JP2001261631A - Hapten compound of flufenoxuron, antibody and method of measuring the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new antibody which reacts with flufenoxuron, to provide a fragment thereof, and to provide a method of preparing the above compounds, giving a hapten compound of the flufenoxuron, a monoclonal antibody having reactivity with the flufenoxuron, etc. SOLUTION: This hapten compound has a structure of the flufenoxuron 1-[4-(2-chloro-α,α,α-trifluoro-p-tolyloxy)-2-fluorophenyl]-3-(2,6-difluorobenzoyl) urea} or another structure in which a spacer arm and a functional group for bonding are attached to the flufenoxuron to make covalent bonds.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 1- [4- (2-
Hapten of chloro-α, α, α-trifluoro-p-tolyloxy) -2-fluorophenyl] -3- (2,6-difluorobenzoyl) urea (hereinafter referred to as “flufenoxuron” in the present specification) It relates to compounds, antigens, antibodies and fragments thereof.

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

[0003]

2. Description of the Related Art Flufenoxuron has the following formula (2):

[0004]

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An insect growth regulator having a structure represented by the following formula: Insect growth regulators are insecticidal agents that inhibit insect chitin synthesis or juvenile hormone-like substances (JHA) and have an insecticidal action that kills pests by disrupting molting and metamorphosis. Insect epidermis contains chitin as a main component, and a compound exhibiting an anti-insect action by inhibiting its synthesis is a chitin synthesis inhibitor. Since chitin is absent from warm-blooded animals, there is little concern for animal toxicity. Species selectivity is high and has little effect on natural enemies. It is highly effective during the larval stage and cannot be molted at the next molting after treatment and dies. Some have no effect during the adult stage, while others have the effect of reducing the number of insects in the next generation. It is slow-acting and its use time is important, but if it is used in a timely manner, it has a long residual effect and helps to reduce the number of control.

Flufenoxuron is a benzophenylurea-based insect growth regulator and is mainly taken orally into larvae to inhibit chitin biosynthesis. As a result, the molting of the larva becomes incomplete and leads to death. It also shows some activity during larval pupation and emergence. The drug is active against insects of the order Pteryophora, spider mites, leafhoppers and thrips. In spider mites and thrips, hatching is suppressed in adult laying eggs incorporating this drug. The insecticidal effect of this drug is slow but has residual effect (Agrochemical Handbook No. 133).
Pages 139-140 and 629 1998
Year Edition (Japan) Plant Protection Association.

[0007] In recent years, there has been great social interest in pesticide residues in the environment such as soil, water, and the atmosphere, and in post-harvest pesticides in imported agricultural products, which have recently increased in particular. With regard to flufenoxuron, the registration standard for pesticide registration is set at 2 ppm for fruits, 0.5 ppm for vegetables, 0.5 ppm for bean, 15 ppm for tea, etc. (Latest Handbook of Agrochemical Regulations, 1995, 311
Page, Japan Plant Protection Association). Therefore, in order to ensure the safety of the environment and foods, it is necessary to measure the amount of flufenoxuron contained therein quickly and accurately.

Conventionally, flufenoxuron is, for example, a fruit,
It has been extracted from vegetables, sugar beet, crude tea, etc., purified, and then analyzed by high performance liquid chromatography (HPLC). That is, for example, after extracting a sample with acetone, performing a coagulation method, transferring the solution to hexane, partitioning with hexane-acetonitrile, further purifying by florisil column chromatography using hexane-acetonitrile as a solvent, and then performing HPLC. Measurement methods are used (“Recent Analytical Methods for Pesticides”, pages 314-316)
Page, published by Chuo Hokyou Shuppan, April 1, 1995)). These methods have problems in that the preparation of the sample is complicated, requires a large amount of procedures and time, requires a high level of skill in the analysis, and requires high costs for measuring devices and equipment. Flufenoxuron measurement requires the analysis of a large number of samples in a short period of time.
There is a need for new measurement methods that are also fast and economical.

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

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

Generally, for a molecule having a large molecular weight,
By inoculating animals without further modification,
An appropriate immune response can be elicited to produce an antibody that recognizes the antigen. However, small molecule compounds such as flufenoxuron usually cannot elicit an immune response when inoculated into animals. These molecules only act as a group of epitopes by binding to immunogenic macromolecules (proteins, polysaccharides, etc.)
Elicits an immune response in the presence of the T cell receptor,
Antibodies are produced by a panel of B lymphocytes. Molecules that produce immunogenicity only when bound to a polymer compound in this way are collectively referred to as “haptens”.

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

[0013] Despite the great need for flufenoxuron, haptens for producing such antibodies as well as suitable antibodies had not been obtained prior to the present invention.

[0014]

An object of the present invention is to provide a novel antibody or a fragment thereof which reacts with flufenoxuron, and a method for producing the same.
As used herein, the term “fragment” of an antibody means a part of an antibody capable of binding to an antigen, for example, a _Fab fragment or the like.

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

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

The present invention further provides a method for immunochemical measurement of flufenoxuron, which comprises using a conjugate of the above-mentioned antibody or fragment thereof and / or the above-mentioned flufenoxuron hapten with a polymer compound or a labeling substance. The purpose is to provide.

[0018]

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

The flufenoxuron of the present invention is represented by the following formula (2):

[0020]

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A compound having a structure represented by the following formula: The antibody of the present invention can be obtained, for example, by using, as an antigen, a derivative in which a spacer arm and a functional group available for binding are introduced into a part of flufenoxuron and bound to an appropriate polymer compound as a hapten. . For example, the following equation (1):

[0022]

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A compound having a structure represented by the formula (1) wherein n is an integer of 1-5 is used as a hapten for preparing an antibody. In the formula (1), n is preferably 2.

The present invention relates to the hapten compound, a conjugate of the hapten compound and a polymer compound, an antibody reactive with flufenoxuron and a method for producing the same, and an immunochemical method for flufenoxuron using the hapten compound or the antibody. Related to the measurement method.

Preparation of Flufenoxuron Hapten The flufenoxuron hapten represented by the formula (1) can be produced according to a known method. Although not limited, for example, the following method can be used.

First, 2,6-difluoro-3-nitrobenzoyl isocyanate which can be synthesized by the method described in JP-A-2-252 is added to an organic solvent of the following formula (X1):

[0027]

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Reaction of 4- (2-chloro-4-trifluoromethylphenoxy) -2-fluoroaniline having a structure represented by the following formula (X2):

[0029]

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1- [4- (2-) having a structure represented by the following formula:
Chloro-4-trifluoromethylphenoxy) -2-fluorophenyl] -3- (2,6-difluoro-3-nitrobenzoyl) urea is obtained. As the organic solvent, for example, dichloromethane, chloroform, acetonitrile,
Examples thereof include tetrahydrofuran, benzene, toluene, and diethyl ether. The reaction is carried out at a temperature between 0 ° C. and the boiling point of the solvent, preferably at 20 ° C. to 80 ° C., for 5 minutes to 20 hours, preferably 30 minutes to 5 hours. Then, using a reducing agent such as sodium borohydride, stannous chloride, or a mixture thereof, or by catalytic hydrogen reduction in the presence of a catalyst such as rhodium carbon or Raney nickel, the nitro compound in the compound of the formula (X2) is obtained. Reducing the group, the following formula (X3):

[0031]

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1- (3-amino-2,6-difluorobenzoyl) -3- [4- (2-
Chloro-4-trifluoromethylphenoxy) -2-fluorophenyl] urea is obtained. The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably from 10 ° C. to 80 ° C., for 5 minutes to 20 hours, preferably 30 minutes to 2 hours.

Finally, a compound of the formula (X3) is prepared by reacting the compound of the formula (X3) in the presence of an organic base such as pyridine in an organic solvent or using an organic base as a solvent.

[0034]

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[In the formula (X4), n is as defined above. The compound of the formula (1) can be obtained by reacting with a compound having a structure represented by the following formula: As the organic solvent, for example, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, benzene,
Examples include toluene and diethyl ether.
The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably 50 ° C.
From 5 to 30 hours, preferably from 5 to 20 hours. By subjecting the compound obtained by the above-mentioned production method to silica gel chromatography or recrystallization operation or the like as necessary, a purified product with higher purity can be obtained.

Hereinafter, the preparation of the antigen and antibody of the present invention and the immunochemical assay will be described. In addition, these preparations can be carried out according to a known method, for example, a method described in Seikagaku Experimental Lecture, Immunobiochemical Research Method (edited by the Japanese Biochemical Society), or the like.

Flufenoxuron hapten and polymer compound
Preparation of a conjugate with a product The flufenoxuron hapten synthesized as described above is bound to an appropriate polymer compound and then used as an antigen for immunization or an antigen for immobilization.

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

The binding between the flufenoxuron hapten and the polymer compound can be performed, for example, by the mixed acid anhydride method (BF Er).
Langer et al. : J. Biol. Che
m. 234 1090-1094 (1954)) or the activated ester method (AEKARU et al .:
J. Agric. Food Chem. 42 301-
309 (1994)).

The acid anhydride used in the mixed acid anhydride method is obtained by a usual Schotten-Baumann reaction,
The desired hapten-polymer compound conjugate is produced by reacting this with a polymer compound. The Schotten-Baumann reaction is performed in the presence of a basic compound.
As the basic compound, a compound commonly used in the Schotten-Baumann reaction can be used. For example, tributylamine, triethylamine, trimethylamine, N-methylmorpholine, pyridine, N, N-dimethylaniline, DBN, DBU, DABCO, etc. And inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The reaction is usually carried out at a temperature between -20 ° C and 150 ° C, preferably between 0 ° C and 1 ° C.
The reaction is performed at 00 ° C., and the reaction time is 5 minutes to 10 hours,
Preferably, it is 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually performed at −20 ° C. to 100 ° C., preferably at 0 ° C. to 50 ° C.,
The reaction time is 5 minutes to 10 hours, preferably 5 minutes to 5 hours. The mixed anhydride method is generally performed in a solvent.
As the solvent, any solvent commonly used in the mixed acid anhydride method can be used, and specifically, ethers such as dioxane, diethyl ether, tetrahydrofuran, and dimethoxyethane, and halogenated solvents such as dichloromethane, chloroform, and dichloroethane. Hydrocarbons, aromatic hydrocarbons such as benzene, toluene and xylene, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, etc. Is mentioned. Examples of the haloformate used in the mixed acid anhydride method include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, and isobutyl chloroformate. The proportions of the hapten, the haloformate and the polymer compound used in the method can be appropriately selected from a wide range.

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

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

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

In a similar manner as described above, a product obtained by binding a labeling substance such as an enzyme to a flufenoxuron hapten can be used in an immunochemical assay. The labeling substance, (hereinafter referred to "HRP") horseradish peroxidase, an enzyme such as alkaline phosphatase, fluorescein isothiocyanate, a fluorescent substance such as rhodamine, radioactive substances such as ³² P, ¹²⁵ I, and the like chemiluminescent substance.

Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of flufenoxuron hapten and a polymer compound. For example, a conjugate of flufenoxuron hapten and KLH is dissolved in a sodium phosphate buffer (hereinafter referred to as "PBS") and mixed with an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or alum. Immunized as an antigen for immunization of an animal. As the animal to be immunized, any of those commonly used in the art can be used. For example, mice, rats, rabbits, goats,
Horses and the like can be mentioned. However, humans are not included.

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

Blood is collected from the immunized animal, and the serum separated therefrom can be used to evaluate the presence of a polyclonal antibody that reacts with flufenoxuron. In the present invention, an antiserum obtained by using a conjugate of a flufenoxuron hapten and a polymer compound as an antigen for immunization is at least about 100% in an indirect competitive ELISA method described later.
It can react with flufenoxuron at a concentration of μg / ml (Example 4, Table 2).

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

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

Hereinafter, a method for preparing a monoclonal antibody against flufenoxuron of the present invention will be described, but it will be apparent to those skilled in the art that the present invention is not limited thereto.
The steps (a) and (b) can be performed by a method substantially similar to the method described for the polyclonal antibody.

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

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

The above-mentioned myeloma cells are subcultured in Dulbecco's modified Eagle's medium (DMEM) or Iscove's modified Dulbecco's medium (IMDM) containing fetal bovine serum, and a cell number of about 1 × 10 ⁶ or more is secured on the day of fusion.

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

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

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

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

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

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

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

The anti-flufenoxuron monoclonal antibody obtained as described above can be determined for its subclass, antibody titer and the like using a known method such as ELISA.

Measurement of Flufenoxuron Using Antibody As a method for measuring flufenoxuron using the antibody used in the present invention, radioisotope immunoassay (RIA),
ELISA method (Engval, E., Methods)
in Enzymol. , 70, 419-439 (1
980)), various methods generally used for detection of antigens such as a fluorescent antibody method, a plaque method, a spot method, an agglutination method, and Ouchterlony (“Hybridoma method and monoclonal antibody”, published by R & D Planning, Inc. Pages 30-53, March 5, 1982
Days). ELIS from the viewpoint of sensitivity, simplicity, etc.
Method A is widely used.

The measurement of flufenoxuron was performed using various ELI
For example, the indirect competitive ELISA method among the SA methods can be performed according to the following procedure. (A) First, a conjugate of flufenoxuron hapten, which is an antigen for immobilization, and a polymer compound is immobilized on a carrier.

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

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

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

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

In the blocking in the step (b), an antibody to be added later besides the flufenoxuron hapten portion can be adsorbed on a carrier on which an antigen (a conjugate of a flufenoxuron hapten and a polymer compound) is immobilized. Parts may be present and are used solely to prevent them.
As a blocking agent, for example, BSA or skim milk solution can be used. Alternatively, Block Ace (“Bl
ock-Ace ", manufactured by Snow Brand Milk Products Co., Ltd., code No. UK-
A commercially available blocking agent such as 25B) can also be used. Specifically, for example, but not limited to, a buffer solution containing a blocking agent [eg, 1% BSA and 60 mM N
85 mM borate buffer (pH 8.
0)], and incubated at about 4 ° C. for 1 to 5 hours, followed by washing with a washing solution. The washing liquid is not particularly limited.
BS can be used.

Next, in step (c), the sample containing flufenoxuron and the antibody are brought into contact with the immobilized antigen, and the antibody is reacted with the immobilized antigen and flufenoxuron, thereby forming the immobilized antigen-antibody complex. Body and flufenoxuron-antibody complexes are formed.

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

The first antibody is dissolved in a buffer and added. Although not limited, the reaction is carried out at 10 to 40 ° C,
Preferably, the heat treatment may be performed at about 25 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the first antibody that has not bound to the immobilized antigen. As the cleaning liquid, for example, PB
S can be used.

Next, a second antibody is added. For example, when a mouse monoclonal antibody is used as the first antibody, it is appropriate to use an antibody against a mouse antibody to which an enzyme (eg, peroxidase or alkaline phosphatase) is bound. It is desirable to react the first antibody bound to the carrier with a second antibody diluted so that the final absorbance is preferably 4 or less, more preferably 0.5 to 3.0. Buffer is used for dilution. Although not limited, the reaction is carried out at room temperature for about 1 hour, and the reaction is followed by washing with a buffer.
By the above reaction, the second antibody binds to the first antibody. Alternatively, a labeled first antibody may be used, in which case the second antibody is not required.

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

When peroxidase is used as the enzyme that binds to the second antibody, for example, hydrogen peroxide and 3,3 ′, 5,5′-tetramethylbenzidine or o
-A chromogenic substrate solution containing phenylenediamine (hereinafter referred to as "OPD") can be used. Although not limited, a chromogenic substrate solution is added and reacted at room temperature for about 10 minutes, and then the enzyme reaction is stopped by adding 1N sulfuric acid. When 3,3 ', 5,5'-tetramethylbenzidine is used, the absorbance at 450 nm is measured.
If OPD is used, measure the absorbance at 492 nm. On the other hand, when alkaline phosphatase is used as the enzyme that binds to the second antibody, the color is developed using, for example, p-nitrophenyl phosphate as a substrate, the enzyme reaction is stopped by adding a 2N NaOH solution, and the absorbance at 415 nm is measured. The method is suitable.

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

Alternatively, the measurement of flufenoxuron can also be performed by a direct competition ELISA method using the monoclonal antibody of the present invention as described below, for example. (A) First, the monoclonal antibody of the present invention is immobilized on a carrier.

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

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

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

The blocking in the step (b) is performed when the carrier on which the antibody is immobilized has a portion where the flufenoxuron and enzyme-bound hapten in the sample to be added later are adsorbed independently of the antigen-antibody reaction. It is done to prevent it. Blocking agent and its method,
The same indirect competitive ELISA method as described above can be used.

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

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

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

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

The monoclonal antibody 48A27- of the present invention
1-1 is preferably used in the direct competition ELISA method,
Reacts with flufenoxuron in a concentration range from about 0.1 ng / ml to about 100 ng / ml, more preferably from about 1 ng / ml to about 50 ng / ml (Example 6, FIG. 1).

Further, as described above, the direct competitive ELISA
In method A, a hapten different from that used for antibody production can be used as a labeling antigen.
Shows intrinsic reactivity in the ISA method.

Cross-reactivity of the Antibodies of the Invention The Direct Competition ELISA or Indirect Competition ELISA described above
By the method, the cross-reactivity of the monoclonal antibody of the present invention can be examined.

For example, monoclonal antibody 48A27-
1-1 shows little reactivity with flufenoxuron analogs and has very high specificity for flufenoxuron (Example 7, Table 3). Hereinafter, the present invention will be described specifically with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description in the present specification, and they are included in the technical scope of the present invention.

[0089]

EXAMPLES Example 1 Combination of Flufenoxuron Hapten
Success

[0090]

Embedded image

1- [4- (2-chloro-4-trifluoro)
2-methylphenoxy) -2-fluorophenyl] -3-
(2,6-difluoro-3-nitrobenzoyl) urea
Synthesis of (1) A mixture of 1.0 g (5.4 mmol) of 2,6-difluoro-3-nitrobenzamide and 0.9 g (7.1 mmol) of oxalyl chloride in 5 ml of toluene was refluxed for 1 hour.
Stir for 5 hours. The reaction mixture was concentrated, 5 ml of toluene was added to the residue, and 1.5 g (4.9 mmol) of 4- (2-chloro-4-trifluoromethylphenoxy) -2-fluoroaniline was added to this solution at 5 to 15 ° C. And stirred at room temperature for 5 hours. After concentrating the reaction mixture, the residual solid was recrystallized from 2-propanol and 1.6 g (yield 55).
%) Of the compound (1).

Melting point: 163-164 ° C. 1- (3-amino-2,6-difluorobenzoyl)-
3- [4- (2-chloro-4-trifluoromethylphen)
Synthesis of 1- [4- (2-chloro-4- )-(oxy) -2-fluorophenyl] urea (2) in 60 ml of ethanol
2. Trifluoromethylphenoxy) -2-fluorophenyl] -3- (2,6-difluoro-3-nitrobenzoyl) urea (1) 1.5 g (2.8 mmol) and tin (II) chloride dihydrate After stirring at 60 ° C. for 15 minutes to a solution of 2 g (14.2 mmol), 590 mg (15.5 mol) of sodium borohydride was added. After stirring at 60 ° C. for 30 minutes, 50 ml of water was added to this solution for 10-15 minutes.
C. and neutralized to pH 7 with 15% aqueous NaOH. Ethanol was distilled off under reduced pressure, and the residue was extracted twice with 40 ml of ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate and concentrated. 2 residue solids
1.1 g (79% yield) recrystallized from propanol
Compound (2) was obtained.

Melting point: 180-182 ° C. 3 ′-[3- (4- (2-chloro-4-trifluoromethyl)
Tylphenoxy) -2-fluorophenyl) ureidoca
Rubonyl)]-2 ', 6'-difluorobenzoyl) s
Synthesis of cincinanilide acid (3) 1- (3-Amino-2,6-difluorobenzoyl) -3- [4- (2-chloro-4-trifluoromethylphenoxy) -2-fluorophenyl] urea in 20 ml of pyridine (2) A mixture of 1.0 g (2 mmol) and 0.24 g (2.4 mmol) of succinic anhydride was added at 80 ° C. for 20 minutes.
Stirred for hours. The reaction mixture was concentrated, 30 ml of water was added to the residue, and the mixture was extracted three times with 30 ml of ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (ethyl acetate: methanol = 5: 1) to obtain 0.61 g (yield 51%) of compound (3).

The flufenoxuron hapten (3)
Physical property data (chemical shift δ) by ¹ H-NMR are shown below.

[0095]

Table 1 ¹ H-NMR (DMSO-D ₆ , 400 MHz) δ 2.53 (2H, m, CH ₂ ), 2.65 (2H, m, CH ₂ ), 7.00 (1 H, d, Ar : H), 7.21 (2H, m, 2Ar: H), 7.31 (1H, d, Ar: H), 7.73 (1H, m, Ar: H), 8.06 (3H, m). , 3Ar: H), 9.87 ( 1H, d, NH), 10.38 (1H, br, NH), 11.73 (1H, br, NH), 12.15 (1H, s, COOH) carried Example 2 Preparation of antigen for immunization and antigen for screening A conjugate of flufenoxuron hapten and BSA was prepared as an antigen for immunization and an antigen for screening by a mixed acid anhydride method.

After 12 mg of flufenoxuron hapten prepared in Example 1 was dissolved in 1 ml of anhydrous dioxane, 25 μl of N-methylmorpholine was added, and the mixture was stirred at room temperature for 15 minutes. Then isobutyl chloro-formate 10
μl was added and stirred at room temperature for 20 minutes. Hereinafter, this is referred to as “Solution A”.

On the other hand, 40 mg of BSA was dissolved in 2 ml of distilled water, and pH 7.0-7.
After adjusting to pH 5, 1.3 ml of anhydrous dioxane was added to adjust the pH to 7.0 to 7.5 again. Add solution A to this solution at pH
Drop while adjusting to 7.0-7.5, 10 ° C
For 4 hours. After the reaction, the mixture was dialyzed against distilled water at 4 ° C., further treated with a 1% K ₂ CO ₃ solution at room temperature for 1 hour, dialyzed again against distilled water at 4 ° C., and obtained with flufenoxuron hapten. A conjugate with BSA (hereinafter, referred to as “flufenoxuron hapten / BSA”) was used as an antigen for immunization.

Further, using the same method, a conjugate of flufenoxuron hapten and RSA (hereinafter referred to as “fluphenoxuron hapten / RSA”) and a conjugate of flufenoxuron hapten and HRP (hereinafter referred to as “fluphenoxuron hapten / RSA”) "Flufenoxuron hapten / HRP") was also prepared.

Example 3 Immunization Balb / c mice were used for immunization. 50 µg of flufenoxuron / BSA prepared in Example 2 was dissolved in 100 µl of PBS as an immunizing antigen, mixed with an equal amount of complete Freund's adjuvant, and inoculated subcutaneously into Balb / c mice. Four weeks later, the cells were mixed in the same manner as described above using Freund's incomplete adjuvant, and boosted subcutaneously into the mice. Two weeks later, 180 μl of PB
The mouse was boosted with 10 μg of the immunizing antigen dissolved in S from the tail vein of the mouse.

Example 4 Antiserum Flufenoxuron
Indirect competitive ELI that immediately before inoculation into mice tail vein in reactive Example 3, was diluted prepare antiserum bled, detailed below against
Flufenoxuron was measured by the SA method, and the antiserum was evaluated.

A flufenoxuron hapten / RSA PBS solution prepared in the same manner as the immunizing antigen (0.5 μg / m
l) was coated on a 96-well microplate in an amount of 50 μl / well (25 ng / 50 μl / well) and diluted 4 times with Block Ace (“Block
Ace ", manufactured by Snow Brand Milk Products Co., Ltd., code No. UK-25B)
To prepare an assay plate. Equal amounts of a 5000-fold diluted antiserum and 20% methanol solution containing flufenoxuron of various concentrations were mixed in this plate, 50 μl of the mixture was added to each well, and reacted at room temperature for 1 hour.

After washing 5 times with PBS, peroxidase-conjugated goat anti-mouse IgG antibody (manufactured by Tago) diluted 2000-fold with Block Ace diluted 10-fold was added in an amount of 50 μl / well, and incubated at room temperature for 1 hour. Reacted. After washing 5 times with PBS, 2 mg / ml O
A 0.1 M citrate-phosphate buffer (pH 5.0) containing PD and 0.02% hydrogen peroxide was added in an amount of 50 μl / well, and reacted at room temperature for 10 minutes to develop color.

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
Table 2 shows an example of the results.

[0104]

[Table 2]

As shown in Table 2, 100 μg of flufenoxuron
/ Ml, it was confirmed that the antiserum used was reactive with flufenoxuron.

Example 5 Preparation of Hybridomas Following Example 3, spleen cells from mice having high anti-flufenoxuron antibody activity in serum and myeloma cells (Sp2 / 0-Ag14) were isolated from S. Yamashita et al. Method (Histocytochemistry: Edited by the Japan Society for Tissue and Cell Chemistry: Interdisciplinary Planning. 1986)
Year) according to the polyethylene glycol method,
Cultured. A culture supernatant in which cell growth was observed was added in an amount of 50 μl / well to the plate coated and blocked in the same manner as in Example 4, and reacted at room temperature for 1 hour.

After washing 5 times with PBS, a peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Tago) diluted 2000-fold with 10-fold diluted Block Ace was added in an amount of 50 μl / well, and the mixture was incubated at room temperature for 1 hour. Reacted. After washing 5 times with PBS, 2 mg / ml OPD
And 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide in an amount of 50 μl / well, and a color reaction was performed at room temperature for 10 minutes.

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
Reactive cells (hybridomas) were selected. Next, the reactivity of each well with flufenoxuron was examined by the indirect competitive ELISA method described in Example 4, and cells producing the target antibody were cloned by the limiting dilution method. As a result, several hybridomas were cloned as cells producing anti-flufenoxuron antibodies. 48A27-1-1 of them was
On March 15, the deposit number FERM P-17733 and the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (〒305-0046)
Deposited at 1-3-1-3 Higashi, Tsukuba City, Ibaraki Prefecture).

Example 6 By Direct Competition Inhibition ELISA
Measurement of Flufenoxuron Hybridoma 48A27-1-1 obtained in Example 5
Was transplanted into the abdominal cavity of a mouse, and the ascites fluid obtained 10 to 15 days later was collected, and the monoclonal antibody 48 was purified by ammonium sulfate fractionation.
A27-1-1 was fractionated. (Hereinafter, monoclonal antibodies have the same names as the hybridomas that produce them.) Using this 48A27-1-1 antibody, the amount of flufenoxuron was measured by a direct competitive ELISA method.

The 48A27-1-1 antibody solution (10
(μg / ml) in a 96-well microplate in an amount of 50 μl / well, coating was allowed to stand at 4 ° C. overnight, and further 4-fold dilution of Block Ace (Yukishira Milk Industry Co., Ltd.)
Was performed to prepare a plate for assay. 50 μl of an equal volume mixture of a 10% methanol solution containing flufenoxuron of each concentration and a PBS solution containing flufenoxuron hapten / HRP prepared in Example 2
Each well was placed in each well and reacted at 25 ° C. for 1.5 hours.

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

Next, 50 μl of 1N sulfuric acid was added to each well to stop the color reaction, and the absorbance at 490 nm was measured. The result is shown in FIG. As shown in FIG.
In the ISA method, the monoclonal antibody 48A of the present invention
27-1-1 is 0.1 ng / m 2 of flufenoxuron.
It can be measured in the range of 1 to 100 ng / ml.

Example 7 Evaluation of Monoclonal Antibody Using the indirect competitive ELISA method described in Example 4, the reactivity of the monoclonal antibody 48A27-1-1 against a flufenoxuron-like compound was examined. Table 3 shows the results. Table 3 shows that the monoclonal antibody 48
A27-1-1 showed high reactivity with flufenoxuron, but showed little reactivity with similar compounds.

[0114]

[Table 3]

[Brief description of the drawings]

FIG. 1 shows the monoclonal antibody 48A2 of the present invention.
7 shows the reactivity with flufenoxuron by a direct competition ELISA method using 7-1-1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 G01N 33/577 B (C12P 21/08 33/577 C12R 1:91) // (C12P 21 / 08 C12N 5/00 B C12R 1:91) 15/00 B (72) Inventor Yoshio Kamata 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Moeda (1) Inside the Institute for Environmental Immunity Technology, Inc. 1-227-14 Hamamatsucho, Minato-ku, Tokyo (72) Inventor Shigeyuki Watanabe Inside the Institute for Environmental Immunity Technology, 1-27-14 Hamamatsucho, Minato-ku, Tokyo ( 72) Inventor Hiroshi Munakata 1-27-14 Hamamatsucho, Minato-ku, Tokyo F-term in the Institute for Environmental Immunity Technology (reference) 4B024 AA05 AA07 AA11 BA53 GA03 HA15 4B064 AG27 CA10 CA20 CC24 DA10 DA11 DA13 4B065 AA92X AB05 AC14 BA08 CA25 CA46 4H006 AA01 AB80 AB84 AB99 BJ50 BM10 BM30 BM71 BM72 BP60 BS10 BV25 BV71 4H045 AA11 AA20 BA72 CA40 DA75 DA76 DA86 EA50 FA41 FA42 FA72

Claims (11)

[Claims] (1) The following formula (1): [In the formula (1), n is an integer of 1-5. ] The compound which has a structure represented by these. 2. The compound according to claim 1, wherein n is 2 in the formula (1). 3. A conjugate of the compound according to claim 1 or 2 with a polymer compound or a labeling substance. 4. An antigen is prepared by binding the compound according to claim 1 or 2 with a polymer compound, and the antigen is used to obtain the following formula (2): A method for producing an antibody or a fragment thereof reactive to a compound represented by the formula (2), characterized by producing an antibody reactive to a compound having a structure represented by the formula: [5] An antibody or a fragment thereof, which is produced by using the conjugate according to [3] as an antigen, and which is reactive with the compound represented by the formula (2). 6. The antibody or fragment according to claim 5, which is a monoclonal antibody. 7. The monoclonal antibody 48A27-1-1 produced from the hybridoma deposited under accession number FERM P-17733.
2. The antibody or fragment according to item 1. 8. A hybridoma producing the antibody according to any one of claims 5 to 7. 9. The hybridoma according to claim 8, which has been deposited under accession number FERM P-17733. 10. A method for immunochemically measuring a compound represented by the formula (2), comprising using the antibody or fragment according to any one of claims 5 to 7. 11. The method according to claim 11, further comprising using the compound according to claim 1 or 2 or the conjugate according to claim 3.
The immunochemical measurement method according to claim 10.