JPH08231591A - Hapten, antigen, antibody specific to carbamate agent and detection of carbamate agent - Google Patents

Abstract

PURPOSE: To obtain a new hapten useful for the preparation of a monoclonal antibody having a specific structure similar to an N-methylcarbamate-type insecticide and necessary for the quick and accurate immunochemical detection of residual agrochemicals such as carbamate agent. CONSTITUTION: This new hapten is useful e.g. for the preparation of a monoclonal antibody specific to a carbamate agent, enabling the quick and accurate immunochemical detection of a carbamate agent remaining in soil, water, food, etc., and expressed by the formula A-O-CO-NH-(CH2 )n COOH [(n) is an integer of 2-5; A is an aromatic group or an N-imino group containing a 5 or 6-membered heterocyclic group having one or more hetero-atoms selected from O, N, S, etc., and expressed by formula I (R1 to R5 are each H, a halogen, nitro, amino, cyano, OH, SH, a 1-6C alkoxy, a 1-6C alkyl, carboxyl, etc.), formula II (R6 to R8 are each same as R1 ), formula III (R9 and R10 are each same as R1 ), formula IV (R11 and R12 are each same as R1 ), etc.].

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hapten, an antigen which is a complex of the hapten and a protein, a monoclonal antibody specific for the carbamate agent, and a method for detecting a carbamate agent using the monoclonal antibody.

[0002]

2. Description of the Related Art In recent years, there has been great social interest in residual pesticide residues in the environment such as soil, water, and the atmosphere, and in post-harvest pesticide residues in imported foods, which has recently been particularly increasing. To ensure the safety of the environment and food,
It is necessary to measure the amount of residual agricultural chemicals contained in these rapidly and accurately.

Conventionally, residual pesticides have been analyzed mainly by extracting the pesticide from a sample, purifying it, and then using gas chromatography or high performance liquid chromatography. Although these methods are excellent in terms of their sensitivity and accuracy, the preparation of samples is complicated and requires a great deal of time and labor, and requires skill in analysis, and expensive equipment such as measuring devices and equipment. There was a problem such as needing.

The measurement of residual pesticides requires a large number of samples,
In particular, analysis of residual agricultural chemicals such as imported foods needs to produce results within a short time, and a new measurement method that is simple, quick, and economical in addition to accuracy is required.

[0005]

Conventionally, for the analysis of N-methylcarbamate insecticides such as aldicarb and mesomil, gas chromatography capable of detecting S atoms contained in the structure thereof is mainly used. Alternatively, a method in which a derivative labeled with a fluorescent substance is measured by high performance liquid chromatography has been adopted, and its analysis requires considerable time and labor.

Therefore, as a simple and rapid method for measuring pesticide residues in environment and food samples, application of immunochemical detection using a specific antibody against the pesticide was attempted, and after intensive studies, the present invention was finally completed. Came to.

That is, the present invention has the general formula A-O-CO-NH- (CH ₂ ) nCOOH (1) [wherein n represents an integer of 2 to 5]. A represents an aromatic group or a N-imino group containing a 5- or 6-membered heterocycle having at least one kind selected from an oxygen atom, a nitrogen atom and a sulfur atom as a hetero atom, and these groups are halogen atoms, Nitro group, amino group, N-substituted amino group, N, N-
Di-substituted amino group, cyano group, hydroxy group, thiol group, alkoxy group, alkylthio group, carboxyl group,
Alkyloxycarbonyl group, carbamoyl group, N-substituted carbamoyl group, N, N-disubstituted carbamoyl group, alkyl group, cycloalkyl group, haloalkyl group, cyano-substituted alkyl group, cyano-substituted alkylthio group, cyano-substituted alkylsulfinyl group, cyano At least one selected from a substituted alkylsulfenyl group, N, N-dialkenyl group, N, N-dialkylaminoimino group, phenylalkyl group and naphthylalkyl group may be substituted. ] A carbamate agent hapten represented by the following formula, an antigen which is a complex of the hapten and a protein, a monoclonal antibody specific to a carbamate agent, and a method for enzyme-linked immunosorbent detection of a carbamate agent using the monoclonal antibody and / or polyclonal antibody.

In the hapten of the above-mentioned general formula (1), as a preferable compound, in the general formula (1), the symbol A is the following general formulas (1-a) to (1-d).

[0009]

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[In the above formulas, R ₁ , R ₂ , R ₃ , R ₄ ,
R ₅ , R ₆ , R ₇ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are the same or different and each represent a hydrogen atom, a halogen atom, a nitro group, an amino group, an N-C _1-6 alkyl-substituted amino group, N, N-di-C _1-6 alkyl-substituted amino group, cyano group, hydroxy group, thiol group, C _1-6 alkoxy group, C _1-6 alkylthio group, C _1-6 alkylsulfinyl group, C _1-6 Alkylsulfonyl group, C _1-6 alkyloxycarbonyl group, C _1-6 haloalkoxy group, C _1-6 haloalkylthio group, C _2-7 alkoxyalkyl group, C _2-8 alkoxyalkoxy group, C _2-7 alkylthio group Alkyl group, CH
₂ N ₃ group, carboxyl group, carbamoyl group, NC
_1-6 alkyl-substituted carbamoyl group, N, N-C _1-6 alkyl-substituted carbamoyl group, C _1-6 alkyl group, C _3-6 cycloalkyl group, C _1-6 haloalkyl group, C _1-6 cyano-substituted alkyl Group, C _1-6 cyano-substituted alkylthio group, C
_1-6 cyano-substituted alkylsulfinyl group, C _1-6 cyano-substituted alkylsulfenyl group, N, N-di-C _1-6 alkenyl group, N, N-di-C _1-6 alkylaminoimino group, phenylalkyl Group or naphthylalkyl group. Further, R ₁₁ and R ₁₂ may combine with each other to form a heterocyclic group. R ₈ represents a hydrogen atom, a C _1-6 alkyl group, a hydroxy group or an oxo group. n shows the integer of 2-5. ] The compound which shows the group represented by these can be illustrated.

Among the haptens represented by the general formula (1), compounds having the following chemical structural formulas are more preferred.

[0012]

[Chemical 9]

[0013]

[Chemical 10]

[0014]

[Chemical 11]

[0015]

[Chemical 12]

[0016]

[Chemical 13]

[In the above formulas, n is the same as above. Among the above compounds, those in which n is 3 or 4 are particularly preferable.

Among the haptens represented by the general formula (1), the compounds represented by the following chemical structural formulas can be mentioned as particularly preferable ones.

[0019]

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The hapten represented by the general formula (1) can be produced by a known method. For example, the general formula A-O-CO-NH- ( CH 2) n-COOR (2) [wherein, R represents a carboxyl protecting group. A and n are the same as above. ] It can manufacture by removing the carboxyl protecting group shown by R from the ester compound represented by these.

In the above general formula (2), the carboxyl protecting group represented by R may be a known one, and specific examples thereof include
For example, tert-butyl group, p-methoxybenzyl group,
Trichloroethyl group, trimethylsilyl group, tert-
Examples thereof include a butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a trimethylsilylethoxymethyl group, a trityl group, a methyl group and an ethyl group.

The carboxyl protecting group represented by R can be removed by a known method. For example, taking the case where the protecting group is a tert-butyl group as an example,
It is carried out by adding a strongly acidic catalyst to an organic solvent containing an ester compound of the general formula (2) and preferably reacting with stirring. As the organic solvent, for example, aromatic hydrocarbons such as benzene, halogenated hydrocarbons such as dichloromethane and 1,2-dichloroethane, or a mixed solvent thereof can be used. As the strongly acidic catalyst, known ones such as carboxylic acid such as trifluoroacetic acid, sulfonic acid such as p-toluenesulfonic acid, mineral acid such as hydrochloric acid and sulfuric acid can be used, and among them, trifluoroacetic acid is preferable. The reaction temperature is usually about 0 to 50 ° C., preferably around room temperature, and the reaction time is usually about 2 to 24 hours.

The removal of the protective group can also be carried out by alkaline hydrolysis. Further removal of the benzyl group
It can also be carried out by hydrogenolysis. The protective group having a silicon atom such as the above-mentioned trimethylsilyl group can be removed by a reagent such as tetrabutylammonium fluoride which generates a fluorine anion.

The ester compound of the general formula (2) can be synthesized by various methods. The methods (a) to (c) are shown below as specific examples.

(B) General formula NH ₂ — (CH ₂ ) n-COOR (3) [In the formula, R and n are the same as above. ] The linear amino acid represented by these and general formula AOH (4) [In formula, A is the same as the above. ] And a hydroxy compound represented by the following formula are reacted in the presence of a base and a carbonylating agent such as haloformic acid esters, phosgene and triphosgene. Examples of the solvent include esters such as ethyl acetate, halogenated hydrocarbons such as dichloroethane and dichloromethane, aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene, chlorobenzene, and alkylnaphthalene, cyclohexanone, methyl ethyl ketone, methyl isobutyl. Ketones and other ketones, dimethylformamide, dimethylsulfoxide, acetonitrile,
Organic solvents such as mineral oil fractions and mixed solvents thereof are preferably used. Known bases, for example, carbonate anhydrides such as sodium carbonate and potassium carbonate, alkali hydroxides such as sodium hydroxide and potassium hydroxide, triethylamine, pyridine, 4-dimethylaminopyridine, 2,
Organic bases such as 4,6-collidine, triethylamine and N-methylmorpholine can be used, and among them, triethylamine is preferable. The reaction temperature may be ice-cooling temperature to around room temperature, and the reaction time may be about 1 to 5 hours. Incidentally, the hydroxy compound of the general formula (4) is different that may be produced by synthetic methods, formulas A-O-CO-NH- CH 3 (5) [wherein A is as defined above. ] It can manufacture easily by hydrolyzing the well-known carbamate agent represented by these. This hydrolysis is performed by stirring an aqueous solution or an aqueous solution containing an organic solvent, usually at room temperature to under heating, in the presence of a conventionally known hydrolysis condition such as an acid or a base.
Examples of the acid include carboxylic acids such as trifluoroacetic acid, sulfonic acids such as p-toluenesulfonic acid, and mineral acids such as hydrochloric acid and sulfuric acid. As the base, known bases such as anhydrous carbonates such as sodium carbonate and potassium carbonate, alkali hydroxides such as sodium hydroxide and potassium hydroxide, triethylamine, pyridine, 4-dimethylaminopyridine, 2,4,6 Examples thereof include organic bases such as collidine, triethylamine and N-methylmorpholine. As the organic solvent, for example, benzene, toluene, xylene, ethylbenzene, chlorobenzene, aromatic hydrocarbons such as alkylnaphthalene, cyclohexanone, methyl ethyl ketone, ketones such as methyl isobutyl ketone, lower alcohols such as methanol, ethanol, isopropanol, Examples thereof include cellosolves such as methyl cellosolve and ethyl cellosolve, dimethylformamide, dimethylsulfoxide, acetonitrile, mineral oil fractions, and mixed solvents thereof. It is also possible to carry out the reaction in a two-phase system of these organic solvent and water. The reaction normally proceeds well at 0 to 50 ° C.

(B) The hydroxy compound of the general formula (4) and the base are heated and stirred for several hours to form a salt of the hydroxy compound, and a carbonylating agent is added at room temperature to give 80
By reacting at a temperature of about 90 ° C for about 1 hour, a compound of the general formula AO-CO-X (6) [wherein A is the same as above. X represents a halogen atom. ] The alkoxy carbonyl halide represented by these is manufactured.
As the base, preferably used are anhydrous carbonates such as sodium carbonate and potassium carbonate, alkali hydroxides such as sodium hydroxide and potassium hydroxide, and organic bases such as triethylamine. Examples of the carbonylating agent include haloformates such as trichloromethyl chloroformate,
Examples thereof include phosgene and triphosgene. Next, the alkoxycarbonyl halide of the general formula (6) is isolated and purified from the reaction system and reacted with the linear amino acid of the general formula (3) to obtain the ester compound of the general formula (2). This reaction is carried out under the same conditions as in the above method (a).

(C) Hydroxy compound of the general formula (4) and general formula Ph-O-CO-NH-COOR (7) [wherein Ph represents a phenyl group. R is the same as above. ]
By reacting the phenyl carbamate represented by the formula (1) in an organic solvent in the presence of a base catalyst,
The ester compound of In this reaction, as the organic solvent, for example, nitriles such as acetonitrile,
A halogenated hydrocarbon such as dichloromethane or a mixed solvent thereof can be used. Known base catalysts such as triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, 1,
5-diazabicyclo [4,3,0] non-5 (DB
N), 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU), 1,4-diazabicyclo [2,2]
2,2] Octane (DABCO) and other organic bases, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate and other inorganic bases can be used, and among these, DBU is preferred. The reaction temperature may be ice-cooling temperature to room temperature, and the reaction time may be 1 to 3 hours.

Next, the antigen, monoclonal antibody and enzyme immunoassay method of the present invention will be described. In addition, these preparations can be performed according to known methods described in, for example, the seminar on biochemistry chemistry, immunobiochemistry research method (edited by the Japanese Biochemical Society) and the like.

The antigen (immunogen) of the present invention, which is a complex of the hapten of the general formula (1) and a protein, is obtained by binding the hapten of the general formula (1) to a carrier-derived protein of animal or plant origin. Can be manufactured by

As the animal-derived protein and the plant-derived protein, any of those commonly used in this field can be used. Among them, pumpkin seed globulin, cannabis seed globulin, keyhole limpet hemocyanin, and pearl mussel Hemocyanin, ovalbumin, bovine serum albumin and the like are preferable, and keyhole limpet hemocyanin and bovine serum albumin are particularly preferable.

The hapten and the protein can be bound by a known method such as a mixed acid anhydride method. The mixed acid anhydride method is a method of reacting a hapten with a halocarboxylic acid ester to form a mixed acid anhydride, and reacting this with a protein.

The mixed acid anhydride used in the mixed acid anhydride method is obtained by a conventional Schotten-Baumann reaction, and the desired hapten-protein conjugate is obtained by reacting this with a protein without isolation. Manufactured. The Schotten-Baumann reaction is carried out in the presence of a basic compound. As the basic compound used, a compound commonly used in the Schotten-Baumann reaction is used, for example, triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, DBN, DB.
Examples thereof include organic bases such as U and DABCO, and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The reaction is usually -20 to 100.
The reaction time is 5 minutes to 10 hours, preferably 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and protein is usually -20 to
It is carried out at 150 ° C, preferably 10 to 100 ° C, and the reaction time is 5 minutes to 10 hours, preferably 5 minutes to 5 hours. The mixed acid anhydride method is generally performed in a solvent. The solvent used may be any solvent commonly used in the mixed acid anhydride method, and specifically, dichloromethane,
Chloroform, dichloroethane, and other halogenated hydrocarbons, benzene, toluene, xylene, and other aromatic hydrocarbons, diethyl ether, dioxane, tetrahydrofuran, dimethoxyethane, and other ethers, methyl acetate, ethyl acetate, and other esters, N, Examples include aprotic polar solvents such as N-dimethylformamide, dimethylsulfoxide, and hexamethylphosphoric triamide. Examples of the alkylhalocarboxylic acid used in the mixed acid anhydride method include, for example, methyl chloroformate, methyl bromoformate,
Examples include ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate and the like. The ratio of the hapten, the alkylhalocarboxylic acid and the protein used in the method can be appropriately selected from a wide range, but it is usually preferable to set them in equimolar amounts.

Among the antigens of the present invention, particularly preferable are those in which the hapten is the compound represented by the above chemical structural formula (1) or (15) and the protein is bovine serum albumin or sky mussel hemocyanin. Can be mentioned.

A monoclonal antibody and a polyclonal antibody are prepared by immunizing a mammal with a solution containing this antigen. As the mammal, any mammal commonly used in this field can be used, but it is preferable to select it in consideration of compatibility with myeloma cells (myeloma cells) used for cell fusion, for example, mouse , Rat,
Rabbits, goats, horses and the like can be mentioned.

More specifically, the monoclonal antibody is prepared, for example, as follows.

First, physiological saline containing an antigen is emulsified and mixed with an equal amount of Freund's complete or incomplete adjuvant, and subcutaneously administered to mice (first immunization). Thereafter, the same operation is performed at intervals of 2 to 4 weeks, and immunization is performed several times. Several days after the final immunization, the mouse spleen is aseptically removed to prepare splenocytes.

Cell fusion is performed using the spleen cells. As the myeloma cell which is the other parent cell of the cell fusion, various known cell lines, various known cells, for example, P3 / X63-Ag8 (X63) (Nature, 256, 495-497 (1975)) ,
P3 / X63-Ag8. U1 (P3U1) (Current Topics in Microbio
logy and Immunology, 81 , 1-7 (1978)), P3 / NSI-1-
Ag4-1 (NS-1) (Eur. J. Immunol., 6, 511-519 (197
6)), Sp2 / 0-Ag14 (Sp2 / 0) (Nature, 276, 269-270 (197
8)), FO (J. Immuno. Meth., 35 , 1-21 (1980)), MPC
-11, X63.6.55.3, S194 etc. and 210.RCY3.
Myeloma cells such as Agl.2.3. (Y3) (Nature, 277, 13l-133, (1979)) can be used.

Cell fusion can be carried out according to known methods, for example, the method of Milstein et al. (Method in Enzymol).
ogy, 73, 3 (1981)) and the like. More specifically, the above fusion reaction is carried out in an ordinary medium in the presence of an ordinary fusion promoter such as polyethylene glycol (PEG) and Sendai virus (HVJ).
If necessary, an auxiliary agent such as dimethyl sulfoxide can be added to the medium to further enhance the fusion efficiency.
In addition, a method using electric treatment (electric fusion) can be appropriately adopted. The ratio of cells to be used may be similar to the known method, and for example, myeloma cells may be used about 5 to 10 times as much as the spleen cells. As the fusion medium, for example, Dulbecco's modified Eagle medium (DMEM) or Iscove's modified Eagle medium (IMEM) containing 40% (W / V) polyethylene glycol can be used. The fusion is performed by thoroughly mixing the immune spleen cells and myeloma cells in the above medium.

Subsequently, cells other than the hybridomas are removed by exchanging half of the culture solution with a selection medium (for example, HAT medium) every day, and the hybridoma culture supernatant is examined for the presence or absence of antibodies specifically reacting with the antigen. The hybridoma producing the desired monoclonal antibody is examined and separated. To examine the presence or absence of the antibody, for example, enzyme-linked immunosorbent assay (ELISA) method (Engvall, E., Meth. Enzymol.,
70, 419-439 (1980)), plaque method, spot method, agglutination method, Ouchterlony method, radioimmunoassay (RIA) method, and other various methods commonly used for antibody detection (“hybridoma method”). And monoclonal antibody ", published by R & D Planning Co., Ltd.
P. 53, March 5, 1982).

The selected hybridomas secreting the desired monoclonal antibody can be subcultured in a general medium, and can be easily stored for a long period of time in liquid nitrogen or the like. As a medium for culturing the hybridoma, for example, DMEM, 10% fetal calf serum (FC
IMEM and the like including S) are used. The hybridoma is cultured, for example, in a medium at a carbon dioxide concentration of about 5 to 7% and at 37 ° C., or a compatible mammal,
For example, it is preferably administered to mice and cultured in the abdominal cavity. The former method is suitable for obtaining highly pure antibody, and the latter method is suitable for mass production of antibody.

The desired monoclonal antibody can be separated and purified from the culture medium in which the hybridoma is cultured or the ascites of the hybridoma-administered mouse. For separation and purification, known methods such as salting out with ammonium sulfate, ion exchange column chromatography, molecular sieve column chromatography using a molecular sieve gel, affinity column chromatography using a protein A or protein G-bound polysaccharide, dialysis, lyophilization, etc. There is.

On the other hand, for the polyclonal antibody, for example, physiological saline containing an antigen is emulsified and mixed with an equal amount of Freund's complete or incomplete adjuvant, and the mixture is subcutaneously administered to a rabbit (first immunization), and thereafter 2 to 4 It can be prepared by immunizing several times by performing the same operation at weekly intervals, collecting blood from the carotid artery or heart of the rabbit one week after the final immunization, and salting out serum deprived of blood with ammonium sulfate.

Since the monoclonal antibody and the polyclonal antibody of the present invention thus obtained show specificity to the carbamate agent, the presence of the carbamate agent in the sample can be detected by using them.

The detection method of the present invention can be performed according to a known method except that the above-mentioned monoclonal antibody and / or polyclonal antibody is used. Examples of the known method include enzyme-linked immunosorbent assay (ELISA), latex agglutination method, immunohistological staining method, radioimmunoassay method (RI).
There may be mentioned conventional immunological means such as A).

When the enzyme immunoassay is performed, for example, (a) an antigen, which is a complex of a carbamate agent hapten and a protein, is immobilized on a carrier, (b) blocking is carried out, and (c) a carbamate is added thereto. And a monoclonal antibody (and / or a polyclonal antibody) are added, and the antibody is competitively bound to the antigen and the carbamate agent to form an antibody-antigen complex and an antibody-carbamate agent complex, and (d) the antigen. -By measuring the amount of the antibody complex, the amount of the carbamate agent in the sample can be determined from the calibration curve prepared in advance.

In the 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. For example, a 96-well microplate made of polystyrene can be mentioned.

To immobilize the antigen on the carrier, for example,
A buffer solution containing the antigen may be placed on the carrier and incubated. A known buffer can be used, for example, 10 mM phosphate buffer (pH 7.5), 100 m
An example of the composition is M NaCl. The concentration of the antigen in the buffer solution is not particularly limited and can be selected from a wide range, but usually about 0.01 to 0.1 μg / ml is suitable. When using a 96-well microplate as a carrier, about 0.1 ml / well is desirable.
Furthermore, the incubation conditions are not particularly limited,
Usually, overnight incubation at about 4 ° C is suitable.

Blocking in the step (b) is carried out in the antigen, which is a complex of hapten and protein immobilized on a carrier, at a site other than the site where the protein binds to the hapten and the site to which an antibody to be added later can be adsorbed. It may be held, and if such a portion is held, the detection accuracy may be deteriorated, so this is performed. Specifically, it is performed by adding an appropriate amount of a 3% skim milk solution to the portion on which the antigen is immobilized, incubating at 25 ° C. for 1 hour, and then washing with a buffer solution. The buffer solution is not particularly limited, but for example, 10 mM phosphate buffer solution (pH 7.2), 0.8% (w / v) NaC
1, 0.02% (w / v) KCl, 0.02% (v /
v) A composition of Tween 20 is suitable.

Then, in the step (c), a sample containing a carbamate agent and a monoclonal antibody and / or a polyclonal antibody are added to the solid phase portion of the antigen, and the antibody is reacted with the solid phase antigen and the carbamate agent on a carrier. , An antigen-antibody complex and a carbamate-antibody complex are formed.

At this time, as the antibody, a monoclonal antibody and / or a polyclonal antibody is added to the first antibody, and an antibody against the first antibody (second antibody) bound with a labeling enzyme is added to these antibodies in order and reacted.

The first antibody is added in a buffer. The reaction may be performed at about 25 ° C. for about 1 hour. After the reaction is completed, the carrier is washed with a buffer solution to remove unreacted primary antibody. As a buffer used in this reaction, 10 mM phosphate buffer (pH 7.2), 0.8% (w / v) NaCl,
A composition of 0.02% (w / v) KCl is preferable.
The concentration of the first antibody can be appropriately selected from a wide range.

Then, the second antibody is added. For example, when a rabbit antiserum is used as the first antibody, an enzyme (for example,
Anti-rabbit IgG goat antibody conjugated with peroxidase or alkaline phosphatase, etc., or anti-mouse Ig when the first antibody from mouse hybridoma is used
Suitably the G goat antibody fraction is used. It is desirable to react the first antibody bound to the carrier with a second antibody diluted to about 5000 to 10000 times, preferably to give a final absorbance of 1.0 to 1.5. A buffer solution is used for dilution. The reaction is performed at 25 ° C. for 1 hour, and after the reaction, it is washed with a buffer.

By this reaction, the second antibody binds to the first antibody. The carbamate agent-antibody complex is removed from the carrier by washing after the two reactions, and only the antigen-antibody complex remains on the carrier.

Next, in step (d), a reagent that develops color by the reaction of the substrate-bound enzyme of the second antibody with the substrate is added, and the absorbance is measured to calculate the amount of the carbamate agent from the calibration curve. .

When peroxidase is used as the enzyme that binds to the second antibody, it is desirable to use hydrogen peroxide as the substrate and o-phenylenediamine as the coloring reagent. After adding the coloring solution and reacting at 25 ° C. for 10 minutes, the enzymatic reaction is stopped by adding 4N sulfuric acid. 492 when using o-phenylenediamine
And the absorbance at 630 nm is measured. On the other hand, when alkaline phosphatase is used as an enzyme that binds to the second antibody, p-nitrophenyl phosphate is used as a substrate for color development, 2N NaOH is added to stop the enzymatic reaction, and 415n
Suitable is the method of measuring the absorbance at m and 630 nm.

The rate of decrease in the absorbance of the solution in which the carbamate agent was added to react with the antibody was calculated as the inhibition rate with respect to the absorbance of the reaction solution in which the carbamate agent was not added. The concentration of the carbamate agent in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which a known concentration of the carbamate agent is added.

The detection method of the present invention comprises, for example, the known carbamate agent represented by the above general formula (5) contained in water such as soil, agricultural products, foods, paddy water, rivers, lakes and ponds. It can be used for detection. Specific preferred examples of the carbamate agent include 2-methyl-2- (methylthio) propionaldehyde O-methylcarbamoyl oxime (generic name: aldicarb), 2-mesyl-2-methylpropionaldehyde O-methylcarbamoyl oxime ( General name: aldoxicarb), 2,2-dimethyl-
1,3-benzodioxol-4-yl methylcarbamate (generic name: bendiocarb), 3- (methylthio) butanone O-methylcarbamoyloxime (generic name: butocoxime), 3-mesylbutanone O-methylcarbamoyloxime (Generic name: butoxycarboxime), 1-naphthyl methyl carbamate (generic name:
Carbaryl), 2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate (generic name:
Carbofuran), 2- (2-chloro-1-methoxyethoxy) phenyl methylcarbamate (generic name: chloetocarb), 2- (1,3-dioxolan-2-yl)
Phenyl methyl carbamate (generic name: dioxacarb), 2-ethylthiomethylphenylmethyl carbamate (generic name: etiophencarb), 2-sec-butylphenyl methyl carbamate (generic name: fenocarb), 3-dimethylaminomethyleneaminophenyl
Methyl carbamate (generic name: formetanato), 2-
Isopropylphenyl methyl carbamate (generic name:
Isoprocarb), 4-methylthio-3,5-xylyl methyl carbamate (generic name: methiocarb), S-
Methyl N- (methylcarbamoyloxy) thioacetimidate (generic name: methomyl), 3-methylphenyl methylcarbamate (generic name: metolcarb), N,
N-dimethyl-2-methylcarbamoyloxyimino-
2- (methylthio) acetamide (generic name: oxamyl), 3-isopropyl-5-methylphenyl methylcarbamate (generic name: promecarb), 2-isopropoxyphenyl methylcarbamate (generic name: propoxur), 3,3- Dimethyl-1-methylthiobutanone O-methylcarbamoyl oxime (generic name: thiophanox), 3,4-xylyl methylcarbamate (generic name: xylylcarb), 3,5-xylylmethylcarbamate, 4-diallylamino-3,5 -Xylyl
Methyl carbamate (generic name: alixicarb), 4-
Dimethylamino-3-methylphenyl methylcarbamate (generic name: aminocarb), 3- (1-methylbutyl) phenyl methylcarbamate (generic name: bufencarb), 3,5-di-tert-butylphenyl methylcarbamate (general name) Name: Butacarb), 6-chloro-3,4-xylyl methyl carbamate (generic name: carbanolate), 3-isopropyl methyl carbamate, 2,3-dihydro-2-methylbenzofuran-7-
Ile methylcarbamate (generic name: decarbofuran), 3,4-dichlorobenzyl methylcarbamate (generic name: dichlormate), 2- (4,5-dimethyl-1,3-dioxolan-2-yl) phenyl methylcarba Mart, 4-ethylthiophenyl methylcarbamate, 3,5-diethylphenyl methylcarbamate (generic name: phenatacarb), 4-dimethylaminomethyleneamino-m-tolylmethylcarbamate (formparanate), 2-methyl ( Prop-2-ynyl) aminophenyl methyl carbamate, 4-methyl (prop-2-ynyl) amino-3,5-xylyl methyl carbamate, 4-dimethylamino-3,5-xylyl
Methyl carbamate (generic name: Mexa culvert),
4,4-Dimethyl-5- (methylcarbamoyloxyimino) valeronitrile (generic name: nitrilacarb), N
-Methyl-1- (3,3,5-trimethyl-4-oxo-1,3-thiazolidine-2-ylideneaminooxy)
Formamide (generic name: Tajimcarb), 2,6-di-
tert-Butyl-p-tolylmethylcarbamate (generic name: terbucarb), 3- [1- (methylcarbamoyloxyimino) ethylthio] propionitrile (generic name: thiocarboxime), 3,4,5-trimethylphenyl Methyl carbamate (generic name: trimetacarb), 2,3,5-trimethylphenyl methylcarbamate (generic name: trimetacarb), etc. can be mentioned. The chemical structural formulas of these carbamate agents are shown below.

[0058]

[Chemical 15]

[0059]

Embedded image

[0060]

[Chemical 17]

[0061]

Embedded image

[0062]

[Chemical 19]

[0063]

Industrial Applicability According to the present invention, it becomes possible to easily and quickly measure the amount of the carbamate agent remaining in water, soil and food in the environment.

[0064]

EXAMPLES The present invention will be described in more detail with reference to the following Reference Examples and Examples.

Reference Example 1

[0066]

Embedded image

(1) Synthesis of 4- (benzyloxycarbonylamino) butyric acid (8) 4-Aminobutyric acid (28 g, 270 mmol) was dissolved in 140 ml of 2N sodium hydroxide solution and cooled with ice. Benzyl chloroformate (70
g, 410 mmol) and 200 ml of 2N sodium hydroxide solution were alternately added dropwise in three divided portions. After completion of the dropping, the mixture was stirred for 30 minutes at ice-cooling temperature, and subsequently at room temperature overnight. Ice-cooled diethyl ether (150m
After unreacted benzyl chloroformate was extracted (1 × 3 times), concentrated hydrochloric acid was added dropwise at an ice-cooling temperature to adjust the pH to about 2. The mixture was extracted with dichloromethane (150 ml × 4 times), the collected dichloromethane extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. When the concentrate was dissolved in a small amount of ethyl acetate and hexane was added thereto, white crystals were precipitated. The white crystals were isolated by suction filtration to obtain the target product (8) (60 g, 93% yield).

(2) Synthesis of tert-butyl 4- (benzyloxycarbonylamino) butyrate (9) 4- (benzyloxycarbonylamino) butyric acid (8)
(28 g, 118 mmol) in dichloromethane (3
(00 ml) was cooled to −16 ° C. with sodium chloride-ice, and a stream of isobutene was slowly poured into the solution for 5 minutes. Next, phosphoric acid (0.92 g, 9.4 mmol) and boron trifluoride diethyl ether complex (2.5 g, 17.7 mmol) were slowly added dropwise with a syringe, and isobutene was slowly poured again for 5 minutes. The solution was stirred as it was until it reached room temperature, and was further stirred overnight. After adding a saturated sodium hydrogen carbonate aqueous solution and stirring for 30 minutes, the mixture was extracted with dichloromethane (150 ml × 4 times). The collected dichloromethane extracts were dried over anhydrous sodium sulfate, suction filtered to remove sodium sulfate, and then concentrated under reduced pressure.
The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 4: 1) to obtain the target product (9) as a colorless transparent liquid (yield 31 g, yield 90).
%).

(3) tert-butyl 4-aminobutyrate (3
Synthesis of a) 4- (benzyloxycarbonylamino) butyric acid tert
-Palladium on carbon (1 g) in a methanol solution (300 ml) of butyl (9) (11.7 g, 40 mmol).
Was added and the mixture was stirred in the presence of hydrogen until the absorption of hydrogen was completed. Then, the reaction solution was filtered, concentrated under reduced pressure, and the solvent was completely removed with a vacuum pump to give a slightly yellowish liquid.
The target product (3) was obtained (amount 6.1 g, yield 100%).

(4) 4- (phenoxycarbonylamino)
Synthesis of tert-butyl butyrate (7a) 4-aminoacetate tert-butyl (3) (6 g, 3.8 mmol) was added to a dichloromethane solution (50 ml) of phenyl chlorocarbonate (8.0 g, 5 mmol) at ice cooling temperature.
Was added dropwise to a dichloromethane solution (50 ml) of triethylamine (5.0 g, 5 mmol) in dichloromethane (50 ml). After the completion of dropping, the solution was stirred as it was until it reached room temperature, and was further stirred overnight. The reaction solution was washed with saturated saline, dehydrated with anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration.
It was concentrated under reduced pressure. The concentrate was dissolved in a small amount of ethyl acetate, hexane was added thereto, and white crystals that crystallized were suction filtered to obtain the target product (7a) (yield 7.4 g, 70% yield).

Example 1 Synthesis of aldicarb hapten

[0072]

[Chemical 21]

(1) Synthesis of aldicarbhapten tert-butyl ester (2a) tert-butyl 4- (phenoxycarbonylamino) butyrate (7a) obtained in Reference Example 1 (21 g, 75 mmol)
And a solution of aldicarboxime (4a) (10 g, 75 mmol) in dichloromethane (100 ml) was added to diazabicyclo [6,5,0] undec-7-ene (DBU, 1
1.4 g, 75 mmol) in dichloromethane (50
ml) was added dropwise at 0 ° C., and the mixture was stirred for 3 hours. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 4: 1), and the obtained liquid was dissolved in ethyl acetate. Hexane was added to the liquid, and the precipitated white crystals were isolated by suction filtration. Thus, the target product (2a) was obtained (12 g, 50% yield).

Melting point: 54-55 ° C. or below Chemical shift (pp) of ¹ H-NMR (CDCl ₃ )
m) and the number of protons are shown.

1.44 (15H), 1.95 (3H),
1.67-2.55 (4H), 3.10-3.52 (2
H), 6.20-6.48 (1H), 7.50 (1
H).

(2) Synthesis of aldicarb hapten (1a) tert-butyl ester of aldicarb hapten (2
a) (12 g, 38 mmol) in benzene (50 m
10 ml of trifluoroacetic acid (TFA) was added to 1) and stirred overnight at room temperature. The reaction solution was directly concentrated under reduced pressure at 40 ° C. or lower, and dichloromethane was added and concentrated under reduced pressure until the pungent odor of TFA disappeared. The concentrate was dissolved in a small amount of ethyl acetate, and hexane was added to the concentrate to isolate a white crystal that was crystallized to obtain the target product (1a) (amount 5.0 g, yield 50%).

Melting point: 112-115 ° C. or lower Chemical shift (pp) of ¹ H-NMR (CDCl ₃ )
m) and the number of protons are shown.

1.45 (6H), 1.95 (3H),
1.70-2.57 (4H), 3.15-3.55 (2
H), 6.00-6.63 (1H), 7.50 (1
H), 8.97 (1H).

Example 2 (Synthesis of Mesomil Hapten)

[0080]

[Chemical formula 22]

(1) Synthesis of tert-butyl ester of mesomil hapten (2b) Mesomil oxime (4b) (3.2 g, 30 mmol)
And tert-butyl 4- (phenoxycarbonylamino) butyrate (7a) obtained in Reference Example 1 (8.4 g, 30 mmol) in acetonitrile solution (100 ml) was added to diazabicyclo [6,5,0] undec-7-ene ( DBU,
4.6 g, 30 mmol) of acetonitrile solution (50
(ml) was added dropwise at 0 ° C. and the mixture was stirred for 3 hours. 40 reaction solution
The mixture was concentrated under reduced pressure at ℃ or below, and the concentrate was extracted with dichloromethane. The dichloromethane extract was washed with saturated brine, dried over anhydrous sodium sulfate, sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. The concentrate was subjected to silica gel column chromatography (hexane-ethyl acetate, 1: 1).
The product (2b) was obtained as a yellow liquid by purification in 1) (yield 8.3 g, yield 90%).

The chemical shift (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

1.42 (9H), 1.75-2.50
(4H), 2.21 (3H), 2.38 (3H), 3.
10-3.47 (2H), 5.99-6.43 (1
H).

(2) Synthesis of Mesomil Hapten (1b) The tert-butyl ester of mesomil hapten (2b)
(8.3 g, 27 mmol) in benzene (100 m
15 ml of trifluoroacetic acid (TFA) was added to 1) and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure as it was at 40 ° C. or lower, and dichloromethane addition and concentration under reduced pressure were repeated until the pungent odor of TFA disappeared. The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 1: 2) to give the desired product (1b) as a brown liquid.
Was obtained (amount 5.5 g, yield 81%).

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

1.55-2.65 (4H), 2.20
(3H), 2.44 (3H), 3.02-3.62 (2
H), 6.15-6.70 (1H), 9.53 (1
H).

Example 3 (Synthesis of Carbofuran Hapten)

[0088]

[Chemical formula 23]

(1) Synthesis of tert-butyl ester of carbofuran hapten (2c) Trichloromethyl chloroformate (TCF, 3.6)
g, 12 mmol) in dichloromethane and ice-cooled, with stirring, a solution of 2,3-dihydro-2,2-dimethylbenzofuran-7-ol (4c) (BFL, 3.3 g, 20 mmol) in dichloromethane (20 ml). ) Was added dropwise, and a dichloromethane solution (20 ml) of triethylamine (2.0 g, 20 mmol) was added dropwise. After the completion of dropping, the mixture was stirred for 30 minutes at ice-cooling temperature, further stirred at 45 ° C for 2 hours, and then cooled to room temperature. Next, tert-butyl 4-aminobutyrate (3a) obtained in Reference Example 1 (1.
6 g, 10 mmol) in dichloromethane (10 m
1) and triethylamine (2.0 g, 20 mmol)
A dichloromethane solution (10 ml) of was added dropwise, and the mixture was heated with stirring at 45 ° C. for 1 hour. The reaction solution was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, suction filtered to remove sodium sulfate, and then concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 4: 1) to obtain the target product (2c) as a colorless transparent liquid (yield 2.5 g, yield 36%).

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

1.41 (15H), 1.60-2.50
(4H), 2.95 (2H), 3.00-3.38 (2
H), 5.52-5.85 (1H), 6.65-7.0.
2 (3H).

(2) Synthesis of Carbofuran Hapten (1c) The tert-butyl ester of carbofuran hapten (2
c) (5.0 g, 14 mmol) was dissolved in 100 ml of anhydrous benzene and 11 ml of trifluoroacetic acid (TF
A) was added and the mixture was stirred at room temperature overnight. The reaction solution was directly concentrated under reduced pressure at 40 ° C. or lower, and addition of dichloromethane and concentration under reduced pressure were repeated until the pungent odor of TFA disappeared. The concentrate was dissolved in ethyl acetate, and hexane was added therein to isolate the precipitated white crystals to obtain the target product (1c) (amount 3.8 g, yield 90%).

Melting point: 136-138 ° C. or below Chemical shift (pp) of ¹ H-NMR (DMSO)
m) and the number of protons are shown.

1.38 (6H), 1.55-1.82
(2H), 2.08-2.90 (2H), 2.95 (2
H), 3.00-3.38 (2H), 6.60-7.1.
0 (3H), 7.53-7.78 (1H).

Example 4 (Synthesis of Oxamylhapten)

[0096]

[Chemical formula 24]

(1) Synthesis of tert-butyl ester of oxamylhapten (2d) tert-butyl 4- (phenoxycarbonylamino) butyrate (7a) (5.6 g, 20 mmol) obtained in Reference Example 1 and oxamyl oxime (2a). 4d) (3.2 g, 20
Diambicyclo [6.5.0] undec-7-ene (DB) in a dichloromethane solution (100 ml).
U, 3 g, 20 mmol) in dichloromethane (50
(ml) was added dropwise at 0 ° C., and the mixture was stirred as it was under ice cooling for 1 hour and further at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 1: 2) to obtain the target product (2d) as a yellow liquid (yield 3.6 g, 53% yield). .

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

1.43 (9H), 1.73-2.01
(2H), 2.16-2.49 (2H), 2.31 (3
H), 3.07 (3H), 3.09 (3H), 3.11
-3.42 (2H), 6.18-6.45 (1H).

(2) Synthesis of Oxamylhapten (1d) The tert-butyl ester of oxamylhapten (2
d) trifluoroacetic acid (TFA) 1 in 1,2-dichloroethane solution (50 ml) of (5.6 g, 16 mmol).
0 ml was added and the mixture was stirred at room temperature overnight. The reaction solution was directly concentrated under reduced pressure at 40 ° C. or lower, and addition of dichloromethane and concentration under reduced pressure were repeated until the pungent odor of TFA disappeared.
The concentrate is purified by silica gel column chromatography (ethyl acetate) to give the desired product (1
d) was obtained (yield 3.9 g, yield 83%).

The chemical shift (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

1.71-2.06 (2H), 2.21-
2.59 (2H), 2.27 (3H), 3.07 (3
H), 3.09 (3H), 3.19-3.50 (2
H), 6.11-6.33 (1H), 7.54 (1
H).

Example 5 (Synthesis of carbaryl hapten)

[0104]

[Chemical 25]

(1) Naphthoxycarbonyl chloride (6
Synthesis of e) 1-naphthol (4e) (14.4 g, 10 mmol) was added to 40 ml of a sodium hydroxide aqueous solution (4 g, 100 mmol), and the mixture was heated with stirring at 85 ° C. for 1 hour. After cooling the reaction solution to room temperature, 50 ml of a toluene solution of trichloromethyl chloroformate (TCF, 10.5 g, 53 mmol) was added dropwise. After 1 hour, the toluene solution was fractionated, dehydrated with anhydrous magnesium sulfate, and magnesium sulfate was removed by suction filtration, followed by concentration under reduced pressure. The concentrate was quickly crudely purified on a short silica gel column (hexane-ethyl acetate, 9: 1), and the eluate was concentrated under reduced pressure. Further, the concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 19: 1) to obtain the target product (6e) as a yellow liquid (yield 6.0 g,
Yield 29%).

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

7.10-8.01 (7H) (2) Synthesis of tert-butyl ester of carbaryl hapten (2e) Naphthoxycarbonyl chloride (6e) (4.0 g, 19 mmol) stirred under ice cooling. 4) in a dichloromethane solution (50 ml) of 4-aminobutyric acid t obtained in Reference Example 1
A solution of ert-butyl (3a) (3.0 g, 19 mmol) in dichloromethane (10 ml) was added dropwise, and further a solution of triethylamine (1.9 g, 19 mmol) in dichloromethane (10 ml) was added dropwise and stirring was continued for 1 hour. . The reaction solution was washed with saturated brine, dried over anhydrous magnesium sulfate, suction filtered to remove magnesium sulfate, and then concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 9: 1 → 1: 1) to obtain the target product (2e) as a colorless transparent liquid (amount 3.7 g, yield 58%). .

The chemical shift (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons thereof are shown below.

1.44 (9H), 1.78-2.06
(2H), 2.22-2.48 (2H), 3.22-
3.46 (2H), 5.32-5.62 (1H), 7.
20-8.01 (7H).

(3) Synthesis of carbaryl hapten (1e) tert-butyl ester of carbaryl hapten (2
e) (2.5 g, 7.6 mmol) in 50 ml of 1,2
-Dissolved in dichloroethane, added 10 ml of TFA and stirred at room temperature for 3 hours. The reaction solution was directly concentrated under reduced pressure at 40 ° C. or lower, and addition of dichloromethane and concentration under reduced pressure were repeated until the pungent odor of TFA disappeared. The concentrate was dissolved in dichloromethane, and ethyl acetate was added to the concentrate to isolate a white crystal that was crystallized to obtain the desired product (1e) (yield 2.1 g, yield 100%).

Melting point: 135-137 ° C. ¹ H-NMR (DMSO) chemical shift (pp)
m) and the number of protons are shown.

1.52-1.97 (2H), 2.11
2.54 (2H), 2.75-3.54 (2H), 7.
20-8.20 (7H), 12.03 (1H).

Example 6 (Synthesis of Fenobucarb Hapten)

[0114]

[Chemical formula 26]

(1) Synthesis of 2- (sec-butyl) phenoxycarbonyl chloride (6f) 40 ml of sodium hydroxide aqueous solution (4.0 g, 100 g)
2- (sec-butyl) phenol (4 mmol)
f) (15 g, 100 mmol) was added, and the mixture was stirred with heating at 85 ° C. for 1 hour. After cooling the reaction solution to room temperature, trichloromethyl chloroformate (TCF, 1
0.5 g, 53 mmol) in 50 ml toluene solution was added dropwise. After the dropping, the mixture was stirred for 1 hour, then the toluene solution was separated, dehydrated with anhydrous magnesium sulfate, and magnesium sulfate was removed by suction filtration, followed by concentration under reduced pressure. The concentrate was passed through a short silica gel column (hexane-ethyl acetate,
4: 1) for rapid crude purification, and the eluate was concentrated under reduced pressure. Further, the concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 19: 1) to obtain the desired product (6f) as a yellow liquid (yield 7.2 g, yield 34%).

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons thereof are shown below.

0.72-1.00 (3H), 1.08-
1.27 (3H), 1.38-1.77 (2H), 2.
68-2.99 (1H), 6.98-7.50 (4
H).

(2) Fenobu Calbu Hapten tert-
Synthesis of butyl ester (2f) tert-butyl 4-aminobutyrate (3) obtained in Reference Example 1
a) (4.3 g, 27 mmol) in ethyl acetate (5
0 ml) with stirring under ice-cooling, while adding 2- (se
c-Butyl) phenoxycarbonyl chloride (6f)
(5.8 g, 27 mmol) in ethyl acetate (50 m
l) was added dropwise and triethylamine (2.7 g, 27
Ethyl acetate solution (50 ml) was added dropwise and 3
The mixture was stirred for 0 minutes and then at room temperature for 1 hour. The reaction solution was washed with saturated brine, dried over anhydrous magnesium sulfate, suction filtered to remove magnesium sulfate, and then concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 9: 1) to obtain the target product (2f) as a colorless transparent liquid (yield 5.8).
g, 64% yield).

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

0.72-0.97 (3H), 1.43
(9H), 1.02-1.99 (7H), 2.11
2.39 (2H), 2.72-3.02 (1H), 3.
06-3.37 (2H), 5.70-5.94 (1
H), 7.02-7.43 (4H).

(3) Synthesis of Fenobucarb hapten (1f) tert-Butyl ester of Fenobu carbhapten (2f) (5.0 g, 15 mmol) in 50 ml of 1
It was dissolved in 2-dichloroethane, 10 ml of TFA was added, and the mixture was stirred at room temperature for 3 hours. Reaction solution as it is at 40 ° C
The solution was concentrated under reduced pressure below, and addition of dichloromethane and concentration under reduced pressure were repeated until the pungent odor of TFA disappeared. The concentrate was subjected to silica gel column chromatography (hexane-
Purification with ethyl acetate, 1: 1) gave the desired product (1f) as a brown liquid. This liquid crystallized when left at 5 ° C. overnight (yield 3.4 g, yield 82%).

Melting point: 62-65 ° C. or lower Chemical shift (pp) of ¹ H-NMR (CDCl ₃ )
m) and the number of protons are shown.

0.72-1.00 (3H), 1.08-
1.27 (3H), 1.38-2.11 (4H), 2.
37-2.68 (2H), 2.68-2.99 (1
H), 3.23-3.52 (2H), 5.12-5.3.
9 (1H), 6.98-7.50 (4H), 8.38
(1H).

Example 7 (Synthesis of Ethiophene Carbuhapten)

[0125]

[Chemical 27]

(1) Ethiophene carb hapten ter
Synthesis of t-butyl ester (2 g) tert-butyl 4-aminobutyrate (3) obtained in Reference Example 1
a) (3 g, 19 mmol) and triethylamine (1.8 g, 18 mmol) in ethyl acetate (50 m
triphosgene (2 g,
A solution of ethyl acetate (6.7 mmol) in ethyl acetate (50 ml) was added dropwise, and the mixture was stirred at 0 ° C. for 30 minutes, and further stirred at room temperature for 1 hour. Then, a solution of 2- (ethylthiomethyl) phenol (4 g) (3 g, 18 mmol) in ethyl acetate was added dropwise under ice-cooling, the mixture was stirred for 30 minutes, and further stirred at room temperature for 1 hour. Water was added to terminate the reaction, and ethyl acetate (150 m
1 × 4 times). The collected ethyl acetate extracts were dried over anhydrous magnesium sulfate, suction filtered to remove magnesium sulfate, and then concentrated under reduced pressure. The concentrate was subjected to silica gel column chromatography (hexane-ethyl acetate,
9: 1 → 1: 1) to obtain the desired product (2 g) as a pale yellow liquid (yield 0.95 g, yield 15%).

The chemical shifts (ppm) of ¹ H-NMR (CDCl ₃ ) and the number of protons are shown below.

1.03 to 1.40 (3H), 1.44
(9H), 1.71-2.05 (2H), 2.03-
2.61 (4H), 3.17-3.42 (2H), 3.
63 (2H), 5.60-5.81 (1H), 7.02.
-7.44 (4H).

(2) Ethiophene carbhapten (1 g)
The tert-butyl ester of ethiophene carbhapten (2 g) (1.5 g, 15 mmol) was dissolved in 50 ml 1,2-dichloroethane and 10 ml TFA.
Was added and the mixture was stirred at room temperature for 2.5 hours. Next, the reaction solution was directly concentrated under reduced pressure at 40 ° C. or lower, and addition of dichloromethane and concentrated under reduced pressure were repeated until the pungent odor of TFA disappeared. The concentrate was purified by silica gel column chromatography (hexane-ethyl acetate, 1: 1) to obtain the desired product (1 g) as slightly brown white crystals (yield 0.4.
g, yield 32%).

Melting point: 46 to 49 ° C. Chemical shift (pp) of ¹ H-NMR (CDCl ₃ ) below
m) and the number of protons are shown.

1.01-1.37 (3H), 1.58-
2.06 (2H), 2.18-2.60 (4H), 3.
04-3.28 (2H), 3.64 (2H), 5.38.
-5.67 (1H), 7.00-7.48 (4H),
9.42 (1H).

Example 8 (Preparation of antigen)

[0133]

[Chemical 28]

Bovine serum albumin (BSA) as immunogen
A conjugate of a carboxylic acid derivative (hapten) of a carbamate agent was prepared by the mixed acid anhydride method. 25 mg of carbamate carboxylic acid (A) (aldicarbhapten) and carboxylic acid derivative (B) (mesomilhapten)
Was dissolved in 2 ml of anhydrous dioxane and 0.05 ml of N 2 was added.
-Methylmorpholine was added and stirred at 10 ° C for 20 minutes,
Further, 0.02 ml of isobutyl chloroformate was added little by little and stirred for 15 minutes to prepare acid anhydrides of (A) and (B). On the other hand, 80 mg of BSA was dissolved in 4.3 ml of distilled water, adjusted to pH 9.5 with 1N NaOH, and 2.6 ml of dioxane was further added dropwise at 10 ° C. The acid anhydride solutions of (A) and (B) prepared above were gradually added dropwise to this BSA solution while maintaining the pH at 9 with 1N NaOH solution, dialyzed at 4 ° C. overnight, and then freeze-dried.
The thus obtained conjugate of aldicarb and mesomil with a hapten was used as an antigen. In addition, for mesomil hapten, a conjugate with keyhole limpet hemocyanin (KLH) instead of BSA was prepared by the same method. In addition, conjugates of (A) and (B) with rabbit serum albumin (RSA) were also prepared in the same manner for use as coating antigens.

Example 9 (Preparation of Polyclonal Antibody) The antigen was adjusted to 1 mg / ml with physiological phosphate buffer (PBS: 10M phosphate buffer (pH 7.2), 0.9).
% NaCl) and mixed with an equal amount of Freund's complete adjuvant to make a water-in-oil emulsion 1
ml was subcutaneously administered to rabbits (NZW, female, 2 kg).
Then, every two weeks, an emulsion of the antigen and Freund's incomplete adjuvant was similarly additionally immunized.

Blood was collected from a rabbit whose antibody titer in the serum was high, and the obtained blood was centrifuged, and the serum was salted out with 33% saturated ammonium sulfate. The obtained precipitate was dialyzed against distilled water and used as the first antibody.

The prepared polyclonal antibody was diluted with PBS, and its reactivity with aldicarb and mesomil was examined by an antigen-immobilized ELISA method. Here, an example of measuring the reactivity of this antibody with aldicarb is shown using an anti-aldicarb antibody.

(1) 0.1 ml / ml of aldicarb-RSA conjugate (100 ng / ml) was added to a 96-well microplate.
The wells were coated and incubated overnight at 4 ° C. for adsorption to the plate. After washing 5 times with wash buffer,
A 0.25 ml / well volume of 3% skim milk solution was added, and the plate was washed after incubation at 25 ° C. for 1 hour.

(2) 500 anti-aldicarb antibody was added to PBS.
It was diluted 0-fold, mixed with an aldicarb solution (sample solution) dissolved in distilled water at a ratio of 1: 1, and incubated at 4 ° C. overnight for reaction. 0.1 ml of this reaction solution
Each well was added to the plate and incubated at 25 ° C. for 1 hour before washing the plate.

(3) An anti-rabbit IgG goat antibody (second antibody) conjugated with horseradish peroxidase was used for 10 times with PBS.
Dilute 000 times, add 0.1 ml / well, add 25
After incubation for 1 hour at ° C, the plates were washed.

(4) 0.2% O-phenylenediamine color developing solution (100 mM phosphate citrate buffer (pH 5.
0), 0.003% hydrogen peroxide solution) was added 0.2 ml / well to stop the enzyme reaction, and 492 nm and 630
Absorbance at nm was measured with a microplate reader.

E when using this anti-aldicarb antibody
The coating antigen concentration by LISA is 0.1 mg
/ Ml, and mix the 5000-fold diluted solution of the antibody and the aldicarb solution at a ratio of 1: 1,
The inhibition curve of aldicarb was determined by using 0-fold diluted one, and it is shown in FIG. As is clear from FIG. 1, it was possible to measure aldicarb at 30 to 10,000 ng / ml. Also, the inhibition curve for Mesomil was determined and is shown in FIG. As is clear from FIG.
Mesomil of 0 to 10000 ng / ml can be measured.

Example 10 (Preparation of monoclonal antibody-producing hybridoma and preparation of antibody) The antigen prepared in Example 8 was dissolved in physiological phosphate buffer at 1 mg / ml and mixed with an equal amount of adjuvant. , Balb / c mice were subcutaneously administered with 0.2 ml.
After that, additional immunity was given every two weeks. The spleen of a mouse with high antibody titer in serum was removed, the spleen removed in a petri dish containing DMEM medium was scratched with scissors, and the medium was injected into the spleen with a syringe to expel the cells. . The medium was transferred to a centrifuge tube and allowed to stand for 5 minutes to sediment large tissue pieces. Gently remove the supernatant of spleen cells,
Single cell suspensions were collected by centrifugation at 300 xg for 4 minutes to prepare spleen cells. Mouse myeloma cells (P3X6
3Ag8.653) was mixed at a cell number ratio of 5: 1 (myeloma cells: spleen cells) to give 300 × g, 4
The cells were collected by centrifugation for a minute. Next, the precipitated cells were suspended in DMEM medium, and 1 ml of a 50% polyethylene glycol (molecular weight 1500) solution warmed to 37 ° C. was gradually added over 60 seconds to perform cell fusion. DMEM medium 10m
After the addition of 1 liter, 1 ml of fetal bovine serum was added. The cells collected by centrifugation were prepared so that the cell number was 5 × 10 ⁵ cells / ml. Dispense 100 ml / well of the cell suspension into a 96-well plastic plate at 37 ° C.
Were incubated in the gas phase of 5% carbon dioxide at. Half of the medium in the wells was replaced with HAT medium every day, and the cells were cultured for 10 to 14 days. The activity of the antibody in the culture solution was examined by the ELISA method, and the cells of the well producing the desired antibody were cloned into a hybridoma by the limiting dilution method using a HAT medium in a 96-well plastic plate. As a result of cloning, 3 hybridoma strains producing anti-aldicarb antibody and 2 hybridoma strains producing anti-mesomil antibody were obtained. DM of cloned hybridoma strain
The cells were cultured in a medium containing 10% fetal bovine serum in EM, and the centrifuged supernatant was used as an antibody solution.

The inhibition curves of aldicarb and methomyl using these antibodies were determined, and are shown in FIGS. 3 and 4, respectively.
As shown. As is clear from FIG. 3, anti-aldicarb antibody I, antibody II and antibody III were 10 to 10,000, respectively.
ng / ml, 10-1000 ng / ml, 5-500 n
It was possible to measure aldicarb in the range of g / ml.
Further, as is clear from FIG. 4, the anti-mesomil antibody I and the antibody II were 30 to 3000 ng / ml and 10 to 50, respectively.
It was possible to measure in the range of 0 ng / ml. The anti-aldicarb antibody III and the anti-mesomil antibody II have a narrow measurement concentration range and can measure an unknown sample more accurately. Other antibodies are suitable for measuring highly concentrated samples.

Example 11 (Cross-reaction to Carbamate Agent Structurally Similar Compounds and Metabolites) Using the prepared anti-aldicarb antibody and anti-mesomil antibody, metabolites of various aldicarb and mesomil and structurally similar compounds were prepared by the method of “Example 2”. Cross-reactivity to carbamate insecticides was measured. As metabolites, S-oxidized form, oxime form and N-demethylated form of aldicarb and mesomemyl were measured, and as structurally similar compounds, alanicalb and oxamyl were measured. The cross-reactivity is the same concentration (1000n) for the inhibition rate of aldicarb and mesomil.
(g / ml) was calculated as the ratio of the inhibition rate of each metabolite or structurally similar compound, and the results are shown in Tables 1 and 2 below.

[0146]

[Chemical 29]

[0147]

Embedded image

[0148]

[Table 1]

[0149]

[Table 2]

In the case of the polyclonal antibody, the anti-aldicarb antibody was 40.5% with respect to N-demethylmethomyl.
However, it showed no reactivity with aldicarb oxime, mesomil sulfone and oxamyl.
It showed 10-20% cross-reactivity with other compounds. The anti-mesomil antibody showed a cross-reactivity of 66.4% with N-demethylmethomyl and about 30% with aldicarb and aldicarb sulfoxide. The reaction was 10 to 20% with respect to other compounds.

In the case of the monoclonal antibody, among the anti-aldicarb antibodies I, II and III, the antibody I was 36%, 34% and 42% of aldicarb oxime, aldicarb sulfoxide and aldicarb sulfone, respectively.
The cross-reactivity was 1% or less with respect to N-demethylmethomyl and alanicarb. It was 10 to 20% with respect to other compounds. The antibody II was 18% with respect to Mesomil, and hardly reacted with other compounds.
For antibody III, aldicarb oxime, mesomyle and N-
26%, 34%, and
22% cross-reactivity, 10 for other compounds
Was ~ 20%. Regarding anti-mesomil antibodies I and II,
The antibody I showed almost no reaction with mesomiloxime, oxamyl, aldicarb and aldicarb sulfone. The reaction was 10 to 20% with respect to other compounds. Antibody II shows a cross-reactivity of 29% with mesomil oxime, 22% with N-demethylmethomesyl, 34% with oxamyl, 105% with aldicarb, 38% with aldicarb oxime, and 28% with aldicarb sulfone. The reaction was about 10% with respect to the above compound.

Among the monoclonal antibodies, antibodies that do not show cross-reactivity (anti-Aldicarb antibody II and anti-mesomil antibody I) and antibodies that show cross-reactivity (anti-Aldicarb antibody I,
Anti-aldicarb antibody III and anti-mesomil antibody II) were obtained. Among these antibodies, anti-mesomil antibody III shows a similar reaction to aldicarb as mesomil, and therefore this antibody can measure both mesomil and aldicarb.

Example 12 (Measurement of Mesomil in Water and Soil) Purified monoclonal anti-mesomil antibody II was used to measure the amount of mesomil in water and soil.

From 10 ng / ml to 500 ng / in tap water
After adding an aqueous solution of Mesomel in the range of ml, 9 volumes of this sample solution and 1 volume of 100 mM PBS buffer were mixed,
This was the mMPBS sample solution. This sample solution was mixed with an equal volume of the antibody diluent, incubated at 4 ° C. overnight for reaction, and measured by ELISA. As a result, mesomil in tap water could be measured in the range of 10 ng / ml to 300 ng / ml.

[0155] Mesomil in soil was measured by 10 ng of methanol solution prepared by dissolving mesomil in air-dried soil / soil 1
The soil was air-dried again and subjected to analysis. Water was added to the soil at a ratio of 2 ml / g of soil, and the mixture was incubated at 4 ° C. overnight, and then shaken at 25 ° C. for 30 minutes. 1500 xg, 10
After centrifuging for minutes, the supernatant was used as a sample solution. 1 / for sample liquid
Ten 100 mM PBS buffers were added, mixed and reacted with an equal volume of antibody diluent, and measured by ELISA. As a result, the amount of mesomil in soil was 50 ng / g to 500 g of soil.
It was possible to measure in the range of ng / g of soil. Here, the reaction between the antibody and the sample solution could be measured even if shortened to 1 hour.

[Brief description of drawings]

FIG. 1 is a graph showing an aldicarb inhibition curve obtained in Example 9.

FIG. 2 is a graph showing an inhibition curve of mesomil in Example 9.

FIG. 3 is a graph showing an inhibition curve of aldicarb in Example 10.

FIG. 4 is a graph showing an inhibition curve of mesomil in Example 10.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 G01N 33/53 G G01N 33/53 J 33/577 B 33/577 9162-4B C12N 15/00 C // (C12P 21/08 C12R 1:91)

Claims (11)

[Claims] 1. A general formula (1) A-O-CO -NH- (CH 2) nCOOH wherein, n represents an integer of 2-5. A represents an aromatic group or a N-imino group containing a 5- or 6-membered heterocycle having at least one kind selected from an oxygen atom, a nitrogen atom and a sulfur atom as a hetero atom, and these groups are halogen atoms, Nitro group, amino group, N-substituted amino group, N, N-
Di-substituted amino group, cyano group, hydroxy group, thiol group, alkoxy group, alkylthio group, carboxyl group,
Alkyloxycarbonyl group, carbamoyl group, N-substituted carbamoyl group, N, N-disubstituted carbamoyl group, alkyl group, cycloalkyl group, haloalkyl group, cyano-substituted alkyl group, cyano-substituted alkylthio group, cyano-substituted alkylsulfinyl group, cyano At least one selected from a substituted alkylsulfenyl group, N, N-dialkenyl group, N, N-dialkylaminoimino group, phenylalkyl group and naphthylalkyl group may be substituted. ] A carbamate agent hapten represented by. 2. The symbol A in the general formula (1) is a group represented by the following general formulas (1-a) to (1-d).
The hapten of the carbamate agent described in. Embedded image [In the above formulas, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ ,
R ₇ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are the same or different and each represents a hydrogen atom, a halogen atom, a nitro group, an amino group, N
-C _1-6 alkyl-substituted amino group, N, N-di-C _1-6 alkyl-substituted amino group, cyano group, hydroxy group, thiol group, C _1-6 alkoxy group, C _1-6 alkylthio group, C
_1-6 alkylsulfinyl group, C _1-6 alkylsulfonyl group, C _1-6 alkyloxycarbonyl group, C _1-6 haloalkoxy group, C _1-6 haloalkylthio group, C _2-7 alkoxyalkyl group, C _{2 -8} alkoxyalkoxy group,
C _2-7 alkylthioalkyl group, CH ₂ N ₃ group, carboxyl group, carbamoyl group, N—C _1-6 alkyl-substituted carbamoyl group, N, N—C _1-6 alkyl-substituted carbamoyl group, C _1-6 alkyl group , C _3-6 cycloalkyl group, C
_1-6 haloalkyl group, C _1-6 cyano-substituted alkyl group, C
_1-6 cyano-substituted alkylthio group, C _1-6 cyano-substituted alkylsulfinyl group, C _1-6 cyano-substituted alkylsulfenyl group, N, N-di-C _1-6 alkenyl group, N, N-
A di-C _1-6 alkylaminoimino group, a phenylalkyl group or a naphthylalkyl group is shown. Further, R ₁₁ and R ₁₂ may combine with each other to form a heterocyclic group. R ₈ represents a hydrogen atom, a C _1-6 alkyl group, a hydroxy group or an oxo group. n shows the integer of 2-5. ] 3. The following chemical structural formula: Embedded image [Chemical 4] Embedded image [Chemical 6] [In each said formula, n shows the integer of 2-5. ] The hapten of the carbamate agent of Claim 1 which is a compound represented by these. 4. An antigen which is a complex of a carbene hapten according to claim 1 and a protein. 5. The protein is pumpkin seed globulin,
Cannabis seed globulin, keyhole limpet hemocyanin,
The antigen according to claim 4, which is at least one kind selected from hemocyanin of limpet, ovalbumin, and serum albumin of bovine. 6. The hapten has the following chemical structural formula: 5. The compound represented by the formula, and the protein is limpet hemocyanin or bovine serum albumin.
The antigen according to. 7. A monoclonal antibody specific for a carbamate agent, which is prepared from the antigen according to claim 4. 8. A method for enzyme-linked immunosorbent detection of a carbamate agent, which comprises using the monoclonal antibody according to claim 7. 9. A monoclonal antibody according to claim 7, wherein (a) the antigen according to claim 4 is immobilized on a carrier, (b) blocking is performed, and (c) a carbamate agent is contained in the sample, and the monoclonal antibody according to claim 7 and / or Alternatively, a polyclonal antibody is added, the antibody is competitively bound to the antigen and the carbamate agent to form an antibody-antigen complex and an antibody-carbamate agent complex, and (d) the antibody-carbamate agent complex is removed.
(E) The enzyme immunoassay method for a carbamate agent according to claim 8, wherein the amount of the antigen-antibody complex is measured and the amount of the carbamate agent in the sample is determined from a calibration curve prepared in advance. 10. The monoclonal antibody used in the step (c) is produced from a culture supernatant of a hybridoma obtained by cell fusion of mouse spleen cells and myeloma cells and producing an antibody highly specific to a carbamate agent. The enzyme immunoassay method for a carbamate agent according to claim 9. 11. 2-Methyl-2- (methylthio) propionaldehyde O-methylcarbamoyl oxime, 2
-Mesyl-2-methylpropionaldehyde O-methylcarbamoyl oxime, 2,2-dimethyl-1,3-
Benzodioxol-4-yl methylcarbamate,
3- (methylthio) butanone O-methylcarbamoyl oxime, 3-mesylbutanone O-methylcarbamoyl oxime, 1-naphthyl methylcarbamate, 2,
3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate, 2- (2-chloro-1-methoxyethoxy) phenyl methylcarbamate, 2-
(1,3-Dioxolan-2-yl) phenyl methylcarbamate, 2-ethylthiomethylphenylmethylcarbamate, 2-sec-butylphenyl methylcarbamate, 3-dimethylaminomethyleneaminophenyl methylcarbamate, 2-isopropyl Phenyl methyl carbamate, 4-methylthio-3,5-xylyl methyl carbamate, S-methyl N- (methylcarbamoyloxy) thioacetimidate, 3-methylphenyl methyl carbamate, N, N-dimethyl-2-
Methylcarbamoyloxyimino-2- (methylthio)
Acetamide, 3-isopropyl-5-methylphenyl
Methyl carbamate, 2-isopropoxyphenyl methyl carbamate, 3,3-dimethyl-1-methylthiobutanone O-methylcarbamoyl oxime, 3,4-xylyl methyl carbamate, 3,5-xylyl methyl carbamate, 4-diallyl Amino-3,5-xylyl methyl carbamate, 4-dimethylamino-3-methylphenyl methyl carbamate, 3- (1-methylbutyl) phenyl methyl carbamate, 3,5-di-
tert-butylphenyl methyl carbamate, 6-
Chloro-3,4-xylylmethyl carbamate, 3-isopropyl methyl carbamate, 2,3-dihydro-
2-methylbenzofuran-7-yl methylcarbamate, 3,4-dichlorobenzyl methylcarbamate,
2- (4,5-dimethyl-1,3-dioxolane-2-
Il) phenyl methylcarbamate, 4-ethylthiophenyl methylcarbamate, 3,5-diethylphenyl methylcarbamate, 4-dimethylaminomethyleneamino-m-tolylmethylcarbamate, 2-methyl (prop-2-ynyl) Aminophenyl methyl carbamate, 4-methyl (prop-2-ynyl) amino-
3,5-xylyl methyl carbamate, 4-dimethylamino-3,5-xylyl methyl carbamate, 4,
4-Dimethyl-5- (methylcarbamoyloxyimino) valeronitrile, N-methyl-1- (3,3,5-
Trimethyl-4-oxo-1,3-thiazolidine-2-
Ylideneaminooxy) formamide, 2,6-di-t
ert-butyl-p-tolyl methyl carbamate, 3
At least one carbamate selected from the group consisting of [1- (methylcarbamoyloxyimino) ethylthio] propionitrile, 3,4,5-trimethylphenyl methylcarbamate and 2,3,5-trimethylphenyl methylcarbamate The enzyme immunoassay method according to claim 8, which is applied to detection of an agent.