

Further, as described above, direct competition ELISA
In method A, a hapten different from that used for antibody production can be used as a labeling antigen.
Shows intrinsic reactivity in the ISA method. Cross-reactivity of the antibodies of the present invention Direct competitive ELISA method or indirect competitive ELISA described above
By the method, the cross-reactivity of the monoclonal antibody of the present invention can be examined.

For example, the monoclonal antibody TCZ1-1
No. 2 shows no reactivity to other tricyclazole analogs having a thiazole ring (mefenacet, thiabendazole) and has very high specificity for tricyclazole (Example 9, FIG. 2). Hereinafter, the present invention will be described specifically with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description in the present specification, and they are included in the technical scope of the present invention.

[0108]

EXAMPLES Example 1 Synthesis of Tricyclazole Hapten

[0109]

Embedded image

2-amino-6-methoxy-4-methyl-
Synthesis of 1,3-benzothiazole (1 ) To a solution of 4.2 g (30 mmol) of 4-methoxy-2-methylaniline in 30 ml of chlorobenzene was added sulfuric acid.
6 g (16 mmol) was gradually added dropwise at room temperature. 2.7 g of sodium thiocyanate (33 mmol)
l), and the mixture was stirred at 100 ° C for 2 hours, cooled to 30 ° C, and further 5.4 g of sulfuryl chloride (40 mmo).
l) was added at 30 ° C to 40 ° C. This mixture is mixed with 50
After stirring at room temperature for 2 hours and returning to room temperature, the solid was collected by filtration.
The obtained solid was dissolved in 100 ml of warm water and filtered. Aqueous ammonia was added to the filtrate until it became alkaline, and the precipitated crystals were collected by filtration, washed with water, washed with n-hexane, and 2.9 g.
(Yield 50%) (1) was obtained. The melting point of this compound (1) was 185 ° C to 188 ° C.

2-hydrazino-6-methoxy-4-methyl
Synthesis of 1,3-benzothiazole (2) 2-amino-6-methoxy-4-methyl-1,3-benzothiazole (1) in 15 ml of ethylene glycol
2.9 g (15 mmol), hydrazine monohydrate 2.3
g (46 mmol) and 0.6 ml of concentrated hydrochloric acid in 1
Stirred at 20 ° C. for 2 hours. 30 ml of water was added to the reaction mixture, and the precipitated solid was collected by filtration. This solid was recrystallized from toluene to obtain 2.3 g (yield 74%) of (2). The melting point of this compound (2) is 167 ° C to 171 ° C.
Met.

7-methoxy-5-methyl-1,2,4-
Synthesis of triazolo [3,4-b] benzothiazole (3)
1. 2-hydrazino-6-methoxy-4-methyl-1,3-benzothiazole (2) in 30 ml of synthetic meta-xylene
To 1 g (10 mmol) of the suspension, 1.5 g (10 mmol) of triethyl orthoformate was added at room temperature, and the mixture was stirred and refluxed for 22 hours. After cooling the reaction mixture to room temperature, the precipitated solid was collected by filtration. This solid was washed with ether and dried to obtain 1.2 g (yield 55%) of (3).

7-Hydroxy-5-methyl-1,2,4
Of triazolo [3,4-b] benzothiazole (4)
7-Methoxy-5-methyl-1,2,4-triazolo [3,4-] in 30 ml of synthetic 48% hydrobromic acid and 10 ml of acetic acid.
b] A suspension of 1.1 g (5 mmol) of benzothiazole (3) was stirred and refluxed for 3 hours. The reaction mixture was concentrated, the residue was dissolved in 30 ml of water, and then made alkaline with a 10% aqueous sodium hydroxide solution. After treating this aqueous solution with activated carbon, the pH was adjusted to 6 with 1N hydrochloric acid, and the precipitated crystals were collected by filtration. The crystals were washed with water and n-hexane in that order and dried to obtain 0.95 g (yield 95%) of (4). The melting point of this compound (4) was over 250 ° C.

6- (5-methyl-1,2,4-triazo
B [3,4-b] benzothiazol-7-yloxy)
Synthesis of ethyl hexanoate (5) 7-hydroxy-5-methyl-1,2,4-triazolo [3,4-
b] 0.9 g of benzothiazole (4) (4.4 mmol
l), 1.2 g of 6-bromohexanoic acid (5.3 mmol
l) and a suspension of 0.7 g (5.1 mmol) of potassium carbonate were stirred at 100 ° C for 1 hour. The reaction mixture was concentrated, 20 ml of water was added to the residue, and the mixture was extracted with ethyl acetate (30 ml × 3). The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was subjected to column chromatography (ethyl acetate; methanol = 10:
Purification was performed in 1) to obtain 1.0 g (yield 67%) of (5). The melting point of this compound (5) was 78 ° C to 79 ° C.

6- (5-methyl-1,2,4-triazo
B [3,4-b] benzothiazol-7-yloxy)
Hexanoic acid (6) 6- (5-methyl-1,2,4 in 15 ml of methanol
0.9 g of ethyl triazolo [3,4-b] benzothiazol-7-yloxy) hexanoate (5) (2.6 m
mol), a solution of 0.5 g (12.5 mmol) of sodium hydroxide in 10 ml of water was added.
Stirred for hours. The methanol was distilled off under reduced pressure.
The solution was adjusted to pH 6 with N hydrochloric acid and extracted with ethyl acetate (30 ml).
× 3). The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate, and concentrated. The residue was recrystallized from ethanol to obtain 0.47 g (yield 57%) of (6). The melting point of this compound (6) was 174 ° C to 176 ° C.

[0116] ¹ of the above-mentioned tricyclazole hapten (6)
Physical property data (chemical shift δ) by H-NMR are shown below.

[0117]

Table 1 ¹ H-NMR (DMSO-D ₆ , 400 MHz) δ 1.42 (2H, m, CH ₂ ), 1.56 (2H,
m, CH ₂ ), 1.73 (2H, m, CH ₂ ), 2.
19 (2H, t, CH ₂ ), 2.66 (3H, s, C
_{H 3), 3.99 (2H,} t, CH 2), 6.96
(1H, d, Ar: H), 7.47 (1H, d, A
r: H), 9.42 (1H, s, CH), 12 (1
(H, br, COOH) Example 2 Preparation of antigen for immunization An antigen for immunization in which tricyclazole hapten was bound to KLH was prepared by the activated ester method.

First, 15 mg of the tricyclazole hapten prepared in Example 1 was dissolved in 1.5 ml of DMF.
23 mg of N, N-carbonyldiimidazole was added and 2
The mixture was stirred at 5 ° C. for 30 minutes (hereinafter, this is referred to as “solution A”).

On the other hand, 20 ml of KLH was added to 2.5 ml of distilled water.
g, and dialyzed overnight with 10 mM PBS (pH 7.4) as an external solution. After dialysis, the mixture was centrifuged at 3000 rpm for 30 minutes, and the above solution A was slowly added to 2.5 ml of the obtained supernatant, followed by reaction at 25 ° C for 2 hours. This reaction solution is 10m
It was dialyzed for 6 days in M PBS (pH 7.4) to prepare a conjugate of tricyclazole hapten and KLH (hereinafter, referred to as “tricyclazole hapten / KLH”). Tricyclazole hapten / KL thus obtained
H was used as an antigen for immunization. Example 3 Preparation of Antigen for Screening A conjugate of tricyclazole hapten and BSA (hereinafter referred to as “tricyclazole hapten / BSA”) was prepared in the same manner as in Example 2 and used as a screening antigen. Example 4 Immunization Balb / c mice were used for immunization. The antigen for immunization prepared in Example 2 (Tricyclazole hapten / KL
For H), each mouse was immunized. 100 μg of the antigen for immunization was dissolved in 100 μl of PBS, mixed with an equal amount of complete Freund's adjuvant, and inoculated intraperitoneally into the mouse. 14 days, 31 days, 58 after the first immunization
On days 79 and 79, mice were boosted with the immunizing antigen prepared using Freund's incomplete adjuvant in the same manner as described above. 115 days later, PB
The mice were boosted with the immunizing antigen dissolved in S. 122
One day later, blood was collected from the tail vein of the immunized mouse, and the serum was separated and used as antiserum. Example 5 Reactivity of antiserum to tricyclazole Indirect competitive ELISA using the screening antigen (tricyclazole hapten / BSA) prepared in Example 3
The reactivity of the antiserum prepared in Example 4 with respect to tricyclazole was examined by the method.

First, the solution of tricyclazole hapten / BSA (5 μg / ml) prepared in Example 3 was
96 μl plates were coated at μl / well. After washing, Block Ace diluted 4 times (“Bl
ock Ace ": Dainippon Pharmaceutical, Code No. UK-2
5B). Next, an equal volume of a 1000-fold dilution of the antiserum of Example 4 and a 10% methanol solution containing various concentrations of tricyclazole or a similar compound were mixed, and 100 μl of the mixture was placed in a well and reacted at 37 ° C. for 1 hour. Was.

After completion of the reaction, 0.05% Tween 20-
After washing once with PBS, peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Cappel) diluted 5000-fold with PBS was added to each well in an amount of 100 μl, and reacted at 37 ° C. for 1 hour. After the reaction is over,
Wash twice with 0.05% Tween 20-PBS,
0.05 M phosphate citrate buffer (pH 4.5) containing 0.4 mg / ml OPD and 0.04% hydrogen peroxide
Was added to each well, and allowed to stand at room temperature for 20 minutes to develop color. After the reaction, 100 μl of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm was measured. Example 6 Preparation of Hybridoma A monoclonal antibody was prepared from the mouse whose antiserum titer was measured in Example 5.

First, following Example 4, cell fusion was performed by electrofusion between mouse spleen cells having increased anti-tricyclazole antibody activity in serum and mouse myeloma cells (P3U1). Antibody activity against tricyclazole was examined by the following method for the culture supernatant in which cell proliferation was observed.

A 96-well plate was coated with a solution of tricyclazole hapten / BSA (5 μg / ml) at 50 μl / well. After washing, blocking was performed with a 4-fold diluted Block Ace, and an equal amount of the culture supernatant was mixed with 10% methanol solution containing various concentrations of tricyclazole or its analogous compound.
1 was placed in a well and reacted at 37 ° C. for 1 hour. After completion of the reaction, the well was washed once with 0.05% Tween 20-PBS, and then, 50 μl of a 5000-fold diluted peroxidase-conjugated anti-mouse IgG goat antibody (Cappel) diluted with PBS at 37 ° C. Allowed to react for hours. After the reaction is completed, 0.05% Tween 20-P
After washing twice with BS, 100 μl of 0.05 M phosphate citrate buffer (pH 4.5) containing 0.4 mg / ml OPD and 0.04% hydrogen peroxide was added to each well, and the mixture was added at room temperature. It was left for 20 minutes to develop color. After the reaction, 100 μl of 2N sulfuric acid was added to each well to stop the reaction.
The absorbance at 490 nm was measured, and those having specific antibody activity were selected.

Next, the cells in the selected wells were subjected to cell cloning using the limiting dilution method. As a result, a hybridoma cell line producing an anti-tricyclazole antibody was cloned. TCZ1-12 of them
Was deposited on September 14, 1999 under the deposit number FERM P-17.
556 as the Institute of Biotechnology, Institute of Industrial Science and Technology (¥ 305
-0046 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture). Example 7 Binding of tricyclazole hapten to HRP
In the same manner as in Example 2, a conjugate of tricyclazole hapten and HRP was prepared by the activated ester method. 15 mg of tricyclazole hapten was dissolved in 1.5 ml of N, N-dimethylformamide, 23 mg of N, N-carbonyldiimidazole was added, and the mixture was stirred at 25 ° C. for 30 minutes (hereinafter referred to as “solution B”).

On the other hand, 10 mM PBS (pH 7.4)
23 mg of HRP was dissolved in 2.5 ml, and solution B was added dropwise thereto. The reaction was performed at 25 ° C. for 2 hours. The reaction solution was dialyzed for 7 days in 10 mM PBS (pH 7.4) to obtain an HRP-bound tricyclazole hapten. Example 8 Tricycla by Direct Competition Inhibition ELISA
Measurement of sol The hybridoma cells (TCZ1-1) obtained in Example 6
2) was transplanted into the abdominal cavity of a mouse, and the ascites fluid obtained 10 to 15 days later was collected, and the monoclonal antibody was purified by ammonium sulfate fractionation. Tricyclazole was measured by the following test method using the obtained purified monoclonal antibody. Hereinafter, each monoclonal antibody has the same name as the monoclonal antibody-producing cells that produce them.

The monoclonal antibody solution (TCZ1-12
Antibody (2 μg / ml) was added to a 96-well plate at 100 μl / well and left at 4 ° C. overnight. The next day, after blocking with Block Ace diluted 4 times, tricyclazole and the appropriately diluted HRP prepared in Example 7 were used.
A 10% methanol-PBS solution containing the bound tricyclazole hapten was added at 50 μl / well, and left at 37 ° C. for 1 hour. After completion of the reaction, 0.05% Tween 20-P
After washing twice with BS, 100 μl of 0.05 M phosphate citrate buffer (pH 4.5) containing 0.4 mg / ml OPD and 0.04% hydrogen peroxide was added to each well, and the mixture was added at room temperature. It was left for 20 minutes to develop color. After the reaction, 100 μl of 2N sulfuric acid was added to each well to stop the reaction.
The absorbance at 490 nm was measured.

The results are shown in FIG. Direct competitive inhibition ELISA
In method A, tricyclazole can be measured, and the measurement range is 0.1 ng / ml of tricyclazole.
To 10 ng / ml. Example 9 Cross-Reactivity of Monoclonal Antibodies The monoclonal antibody TCZ1-12 produced by the cloned hybridoma TCZ1-12, derived from the tricyclazole hapten, has a tricyclazole and other thiazole ring analogous methods using the same method as in Example 8. The cross-reactivity to the compound was determined.

The results are shown in FIG. Monoclonal antibody T
CZ1-12 does not react with mefenacet, a similar compound having another thiazole ring, but reacts specifically with tricyclazole.

[Brief description of the drawings]

FIG. 1 shows the monoclonal antibody TCZ1 of the present invention.
12 shows the reactivity of -12 with tricyclazole by a direct competitive ELISA method.

FIG. 2 shows the monoclonal antibody TCZ1 of the present invention.
FIG. 3 shows the measurement of tricyclazole and other analogous compounds having a thiazole ring by a direct competition ELISA method using -12.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12N 15/02 C12N 15/00 C (72) Inventor Kazuaki Watanabe 1-chome, Hamamatsucho, Minato-ku, Tokyo 27-14 Inside the Institute for Environmental Immunity Technology (72) Inventor Yasuhiro Kagawa 1-14-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Megumi Saito Minato-ku, Tokyo 1-27-14 Hamamatsucho F-term in the Institute for Environmental Immunity Technology (Reference) 4B024 AA07 AA11 BA53 GA03 GA08 GA10 GA18 GA27 HA03 HA15 4B064 AG27 CA10 CA20 CC01 CC24 CE04 DA11 DA13 4B065 AA91X AA99Y AB05 AC14 AC15 BA08 BA24 BC50 BD14 BD50 CA25 CA46 CA47 4C072 AA01 AA06 CC03 CC16 EE13 FF02 GG07 UU08 4H045 AA11 AA20 AA30 BA42 CA40 CA50 DA76 EA50 FA72 GA06

Claims (13)

[Claims] (1) The following formula (1): [In the formula (1), R is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms; and n is an integer of 1 to 10.] Compound. 2. In the formula (1), R is a methyl group,
The compound according to claim 1, wherein the oxygen atom bonded to the methylene group is bonded to the 7-position of the fused ring. 3. The compound according to claim 1, wherein n is 5 in the formula (1). 4. A conjugate of the compound according to any one of claims 1 to 3 with a polymer compound or a labeling substance. 5. An antigen is prepared by binding the compound according to any one of claims 1 to 3 to a polymer compound, and the antigen is used to obtain the following formula (2): ] [Wherein, in the formula (2), R is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms]. A method for producing an antibody or a fragment thereof, which is reactive to a compound having a structure represented by the formula (2). 6. In the formula (2), R is a methyl group.
The method according to claim 5. 7. An antibody or a fragment thereof, which is produced by using the conjugate according to claim 4 as an antigen, and which is reactive with the compound of the formula (2). 8. The antibody or fragment according to claim 7, which is a monoclonal antibody. 9. The monoclonal antibody TCZ1-12 produced by the hybridoma deposited under accession number FERM P-17556.
2. The antibody or fragment according to item 1. A hybridoma that produces the antibody or fragment according to any one of claims 7 to 9. 11. The hybridoma according to claim 10, which has been deposited under accession number FERM P-17556. 12. A method for immunochemically measuring a compound represented by the formula (2), comprising using the antibody or fragment according to any one of claims 7 to 9. 13. The method according to claim 1, further comprising:
13. The immunochemical assay method according to claim 12, comprising using the compound according to claim or the conjugate according to claim 4.