JPH11246536A - Mefenacet compound, immunological reactive material, hybridoma and determination of mefenacet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new mefenacet compound capable of largely simplifying the mefenacet analysis and shortening the measuring time, and quickly, readily and economically measuring various samples. SOLUTION: This compound is expressed by formula I (n) is 1-9}, e.g. N-(benzothiazolyl oxymethyl)-N-phenylcarbamoyl butyrate. The compound of the formula I is obtained by, e.g. reacting 2-halogenobenzothiazole of formula II (X is a halogen) with an α-hydroxyacetoanilide compound of formula III (R is a carboxyl protecting group) in an inert solvent such as N,N- dimethylformamide in the presence of a base such as 2-halogenobenzothiazole at room temperature.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a mefenacet compound, an immunological reactant, a hybridoma, and a method for measuring mefenacet.

[0002]

2. Description of the Related Art Mefenacet, an acid amide herbicide, that is, 2- (1,3-benzothiazole-2-)
(Iloxy) -N-methylacetanilide is represented by the formula (2)

[0003]

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A compound having a structure represented by the following formula: The compound is useful as a herbicide used in paddy fields and has a prominent effect on barnyard millet.

[0005] At present, mefenacet is widely used as a "one-shot treatment" which is effective in a single application before and after rice transplantation in a mixed preparation with other paddy herbicides. It is expected that soil and soil will be contaminated, and its monitoring is an important issue. In particular, since this formulation is applied in a concentrated manner at the time of rice transplanting, it is inevitable that the concentration of mefenacet in the drainage ditch or river of the paddy field is increased due to runoff from the paddy field. Therefore, it is particularly important to monitor the water concentration at this time.

Conventionally, instrumental analysis has been used for the analysis of these herbicides. For example, in the case of monitoring the concentration in water, measurement using gas chromatography is performed after extracting and purifying pesticides from a sample. I have been. Although these methods have no problem in terms of accuracy, the operation is complicated and the measurement takes time, so that mefenacet remaining in the environment and in food can be monitored more quickly, easily and economically. The development of a new measurement method was required.

[0007] On the other hand, the immunological measurement method, which measures an antigen using an antigen-antibody reaction, is not only excellent in measurement accuracy, but also a quick, simple and economical measurement method. Conventionally, immunoassays have played a major role in the field of clinical diagnosis as one of the methods for analyzing the condition of patients, but are gradually applied to the measurement of substances such as pesticides remaining in the environment. It has become. However, despite its high necessity for application to mefenacet, it has not been conventionally performed at all, and haptens for preparing antibodies as well as suitable antibodies have not been obtained.

[0008]

Agrochemicals play an extremely important role in facilitating the cultivation of agricultural crops, ensuring stable productivity, and ensuring the quality of products.
However, for the general consumer, the use of pesticides, such as environmental pollution, residues in foods, and their effects on the human body are of great concern. In addition, Japan's food self-sufficiency rate has gradually declined, and the fact is that many rely on imports. Concerns about the use of post-harvest pesticides in pesticide residues have also increased in such imported agricultural products. In order to ensure the safety of the environment and food, it is necessary to quickly and accurately measure the amount of residual pesticides contained in these.

Conventionally, in measuring pesticide residues, pesticides are mainly drawn from a sample, purified, and then analyzed using gas chromatography or high-performance liquid chromatography. Although these methods are excellent in terms of sensitivity and accuracy, preparation of a sample is complicated and requires a great deal of labor and time, skill is required for analysis, and expensive measurement equipment and equipment costs are required. Need expense. In the analysis of pesticides remaining in the environment and agricultural products, it is necessary to obtain results within a short time, and there is a demand for a new measurement method that is simple, quick, and economical in addition to accuracy.

Accordingly, the present inventors have developed an immunochemical method using an antibody reactive with mefenacet as a simple and rapid method for measuring the concentration of mefenacet in environmental water such as soil, paddy drains and rivers. We have considered the application of the detection method. As a result, a mefenacet compound represented by the following general formula (1) is synthesized, and a conjugate of a mefenacet compound and a carrier obtained by using the compound as a hapten is suitable for preparing an immunological reactant reacting with mefenacet. I found that In addition, the immunological reactants prepared in this manner, for example, monoclonal antibodies specifically react with mefenacet, and the mefenacet is accurately measured by an immunoassay using these immunological reactants. I found that I could do it. The present invention has been completed based on such findings.

[0011]

According to the present invention, the general formula (1)

[0012]

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[Wherein, n represents an integer of 1 to 9. And a mefenacet compound represented by the formula:

According to the present invention, the above-mentioned general formula (1)
And a conjugate of a mefenacet compound represented by the formula: and a carrier or a labeling substance.

Further, according to the present invention, there is provided an immunological reactant, for example, a monoclonal antibody, which is reactive with mefenacet and the mefenacet compound represented by the general formula (1).

Further, according to the present invention, there is provided a hybridoma which produces an immunological reactant, for example, a monoclonal antibody, which is reactive with mefenacet and the mefenacet compound represented by the general formula (1).

Further, according to the present invention, there is provided a method for immunologically measuring mefenacet, which comprises using the immunological reactant.

According to the method of the present invention, the mefenacet analysis can be greatly simplified and the measurement time can be shortened, and a large number of samples can be measured quickly, easily and economically.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a mefenacet compound of the present invention and a method for synthesizing the same, a conjugate of a mefenacet compound with a carrier or a labeling substance and a method for preparing the same, a method for preparing a polyclonal antibody, separation of a hybridoma producing a monoclonal antibody and A method for preparing a monoclonal antibody produced by a hybridoma, a method for preparing these antibody fragments, and an immunoassay will be described in this order.

The mefenacet compound of the present invention represented by the general formula (1) is a novel compound not described in any literature. In the general formula (1), n is an integer of 1 to 9, preferably 2 to
It is an integer of 5.

The compound of the present invention represented by the general formula (1)
It can be manufactured according to a known method. For example, the general formula (3)

[0022]

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[Wherein, X represents a halogen atom. 2-halogenobenzothiazole represented by the general formula (4)

[0024]

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[Wherein, R represents a carboxyl protecting group.
n is the same as above. With an α-hydroxyacetanilide compound represented by the general formula (5)

[0026]

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Wherein R and n are as defined above. From the protected mefenacet compound represented by the formula:

In the general formula (3), examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

In the formula (4), the carboxyl-protecting group may be a conventionally known carboxyl group, and is not particularly limited. Examples thereof include a methyl group, an ethyl group and a t-group.
Examples thereof include an tert-butyl group, a benzyl group, a tert-butyldimethylsilyl group, and a trimethylsilyl group.

Using a 2-halogenobenzothiazole represented by the general formula (3) and an α-hydroxyacetanilide compound represented by the general formula (4), appropriately using a known method available in the art. As a result, a protected mefenacet compound represented by the general formula (5) is produced.

For example, 2-halogenobenzothiazole represented by the general formula (3) and the compound represented by the general formula (4) in the presence of a base such as sodium hydride in an inert solvent such as N, N-dimethylformamide. By reacting the compound with an α-hydroxyacetanilide compound at room temperature.
The protected mefenacet compound represented by is easily produced.

Further, the method described in JP-B-61-4395, which is carried out in the presence of an alkali metal hydroxide, an alkali metal carbonate, an alkali metal alcoholate or an amine as an acid acceptor, is disclosed in JP-B-41-6333. It may be carried out according to the method described in the publication in a high-concentration sodium hydroxide solution in the presence of a phase transfer catalyst.

As a method for removing the carboxyl protecting group R, for example, a compound represented by the general formula (5) in the presence of an alkali or an acid:
A method of hydrolyzing the compound of the formula (1). Here, examples of the basic catalyst include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and organic metal bases such as potassium tert-butoxide. Examples of the acidic catalyst include mineral acids such as hydrochloric acid and sulfuric acid, carboxylic acids such as acetic acid and trifluoroacetic acid, and sulfonic acids such as p-toluenesulfonic acid. In this reaction, as a solvent used, for example, alcohols such as methanol and ethanol, ethers such as tetrahydrofuran and dioxane, sulfoxides such as dimethyl sulfoxide, aromatic hydrocarbons such as benzene and toluene, and 1,2- Examples thereof include halogenated hydrocarbons such as dichloroethane, water, and a mixed solvent thereof. The reaction usually proceeds in a temperature range from 0 ° C. to the boiling point of the solvent used, preferably at about room temperature, and is generally completed in about 0.5 to 24 hours.

When the carboxyl protecting group R is a benzyl group, the removal can also be carried out by hydrogenolysis.

Thus, the mefenacet compound of the present invention is produced.

The target compound of the present invention obtained in each of the above reactions can be separated from the reaction system by a usual separation means and further purified. As the separation and purification means, for example, distillation, recrystallization, column chromatography, ion exchange chromatography, gel filtration chromatography, affinity chromatography, preparative thin-layer chromatography, solvent extraction, and the like can be employed.

The 2-halogenobenzothiazole represented by the general formula (3) used as a starting material in the above reaction is a known compound that is easily available.

The α-hydroxyacetanilide compound represented by the general formula (4) can be produced by various methods by appropriately utilizing a known method. As one of them, a compound of the general formula (4) where R = ethyl group and n = 3 will be described as an example.
Ethyl 4-bromobutyrate is condensed with commercially available N-benzylaniline according to a commonly used N-alkylation reaction to obtain ethyl 4- (N-benzylanilino) butyrate, and debenzylation such as hydrogenolysis. According to the N-acylation reaction, ethyl 4-anilinobutyrate was obtained.
The above-mentioned target compound can be obtained by introducing ethyl ((benzyloxyacetyl) anilino] butyrate and finally performing a debenzylation reaction.

The mefenacet compound represented by the general formula (1) is capable of binding to an immunological reactant (eg, an antibody, more specifically, a polyclonal antibody or a monoclonal antibody, or a mefenacet) that specifically reacts with mefenacet. Hapten in the preparation of fragments thereof containing various sites, or antiserum, etc.), and can be used for the preparation of labeled antigens necessary for the development of various immunological assay methods. Here, “hapten” refers to a substance that has an ability to bind to an antibody, but has no immunogenicity by itself and expresses immunogenicity when bound to a carrier. Therefore, haptens generally have a functional group for binding to a carrier.

Preparation of a conjugate of the mefenacet compound of the present invention represented by the general formula (1) and a carrier or a labeling substance, preparation of antiserum and polyclonal antibody, separation of a hybridoma producing a monoclonal antibody, and production of a hybridoma The method of preparing a monoclonal antibody
Any of these methods can be performed by a conventional method, for example, a method described in the Seikagaku Experimental Course (edited by the Biochemical Society of Japan) or the Immunobiochemical Research Method (edited by the Japanese Biochemical Society).

In preparing the conjugate with the mefenacet compound represented by the general formula (1), a known carrier generally used can be used. Here, the carrier is bound to the hapten (conjugation)
Means that imparts immunogenicity to the hapten. As the carrier, for example, a biopolymer compound, for example, a protein having a molecular weight of about 10,000 or more, preferably about 40,000 to 1,000,000 (eg, serum albumin, immunoglobulin, ovalbumin, polylysine, keyhole limpet hemocyanin, etc.) Or polysaccharides (eg, dextran, amylose, amylopectin, etc.) or cells (eg, mammalian erythrocytes, BCG bacteria, etc.).

On the other hand, the mefenacet compound represented by the general formula (1) has a functional group capable of binding to the carrier. The functional group may be any as long as it can bind to the carrier, and can be appropriately selected depending on the carrier used.

For example, when a protein, which is a high molecular compound, is used as a carrier, a functional group capable of binding to the amino group includes, for example, a carboxyl group, an aldehyde group, an amino group, an epoxy group, and the like. Examples of the functional group capable of binding to a group include a thiol group and a maleimide group.
In the case of polysaccharides, as a functional group capable of binding to a hydroxyl group, for example, a carboxyl group, an aldehyde group,
Epoxy groups and the like can be mentioned.

The binding of the mefenacet compound of the present invention to a carrier can be carried out by a conventionally known method. For example, when a protein which is a polymer compound is used as a carrier, a conjugate of the present mefenacet compound and a protein is prepared by a conventionally known bonding method such as a mixed acid anhydride method, an active ester method, and a carbodiimide method. Can be.

In the present invention, the mefenacet compound represented by the general formula (1) can be bound to a labeling substance. As the labeling substance, known labels can be used. For example, enzymes (eg, peroxidase, alkaline phosphatase, etc.), luminescent substances (eg, luminol, acridinium derivatives, etc.), fluorescent substances (eg, fluorescein, europium chelate, etc.) Can be mentioned. Such a labeling substance can be bound to the mefenacet compound of the present invention by the same operation as described above. The obtained conjugate can be used for a competitive reaction with mefenacet to be measured.

The immunological reactant of the present invention is prepared, for example, as follows.

For example, in preparing an antiserum or polyclonal antibody according to the present invention, a conjugate of a mefenacet compound represented by the above general formula (1) and a carrier can be used as an immunogen. Immunization is performed by inoculating a mammal or bird (eg, mouse, rabbit, or chicken) with a usual method, for example, by emulsifying and mixing an immunogen solution with an equal amount of Freund's complete adjuvant or incomplete adjuvant (first time). Immunity)
Thereafter, immunization can be performed several times at intervals of 2 to 4 weeks. Thereafter, blood is collected from the immunized animal, and an antiserum containing a polyclonal antibody is prepared. Then, by DEAE ion exchange chromatography or protein G affinity chromatography, etc.
A polyclonal antibody can be purified from the antiserum.

The hybridoma producing the monoclonal antibody according to the present invention can be isolated by using the conjugate of the mefenacet compound represented by the general formula (1) and a carrier as an immunogen.

For example, a spleen is aseptically removed from an animal (eg, a mouse) several days after the final immunization, and crushed with a stainless steel mesh or the like to prepare spleen cells.
Used for the cell fusion step. Myeloma cells (myeloma cells), which are the other parent cell of the cell fusion, are various known cell lines, for example, P3 NS-1 / 1 Ag 4.1 [Eu
r. J. Immunol. , 5; 511-517 (19
75)], SP2 / 0-Ag14 [Nature, 27
6; 269-270 (1978)], P3-X63-A
g8.653 [J. Immunol. , 123; 154.
8-1550 (1979)].

Cell fusion can be carried out by a conventional method, for example, by spleen cells and myeloma cells in a medium containing a known fusion promoter [eg, commercially available polyethylene glycol for cell fusion (molecular weight: 1500; manufactured by Boehringer Mannheim)]. The myeloma cells can be mixed with the spleen cells of the mouse at a ratio of about 1/5 to 1/10 according to a conventional method. After fusion, a selection medium (eg, HAT
Medium) to grow only the hybridomas. Among them, hybridomas secreting the desired monoclonal antibody in the culture supernatant can be used, for example, to measure the reactivity of the culture supernatant with mefenacet by enzyme-linked immunosorbent assay (ELIS).
It can be confirmed and selected by screening in A). In addition, these hybridomas can be usually separated using a cell cloning method, for example, a limiting dilution method, can be subcultured in a known medium, and can be easily stored in liquid nitrogen for a long period of time.

A monoclonal antibody produced by a hybridoma (monoclonal antibody-producing cell) can be easily prepared by culturing it. In particular, in the in vitro culture, for example, the culture can be performed using 5% carbon dioxide and any medium suitable for culture under the conditions of 37 ° C., and preferably, 10% fetal bovine serum (hereinafter referred to as “Dalbeco medium”). (Hereinafter abbreviated as “FBS”)
"Dulbeco / 10% FBS medium" can be used. In the in vivo culture, it is preferable to culture in the peritoneal cavity of an animal of the same species as the myeloma used, for example, a mouse. Each of these culture supernatants or ascites can be used as a monoclonal antibody solution.

[0052] Antibodies can also be purified using antiserum, culture supernatant, or ascites as a starting material. For antibody purification, a method generally used for protein purification, such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography, affinity chromatography using a protein A or protein G binding polymer, or a method such as dialysis. They can be used in appropriate combination.

According to the method for immunologically measuring mefenacet of the present invention, the immunological reactant (polyclonal antibody or monoclonal antibody, or antiserum) is used, so that the mefenacet can be accurately analyzed. it can. That is, detection of the presence of mefenacet, semi-quantitative measurement, or quantitative measurement can be performed by the immunological measurement method of the present invention. The immunological assay method according to the present invention, except that an immunological reactant specifically reacting to mefenacet is used, otherwise, a conventionally known immunological assay method, for example, an enzyme immunoassay method, A fluorescent immunoassay, a radioimmunoassay, or the like can be applied.

According to the method of the present invention, mefenacet, a herbicide that may possibly remain in environmental water or the like, can be measured. The test sample is not particularly limited as long as it is a sample containing mefenacet.

The immunoassay according to the present invention is carried out, for example, by a method usually called an antigen labeling method, that is, after immobilizing the above-mentioned antibody or antibody fragment, labeling which is a conjugate of a mefenacet compound and a labeling substance. The present invention can be applied to a method of causing a competitive inhibition reaction between a mefenacet compound (standard) and a test compound mefenacet in a sample. Here, among the immunological measurement methods according to the present invention, the antigen labeling method will be described below.

The method of the present invention includes, for example, (1) a step of immobilizing an immunological reactant specifically reacting with mefenacet, particularly an antibody or an antibody fragment (hereinafter, abbreviated as “immobilized antibody”); (2) When the test sample is an aqueous sample, the test sample is filtered with a filter paper or the like. When the test sample is soil or the like, a mephena set that may be contained in the test sample is used. Extracting the test sample with an organic solvent capable of extracting
A step of preparing a specimen; (3) a step of bringing the solid-phased antibody and the specimen into contact with a labeled mefenacet compound to cause a reaction; and (4) a step of not binding to the labeled mefenacet compound bound to the solid-phased antibody. Separating a labeled mefenacet compound from the labeled mefenacet compound; and (5) measuring a signal derived from the labeled mefenacet compound bound to one or the other, preferably an immobilized antibody, separated in the step (4).

When the method of the present invention is carried out, as described above, mefenacet can be confirmed or quantified using a labeled mefenacet compound. For labeling the mefenacet compound, known labels, for example, radioisotopes (for example: ³² P, ³⁵ S, ³ H, etc.), enzymes (for example, peroxidase, alkaline phosphatase, etc.), vitamins (for example, biotin, etc.) , A fluorescent substance (for example, FITC or the like), or a chemiluminescent substance (for example, acridinium) can be used.

In the method of the present invention, after the reaction between the immobilized antibody and the labeled mefenacet compound is completed, the labeled mefenacet compound not bound to the immobilized antibody is separated. Separation can be performed, for example, by filtration, centrifugation, or washing with a buffer.

After separation, for example, a signal from the labeled mefenacet compound bound to the immobilized antibody can be measured. When measuring a signal, it is preferable to change the reaction system to conditions suitable for signal measurement. For example, as a sign,
When a fluorescent or chemiluminescent substance is used, a signal is detected under conditions where quenching does not occur. In the above immunoassay, an appropriate control solution (for example, a conjugate of an immobilized antibody and a mefenacet compound) can be used as a control.

[0060]

EXAMPLES The present invention will now be described specifically with reference to examples, but these do not limit the scope of the present invention.

Example 1 4- [N- (benzyloxyacetyl) anilino] butyric acid
Synthesis of ethyl (12)

[0062]

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N-benzylaniline (6) (6.4 g,
35 mmol) and ethyl 4-bromobutyrate (7) (8.6)
g, 42 mmol) and potassium carbonate (7.3 g, 54 mmol).
(Mmol) in acetonitrile (100 ml) for 88 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The obtained residue is dissolved in ethyl acetate, and 1N
After washing with hydrochloric acid, the ethyl acetate solution was concentrated under reduced pressure to give 4
Ethyl- (N-benzylanilino) butyrate (8) was obtained.
Orange-yellow oil, yield 7 g.

Ethyl 4- (N-benzylanilino) butyrate (8) (4.9 g, 16 mmol) was added to methanol (50
ml), 10% palladium on carbon (0.1 g) was added, and the mixture was stirred under a hydrogen atmosphere. After completion of the reaction, the mixture is filtered,
The filtrate is concentrated under reduced pressure, and ethyl 4-anilinobutyrate (9)
I got Pale red oil, yield 3.7 g.

Ethyl 4-anilinobutyrate (9) (3.7)
g, 17 mmol) and pyridine (1.3 g, 17 mmol) were dissolved in benzene (30 ml) and stirred under water cooling. To this solution was added benzyloxyacetyl chloride (1
0) (3.2 g, 17 mmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. Water was added to the reaction solution, the layers were separated, and the organic layer was washed successively with 1N hydrochloric acid, water, a saturated aqueous solution of sodium hydrogen carbonate and saturated saline, dried over anhydrous magnesium sulfate and concentrated. The obtained oil was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 2).
Ethyl 4- [N- (benzyloxyacetyl) anilino] butyrate (11) was obtained. Yellow oil, yield 2.9 g.

Ethyl 4- [N- (benzyloxyacetyl) anilino] butyrate (11) (2.7 g, 7.6 mmol) was dissolved in methanol (40 ml), 10% palladium on carbon (0.1 g), catalytic amount Was added and stirred under a hydrogen atmosphere. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The obtained oil is subjected to silica gel column chromatography (hexane: ethyl acetate = 3:
Purification in 2) gave the title compound (12). Colorless oil, yield 1.6 g.

Example 2 Benzothiazolyloxy-N- [3- (ethoxycarbo
Synthesis of [nyl) propyl] acetanilide (14)

[0068]

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60% oily sodium hydride (0.18
g, 4.5 mmol) was washed with hexane, N, N-dimethylformamide (5 ml) was added, and the mixture was stirred under a nitrogen atmosphere. Ethyl 4- [N- (benzyloxyacetyl) anilino] butyrate (12) (1.0 g,
(3.8 mmol) in N, N-dimethylformamide (5 ml) was added under ice-cooling. Return to room temperature and after 30 minutes,
2-chlorobenzothiazole (13) (0.70 g,
4.2 mmol) in N, N-dimethylformamide (10 ml) was added. 1.5 hours later, dilute hydrochloric acid was added,
Extracted with ether (60 ml, 3 times). The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained oil was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 2) to obtain the title compound (14). Pale yellow oil, yield 0.3 g.

Example 3 N- (benzothiazolyloxymethyl) -N-phenyl
Synthesis of carbamoyl butyric acid (15)

[0071]

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Benzothiazolyloxy-N- [3- (ethoxycarbonyl) propyl] acetanilide (14)
(0.3 g, 0.75 mmol) in ethanol (20 m
l), a 0.6% (w / w) aqueous sodium hydroxide solution (10 ml, 1.5 mmol) was added, and the mixture was stirred at room temperature. After 6 hours, the reaction solution was concentrated under reduced pressure, water was added to the residue, and the mixture was washed with ethyl acetate (60 ml, 3 times). The aqueous layer was acidified with diluted hydrochloric acid, and ethyl acetate (60 ml, 3 times)
Extracted. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated. The solid residue obtained was purified by silica gel column chromatography (chloroform: methanol = 15: 1) to obtain the title compound (15). Was. White solid, yield 0.2 g.

¹ H-NMR: 1.75 to 2.08 (2
H, m), 2.43 (2H, t), 3.81 (2H,
t), 4.83 (2H, s), 7.08-7.72 (9
H, m).

Example 4 Preparation of Conjugate of Mefenacet Compound and Carrier Protein
Mefenacet compounds of the present invention with bovine serum albumin (BSA) as a manufacturing immunogen was prepared using a mixed acid anhydride method a conjugate of (hapten).

25 mg of N- (benzothiazolyloxymethyl) -N-phenylcarbamoylbutyric acid (15) prepared in Example 3 was dissolved in 2 ml of anhydrous dioxane, and 0.05 ml of N-methylmorpholine was added. Ten
At 20 ° C. for 20 minutes, further add 0.02 ml of isobutyl chloroformate little by little, and stir for 15 minutes to obtain a mixed anhydride of N- (benzothiazolyloxymethyl) -N-phenylcarbamoylbutyric acid (15). Was prepared. On the other hand, 8
After dissolving 0 mg of BSA in 4.3 ml of distilled water,
The pH was adjusted to 9.5 with 1N NaOH, and 2.6 ml of dioxane was added dropwise at 10 ° C. In this BSA solution,
While maintaining the pH at 9 with a 1N NaOH solution, the mixed acid anhydride solution of (15) prepared above was gradually dropped, and the solution was added at 4 ° C for 4 hours.
Stirred for hours. After completion of the reaction, the mixture was dialyzed against distilled water at 4 ° C. overnight, lyophilized, and stored in a freezer. The conjugate of the thus obtained mefenacet compound and BSA was used as an immunogen.

For use as a coating antigen, a conjugate of N- (benzothiazolyloxymethyl) -N-phenylcarbamoylbutyric acid (15) and rabbit serum albumin (RSA) was prepared in the same manner.

Example 5 Preparation of monoclonal antibody-producing hybridoma and antibody production
The immunogen prepared in Preparation Example 4 was dissolved in a physiological phosphate buffer to a concentration of 1 mg / ml, mixed with an adjuvant in an equal amount, and then 100 μl of Balb / c mice was intraperitoneally administered to the mice. Thereafter, a booster was given every two weeks. The spleen of the mouse whose antibody titer in the serum of the blood collected from the tail blood vessel became high was excised, and the spleen excised in a Petri dish containing DMEM medium (Dulbeco's modified Eagle medium) was cut with scissors, The medium was injected into the spleen to drive out the cells. The medium was transferred to a centrifuge tube and left for 5 minutes to sediment large pieces of tissue. The supernatant in which the spleen cells were floating was gently removed, and the single cell suspension was centrifuged at 300 g for 4 minutes to collect the cells, thereby preparing spleen cells. Mouse myeloma cells (P3X63Ag8.653) were mixed at a cell ratio of 5: 1 (myeloma cells: spleen cells), and the cells were collected by centrifugation at 300 g for 4 minutes. next,
Precipitated cells are suspended in DMEM medium and warmed to 37 ° C. in 50% polyethylene glycol (molecular weight 1,500).
1 ml of the solution was slowly added over 60 seconds to perform cell fusion. After adding 10 ml of DMEM medium, 1 m of fetal bovine serum
1 was added. The cells collected by centrifugation were prepared so that the cell number was 5 × 10 ⁵ cells / ml. The cell suspension was dispensed into a 96-well plastic plate in an amount of 100 μl / well and incubated at 37 ° C. in a gas phase of 5% carbon dioxide. Half of the medium in the wells is
The medium was replaced with AT medium and cultured for 10 to 14 days. The activity of the antibody in the culture solution was examined by ELISA, and the cells in the wells producing the desired antibody were cloned by a limiting dilution method using a HAT medium in a 96-well plastic plate using a HAT medium. As a result of cloning, finally, two stable hybridoma strains producing an anti-mefenacet antibody were obtained. These strains were named K73214 and K73354, respectively, and the hybridoma strains were cultured in a medium containing 10% fetal calf serum in DMEM. The centrifuged supernatant of the culture solution was used as a monoclonal antibody solution.
14 "(hereinafter simply referred to as" antibody K73214 ") and" monoclonal antibody K73354 "(hereinafter simply referred to as" antibody K73354 "). The prepared monoclonal antibody solution was diluted with PBS, and the reactivity of mefenacet was examined by an antigen-immobilized ELISA method.

An example of the measurement of the reactivity between the monoclonal antibody and mefenacet is shown below.

1) 100 conjugates of mefenacet and RSA (100 ng / ml) were placed in a 96-well microplate.
A volume of μl / well was added and incubated overnight at 4 ° C. to adsorb to the plate. After washing 5 times with a washing buffer, 250 μl / well volume of a 3% skim milk solution or a 4-fold diluted Block Ace (Block-Ace, manufactured by Dainippon Pharmaceutical Co., Ltd.) was added, and the mixture was incubated at 25 ° C. for 1 hour. The plate was washed.

2) Monoclonal antibody was added to PBS at 3,0
50 times each of the 100-fold diluted solution and the mefenacet solution
μl was added to the wells and after 1 hour incubation, the plate was washed.

3) Horseradish peroxidase-conjugated anti-mouse IgG goat antibody (second antibody) was
Dilute 000-fold, add 100 μl / well, and add
After incubation for 1 hour, the plate was washed.

4) 0.2% o-phenylenediamine coloring solution (100 mM phosphate citrate buffer (PH5.
0), 0.003% aqueous hydrogen peroxide), 200 μl / well, and incubated at 25 ° C. for 10 minutes. Then, 50 μl / well of 4N sulfuric acid was added to stop the enzyme reaction, and the absorbance at 492 nm and 630 nm was measured using a microplate reader. Was measured. The concentration of a conjugate of mefenacet and RSA by ELISA when an anti-mefenacet antibody was used was 100 ng / ml.
A 1000-fold diluted solution and a mefenacet standard stock solution dissolved in methanol were prepared from 0.1 ng / ml to 3000
FIG. 1 shows a standard inhibition curve of mefenacet prepared by adding a solution serially diluted with PBS to a concentration of ng / ml to a well and using a 5,000-fold dilution of the second antibody. . The inhibition rate was calculated and shown by the following equation.

Inhibition ratio (%) = (absorbance of standard sample / absorbance of control) × 100 As is clear from FIG. 2, both monoclonal antibodies show very similar properties, and are in the range of 1 to 50 ng / ml. Measurement of mefenacet was possible, and the titer showing 50% inhibition (IC ₅₀ value) was 7 ng / ml.

Example 6 A monoclonal antibody having a structure similar to the mefenacet structure
The cross-reactivity of the antibody K73214 and the antibody K73354 was examined for a compound having a benzothiazole group or an aniline group, which forms part of the structure of the cross-reactive mefenacet. The cross-reactivity was calculated from the following formula by determining the IC ₅₀ value of the test compound.

Cross-reactivity (%) = (IC _{50 of} mefenacet)
Value / IC ₅₀ value of test compound) × 100 The results of calculating the cross-reactivity rate using the above formula are shown in Table 1. Table 1 is a table showing the results of examining the cross-reactivity of two kinds of antibodies K73214 and K73354 according to the present invention with compounds having an analogous structure of mefenacet.

[0086]

[Table 1]

The following can be seen from Table 1. That is, the antibodies K73214 and K73354 hardly reacted with the mefenacet structural analog compound tested. From these results, it is assumed that the binding mode of both monoclonal antibodies to mefenacet is to bind to the entire molecule including the benzothiazole group and the methylanilide group possessed by this molecule, indicating that the monoclonal antibody is highly specific to mefenacet. I can say.

Example 7 Addition and Recovery Test of Mefenacet to Water Samples Water samples were mefenacet for tap water in Kobe City, river water (brook) flowing near Kobe University's Faculty of Agriculture, and paddy water collected from a paddy field in an experimental field in the school. A recovery test was carried out. For tap water and river water, the collected sample was used as it is, and for paddy water, a sample filtered with a membrane filter having a pore size of 0.45 μm was used. For each sample, mefenacet was used in the range of 1 ng / ml to 500 ng / ml. It was added to a concentration. The sample to which mefenacet was added was measured by the ELISA method described in Example 5 using the antibody K73214, and the obtained recovery was shown in Table 2.
Shown in Table 2 is a table showing the results of the recovery rate when a mefenacet of a known concentration was added to a water sample and analyzed using the ELISA method according to the present invention.

[0089]

[Table 2]

The following can be seen from Table 2. That is, the recovery rate of tap water was 86% to 110%, and good results were obtained. The river water recovery was 68% to 93%, which was slightly lower. The recovery rate of paddy water was 68% to 104%. The low recovery rate from river water and paddy water depends on the substances contained in the sample, depending on the immobilized antigen and antibody K73214.
Is thought to be hindered.

Example 8 The concentration of water in a paddy field sprayed with a mefenacet preparation was measured.
Measurement Rice seedlings were transplanted to paddy fields in the experimental field of Kobe University. Transplant 3
A day later, a mixed preparation with another paddy field herbicide (trade name: Wolface Granule 17: 0.17% bensulfuron-methyl, 5%
Benthiocarb (containing 1% mefenacet) was applied according to conventional usage. Water was supplied from one place in the paddy field so that the depth of the paddy water was 5 cm, water was sampled from several places in the paddy field, and collected in one container. A part of the sample filtered through the membrane filter was directly measured by the ELISA method. On the other hand, measurement by high-performance liquid chromatography (HPLC) was also performed to confirm the measured value. H
A sample of the PLC was extracted twice from 100 ml of the filtrate with 50 ml of dichloromethane, water was removed by adding anhydrous sodium sulfate, and the mixture was evaporated to dryness using a rotary evaporator.
A sample to which 5 ml of methanol was added was measured by HPLC. The concentration of the sample was calculated by preparing a calibration curve from each of the mefenacet standard solutions. HPLC is Shimadzu LC-10A
Using S and Cosmozil 5C-18-AR column, mobile phase: acetonitrile: water = 50: 50, column temperature: 4
0 ° C., mobile phase flow rate: 1 ml / min, detection wavelength: 216 n
The measurement was performed under the measurement conditions of m. FIG. 2 shows the results of a test conducted on two test paddy fields that have not been used in mefenacet. According to FIG. 2, the concentration of mefenacet in two paddy waters decreased daily, and ELISA and HPLC
The values measured in the above are well in agreement.

From the above results, the following became clear. By fusing spleen cells and myeloma cells obtained by immunizing mice with a conjugate of mefenacet and a carrier protein, a fusion cell producing a monoclonal antibody that binds to mefenacet was selected. Using the culture supernatant obtained by culturing the cells as a monoclonal antibody, the optimal conditions of the ELISA method for measuring mefenacet were determined. As a result of the addition test of mefenacet to river and paddy water, a good recovery rate was obtained by this ELISA method, and the mefenacet concentration of paddy water was also measured. In monitoring the concentration of mefenacet in river water and paddy water, the EL
A simple and quick method by the ISA method was suitable.

[Brief description of the drawings]

FIG. 1 is a graph showing the results obtained by examining the reactivity of two kinds of monoclonal antibodies K73214 and K73354 according to the present invention with mefenacet using the ELISA method.

[FIG. 2] FIG. 2 shows the results of the ELISA method and the HP method for the daily changes in the concentration of mefenacet in paddy water in two test paddy fields.
It is a graph which shows the result of having compared by LC method.

Claims (6)

[Claims] 1. A compound of the general formula (1) [In the formula, n represents an integer of 1 to 9. ] The mefenacet compound represented by these. 2. A conjugate of the mefenacet compound according to claim 1 and a carrier or a labeling substance. 3. An immunological reactant which is reactive to mefenacet and the mefenacet compound according to claim 1. 4. The immunological reactant according to claim 3, wherein the immunological reactant is a monoclonal antibody. A hybridoma producing the immunological reactant according to claim 3. 6. An immunoassay for mefenacet, comprising using the immunological reactant according to claim 3 or 4.