JP2000095787A - Hapten compound for butamifos, antibody and assay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound having a structure in which a spacer arm and a functional group usable for binding butamifos, to a polymeric compound are introduced into the butamifos, and useful as a hapten compound for constructing an antigen for preparing a new antibody having reactivity against butamifos. SOLUTION: A compound of formula I (R1 is a 1-4C alkyl; R2 is H or methyl; A is a 1-10C alkylene), for example, a compound of formula II. The compound of formula I is obtained, for example, by reacting a compound of formula III (L1 and L2 are each a halogen) (e.g. ethyl dichlorothiophosphate) with a compound of formula IV (e.g. 5-methyl-2-nitrophenol) in an organic solvent such as benzene in the presence of a base such as potassium carbonate, and further reacting the resultant compound with a compound of the formula H2NACOOZ (Z is a protecting group for a carboxyl) (e.g. tert-butyl 6-aminocaproate) in the same organic solvent as above in the presence of the same base as above followed by the removal of the protection group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an O-ethyl-O-
The present invention relates to a hapten compound, an antigen, an antibody and a fragment thereof of (3-methyl-6-nitrophenyl) secondary butyl phosphoramidothioate (hereinafter, referred to as "butamiphos").

[0002] The present invention further relates to an immunoassay method using the above-mentioned antigens, antibodies and fragments thereof.

[0003]

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

Embedded image An organic phosphorus compound having a structure represented by the following formula, which is used as a soil treatment agent. It shows a high effect on annual weeds, especially grasses. It is used for controlling weeds on lawns.

[0004] More specifically, butamiphos is a non-hormonal herbicide, and is used as a soil treatment agent before weed emergence.
It is effective for annual grasses and broadleaf weeds such as Poa annua. Less effective against Asteraceae and perennial weeds.
This agent is mainly absorbed into the germ area and causes abnormal growth and further cessation of growth due to inhibition of cell division, causing weeds to die. Mobility in soil is small, potency duration is 30 to 40 days (Agrochemical Handbook 1994)
624, 410-411, Japan Plant Protection Association; "Residual Analysis Method for the Latest Pesticides", pp. 97-98, edited by the Research Group for Pesticide Residue Analysis Method, Chuo Law Publishing.
95.4.1).

[0005] In recent years, there has been great social interest in the effects of pesticide residues in the environment, such as water, soil, and air, and foods on human health. Butamiphos has a pesticide residue standard based on the Food Sanitation Law, which is based on rice, sea cucumber, potatoes such as sweet potato, yam, etc. Lily vegetables, carrots and other vegetables, tomatoes,
Eggplant vegetables such as eggplant, cucumbers, cucurbits such as pumpkins and fruits such as watermelon, melon 0.05ppm,
It is specified as 0.2 ppm etc. in potatoes (Revision table of "Standards for Pesticide Residues"-Revised by notification on September 2, 1996-Food Chemistry Division, Ministry of Health and Welfare, Ministry of Health and Welfare, Japan Food Sanitation Association). In addition, regarding the pesticide registration suspension standard, 0.05 ppm for rice, 0.1 ppm for fruits, 0.05 ppm for vegetables, 0.2 ppm for potatoes, and 0.1 ppm for beans.
The reference value is set at 05 ppm, etc. (Handbook of Regulations and Reference Values for the Latest Agrochemicals, 1995 edition, p. 299, Japan Plant Protection Association).

[0006] Regarding the water quality, the provisional guidance guideline on the prevention of water pollution by the pesticides used in golf courses by the Environment Agency in 1990 (1990) shows that the provisional guidance guideline on the drainage of golf courses by butamiphos is 0. It is determined to be 04 mg / l. The nature of the guideline value is set as an upper limit value from the viewpoint of preventing water pollution due to the outflow of pesticides used in the golf course. Further, as a guideline for evaluating the water quality of pesticides in public water bodies, butafomis has a standard value of 0.004 mg / l. As part of water quality preservation measures for public water bodies, in addition to environmental standards related to water pollution (so-called water quality environmental standards),
These are guidelines provided by the Environment Agency for pesticide components (the latest handbook of regulations and standard values for pesticides).

Therefore, in order to ensure the safety of foods and the environment, it is necessary to quickly and accurately measure the amount of butamiphos contained therein.

Conventionally, for example, butamiphos in agricultural products has been extracted from samples of rice, fruits, vegetables, potatoes, beans, etc., purified, and then analyzed by gas chromatography (GC). That is, a method has been adopted in which a sample is extracted with acetone, the concentrate is purified by a C18 mini column and a silica gel mini column, and then analyzed by GC ("Residual analysis method of latest agricultural chemicals", supra). This method has problems that the preparation of the sample is complicated and requires a great deal of labor and time, that the analysis is skilled, and that the measuring device and the equipment require high costs. In the measurement of butamiphos, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and a new measurement method having not only accuracy but also simplicity, speed, and economy has been demanded.

The immunoassay is a method for detecting an antigen based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen. Due to its excellent accuracy, simplicity, rapidity, and economical efficiency, it has recently It's getting attention. In immunoassays, a great variety of labels have been applied as detection methods, for example enzymes, radiotracers, chemiluminescent or fluorescent substances, metal atoms, sols, latexes and bacteriophages.

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

Generally, for a molecule having a large molecular weight,
By inoculating animals without further modification,
An appropriate immune response can be elicited to produce an antibody that recognizes the antigen. However, small molecule compounds such as butamiphos usually cannot elicit an immune response when inoculated into animals. For the first time, these molecules act as a panel of epitopes by binding to immunogenic macromolecules and elicit an immune response in the presence of the T cell receptor, resulting in the production of antibodies by a group of B lymphocytes. Is done. Molecules that produce immunogenicity only when bound to a polymer compound in this way are collectively referred to as “haptens”.

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

[0013] However, despite the very high need for butamiphos, not only a suitable antibody but also a hapten for producing the antibody had not been obtained before the present invention.

[0014]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antibody or a fragment thereof that reacts with butamiphos, 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 butamifos.

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

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

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

Another object of the present invention is to provide a method for immunological measurement of butamiphos, which comprises using a conjugate of the above-mentioned antibody or fragment thereof and / or the above-mentioned butamiphos derivative with a polymer compound. .

[0020]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a derivative of butamiphos in which a functional group which can be used for binding to a spacer arm and a polymer compound has been introduced into butamiphos or a portion thereof. As a result, an antibody having reactivity with the compound was successfully obtained, and the present invention was completed.

The butamiphos to be used in the present invention has the following formula (2):

Embedded image It is a compound represented by these.

The antibody of the present invention can be obtained, for example, by using, as an antigen, butamiphos or a derivative obtained by introducing a spacer arm and a functional group available for bonding into a portion thereof, with a suitable polymer compound as a hapten. Can be. For example, the following equation (1):

Embedded image [In the formula (1), R ¹ is an optionally branched alkyl group having 1 to 4 carbon atoms; R ² is a hydrogen atom or a methyl group; and A is optionally branched. A good alkylene group having 1 to 10 carbon atoms] is used as a hapten for preparing an antibody.

R ¹ is an alkyl group having 1 to 4 carbon atoms which may be branched if desired, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group and a tert-butyl group. And preferably an ethyl group.

R ² is a hydrogen atom or a methyl group,
Preferably it is a methyl group.

A is an alkylene group having 1 to 10 carbon atoms which may be branched if desired, and includes, for example, those listed in Table 1 below, preferably a polymethylene group having 1 to 10 carbon atoms, It is preferably a pentamethylene group having 5 carbon atoms.

[0026]

TABLE 1 polymethylene groups -CH carbon number _{1-10 (CH 3) - (CH} 2) n - (n = 1-8) -C (CH 3) 2 - (CH 2) n - (n _{= 1-7) -C (CH 3)} (C 2 H 5) - (CH 2) n - (n = 1-6) -C (CH 3) (C 3 H 7) - (CH 2) n - (n = 1-5) -C (C 2 H 5) 2 - (CH 2) n - (n = 1-5) -C (C 2 H 5) (C 3 H 7) - (CH 2) n - (n = 1-4) -C ( C 2 H 5) (C 4 H 9) - (CH 2) n - (n = 1-3) -C (C 3 H 7) 2 - (CH 2) _n − (n = 1-3) In the present invention, the stereoisomer of the compound is not particularly limited and includes all stereoisomers.

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

Preparation of Derivative of Butamiphos The butamiphos derivative represented by the formula (1) can be prepared according to a known method. Although not limited, for example, there is a method as described below.

First, the following equation (X1):

Embedded image [In the formula (X1), L ¹ and L ² are halogen atoms (the halogen atom means F, Cl, Br or I in the present specification), and may be the same or different. And R ¹ is as defined above], with the following formula (X2):

Embedded image By reacting the compound represented by the formula (X2), wherein R ² is as defined above, in an organic solvent in the presence of a base, the following formula (X3):

Embedded image [In the formula (X3), R ¹ , R ² and L ¹ (L ² ) are as defined above].

Examples of the organic solvent for synthesizing the compound of the formula (X3) include acetonitrile, acetone, hexane, pentane, benzene, toluene, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, diglyme, N, N-dimethylformamide, hexamethylphosphoric triamide, or the like, or a mixed solution thereof can be used. Examples of the base include potassium carbonate, sodium carbonate, lithium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate, triethylamine, N, N-dimethylaniline, and hydroxide. Sodium, lithium diisopropylamide and the like can be used.

The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably at 0 ° C. to 80 ° C., for 1 hour to 40 hours, preferably for 1 hour to 24 hours.

Next, a compound of the formula (X3) and the following formula (X4):

Embedded image [In the formula (X4), Z is a carboxyl-protecting group;
And A is as defined above] in an organic solvent in the presence of a base,
The following formula (X5):

Embedded image [In the formula (X5), Z, R ¹ , R ² and A are as defined above].

The ester compound represented by the formula (X4) is
Known methods, for example, Skerrit et al.
gric. Food Chem. 1992, 40 , 14
66-1470), and can be easily synthesized.

The organic solvent and the base are represented by the formula (X3) described above.
The same compounds as in the case of synthesizing the compound can be used.

The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably at 0 ° C. to 60 ° C., for 1 hour to 50 hours, preferably for 1 hour to 24 hours.

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

The compound of the formula (X1) is converted to the compound of the formula (X5)
The compound of formula (X1) may be synthesized by first reacting a compound of formula (X1) with an ester compound represented by formula (X4), and then reacting a compound of formula (X2).

Further, the compound of the formula (1) can be obtained by removing the protecting group for the carboxyl group represented by Z from the compound of the formula (X5). The removal of the carboxyl-protecting group can be carried out by a known method such as alkali hydrolysis and acid hydrolysis.

That is, in the case of acid hydrolysis, the formula (X)
The compound of 5) is preferably dissolved in an organic solvent such as acetic acid, formic acid, benzene, dichloromethane, 1,2-dichloroethane and the like, and then hydrochloric acid, sulfuric acid, boron trifluoride diethyl ether complex, trifluoroacetic acid, trifluoromethanesulfonic acid , P-toluenesulfonic acid or the like, and the mixture is heated at 0 ° C to the boiling point of the solvent, preferably at 0 ° C to 50 ° C.
The compound of formula (1) can be obtained by stirring and reacting for 1 to 5 hours, preferably for 1 to 5 hours.

In the case of alkali hydrolysis, the formula (X
The compound of 5) is preferably methanol, ethanol,
Dissolve in an organic solvent such as tetrahydrofuran and ethylene glycol, and then add sodium hydrogencarbonate, sodium carbonate, potassium carbonate, lithium hydroxide, sodium hydroxide or potassium hydroxide aqueous solution and the like, and the temperature is preferably from 0 ° C to the boiling point of the solvent, preferably. From 0 ° C to room temperature for 5 minutes to 10
The compound of formula (1) can be obtained by stirring and reacting for 1 hour to 2 hours.

Further, when Z is a benzyl group, the removal can be carried out by hydrogenolysis with hydrogen.

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

By subjecting the compound obtained by the above-described production method to silica gel chromatography or recrystallization as required, a purified product having a 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.

Bonding between butamiphos derivative and polymer compound
Preparation of body The butamiphos derivative synthesized as described above is used as an immunizing antigen after binding to an appropriate polymer compound.

Examples of preferred high molecular 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 butamiphos derivative and the polymer compound can be performed, for example, by the mixed acid anhydride method (BF Erlange).
r et al. : J. Biol. Chem. 234 1
090-1094 (1954)) or the activated ester method (AEKARU et al .: J. Agric.
Food Chem. 42 301-309 (199
It can be performed by a known method such as 4)).

The mixed acid anhydride used in the mixed acid anhydride method is obtained by a usual Schotten-Baumann reaction, and this is reacted with a high molecular compound to produce a desired hapten-high molecular compound conjugate. You. 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, tri-n
Organic bases such as -butylamine, triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, DBN, DBU, and DABCO; and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogencarbonate, and sodium hydrogencarbonate. The reaction is usually carried out at a temperature between -20 ° C and 100 ° C, preferably between 0 ° C and 5 ° C.
The reaction is performed at 0 ° C., and the reaction time is 5 minutes to 10 hours, preferably 5 minutes to 2 hours. The reaction between the resulting mixed acid anhydride and the polymer compound is usually carried out at a temperature of -20 ° C to 1 ° C.
Carried out at 50 ° C, preferably at 0 ° C to 100 ° 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, dichloromethane,
Halogenated hydrocarbons such as chloroform and dichloroethane; aromatic hydrocarbons such as benzene, toluene and xylene; ethers such as diethyl ether, dioxane, tetrahydrofuran and dimethoxyethane; esters such as methyl acetate and ethyl acetate; Aprotic polar solvents such as N-dimethylformamide, dimethylsulfoxide, hexamethylphosphoric triamide and the like; Examples of the haloformate used in the mixed acid anhydride method include 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 produce an N-hydroxysuccinimide activated ester.

As the coupling agent, an ordinary coupling agent commonly used in a condensation reaction can be used.
Dicyclohexylcarbodiimide, carbonyldiimidazole, water-soluble carbodiimide and the like. Examples of the organic solvent include N, N-dimethylformamide (DM
F), dimethylsulfoxide, dioxane and the like can be used. The molar ratio of the hapten compound and N-hydroxysuccinimide used in the reaction is preferably from 1:10 to 10:
1, most preferably 1: 1. The reaction temperature is 0 ° C to 50 ° C, preferably 22 ° C to 27 ° C, and the reaction time is 5 minutes to 24 hours, preferably 1 hour to 2 hours.
The reaction can be carried out at a temperature from the melting point to the boiling point.

After the coupling reaction, the reaction solution is centrifuged, and the supernatant is added to a solution in which the polymer compound is dissolved. When the polymer compound has a free amino group, for example, the amino group and the hapten compound An acid amide bond is formed between the carboxyl groups of The reaction temperature is 0 ° C to 60 ° C, preferably 5 ° C to 40 ° C, more preferably 2 ° C to 40 ° C.
At 2 ° C. to 27 ° C., the reaction time is 5 minutes to 24 hours, preferably 1 hour to 16 hours, more preferably 1 hour to 2 hours. The reaction product is purified by dialysis, a desalting column, or the like to obtain a conjugate of a butamiphos derivative 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 butamiphos derivative can be used in an immunological assay. As a labeling substance, horseradish peroxidase (hereinafter, “HR”)
P "), enzymes such as alkaline phosphatase, fluorescent substances such as fluorescein isocyanate and rhodamine, radioactive substances such as ³² P and ¹²⁵ I, and chemiluminescent substances.

Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of a butamiphos derivative and a polymer compound. For example, a butamiphos derivative-KLH conjugate is added to a sodium phosphate buffer (hereinafter referred to as “P
BS) and mixed with an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or alum, as an antigen for immunization. As the animal to be immunized, any of those commonly used in the art can be used,
For example, mouse, rat, rabbit, goat, horse and the like can be mentioned.

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

[0055] 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 butamifos.

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

In producing a monoclonal antibody,
At least the following work steps are required.

(A) Preparation of a conjugate of a butamiphos derivative and a polymer compound used as an antigen for immunization (b) Immunization of animals (c) Collection of blood, assay, and preparation of antibody-producing cells (d) Myeloma Preparation of cells (e) Cell fusion between antibody-producing cells and myeloma and selective culture of hybridomas (f) Screening and cloning of hybridomas producing the desired antibody (g) Culture of hybridomas or transplantation of hybridomas into animals (H) Measurement of reactivity of the prepared monoclonal antibody and the like.

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

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

The steps (a) and (b) can be performed by a method substantially similar to the method described for the polyclonal antibody.

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

The myeloma cells that can be used in the step (d) include, for example, P3 / X63-Ag8 (X63) (Nat) of a myeloma cell line derived from a Balb / c mouse.
ure, 256 , 495-497 (1975)), P3
/ X63-Ag8. U1 (P3U1) (Current
Topics. in Microbiology an
d Immunology, 81 , 1-7 (198
7)), P3 / NSI-1-Ag 4-1 (NS-1)
(Eur. J. Immunol., 6 , 511-519).
(1976)), Sp2 / O-Ag14 (Sp2 / O)
(Nature, 276 , 269-270 (197
8)), FO (J. Immuno. Meth., 35 ,
1-21 (1980)), MPC-11, X63.65.
3, myeloma cell line such as S194, or rat-derived cell line 210. RCY3. Ag 1.2.3. (Y3) (N
ature, 277 , 131-133, (1979))
Etc. can be used.

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

The cell fusion in the step (e) is performed by a known method, for example, the method of Milstein et al. (Met
hods in Enzymology, 73 , 3 (19
81)) and the like. Currently the most commonly used is polyethylene glycol (PEG)
It is a method using. The PEG method is described in, for example, Cell Histochemistry, Shuji Yamashita et al. (Supra). As another fusion method, a method using electric processing (electric fusion) can be adopted (Etsuko Okochi et al., Experimental Medicine)
5 . 1315-19, 1987). Other methods can be appropriately adopted. In addition, the ratio of cells used may be the same as in a known method. For example, spleen cells may be used about 3 to 10 times as much as myeloma cells.

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

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

Further, cell cloning of a hybridoma which has been found by measurement to produce an antibody reactive with butamiphos is performed. As this cell cloning method,
Method for limiting one hybridoma to one well by limiting dilution "Limiting dilution method"; Method for collecting colonies on a soft agar medium; Method for removing one cell with a micromanipulator; One method using a cell sorter "Sorter clone method" for separating the cells. The limiting dilution method is simple and often used.

For the wells in which the antibody titer has been recognized, cloning is repeated 1-4 times, for example, by the limiting dilution method, and those with a stable antibody titer are selected as anti-butamiphos monoclonal antibody-producing hybridoma strains. As a medium for culturing the hybridoma, for example, DMEM or IMDM containing fetal calf serum (FCS) is used. The culture of the hybridoma is performed, for example, at a carbon dioxide concentration of about 5 to 7% and at 37 ° C. (in a thermostat at 100% humidity).
It is preferable to culture the cells.

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-butamiphos monoclonal antibody. The antibody fraction is further subjected to dialysis, salting out with ammonium sulfate, gel filtration, lyophilization, etc. By collecting and purifying, an anti-butamiphos monoclonal antibody can be obtained. Further, when purification is necessary, ion exchange column chromatography, affinity chromatography, high performance liquid chromatography (HPL)
It can be carried out by combining commonly used methods such as C).

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

Measurement of Butamiphos with Antibodies As a method for measuring butamiphos with antibodies used in the present invention, radioisotope immunoassay (RIA), ELISA
Method A (Engval, E., Methods in E)
nzymol. , 70 , 419-439 (198
0)), fluorescent antibody method, plaque method, spot method, agglutination method, Ouchterlony, etc., various methods generally used for detection of antibodies ("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 butamiphos can be carried out by the following procedure using, for example, an indirect competitive inhibition ELISA method among various ELISA methods.

(A) First, a conjugate of a butamiphos derivative as an antigen and a polymer compound is immobilized on a carrier.

(B) The solid surface on which the antigen is not adsorbed is blocked by a protein unrelated to the antigen, for example, a protein.

(C) A sample containing various concentrations of butamiphos and an antibody are added thereto, and the antibody is competitively bound to the immobilized antigen and butamyphos to form an immobilized antigen-antibody complex and butamyphos-antibody. Allow the complex to form.

(D) By measuring the amount of the immobilized antigen-antibody complex, the amount of butamiphos in the sample can be determined from a previously prepared calibration curve.

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

For immobilizing an antigen on a carrier, for example,
A buffer containing an antigen may be added to the carrier and incubated. Known buffers can be used as the buffer, for example, 10 mM PBS containing 145 mM NaCl. The concentration of the antigen in the buffer can be selected from a wide range, but is usually from 0.01 μg / ml to 100 μg / ml.
μg / ml, preferably from 0.05 μg / ml to 5 μg / ml.
μg / ml is suitable. When a 96-well microtiter plate is used as a carrier, 30
20 μl / well to 150 μl below 0 μl / well
/ Well is desirable. 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 antibody includes:
Not only the conjugate of the butamiphos derivative and the polymer compound prepared in the antibody itself, but also the conjugate of another derivative represented by the formula (1) and the polymer compound can be used. For example, antigens having different R ¹ , R ² or A in the compound represented by the formula (1) can be used for antibody production and solid phase immobilization, respectively. Furthermore, other butamiphos-like compounds not included in the formula (1) can also be used as the immobilized antigen.

In the blocking in the step (b), in the carrier on which the antigen (combination of the butamiphos derivative and the high molecular compound) is immobilized, there is a portion other than the butamiphos derivative portion to which the antibody to be added later can be adsorbed. May be done solely to prevent it. As a blocking agent, for example, BSA or skim milk solution can be used.
Or, Block Ace ("Block-Ace",
Dainippon Pharmaceutical, code No. A commercially available blocking agent such as UK-25B) can also be used. Specifically, the method is carried out by, for example, but not limited to, adding an appropriate amount of Block Ace to a portion where the antigen is immobilized, incubating at about 4 ° C. overnight, and then washing with a buffer. The buffer is not particularly limited, but may be, for example, 10 mM PBS (pH 7.2), 0.1 mM PBS.
8% (w / v) NaCl, 0.02% (w / v) KC
1, a composition of 0.02% (v / v) Tween 20 is suitable.

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

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

The first antibody is added after being dissolved in a buffer. Although not limited, the reaction may be performed at about 37 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the first antibody that has not bound to the immobilized antigen. As a reagent used in this reaction, 10 mM PBS (pH 7.
2), 0.8% (w / v) NaCl, 0.02% (w / v)
v) KCl compositions are preferred.

Next, a second antibody is added. For example, when a mouse monoclonal antibody is used as the first antibody, it is appropriate to use an anti-mouse-goat antibody conjugated with an enzyme (for example, peroxidase or alkaline phosphatase). About 500-1000 for the first antibody bound to the carrier
A second antibody diluted to a 0-fold, preferably a final absorbance of 4 or less, more preferably 0.5 to 3.0 is reacted. Buffer is used for dilution. The reaction is carried out, but not limited to, at about 37 ° C. for about 1 hour, followed by washing with a buffer after the reaction. 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 butamiphos 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 about 25 ° C. for about 20 minutes, and then the enzyme reaction is stopped by adding 2N sulfuric acid. 480-50 when using OPD
Measure the absorbance at 0 nm. When 3,3 ', 5,5'-tetramethylbenzidine is used, the absorbance at 450 nm is measured. On the other hand, when alkaline phosphatase is used as the enzyme that binds to the second antibody, for example, p-
The color is developed using nitrophenyl phosphate as a substrate and 2N N
A method of stopping the enzymatic reaction by adding aOH and measuring the absorbance at 415 nm is suitable.

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

Alternatively, the measurement of butamiphos can be carried out, for example, by a direct competitive inhibition ELISA method using the monoclonal antibody of the present invention as described below.

(A) First, the monoclonal antibody of the present invention is immobilized on a carrier.

(B) The surface of the carrier on which the antibody is not immobilized is blocked with a protein unrelated to the antigen, for example, a protein.

(C) a sample containing various concentrations of butamiphos,
And an enzyme-bound hapten in which a butamiphos derivative is bound to an enzyme is reacted with an antibody immobilized on a carrier.

(D) By measuring the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of butamiphos in the sample is determined from a previously prepared calibration curve.

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

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

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

The hapten to be bound to a labeling substance such as an enzyme is not limited to the butamiphos derivative used for preparing the antibody, but may be represented by the formula (1) as in the case of the immobilized antigen in the indirect competitive inhibition ELISA method. Other derivatives described can also be used. For example, antigens having different R ¹ , R ² or A in the compound represented by the formula (1) can be used for antibody production and label competition, respectively. Further, other butamiphos-like compounds not included in the formula (1) can be used as haptens to be bound to the enzyme.

In the step (c), the sample containing butamiphos and the enzyme-bound hapten are brought into contact with the antibody-immobilized carrier, and the competition inhibition reaction between butamiphos and the enzyme-bound hapten is performed.
A complex of these and the solid-phased carrier is formed. The sample containing butamiphos 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 buffer, for example, PBS can be used.

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

The monoclonal antibody BTF2-7 of the present invention
No. 7 can measure the amount of butamiphos in the range of 0.01 ng / ml to 1000 ng / ml, preferably 0.1 ng / ml to 100 ng / ml by the direct competitive inhibition ELISA method (Example 9, FIG. 1).

Cross-reactivity of the Antibody of the Present Invention The above-described direct competitive inhibition ELISA or indirect competitive inhibition E
The cross-reactivity of the monoclonal antibody of the present invention can be examined by the LISA method.

For example, the monoclonal antibody BT of the present invention
F2-77 does not show cross-reactivity with butamifos oxone, a metabolite of butamiphos, and the related organophosphorus compounds amiprofos and anilofos (Example 1).
0, FIG. 2).

Hereinafter, the present invention will be described in detail 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.

[0105]

EXAMPLES Example 1 Synthesis of Butamiphos Derivative-1

Embedded image 3.6 g of ethyl dichlorothiophosphate (1) (20 mm
l) and 3.1 g of 5-methyl-2-nitrophenol (2
0 mmol) was dissolved in 100 ml of benzene, 8.4 g (61 mmol) of potassium carbonate was added, and the mixture was stirred at 80 ° C for 2 hours. The reaction solution was filtered through celite, concentrated, and then concentrated on a silica gel column (n-hexane: ethyl acetate = 19:
Purification in 1) gave 2.0 g (yield 3) as a clear oil.
4%) of (2) was obtained.

2.0 g of chlorothiophosphoric acid derivative (2)
(6.77 mmol) and 6-aminocaproic acid tert
To a solution of 1.27 g (6.77 mmol) of -butyl in 50 ml of acetonitrile was added 1.0 g of potassium carbonate (7.25 mmol).
mmol) and stirred at 60 ° C. for 1 hour. The reaction solution was filtered through celite, and the filtrate was concentrated and then subjected to silica gel chromatography (n-hexane: ethyl acetate = 19: 1 →
Purification in 9: 1) gave 2.43 g as a clear oil.
(Yield 80%) (3) was obtained.

The hapten ester (3) 2.43 g (5.
4 mmol) in 50 ml of dichloromethane,
1. Add 5 ml of trifluoroacetic acid (TFA) and add at room temperature.
Stir for 5 hours. The reaction solution was concentrated under reduced pressure and subjected to silica gel chromatography (n-hexane: ethyl acetate = 4:
1 → 1: 1) to give 1.94 as a clear oil.
g (yield 91%) of (4) was obtained.

The chemical shift data of the butamiphos derivative-1 by ¹ H-NMR is shown below.

¹ H-NMR (CDCl ₃ ) 1.19-1.46 (overlap, 5H), 1.46-1.72 (overlap, 4H) 2.30-2.41 (t, 2H), 2 .43 (s, 3H) 3.02-3.22 (m, 2H), 3.22-3.42 (m, 1H) 4.04-4.21 (m, 2H), 7.01-7 .10 (d, 1H) 7.41 (s, 1H), 7.77-7.85 (d, 1H) Example 2 Synthesis of butamiphos derivative-2

Embedded image 4.1 g of ethyl dichlorothiophosphate (1) (23 mmol)
l) and 3.8 g of tert-butyl 6-aminocaproate
(20 mmol) was dissolved in 100 ml of acetonitrile, 8.4 g (61 mmol) of potassium carbonate was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was filtered through Celite,
After concentration, silica gel column (n-hexane: ethyl acetate
= 9: 1 → 4: 1) to give 3.9 g (58% yield) of (2) as a clear oil.

3.3 g of chlorothiophosphoric acid derivative (2)
(10 mmol) and 1.5 g of 2-nitrophenol (1
0.8 mmol) in 100 ml of acetonitrile solution,
Add 4.3 g (31 mmol) of potassium carbonate and add 80 ° C
For 2 hours. The reaction solution was filtered through celite, and the filtrate was concentrated and purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1) to give 1.0 g (yield 23%) of (3) as a transparent oil. Obtained.

Hapten ester (3) 1.0 g (2.3
mmol) in 100 ml of dichloromethane,
10 ml of trifluoroacetic acid was added and the mixture was stirred at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure, and silica gel chromatography (n-hexane: ethyl acetate = 4: 1 → 1: 1)
And 0.77 g as a clear oil (yield 89
%) Of (4) was obtained.

The chemical shift data of the butamiphos derivative-2 by ¹ H-NMR is shown below.

¹ H-NMR (CDCl ₃ ) 1.19-1.44 (duplicate, 5H), 1.44-1.72 (duplicate, 4H) 2.27-1.40 (t, 2H), 3 0.000-3.20 (m, 2H) 3.22-3.40 (m, 1H), 4.02-4.22 (m, 2H) 7.20-7.33 (m, 1H), 7 .50-7.69 (m, 2H) 7.83-7.94 (d, 1H) Example 3 Preparation of Antigen for Immunization Using the butamiphos derivative-1 prepared in Example 1 as a hapten as a hapten, a mixed acid was prepared. It was prepared by the anhydride method.

First, 7 mg of butamiphos derivative-1 was dissolved in 0.7 ml of anhydrous dioxane, cooled to 10 ° C. to 12 ° C., and 4 μl of tri-n-butylamine and 24 μl of isobutyl chloroformate were added. ° C
For 30 minutes (hereinafter referred to as “solution A”).

On the other hand, 20 mg of KLH was dissolved in 1 ml of distilled water, and dialyzed overnight using 0.5% NaHCO ₃ (pH 9.4) as an external solution. After the dialysis, the solution A was slowly added to 1.5 ml of the supernatant obtained by centrifugation at 3000 rpm for 30 minutes.
After reacting at 4 ° C. for 2 hours, one spatula of glycine was added, and the mixture was further stirred at 4 ° C. for 30 minutes to complete the reaction. This reaction solution was 145 mM Na
The antigen for immunization was obtained by dialysis for one week in Cl-10 mM PBS (pH 7.4).

The conjugate of the thus obtained butamiphos derivative-1 and KLH (hereinafter referred to as "butamiphos derivative 1")
-KLH conjugate ") was used as the antigen for immunization.

Example 4 Preparation of Antigen for Screening A butamiphos derivative 1-BSA conjugate was obtained as a screening antigen in the same manner as in Example 3.

Example 5 Immunization The butamiphos derivative 1-KLH prepared in Example 3
Mice were immunized using the conjugate as an antigen for immunization.
After 100 μg of the antigen for immunization was dissolved in 100 μl of PBS and mixed with an equal volume of Freund's complete adjuvant, Bal
b / c mice were inoculated. Seventeen days later, mice were boosted with the immunizing antigen prepared using Freund's incomplete adjuvant in the same manner as described above. Also,
Forty-one days later, mice were boosted with an immunizing antigen dissolved in PBS.

Example 6 Measurement with Antiserum The titer of the antiserum prepared in Example 5 was determined by an indirect competitive inhibition ELISA using the screening antigen prepared in Example 4.
The evaluation was performed by the method A.

First, a solution (0.1 μg / ml) of the butamiphos derivative 1-BSA conjugate prepared in Example 4 was added to 10
Coated to 96-well plate at 0 μl / well. After washing, Block Ace diluted 4 times (“B
lock Ace ": Dainippon Pharmaceutical, code No. UK-
After blocking with 25B), an equal amount of an antiserum diluent and 10% methanol solution containing various concentrations of butamiphos or a similar compound were mixed, 100 μl of the mixture was added to a well, and reacted at 37 ° C. for 1 hour. Was. After the reaction,
After washing once with 0.05% Tween 20-PBS,
100 μl of peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Kappel) diluted 5000 times with PBS
l of the mixture was added to each well and reacted at 37 ° C. for 1 hour. After the reaction is completed, 0.05% Tween 20-P
After washing twice with BS, 10% of a 0.05 M phosphate citrate buffer (pH 4.5) containing 0.4 mg / ml o-phenylenediamine (OPD) and 0.04% hydrogen peroxide was added.
0 μl was added to each well and left at room temperature for 20 minutes to develop color. After the reaction, 100 μl of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm was measured.

Example 7 Preparation of Hybridoma Cells Following Example 5, spleen cells of a mouse with increased anti-butamiphos antibody activity in serum and mouse myeloma cells (P3U1) were electrofused. Cell fusion was performed. The culture supernatant in which cell proliferation was observed was examined for antibody activity against butamiphos by the following method. A solution of the butamiphos derivative 1-BSA conjugate (0.1 μg / ml) was coated on a 96-well plate at 50 μl / well. After washing, after blocking with Block Ace diluted 4 times, equal amounts of the culture supernatant and 10% methanol solutions containing various concentrations of butamiphos or a similar compound are mixed, and 100 μl of the mixture is placed in a well. , 37 ° C
For 1 hour. After completion of the reaction, 0.05% Tween
After washing once with en20-PBS, using PBS,
Peroxidase-conjugated anti-mouse Ig diluted 5000-fold
A G goat antibody (manufactured by Kappel) was reacted in an amount of 50 μl / well in each well at 37 ° C. for 1 hour. After the reaction is completed, 0.0
After washing twice with 5% Tween 20-PBS, 0.4
mg / ml OPD and 0.05 M phosphate citrate buffer (pH 4.5) containing 0.04% hydrogen peroxide
0 μl was added to each well and left at room temperature for 20 minutes to develop color. After the reaction, 100 μl of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm was measured to select those having specific antibody activity.

Next, the cells in the selected wells were subjected to cell cloning using the limiting dilution method. As a result, the hybridoma cell line producing the anti-butamiphos antibody was cloned. Among them, BTF2-77 was deposited on July 23, 1998 under accession number FERM-16907, by the Institute of Biotechnology and Industrial Science, National Institute of Advanced Industrial Science and Technology (〒305-0046).
Deposited at 1-3-1 Tsukuba City, Ibaraki Prefecture).

Example 8 Production of Butamiphos Derivative with HRP
Conjugate Preparation A conjugate of butamiphos derivative-1 and HRP prepared in Example 1 was prepared by the same mixed anhydride method as in Example 3.
1 mg of butamiphos derivative in 0.2 ml of anhydrous dioxane
Then, 0.5 μl of tri-n-butylamine and 0.3 μl of isobutyl chloroformate were added thereto, and
Stirred at 2 ° C. for 30 minutes. (Hereinafter, this is referred to as “solution B”) On the other hand, 0.5% NaHCO ₃ is pH-adjusted with NaOH.
Dissolve 5 mg of HRP in 1 ml of the solution adjusted to 9.4,
Liquid B was dropped into this. The mixture was stirred at 4 ° C. for 2 hours, glycine was further added, and the mixture was stirred for 30 minutes to complete the reaction. The reaction product was dialyzed against PBS to obtain a purified butamiphos derivative 1-HRP conjugate.

Example 9 By direct competitive inhibition ELISA
Determination of butamiphos The hybridoma cells obtained in Example 7 (BTF2-7
7) was transplanted into the abdominal cavity of a mouse, and the ascites fluid obtained 10 to 15 days later was collected, and the monoclonal antibody was purified by ammonium sulfate fractionation. By this operation, anti-butamiphos antibody, B
TF2-77 was obtained (hereinafter, monoclonal antibodies use the same names as the hybridomas that produce them). Using the anti-butamiphos antibody BTF2-77, butamiphos was measured by the following test method.

Monoclonal antibody solution (BTF2-7
7 antibody (40 μg / ml) at 100 μl / well for 96
After addition to the well plate, the mixture was allowed to stand at 4 ° C. overnight, and then blocked with a block ace diluted 4 times the next day. Then, butamiphos and the appropriately diluted butamiphos derivative 1-HRP conjugate prepared in Example 8 were ligated. Containing 10% methanol-PB
The S solution was added at 50 μl / well and left at 37 ° C. for 1 hour. After the completion of the reaction, the cells were washed twice with 0.05% Tween 20-PBS, and then 0.4 mg / ml OPD and 0.0%
100 μl of a 0.05 M phosphate citrate buffer (pH 4.5) containing 4% hydrogen peroxide was added to each well, and allowed to stand at room temperature for 20 minutes to develop color. After the reaction, 2N sulfuric acid 10
After adding 0 μl to each well to stop the reaction,
The absorbance at nm was measured.

The results are shown in FIG.こ こ Here, the% maximum absorbance was calculated by the following equation.

[0127]

Embedded image Monoclonal antibody BTF2-77 is a direct competitive inhibition EL
Butamiphos can also be measured by the ISA method,
The measurement range is from 0.01 ng / ml of butamiphos to 10
00 ng / ml (FIG. 1).

Example 10 Evaluation of Monoclonal Antibody Using the same method as in Example 9, butamiphos, its metabolites, and analogs were obtained for monoclonal antibody BTF2-77 obtained using butamiphos derivative-1. The reactivity to organic phosphorus compounds was investigated.

The results are shown in FIG. Where the cross-reactivity is
% Butamifos concentration when the maximum absorbance is 50 (n
g / ml) by the concentration (ng / ml) of the target compound (amiprofos or the like) when the% maximum absorbance shows 50, and 1
Judgment is made by the numerical value (%) multiplied by 00.

The monoclonal antibody BTF2-77 had a cross-reactivity of 0.5% or less with both butamifos oxone, a metabolite of butamiphos, and the related organophosphorus compounds amiprofos and anilofos. (Fig. 2).

[Brief description of the drawings]

FIG. 1 shows the monoclonal antibody BTF2 of the present invention.
Fig. 3 shows the measurement of butamiphos by the direct competitive inhibition ELISA method of -77.

FIG. 2 shows cross-reactivity with butamifos and its metabolites and related compounds by a direct competitive inhibition ELISA using the monoclonal antibody BTF2-77.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 C12N 5/00 B 33/531 15/00 A 33/577 C // (C12P 21/08 (72) Inventor Shozo Kanai 1-27-114 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Yasuhiro Kagawa 1-27-114 Hamamatsucho, Minato-ku, Tokyo No. Inside the Institute for Environmental Immunity Technology (72) Inventor Kazuaki Watanabe 1-27-114 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Megumi Saito, Minato-ku, Tokyo 1-27-14, Inside the Institute for Environmental Immunity Technology

Claims (11)

[Claims] (1) The following formula (1): [In the formula (1), R ¹ may have 1 to 4 carbon atoms which may be branched if desired.
R ² is a hydrogen atom or a methyl group; and A is an optionally branched alkylene group having 1 to 10 carbon atoms]. 2. A conjugate of the compound according to claim 1 with a polymer compound or a labeling substance. 3. An antigen is prepared by binding the compound according to claim 1 with a polymer compound, and the antigen is used to obtain the following formula (2): A method for producing an antibody or a fragment thereof reactive to a compound of the formula (2), characterized by producing an antibody reactive to a compound having a structure represented by the formula: 4. An antibody or a fragment thereof, which is produced by using the conjugate according to claim 2 as an antigen, and which is reactive with the compound of the formula (2). 5. A compound according to claim 1, wherein R ¹ is an ethyl group, R ² is a methyl group, and A is a conjugate according to claim ^2. 5. The antibody or fragment according to claim 4, which is a pentamethylene group. 6. The antibody or fragment thereof according to claim 4, which is a monoclonal antibody. 7. The antibody or a fragment thereof according to claim 4, which is a monoclonal antibody BTF2-77. 8. A hybridoma producing the antibody according to any one of claims 4 to 7. 9. The hybridoma according to claim 8, which has been deposited under accession number FREM P-16907. 10. A method for immunologically measuring a compound represented by the formula (2), comprising using the antibody or fragment according to any one of claims 4 to 7. 11. The method according to claim 10, further comprising using the compound according to claim 1 or the conjugate according to claim 2.
The immunological measurement method according to the above.