JP2000086654A - Hapten compound of isoprothiolane, antibody and assay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound useful as a hapten compound constituting an antigen for making a new monoclonal antibody reactive to isoprothiolane as a dithiolane-based rice blight-controlling agent. SOLUTION: This new compound is represented by formula I (R is a 1-5C alkyl; (n) is 1-15), e.g. a compound of formula II. The compound is obtained by the following steps: malonic acid is first reacted with an alcohol of the formula ROH in an organic solvent in the presence of a mineral acid or Lewis acid to effect esterification, the resulting malonic ester is then reacted with an ester compound of formula III (Z is a carboxyl-protecting group) to form the corresponding malonic diester derivative, which, in turn is reacted with carbon disulfide and a dihaloethane of the formula L1CH2CH2L2 (L1 and L2 are each a halogen) in an organic solvent in the presence of a base followed by eliminating the carboxyl-protecting group from the resulting product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to diisopropyl
1,3-dithiolan-2-ylidenemalonate (hereinafter, referred to as
(Hereinafter referred to as "isoprothiolane"), an antigen, an antibody and a fragment thereof.

[0002] The present invention further relates to an immunoassay method using the above antigen, antibody and fragment thereof.

[0003]

BACKGROUND OF THE INVENTION Isoprothiolane has the following formula (2):

Embedded image A dithiolane-based blast control agent having the structure represented by Isoprothiolane agents are effective for water application as well as spraying because they have the ability to penetrate and transfer to rice bodies. In addition, it also has an effect as a growth regulator, such as a growth-suppressing effect of brown planthoppers and prevention of sapling.

[0004] It has a therapeutic effect and a prophylactic effect. It has systemic translocation and is effective. There is almost no effect on spore germination of blast fungus,
It strongly inhibits the growth of invading hyphae after appressorium formation.
The mechanism of action of isoprothiolanes is thought to be the same inhibition of phospholipid synthesis as organophosphorus fungicides. There is almost no harm to various crops, and it can be mixed with fungicides and insecticides. In addition, although the effect of inhibiting the growth of brown planthopper is observed in the application of granules, it is used prophylactically because it takes two weeks or more to achieve the effect. The granules are also effective for stuffy seedlings (physiological acute whiplash disorder) caused by imbalance between water absorption and transpiration, such as a decrease in root water absorption due to low temperature during seedling raising and an increase in transpiration due to high temperature (Agricultural Chemical Handbook 19).
1994 version, p. 583, p. 247-p. 250, p. 120-
121, p. 471, Japan Plant Protection Association; "Recent Analytical Methods for Pesticide Residues", pp. 374-375, edited by the Research Group for Pesticide Residue Analytical Methods, published by Chugoku-Honki '95 .4.1).

[0005] In recent years, there has been great social interest in the effects of pesticide residues in the environment, such as water, soil, and air, and foods on human health. Therefore, it is necessary to quickly and accurately measure the amount of isoprothiolane remaining in such widely used pesticides, i.e., isoprothiolane, in order to ensure environmental and food safety.

[0006] Isoprothiolane does not currently have a standard for pesticide residues based on the Food Sanitation Law. However, since it is a widely used pesticide such as rice, pesticide registration suspension standards have been established. The standard values are 2 ppm for rice and 0.1 ppm for fruit.

[0007] Various guidelines for isoprothiolane regarding water quality are determined and monitored. For example, it is listed as a monitoring item of the water quality environmental standard based on the Basic Environmental Law and a monitoring item of the tap water quality standard based on the Water Supply Law, and the guideline value is 0.04 mg /
1 or less. In the "Provisional Guidance on Prevention of Water Pollution Caused by Pesticides Used at Golf Courses" (1990) by the Environment Agency, the provisional guidance value on golf course pesticide drainage was set at 0.4 mg / l. (The latest pesticide regulation and standard value handbook, 1995 edition)
(Japan) Plant Protection Association. Therefore, in order to ensure the safety of foods and the environment, it is necessary to measure the amount of isoprothiolane contained therein quickly and accurately.

Conventionally, for example, isoprothiolane in agricultural products has been extracted and purified from samples of rice and fruits, and then analyzed by gas chromatography (GC). That is, a method has been adopted in which a sample is extracted with acetone, the concentrated solution is purified with a porous diatomaceous earth column, partitioned with hexane-acetonitrile, further purified by column chromatography, and then analyzed by GC (""). Residue analysis method for the latest pesticides ”, supra). This method has problems that the preparation of the sample is complicated and requires a great deal of labor and time, that the analysis is skilled, and that the measuring device and the equipment require high costs. In the measurement of isoprothiolane, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and a new measurement method not only in terms of accuracy but also in simplicity, speed, and economy has been demanded.

The immunoassay is a method for detecting an antigen based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen. Due to its excellent accuracy, simplicity, rapidity, and economical efficiency, it has recently It's getting attention. In immunoassays, a great 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 immunoassays, 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 molecules such as isoprothiolane usually cannot elicit an immune response when inoculated into animals. For the first time, these molecules act as a panel of epitopes by binding to immunogenic macromolecules and elicit an immune response in the presence of the T cell receptor, resulting in the production of antibodies by a group of B lymphocytes. Is done. 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] However, although the need for isoprothiolane was very high, not only a suitable antibody but also a hapten for producing the antibody had not been obtained before the present invention.

[0014]

An object of the present invention is to provide a novel antibody or a fragment thereof which reacts with isoprothiolane, and a method for producing the same.
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 isoprothiolane.

Another object of the present invention is to provide a derivative of the compound, which is a hapten compound constituting an antigen for preparing a novel antibody reactive with isoprothiolane.

Another object of the present invention is to provide a conjugate of an isoprothiolane derivative and a polymer compound. The conjugate serves as an antigen for producing an antibody reactive with isoprothiolane.

[0018] It is still another object of the present invention to provide a hybridoma that produces the antibody and its fragment.

Another object of the present invention is to provide a method for immunologically measuring isoprothiolane, which comprises using a conjugate of the antibody or a fragment thereof and / or the isoprothiolane derivative with a polymer compound. .

[0020]

Means for Solving the Problems As a result of intensive studies, the present inventors have used isoprothiolane derivatives in which a spacer arm and a functional group available for bonding to a polymer compound have been introduced into isoprothiolane as a hapten. As a result, an antibody having reactivity with the compound was successfully obtained, and the present invention was completed.

The isoprothiolane of the present invention is
The following equation (2):

Embedded image It is a compound represented by these.

The antibody of the present invention can be obtained, for example, by using a derivative obtained by introducing a spacer arm and a functional group available for binding into isoprothiolane as a hapten and binding to an appropriate polymer compound as an antigen. For example, the following equation (1):

Embedded image [Wherein, in the formula (1), R is an optionally branched alkyl group having 1-5 carbon atoms, preferably an isopropyl group; and n is an integer of 1-15, preferably 8,]. Is used as a hapten for antibody production. In the present invention, the stereoisomers of the compound are not particularly limited, and include all stereoisomers.

The present invention relates to the hapten compound, a conjugate of the hapten compound and a polymer compound, an antibody reacting with isoprothiolane, a method for producing the same, and a method for immunological measurement of isoprothiolane using the hapten compound or the antibody.

Preparation of Isoprothiolane Derivative The isoprothiolane derivative represented by the formula (1) can be prepared according to a known method. For example, but not limited to, the methods described below.

First, the following equation (X1):

Embedded image And malonic acid represented by the following formula (X2):

Embedded image [In the formula (X2), R is as defined above] and an inorganic acid such as concentrated sulfuric acid, sulfuric acid and hydrochloric acid, or a Lewis such as boron trifluoride diethyl ether complex in an organic solvent. The esterification is carried out by reacting in the presence of an acid to give the following formula (X3):

Embedded image [In the formula (X3), R is as defined above] to obtain a malonic acid derivative represented by the following formula:

In the synthesis of the formula (X3), the organic solvent includes
For example, dichloromethane, acetonitrile, acetone,
Hexane, pentane, benzene, toluene, chloroform, 1,2-dichloroethane, ethyl acetate, diglyme,
N, N-dimethylformamide, hexamethylphosphoric triamide and the like, or a mixed solvent thereof can be used. The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably 60 ° C. to 90 ° C., for 1 hour to 20 hours, preferably 2 hours.
Time-Perform for 5 hours.

Next, the malonic acid derivative of the formula (X3) is converted into the following formula (X4):

Embedded image [In the formula (X4), Z is a carboxyl-protecting group;
And n is as defined above] with an ester compound represented by the following formula (X
5):

Embedded image [In the formula (X5), R, Z and n are as defined above] to obtain a malonic acid diester derivative represented by the following formula:

The protecting group for the carboxyl group represented by Z may be a known one, and specific examples include a methyl group, an ethyl group, a tert-butyl group, a benzyl group, a p-methoxybenzyl group and a 3,4-dimethoxy group. Benzyl group, trichloroethyl group, trimethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triethylsilyl group, triisopropylsilyl group,
And a trimethylsilylethoxymethyl group.

In the synthesis of the compound of the formula (X5), as an organic solvent, for example, tetrahydrofuran (THF), dichloromethane, 1,2-dichloroethane, diethyl ether, benzene, toluene, xylene, N, N-dimethylformamide (DMF) ) And the like, or a mixed solvent thereof can be used. In the reaction solution, a condensing agent such as N, N-dicyclohexylcarbodiimide (DCC), N, N-diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide,
A base such as N-dimethylaminopyridine (DMAP);
It is desirable to coexist an acid such as camphorsulfonic acid (CSA). The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably at 20 ° C. to 35 ° C., for 1 to 40 hours, preferably for 10 to 30 hours.

The ester compound of the formula (X4) can be synthesized, for example, as follows.

First, the following equation (X6):

Embedded image By introducing a protective group into the carboxyl group of the alkene carboxylic acid represented by the formula (X6), n is as defined above, the following formula (X7):

Embedded image [In the formula (X7), Z and n are as defined above].

Many known methods for synthesizing the ester compound of the formula (X7) are known. For example, a method of adding an acid catalyst such as hydrogen chloride, thionyl chloride, sulfuric acid, and p-toluenesulfonic acid to carry out dehydration condensation, or a method of adding an equal amount or more of thionyl chloride to a solution or suspension of an alcohol corresponding to an amino acid is exemplified. Can be After protecting the amino group of the amino acid with an appropriate protecting group, dichloromethane, 1,2-dichloroethane, tetrahydrofuran (T
HF), diethyl ether, benzene, toluene, xylene, N, N-dimethylformamide (DMF) in the presence of a suitable solvent such as N, N-dicyclohexylcarbodiimide (DCC), N, N-diisopropylcarbodiimide (DIPC), A method of using a carbodiimide-based condensing agent such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), or an azodicarboxylic acid ester such as triphenylphosphine and diethyl azodicarboxylate for dehydration condensation with alcohol, or BO
Method for synthesizing esters using other condensing agents such as P reagent and DPPA reagent (Experimental Chemistry Lecture 22, p. 43-51)
Page, edited by The Chemical Society of Japan, Maruzen Co., Ltd.) to obtain an amino acid ester by removing the protecting group of the amino acid derivative. Alternatively, a method of removing the protecting group of an amino acid derivative obtained by a method of reacting an alkyl halide in the presence of a base or the like can also be mentioned. For tert-butyl ester of amino acid, dichloromethane, 1,2-dichloroethane, benzene, toluene,
In a suitable solvent such as THF, in the presence of phosphoric acid, sulfuric acid, boron trifluoride diethyl ether complex or the like, the amino group-protected amino acid and isobutene are stirred to remove the protective group of the obtained amino acid derivative. The method of obtaining is also mentioned.

Examples of the amino-protecting group include a benzyloxycarbonyl group, a tert-butoxycarbonyl group and a 9-fluorenylmethoxycarbonyl group. Examples of the method for removing the amino-protecting group include a method by hydrolysis, a method using a mineral acid such as hydrochloric acid and sulfuric acid, a method using an organic acid such as trifluoroacetic acid, and a method using a base such as pyridine and piperidine.

Next, mercury (II) acetate is added to the ester compound of the formula (X7), and the carbon-mercury bond is reductively cleaved (mercury-demerch reaction) to give the ester of the formula (X4) A compound can be obtained. The solvent in this case, it is possible to use a THF-H ₂ O and the like.
The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably 20 ° C.
At -30 ° C for 30 minutes to 10 hours, preferably 30 minutes
Perform for 2 hours.

The malonic diester derivative of the above formula (X5) is then added to carbon disulfide and the following formula (X8) in an organic solvent in the presence of a base:

Embedded image [In the formula (X8), L ¹ and L ² are a halogen atom (in the present specification, the halogen atom means F, Cl, Br or I), and may be the same or different]
By reacting a dihalogenated ethane represented by the following formula (X9):

Embedded image [In the formula (X9), R, Z and n are as defined above).

As the organic solvent, the same organic solvents as those used for synthesizing the compound of the formula (X5) can be used.
As the base, for example, sodium hydride, sodium methylate, sodium ethylate, lithium diisopropylamide and the like can be used. The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably 20 ° C.-8
The reaction is performed at 0 ° C. for 1-20 hours, preferably 4-6 hours.

Further, the isoprothiolane derivative of the formula (1) can be obtained by removing the protecting group of the carboxyl group represented by Z from the ester compound of the formula (X9). The removal of the carboxyl-protecting group can be carried out by a known method such as alkali hydrolysis and acid hydrolysis.

That is, in the case of acid hydrolysis, the formula (X
The ester compound 9) is preferably dissolved in an organic solvent such as benzene, dichloromethane, 1,2-dichloroethane and the like, and then hydrochloric acid, sulfuric acid, boron trifluoride, trifluoroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid At 0 ° C. to the boiling point of the solvent, preferably 0 ° C. to room temperature, for 5 minutes to 10 hours, preferably 1-
The isoprothiolane derivative of the formula (1) can be obtained by stirring and reacting for 5 hours.

In the case of alkali hydrolysis, the formula (X)
The ester compound of 9) is preferably dissolved in an organic solvent such as methanol, ethanol, tetrahydrofuran or ethylene glycol, and then an aqueous solution of sodium hydroxide or potassium hydroxide is added thereto. The isoprothiolane derivative of the formula (1) can be obtained by stirring and reacting at a temperature of from 5 ° C. to 5 hours, preferably 1-2 hours.

Further, when Z is a benzyl group, the removal can be carried out by a hydrogenolysis reaction using a catalyst such as palladium carbon or a Birch reduction using liquid ammonium-sodium.

Further, when Z is a silyl group, deprotection can also be carried out using a reagent that generates a fluorine anion such as tetra-n-butylammonium fluoride or pyridinium fluoride.

The compound obtained by the above production method is
By performing a silica gel chromatography or a recrystallization operation as needed, a purified product with higher purity can be obtained.

Hereinafter, the preparation of the antigen and the antibody of the present invention and the immunological measurement method 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.

An isoprothiolane derivative and a polymer compound
Preparation of the conjugate The isoprothiolane derivative synthesized as described above is bound to a suitable polymer compound and then used as an antigen for immunization.

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.

The bond between the isoprothiolane derivative and the polymer compound can be formed, for example, by the activated ester method (AEKA).
RU et al. : J. Agric. Food
Chem. 42 301-309 (1994)),
Or a mixed acid anhydride method (BF Erlanger e)
t al. : J. Biol. Chem. 234 10
90-1094 (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 produce an N-hydroxysuccinimide ester.

As the coupling agent, an ordinary coupling agent commonly used in a condensation reaction can be used.
Dicyclohexylcarbodiimide, carbonyldiimidazole, water-soluble carbodiimide and the like. As the organic solvent, for example, N, N-dimethylformamide (hereinafter, referred to as “DMF”), dimethylsulfoxide (hereinafter, referred to as “DMSO”), dioxane, and the like can be used. The molar ratio of the hapten compound and N-hydroxysuccinimide used in the reaction is preferably from 1:10 to 10:
1, more preferably 1: 1 to 1:10, most preferably 1: 1. The reaction temperature is from 0 ° C to 100 ° C, preferably from 5 ° C to 50 ° C, more preferably from 22 ° C to 2 ° C.
At 7 ° C., the reaction time is from 5 minutes to 24 hours, preferably 30 minutes.
It is from minutes to 6 hours, more preferably from 1 hour to 4 hours. The reaction can be carried out at a temperature from the melting point to the boiling point.

After the coupling reaction, the reaction supernatant is added to the solution of the polymer compound and reacted. For example, when the polymer compound has a free amino group, the amide is located between the amino group and the carboxyl group of the hapten compound. A bond is created. The reaction temperature is 0 ° C to 60 ° C, preferably 5 ° C to 40 ° C, more preferably 22 ° C to 27 ° C, and the reaction time is 5 minutes to 24 hours, preferably 1 hour to 16 hours. The reaction product is purified by dialysis, a desalting column, or the like to obtain a conjugate of the isoprothiolane derivative and the polymer compound.

On the other hand, the mixed acid anhydride used in the mixed acid anhydride method is obtained by a reaction between a carboxylic acid and a haloformate, and the resulting hapten-polymer compound is reacted with the polymer compound. A conjugate is produced. This reaction is performed in the presence of a basic compound.
Examples of the basic compound include N-methylmorpholine,
Triethylamine, trimethylamine, pyridine, N,
Organic bases such as N-dimethylaniline, DBN, DBU, and DABCO; and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The reaction is usually carried out at a temperature of −20 ° C. to 100 ° C., preferably 0 ° C. to 50 ° C., for a reaction time of 5
Minutes to 10 hours, preferably 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually carried out at a temperature of −20 ° C. to 150 ° C., preferably 0 ° C. to 100 ° C.
C. and the reaction time is from 5 minutes to 10 hours, preferably from 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 compounds 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-dimethylformamide, dimethylsulfoxide, hexamethylphosphoric triamide and the like; Examples of the haloformate used in the mixed acid anhydride method include isobutyl chloroformate, methyl chloroformate, methyl bromoformate, ethyl chloroformate, and ethyl bromoformate. 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 an isoprothiolane derivative can be used in an immunoassay. As a labeling substance, horseradish peroxidase (hereinafter referred to as "H
RP ") or enzymes such as alkaline phosphatase,
There are coloring substances such as fluorescein isothiocyanate and rhodamine, and radioactive substances such as ³² P and ¹²⁵ I.

Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of an isoprothiolane derivative and a polymer compound. For example, an isoprothiolane derivative-BSA conjugate is added to a phosphate buffer (hereinafter referred to as “P
BS)) and immunized animals with adjuvants such as Freund's complete or incomplete adjuvant or alum. 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.

The administration method 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. Immunity is 1
It can be performed multiple times or at appropriate intervals, preferably one to five weeks.

Blood is collected from the immunized animal, and the serum separated therefrom can be used to evaluate the presence of a polyclonal antibody that reacts with isoprothiolane.

Preparation of Monoclonal Antibody The monoclonal antibody of the present invention can be prepared by a known method using a conjugate of an isoprothiolane derivative and a polymer compound.

In preparing a monoclonal antibody,
At least the following work steps are required. (A) Preparation of a conjugate of an isoprothiolane derivative to be 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 antibody of interest (g) Monoclones by culturing hybridomas or transplanting hybridomas into animals Preparation of antibodies (h) Measurement of reactivity of prepared monoclonal antibodies

A conventional method for producing a hybridoma producing a monoclonal antibody is described in, for example, Hybridoma Techniq.
es), Cold Spring Harbor Lab
laboratory, 1980 edition), cytohistochemistry (Syuji Yamashita et al., edited by the Japanese Society for Tissue and Cellular Chemistry; interdisciplinary project, 1986
Year).

Hereinafter, a method for producing a monoclonal antibody against isoprothiolane 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 the step (c) are lymphocytes, which can be generally obtained from the spleen, thymus, lymph nodes, peripheral blood or a combination thereof, but spleen cells 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 that can be used in the step (d) include, for example, P3 / X63-Ag8 (X63) (Nat) of a myeloma cell line derived from Balb / c mouse.
ure, 256 , 495-497 (1975)),
P3 / X63-Ag8. U1 (P3U1) (Curre
nt Topics. in Microbiology
and Immunology, 81 1-7
(1987)), P3 / NSI-1-Ag4-1 (NS
-1) (Eur. J. Immunol., 6 , 511).
-519 (1976)), Sp2 / 0-Ag14 (S
p2 / 0) (Nature 276 , 269-270)
(1978)), FO (J. Immuno. Me)
th. , 35 , 1-21 (1980)), MPC
Myeloma cell lines such as -11, X63.653, S194, or rat-derived 210. RCY3. Ag1.
2.3. (Y3) (Nature 277 , 131-
133, (1979)) and the like.

The above-mentioned cell line is 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 1 × 10 ⁶ or more is secured on the day of fusion.

The cell fusion in the step (e) is performed by a known method, for example, the method of Milstein et al.
articles in Enzymology, 73 ,
3 (1981)). The most commonly used method at present is a method using polyethylene glycol (PEG). For the PEG method,
For example, it is described in Cell Histochemistry, Shuji Yamashita et al. Another fusion method is electrical processing (electric fusion)
(Etsuko Okochi et al., Experimental Medicine 5.1135-19, 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 isoprothiolane is measured by the LISA method.

Further, cell cloning of a hybridoma which has been found by measurement to produce an antibody reactive with isoprothiolane 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 once to four times by, for example, limiting dilution, and a stable antibody titer is obtained, and the well is selected as an anti-isoprothiolane monoclonal antibody-producing hybridoma strain. Examples of a medium for culturing a hybridoma include DMEM or IM containing fetal calf serum (FCS).
DM or the like 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, Ba
lb / c) or hybridomas can be grown in the abdominal 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-isoprothiolane 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-isoprothiolane monoclonal antibody can be obtained. If further purification is required, the operation of collecting the antibody fraction using a commonly used method such as ion exchange column chromatography, affinity chromatography, open column chromatography, high performance liquid chromatography (HPLC), etc. is performed. It can be carried out by performing one or more times, or a combination thereof.

The anti-isoprothiolane monoclonal antibody obtained as described above can be used, for example, in the ELISA described below.
The subclass, antibody titer, and the like can be determined using a known method such as a method.

Measurement of Isoprothiolane Using Antibody As a method for measuring isoprothiolane using the antibody used in the present invention, a radioisotope immunoassay (RIA)
Method), ELISA method (Engval, E., Meth)
ods in Enzymol. , 70 , 419-
439 (1980)), fluorescent antibody method, plaque method, spot method, agglutination method, Ouchterlo (Ouchterlo)
ny) method and the like, which are generally used for detecting antigens ("Hybridoma method and monoclonal antibody", published by R & D Planning, pp. 30-53, March 5, 1982). Can be The ELISA method is widely used from the viewpoints of sensitivity, simplicity, and the like.

The measurement of isoprothiolane was performed by various ELISAs.
Among the methods A, the indirect competitive inhibition ELISA method can be used for the following procedures. (A) First,
A conjugate of an isoprothiolane derivative as an antigen and a polymer compound is immobilized on a carrier. (B) blocking the surface of the solid phase on which the antigen is not adsorbed by a protein unrelated to the antigen, for example, a protein; (C) A sample and an antibody containing various concentrations of isoprothiolane are added thereto, and the antibody is allowed to react competitively with the immobilized antigen and isoprothiolane to form an immobilized antigen-antibody complex and an isoprothiolane-antibody complex. Generate. (D) By measuring the amount of the immobilized antigen-antibody complex, the amount of isoprothiolane in the sample can be determined from a previously prepared calibration curve.

In step (a), the carrier on which the antigen is immobilized is not particularly limited, and any carrier commonly used in the ELISA method can be used. An example is a 96-well microtiter plate made of polystyrene.

To immobilize an antigen on a carrier, for example,
A buffer containing an antigen may be placed on a carrier and incubated. Known buffers can be used, and examples thereof include a phosphate buffer. The concentration of the antigen in the buffer can be selected from a wide range.
g / ml to about 100 μg / ml, preferably 0.0
5 μg / ml to 10 μg / ml is suitable. Also,
When a 96-well microtiter plate is used as a carrier, 50 μl / well at 300 μl / well or less.
From the well, about 150 μl / well is desirable. Furthermore,
Incubation conditions 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 isoprothiolane derivative and the polymer compound used for preparing the antibody itself, but also the conjugate of another derivative represented by the formula (1) and the polymer compound can be used. For example, antigens having different R and / or n in the compound represented by the formula (1) can be used for antibody production and immobilization, respectively. Further, other isoprothiolane-like compounds not included in the formula (1) can also be used as the immobilized antigen.

In the blocking in the step (b), there is a portion to which an antibody to be added later can be adsorbed in addition to the isoprothiolane derivative portion in the carrier on which the antigen (combination of the isoprothiolane derivative and the polymer compound) is immobilized. May be done solely to prevent it. As a blocking agent, for example, BSA or skim milk solution can be used. Or, Block Ace ("Block
Ace ", manufactured by Snow Brand Milk Products Co., Ltd., code No. UK-25B)
Commercially available blocking agents can also be used. Specifically, for example, but not limited to, an appropriate amount of Block Ace diluted 4-fold with PBS is added to a portion where the antigen is immobilized, and the mixture is incubated at about 4 ° C. overnight, and then washed with a buffer. It is done by doing. Although there is no particular limitation on the buffer, for example, PB
S is suitable.

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

At this time, as the antibody, an antibody against the isoprothiolane 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 to react.

The first antibody is dissolved in a buffer and added. Although not limited, the reaction may be performed at about 25 ° 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 the washing liquid, for example, PBS is preferable.

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 anti-mouse antibody-goat antibody conjugated with an enzyme (for example, peroxidase or alkaline phosphatase). It is desirable to react the first antibody bound to the carrier with a second antibody diluted so that the final absorbance is 4 or less, preferably 0.3 to 3.0. Buffer is used for dilution. Although not limited, the reaction is carried out at room temperature 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 enzyme of the second antibody bound to the carrier is added, and the amount of isoprothiolane can be calculated from the calibration curve by measuring the absorbance.

When peroxidase is used as the 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 room temperature for about 10 minutes, and then the enzyme reaction is stopped by adding 1N sulfuric acid. If OPD is used, measure the absorbance at 490 nm. When 3,3 ', 5,5'-tetramethylbenzidine is used, the absorbance at 450 nm is measured.
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 2N NaOH, and the absorbance at 415 nm is measured. The method is suitable.

With respect to the absorbance of the reaction solution without the addition of isoprothiolane, the rate of decrease in the absorbance of the solution in which isoprothiolane was added and reacted with the antibody was calculated as the inhibition rate. The isoprothiolane concentration in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which isoprothiolane of a known concentration has been added.

Alternatively, the measurement of isoprothiolane can be carried out, for example, by a direct competitive inhibition 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 by a protein unrelated to the antigen, for example, a protein.

(C) A sample containing various concentrations of isoprothiolane and an enzyme-bound hapten obtained by binding an enzyme to an isoprothiolane derivative are reacted with an antibody immobilized on a carrier.

(D) By measuring the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of isoprothiolane 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 can be the same as in the indirect competitive inhibition ELISA method described above. Alternatively, an antibody bonded to an amino group-bonded microtiter plate by a chemical bonding method can be used.

The blocking in the step (b) is carried out because, in the carrier on which the antibody is immobilized, there may be a portion where isoprothiolane and enzyme-bound hapten in the sample to be added later are adsorbed independently of the antigen-antibody reaction. , To prevent it. As the blocking agent and the method therefor, those similar to the indirect competitive inhibition ELISA method described above can be employed.

The preparation of the enzyme-bound hapten used in the step (c) may be carried out by any method without particular limitation as long as it is a method of binding an isoprothiolane derivative to an enzyme. For example, the activated ester method described above can be employed. The prepared enzyme-linked hapten is mixed with a sample containing isoprothiolane.

The hapten to be bound to the enzyme is not limited to the isoprothiolane derivative used for the production of the antibody, but also to the other hapten represented by the formula (1), as in the case of the immobilized antigen in the indirect competitive inhibition ELISA method. Derivatives can also be used. For example, compounds having different numbers of R and / or n in the formula (1) can be used for antibody production and as a labeling competitor, respectively. Further, equation (1)
Other isoprothiolane analogs not included in can be used as haptens to bind to enzymes. In step (c), a sample containing isoprothiolane and an enzyme-bound hapten are brought into contact with an antibody-immobilized carrier, and a complex of these and an immobilized antibody is formed by a competitive inhibition reaction between isoprothiolane and the enzyme-bound hapten. The sample containing isoprothiolane is used after being diluted with an appropriate buffer. Without limitation, the reaction is performed, for example, at room temperature for about 1 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 buffer, for example, PBS can be adopted.

Further, in step (d), a chromogenic substrate solution that reacts with the enzyme of the enzyme-bound hapten is added in the same manner as in the above-described indirect competitive inhibition ELISA method, and the absorbance is measured to calculate the amount of isoprothiolane from the calibration curve. Can be.

The monoclonal antibody 27A1-1 of the present invention
-1 is about 0.1- in the direct competitive inhibition ELISA method.
1000 ng / ml, preferably 1-100 ng / ml
(Example 7 and FIG. 1).

Cross-reactivity of the Antibody of the Present Invention The cross-reactivity of the monoclonal antibody of the present invention can be examined by the above-described direct competitive inhibition ELISA or indirect competitive inhibition ELISA. For example, 27A1-1
-1 showed little reactivity with other similar compounds in the indirect competitive inhibition ELISA (Example 6).
And Table 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.

[0097]

EXAMPLES Example 1 Synthesis of Isoprothiolane Derivative

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Malonic acid (1) 52 g (500 mmol)
And 7.5 g (125 mmol) of 2-propanol were added to 500 ml of dichloromethane, and concentrated sulfuric acid was added.
For 3 hours. After washing the reaction solution with water, the dichloromethane layer was dehydrated (anhydrous magnesium sulfate), filtered and concentrated, and then subjected to a silica gel column (n-hexane: ethyl acetate = 4: 1 →
Purification by 2: 1) yielded 3.0 g (16% yield) of (2) as a transparent liquid.

Malonic acid monoisopropyl ester (2)
1.5 g (10 mmol), 2.6 g (10 mmol) of tert-butyl 10-hydroxyundecanoate and
0.77 g (3.3 mmol) of camphorsulfonic acid (CSA) and 0.4 g (3.3 mmol) of N, N-dimethylaminopyridine (DMAP) are suspended in 50 ml of tetrahydrofuran (THF) and 3.1 g ( 15mm
ol) of N, N-dicyclohexylcarbodiimide (D
20 ml of a THF solution of CC) was added dropwise at room temperature. After stirring for 1 day, hexane was added and the white precipitate was filtered off. The filtrate was concentrated and purified by silica gel chromatography (n-hexane: ethyl acetate = 19: 1) to give 2.38 g of a transparent oil ( (3) of (62% yield) was obtained.

Sodium hydride (oil-based, 60%) 0.8
g (20 mmol) were suspended in 20 ml of THF, and 3.86 g (10 mmol) of malonic acid diester (3) was dissolved in THF.
10 ml of HF solution was added dropwise at room temperature. After stirring at room temperature for 20 minutes, 5 ml of a THF solution containing 0.76 g (10 mmol) of carbon disulfide was added dropwise. After stirring for 1 hour, a solution of 2.5 g (13.3 mmol) of 1,2-dibromoethane in THF 10
Then, the mixture was heated and stirred at room temperature for 2 hours and at 80 ° C. for 2 hours. The reaction solution is concentrated, the residue is dissolved in dichloromethane, washed with water, dehydrated (anhydrous magnesium sulfate), filtered, concentrated, and then roughly purified by silica gel column chromatography (n-hexane: ethyl acetate = 9: 1 → 4: 1). , (4)
I got

The crude product containing hapten ester (4) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution is concentrated under reduced pressure and subjected to silica gel chromatography (n
-Hexane: ethyl acetate = 4: 1 → 2: 1) gave 2.06 g (51% of the total yield from (3)) of (5) as a yellow oil.

¹ H—N of the isoprothiolane derivative (5)
Physical property data by MR are shown below. _{^{1 H-NMR (CDCl 3)}} 1.13-1.70 ( overlap, 23H), 2.27-2.
39 (t, 2H) 3.37 (s, 4H), 4.96-5.19 (duplicate, 2
H)

Note that 10-hydroxyundecanoic acid te
rt-butyl was synthesized as follows.

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5.5 g (30 ml) of 10-undecenoic acid
Was dissolved in 200 ml of dichloromethane, and after cooling with salt-ice, isobutene was bubbled and 0.3 ml of concentrated sulfuric acid was added. After stirring overnight, the reaction solution was concentrated and purified by a silica gel column (hexane: ethyl acetate = 49: 1 → 19: 1) to obtain 3.4 g (47%) of tert-butyl undecenoate as a transparent oil. .

2.8 g of mercury (II) acetate (8.8 mmol)
l) was dissolved in 20 ml of THF-water (1: 1). 10
Tert-butyl undecenoate 2.1 g (8.8 mm
ol) in THF (10 ml) was added dropwise. After stirring at room temperature for 30 minutes, 9 ml of a 3N NaOH aqueous solution, and then 180 mg of sodium borohydride were added to a 3N NaOH aqueous solution 9
What was dissolved in ml was dropped at room temperature. After stirring at room temperature for 1 hour, 50 ml of THF was added and potassium chloride was added until saturation, and the mixture was transferred to a separating funnel and allowed to stand. The THF layer was separated, dehydrated (anhydrous magnesium sulfate), filtered and concentrated, and then subjected to a silica gel column (hexane: ethyl acetate = 9:
Purification in 1) yielded 1.8 g (80%) as a clear oil.
%) Tert-butyl 10-hydroxyundecanoate.

Example 2 Isoprothiolane Derivatives
Preparation of Conjugate with Child Compound or Labeled Compound As an immunogen and an antigen for screening, a conjugate of an isoprothiolane derivative and BSA was prepared by the activated ester method.

0.2 mmol of the isoprothiolane derivative prepared in Example 1 was dissolved in 1.0 ml of DMSO,
0.3 mmol of hydroxysuccinimide and 0.3 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide were added, and the mixture was stirred at room temperature for 3.5 hours.
After the reaction, the mixture was centrifuged at 10,000 rpm for 15 minutes to separate into a supernatant and a precipitate.

On the other hand, 50 mg of BSA was added to 145 mM Na
Cl-0.01 M phosphate buffer (pH 7.2: hereinafter referred to as “P
BS)) and dissolved in 5.0 ml of DMSO 1.05
A solution to which ml was added was prepared, and 0.25 ml of the above supernatant was added to this solution, and reacted at room temperature for 16 hours.
After the reaction, an isoprothiolane derivative-BSA conjugate dialyzed against distilled water at 4 ° C. was used as an antigen for immunization.

Further, a conjugate of isoprothiolane derivative-RSA and a conjugate of isoprothiolane derivative-HRP were prepared by the same method.

Example 3 Immunization 100 μg of the immunizing antigen prepared in Example 2 was added to PBS50.
It was dissolved in μl, mixed with an equal volume of Freund's complete adjuvant, and inoculated subcutaneously into Balb / c mice. Further, four weeks later, an incomplete immunizing adjuvant was used to additionally immunize with an immunizing antigen prepared in the same manner as described above. On the 6th week, 30 μg of the immunizing antigen dissolved in 180 μl of PBS was boosted through the tail vein of the mouse.

Example 4 Isoprothiolane with antiserum
Immediately before inoculation into mice tail vein in reactive Example 3, the antiserum bled diluted prepared, indirect competitive inhibition E to be described in detail below
Isoprothiolane was measured by LISA, and antiserum was evaluated.

A solution of the isoprothiolane derivative-RSA conjugate prepared in the same manner as the immunizing antigen (0.5 μg / ml)
To a 96-well microplate in an amount of 50 μl / well (25 ng / 50 μl / well),
Block Ace diluted 4 times (“Block Ac
e ", manufactured by Snow Brand Milk Products Co., Ltd., code No. The plate was blocked with UK-25B) to prepare an assay plate. An equal volume of a 10000-fold diluted antiserum and a 20% methanol solution containing various concentrations of isoprothiolane were mixed, 50 μl of the mixture was added to each well, and reacted at room temperature for 1 hour.

After washing 5 times with PBS, 50 μl / well of peroxidase-conjugated goat anti-mouse IgG antibody (manufactured by Tago) diluted 2000-fold with 10-fold diluted Block Ace was added, and the mixture was added at room temperature for 1 hour. Reacted.

After washing 5 times with PBS, 0.1 M citrate-phosphate buffer (pH 5.0) containing 2 mg / ml OPD and 0.02% hydrogen peroxide was added in an amount of 50 μl / well. The reaction was allowed to proceed at room temperature for 10 minutes to develop color.

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
Table 1 shows an example of the results.

[0116]

Table 1 Measurement of isoprothiolane using mouse antiserum Isoprothiolane concentration Absorbance (490 nm) Difference in absorbance 0 ng / ml 0.231 10 ng / ml 0.152 0.079 From Table 1, inhibition reaction at 10 ng / ml of isoprothiolane Was confirmed, indicating that the antiserum used was reactive with isoprothiolane.

Example 5 Preparation of Hybridomas Following Example 3, spleen cells from mice with high anti-isoprothiolane antibody activity in serum and myeloma cells (S
p2 / 0-Ag14) and the method of Shuji Yamashita et al. (Histocytochemistry: Japan Society for Histocellular Chemistry: Interdisciplinary Planning, 1986)
And fused by the polyethylene glycol method according to the above. To the plate coated and blocked in the same manner as in Example 4, 50 μl / well of the culture supernatant in which cell growth was observed was added at room temperature.
Allowed to react for hours.

After washing 5 times with PBS, a peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Tago) diluted 2000-fold using Block Ace diluted 10-fold was added in an amount of 50 μl / well, and incubated at room temperature for 1 hour. Reacted.

After washing 5 times with PBS, 0.1 M citrate-phosphate buffer (pH 5.0) containing 2 mg / ml OPD and 0.02% hydrogen peroxide was added in an amount of 50 μl / well. The color was developed at room temperature for 10 minutes.

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
Reactive cells (hybridomas) were selected. Next, the reactivity of each well with isoprothiolane was determined in Example 4.
The cells were produced by the indirect competitive inhibition ELISA described in (1) above, and cells producing the desired antibody were cloned by the limiting dilution method. As a result, several hybridomas were cloned as cells producing anti-isoprothiolane antibodies. Among them, 27A1-1-1 was deposited on July 2, 1998 under the deposit number FERM P-16868 under the name of 1-3-1 Higashi, Tsukuba City, Ibaraki Pref. Deposited.

Example 6 Cross-Reaction of Monoclonal Antibody
The reactivity of the monoclonal antibody 27A1-1-1 derived from the cloned hybridoma 27A1-1-1 with respect to two kinds of isoprothiolane analogs was examined using the indirect competitive ELISA method described in Example 4. . (Hereinafter, monoclonal antibodies use the same names as the hybridomas that produce them.) The results are shown in Table 2.

[0122]

[Table 2]

Here, IC ₅₀ (nmol / L) indicates the concentration of the target compound in the reaction solution that reduces the absorbance by 50% with respect to the absorbance of the reaction solution to which no compound such as isoprothiolane is added. Furthermore, cross-reactivity rate (%) is, (IC ₅₀ of IC ₅₀ / subject compound Isoprothiolane) X100
Defined. An antibody that specifically reacts with isoprothiolane as the numerical value of the cross-reaction rate between isoprothiolane and the similar compound deviates.

As shown in Table 2, the monoclonal antibody 27A1-1-1 showed reactivity with isoprothiolane, but showed little reactivity with analogous compounds.

Example 7 Direct competitive inhibition ELISA
Measurement of isoprothiolane The hybridoma 27A1-1-1 obtained in Example 5 was transplanted into the peritoneal cavity of a mouse, the ascites obtained 10-15 days later was collected, and the monoclonal antibody 27A1-1-1 was purified by affinity chromatography. Purified. This 2
The amount of isoprothiolane was measured by direct competitive inhibition ELISA using the 7A1-1-1 antibody.

The 27A1-1-1 antibody solution (10 μl
g / ml) in a 96-well microplate in an amount of 50 μl / well, coating was allowed to stand overnight at 4 ° C., and further 4-fold dilution of Block Ace (Yukishira Milk Industry Co., Ltd.)
Was performed to prepare a plate for assay. An equal volume mixture of a 20% methanol solution containing each concentration of isoprothiolane and a PBS solution containing the HRP-bound isoprothiolane derivative prepared in Example 2 was added to each well in an amount of 50 μl, and reacted at 25 ° C. for 1.5 hours.

After the reaction, the plate was washed 5 times with PBS, and then 2 mg
50 μl of a citrate-phosphate buffer (pH 5.0) containing 0.02% / ml OPD and 0.02% hydrogen peroxide was added to each well, and the mixture was allowed to stand at room temperature for 10 minutes to perform a color reaction.

Next, 50 μl of 1N sulfuric acid was added to each well to stop the color reaction, and the absorbance at 490 nm was measured. The result is shown in FIG. This direct competitive inhibition EL
Using the ISA method, the monoclonal antibody 27 of the present invention
A1-1-1 was able to measure isoprothiolane in the range of 1 ng / ml to 100 ng / ml.

[Brief description of the drawings]

FIG. 1 shows the monoclonal antibody 27A1 of the present invention.
The reactivity with isoprothiolane by the direct competitive inhibition ELISA method using -1-1 is shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 C12N 15/00 C // (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91) (72) Inventor Masao Hayashi 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Jun Fujii 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Insider of Inventor Uchida 1-27-14, Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Yoshio Kamada Inventor Yoshio Kamata, Minato-ku, Tokyo 1-27-14 Inside the Institute for Environmental Immunity Technology (72) Inventor Shigeyuki Watanabe 1-27-14 Hamamatsucho, Minato-ku, Tokyo F-term inside the Institute for Environmental Immunity Technology 4B024 AA05 AA07 AA11 B A53 GA03 HA15 4B064 AG27 CA10 CA20 DA10 DA13 4B065 AA92X AB05 AC14 BA08 CA25 CA41 CA46 CA47 4C023 NA06 4H045 AA11 AA20 AA30 BA10 BA51 DA76 DA86 EA50 FA41 FA72

Claims (11)

[Claims] (1) The following formula (1): [In the formula (1), R is an optionally branched alkyl group having 1 to 5 carbon atoms; and n is an integer of 1 to 15]
A compound having a structure represented by the formula: 2. A conjugate of the compound according to claim 1 with a polymer compound or a labeling substance. 3. An antigen is prepared by binding the compound according to claim 1 with a polymer compound, and the antigen is used to obtain the following formula (2): A method for producing an antibody or a fragment thereof reactive to a compound of the formula (2), characterized by producing an antibody reactive to a compound having a structure represented by the formula: [4] An antibody or a fragment thereof, which is produced by using the conjugate according to [2] as an antigen, and which is reactive with the compound of the formula (2). 5. The antibody or claim of claim 4, wherein in the compound of formula (1) according to claim 1 of the conjugate according to claim 2, R is isopropyl and n is 8. Fragment. 6. The antibody or fragment thereof according to claim 4, which is a monoclonal antibody. 7. The antibody or fragment thereof according to any one of claims 4 to 6, which is the monoclonal antibody 27A1-1-1. 8. A hybridoma producing the antibody according to any one of claims 4 to 7. 9. The hybridoma according to claim 8, which has been deposited under accession number FERM P-16868. 10. Use of the antibody or fragment according to any one of claims 4 to 7,
An immunological measurement method for the compound represented by the formula (2). 11. The immunoassay according to claim 10, further comprising using the compound or the conjugate according to claim 1 or 2.