JP2001139552A - Haptenic compound and antibody to acetamiprid and method for assaying - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a haptenic compound and an antibody to acetamiprid and to provide a method for assaying. SOLUTION: This haptenic compound has a structure in which a functional group for a spacer arm and binding is covalently bonded to the acetamiprid or a part thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to (E) -N-
Hapten compounds, antigens, antibodies and fragments thereof of [(6-chloro-3-pyridyl) methyl] -N'-cyano-N-methylacetamidine (hereinafter referred to as "acetamipride") and its analogous compounds About.

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

[0003]

2. Description of the Related Art Acetamiprid has the following formula (3):

[0004]

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A chloronicotinyl insecticide having a nitromethylene skeleton having a structure represented by the following formula: Chloronicotinyl insecticides are acetylcholine receptors whose site of action is the same as nicotine, which is a natural product. In other words, chloronicotinyl insecticides block nervous excitement by binding to nicotinic acetylcholine receptors in the postsynaptic membrane of nerves, resulting in paralysis and death of insects. As described above, chloronicotinyl compounds are referred to as “neonicotinoids” because they have the same action point as nicotine and are similar in chemical structure.
Also referred to collectively.

[0006] Acetamiprid is a kind of chloronicotinyl insecticide, and its insecticidal spectrum is broad, such as Hemiptera, Lepidoptera, Thripidae, and some Coleoptera pests.
It works more quickly through the skin. In addition, since it has a high osmotic transferability, it is effective against pests that infest the back of leaves even if only the surface of the leaves is treated, and it is also possible to control pests that damage the above-ground parts by soil treatment. The mechanism of action of acetamiprid is thought to act on the acetylcholine receptor in the postsynaptic membrane and block excitatory transmission (Agrochemical Handbook No. 126).
Page-Page 131 and Page 626 1998 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. For acetamiprid, the standard for pesticide residues by the Food Sanitation Act is 0.5 ppm for potatoes, roots of Japanese radish, and watermelon, 1 ppm for oranges, 5 ppm for leaves of Japanese radish, potatoes, cabbage, tomatoes, peppers, eggplants, and cucumbers. 5ppm for fruits such as natsumikan, lemon and grapefruit, and 50pp for tea
(Revised 3rd edition Agricultural Chemical Registration Reservation Standard Handbook pages 26-28, September 25, 1998)
Published by The Chemical Daily.) Therefore, in order to ensure the safety of the environment and food, it is necessary to measure the amount of acetamiprid contained therein rapidly and accurately.

Conventionally, for example, acetamiprid in agricultural products has been extracted from fruits, vegetables and the like, purified, and then analyzed by gas chromatography (GC). That is, for example, a method in which a sample is extracted with acetone, purified by magnesium silicate column chromatography using acetone-hexane as a solvent, and then measured by GC has been adopted (Revised 3rd edition Agricultural Chemical Registration Reservation Standard Handbook, Above). In these methods, the preparation of the sample is complicated, requires a large amount of procedures and time, requires skill in analysis, and
There is a problem that a large amount of cost is required for the measuring device and the equipment. In the measurement of acetamiprid, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and a new measuring method having not only accuracy but also simplicity, speed, and economy is required.

[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 molecules such as acetamiprid 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 (proteins, polysaccharides, etc.) and elicit an immune response in the presence of the T cell receptor, resulting in a group of Antibodies are produced by 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 necessity of acetamiprid, no hapten for producing such an antibody as well as a suitable antibody had been obtained before the present invention.

[0014]

DISCLOSURE OF THE INVENTION The present invention provides a novel antibody or a fragment thereof which reacts with acetamiprid,
And a method for manufacturing 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 to acetamiprid.
Another object of the present invention is to provide a hapten compound (acetamiprid hapten) which constitutes an antigen for producing a novel antibody reactive with acetamiprid.

Another object of the present invention is to provide a conjugate of acetamiprid 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 immunochemically measuring acetamiprid, which comprises using a conjugate of the above-mentioned antibody or fragment thereof and / or the above-mentioned acetamiprid hapten with a polymer compound or a labeling substance. Aim.

[0018]

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

Acetamiprid which is the object of the present invention is represented by the following formula (3):

[0020]

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A compound having a structure represented by the following formula: Further, the antibody of the present invention recognizes, in acetamiprid of the formula (3), an acetamiprid analog compound in which a chlorine atom is substituted by another halogen atom and an acetamiprid analog compound in which a methyl group is substituted by another alkyl group. Including things. That is, the antibody of the present invention has the following formula (2):

[0022]

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[In the formula (2), L ¹ is a halogen compound selected from the group consisting of Cl, Br and I; and R ¹ and R ² each have 1 carbon atoms which may be different from each other. Which is an alkyl group having a structure represented by the following formula:
As used herein, “acetamipride” means acetamiprid represented by the formula (3) and / or acetamiprid analog compound represented by the formula (2) depending on the context.

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

[0025]

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[In the formula (1), A is a sulfur atom, an oxygen atom, or an -NH group; R ¹ and R ² are each an alkyl group having 1 to 3 carbon atoms which may be different from each other. And n is an integer of 1 to 5], which is used as a hapten for preparing an antibody.

In the formula (1), preferably, both R ¹ and R ² are methyl groups. Preferably, A is a sulfur atom. Preferably, n is 2. The present invention provides the hapten compound, a conjugate of the hapten compound and a polymer compound,
The present invention relates to an antibody reacting with acetamiprid and a method for producing the same, and a method for immunochemical measurement of acetamiprid using the hapten compound or the antibody.

Preparation of Acetamiprid Hapten Acetamiprid 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, the following equation (X1):

[0030]

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In the formula (X1), L ² is a halogen atom selected from the group consisting of Cl, Br and I. Formula (X2):

[0032]

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In the formula (X2), A and n are as defined above, and a compound having a structure represented by the following formula (X3) is reacted:

[0034]

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A compound having a structure represented by the formula (X3), wherein A and n are as defined above, is obtained.
The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably 10 ° C.
To 100 ° C. for 5 minutes to 10 hours, preferably 30 minutes to 2 hours.

As a solvent for synthesizing the compound of the formula (X3), for example, methanol, ethanol, benzene,
Toluene, xylene, dichloromethane, chloroform,
Carbon tetrachloride, diethyl ether, tetrahydrofuran,
Dioxane, acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, dimethylformamide, dimethylsulfoxide, water and the like can be used. Examples of the base include sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate and the like.

Next, the compound of the formula (X3) is added to the compound of the formula (X3) in an organic solvent, preferably in the presence of a condensing agent.

[0038]

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In the formula (X4), P is a compound having a structure represented by the following formula (X5):

[0040]

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A compound having a structure represented by the formula (X5), wherein A, P and n are as defined above, is obtained. The protecting group for the carboxyl group represented by P may be a known group, and specific examples include, for example, a methyl group, an ethyl group,
tert-butyl group, benzyl group, p-methoxybenzyl group, 3,4-dimethoxybenzyl group, trichloroethyl group, trimethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triethylsilyl group, triisopropylsilyl And a trimethylsilylethyl group.

The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably at 20 ° C. to 100 ° C., for 5 minutes to 20 hours, preferably 30 minutes to 4 hours. As a solvent for synthesizing the compound of the formula (X5), for example, dichloromethane, tetrahydrofuran, benzene, toluene, diethyl ether and the like can be mentioned. As the condensing agent, for example, dicyclohexylcarbodiimide (DCC), trifluoroacetic anhydride, 1,1′-carbonyldiimidazole and the like can be used. The reaction can be promoted by adding a catalyst such as N, N-dimethylaminopyridine.

Next, the compound of formula (X5) is reduced to give the following formula (X6):

[0044]

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In the formula (X6), A, P and n are as defined above to obtain a compound having a structure represented by the following formula: The reduction reaction can be performed using a known method. For example, the reaction is performed using a reducing agent such as sodium borohydride or lithium aluminum hydride in a solvent such as methanol, ethanol, benzene, toluene, xylene, diethyl ether, tetrahydrofuran, dioxane, acetonitrile, ethyl acetate, acetic acid and water. The reaction is carried out at a temperature from −80 ° C. to the boiling point of the solvent, preferably 0 ° C. to 50 ° C., with stirring for 5 minutes to 10 hours, preferably 30 minutes to 5 hours.

Next, the compound of the formula (X6) is reacted with a halogenating agent such as thionyl chloride in an organic solvent such as chloroform or dichloromethane, or using a halogenating agent as a solvent, to give the following compound of the following formula: (X7):

[0047]

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In the formula (X7), L ³ is a halogen atom selected from the group consisting of Cl, Br and I; and A, P and n are as defined above. To obtain a compound having the structure:

The reaction is carried out at a temperature from 0 ° C. to the boiling point of the solvent, preferably at room temperature to 100 ° C., for 5 minutes to 10 hours, preferably 30 minutes to 3 hours. Further, the compound of the formula (X7) is added to a solvent such as acetonitrile, methanol, ethanol, diethyl ether, tetrahydrofuran, benzene, toluene, dichloromethane, water or the like to obtain a compound having 1 to 3 carbon atoms.
By reacting an alkylamine of the following formula (X8):

[0050]

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In the formula (X8), A, P, R ¹ and n
Is as defined above] to obtain a compound having a structure represented by the following formula: The reaction is carried out at a temperature between 0 ° C. and the boiling point of the solvent, preferably at 10 ° C. to 100 ° C., for 5 minutes to 20 hours, preferably 30 minutes to 3 hours.

Then, the compound of the formula (X8) is converted to a compound of the following formula (X9) in an inert solvent, especially an alcohol solvent such as methanol or ethanol.

[0053]

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In the formula (X9), R ² is the same as defined above by reacting with an N-cyanoimidate having a structure represented by the following formula (X1).
0):

[0055]

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A compound having a structure represented by the formula (X10), wherein A, P, R ¹ , R ² and n are as defined above, 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 100 ° C., for 5 minutes to 30 hours, preferably 30 minutes to 16 hours.

Further, by removing the protecting group of the carboxyl group represented by P from the compound of the formula (X10),
The compound of the formula (1) can be obtained. 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 compound represented by the formula (X1
The compound of 0) 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 10) is preferably dissolved in an organic solvent such as methanol, ethanol, tetrahydrofuran, ethylene glycol and the like, and then an aqueous solution of sodium hydrogencarbonate, sodium carbonate, potassium carbonate, lithium hydroxide, sodium hydroxide or potassium hydroxide is added. From 0 ° C. to the boiling point of the solvent, preferably from 0 ° C. to room temperature for 5 minutes to 1 hour.
The compound of formula (1) can be obtained by stirring and reacting for 0 hour, preferably for 1 to 2 hours.

Further, when P is a benzyl group, the removal can be carried out by hydrogenolysis with hydrogen. Furthermore, when P is a group containing a silyl atom, the deprotection is
The reaction can also be performed using a reagent that generates a fluorine anion such as n-butylammonium fluoride and pyridinium fluoride. 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.

Acetamiprid hapten and polymer compound
Preparation of a conjugate The acetamiprid 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 preferable 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 acetamiprid hapten and the polymer compound can be performed, for example, by the activated ester method (AEKAR).
U et al. : J. Agric. Food Che
m. 42 301-309 (1994)) or the mixed acid anhydride method (BF Erlanger et al .:
J. Biol. Chem. 234 1090-1094
(1954)) and the like.

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, 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 can be purified by dialysis, a desalting column, or the like to obtain a conjugate of acetamiprid hapten and a polymer compound.

On the other hand, the acid anhydride used in the mixed acid anhydride method is obtained by a usual Schotten-Baumann reaction, and the desired hapten-polymer compound conjugate is produced by reacting it with a polymer compound. Is done. 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,
Organic bases such as N-methylmorpholine, pyridine, N, N-dimethylaniline, 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 150 ° C, preferably at 0 ° C.
To 100 ° C., and the reaction time is 5 minutes to 10
Time, preferably from 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually carried out at minus 20.
C. to 100.degree. C., preferably 0.degree. C. to 50.degree. C., and 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.

Further, a substance obtained by binding a labeling substance such as an enzyme to acetamiprid hapten by the same method as described above 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 A polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of acetamiprid hapten and a polymer compound. For example, a conjugate of acetamiprid hapten and KLH is converted to a sodium phosphate buffer (hereinafter, referred to as a sodium phosphate buffer).
It can be obtained by immunizing an animal with an adjuvant as an immunizing antigen, which is dissolved in "PBS") and mixed with an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or alum. As the animal to be immunized, any of those commonly used in the art can be used, and examples thereof include mice, rats, rabbits, goats, and horses. However, humans are not included.

The method of administration for 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 serum separated therefrom can be used to evaluate the presence of a polyclonal antibody that reacts with acetamiprid. In the present invention, an antiserum obtained by using a conjugate of acetamiprid hapten and a polymer compound as an antigen for immunization is at least about 10 ng / ml in an indirect competitive ELISA method described below.
Can be reacted with acetamiprid (Example 4, FIG. 1).

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

In producing a monoclonal antibody,
At least the following work steps are required. (A) Preparation of a conjugate of acetamiprid hapten used as an antigen for immunization and a polymer compound (b) Immunization of an animal (c) Collection of blood, assay, and preparation of antibody-producing cells (d) Preparation of myeloma cells (E) Cell fusion between antibody-producing cells and myeloma cells and selective culture of hybridomas (f) Screening and cell cloning of hybridomas producing the desired antibody (g) Monoclones by culturing hybridomas or transplanting hybridomas into animals Preparation of Antibodies (h) Measurement of Reactivity of Prepared Monoclonal Antibodies Conventional methods for producing hybridomas that produce monoclonal antibodies include, for example, Hybridoma Technologies, Cold Spring Harbor Laboratories (Co.)
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 acetamiprid 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. (A)
Step (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, the myeloma cell line P3 / X63-Ag8 (X63) (Nat) 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-described 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 3 × 10 ³ or more is secured on the day of fusion.

The cell fusion in the step (e) can be performed 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.

[0080] Selection of a hybridoma group in which spleen cells and myeloma cells are fused to obtain antibody secretion ability and proliferation ability is performed, for example, when a hypoxanthine guanine phosphoribosyltransferase-deficient strain is used as a myeloma cell line, for example, as described above. It can be performed by using a HAT medium prepared by adding hypoxanthine / aminopterin / thymidine to DMEM or IMDM.

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

Further, cell cloning of a hybridoma that has been found by measurement to produce an antibody reactive with acetamiprid 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: "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 1-4 times, for example, by the limiting dilution method, and a stably obtained antibody titer is selected as an anti-acetamipride monoclonal antibody-producing hybridoma strain. As a medium for culturing a hybridoma, for example,
DMEM or IMDM containing fetal calf serum (FCS) 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-acetamipride monoclonal antibody. Further, dialysis, salting out with ammonium sulfate, gel filtration, freeze-drying, etc. are performed to collect and purify the antibody fraction. By doing so, an anti-acetamipride 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 subclass, antibody titer and the like of the anti-acetamipride monoclonal antibody obtained as described above can be determined by using a known method such as ELISA.

Assay of Acetamiprid with Antibody Assays of acetamiprid with antibodies used in the present invention include radioisotope immunoassay (RIA), EL
ISA method (Engval, E., Methods i)
n Enzymol. , 70, 419-439 (198).
0)), 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.

Acetamiprid was measured by various ELISAs.
The method can be performed by the following procedure, for example, by an indirect competitive ELISA method. (A) First, a conjugate of acetamiprid 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 acetamiprid are added thereto, and the antibody is allowed to react competitively with the immobilized antigen and acetamiprid to form an immobilized antigen-antibody complex and an acetamiprid-antibody complex Is generated.

(D) By measuring the amount of the immobilized antigen-antibody complex, the amount of acetamiprid 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.

For immobilizing 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 acetamiprid hapten that produced the antibody 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. is there. For example, in equation (1), A
Alternatively, a compound in which n is different from that for antibody production can also be used as the immobilized antigen. Furthermore, other acetamiprid analog compounds not included in the formula (1) can be used as the immobilized antigen.

In the blocking in the step (b), in the carrier on which the antigen (conjugate of acetamiprid hapten and a high molecular compound) is immobilized, there is a portion other than the acetamiprid hapten 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 ", manufactured by Dainippon Pharmaceutical Co., Ltd., code No. UK-25
A commercially available blocking agent such as B) can also be used. Specifically, for example, but not limited to, a buffer solution containing a blocking agent at the portion where the antigen is immobilized [eg, 1% BSA and 60 mM NaC
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 solution is not particularly limited.
Can be used.

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

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

The first antibody is dissolved in a buffer and added. Although not limited, the reaction 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 acetamiprid 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 acetamiprid 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 acetamiprid 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 acetamiprid has been added.

Alternatively, acetamiprid can be measured by, for example, a direct competitive 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 carrier surface on which the antibody is not immobilized is blocked with a substance unrelated to the antigen, for example, a protein. (C) Apart from the above steps, a mixture is prepared by adding an acetamiprid hapten and an enzyme-linked hapten to an enzyme to a sample containing various concentrations of acetamiprid.

(D) The mixture is reacted with the antibody-immobilized carrier. (E) The amount of acetamiprid in the sample is determined from the calibration curve prepared in advance by measuring the amount of the immobilized antibody-enzyme-bound hapten complex.

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.

In the blocking in the step (b), there may be a case where acetamiprid and an enzyme-bound hapten in a sample to be added later are adsorbed irrespective of the antigen-antibody reaction in 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 ELISA method described above can be used.

The preparation of the enzyme-bound hapten used in the step (c) is not particularly limited as long as it is a method of binding the acetamiprid 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 acetamiprid.

The hapten to be bound to a labeling substance such as an enzyme is not limited to the acetamiprid hapten itself used for the production of the antibody, but is also represented by 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 as an antigen for labeling. For example, a compound in which A or n in formula (1) is different from that for antibody production can be used as the labeling antigen. Furthermore, other acetamiprid analog compounds not included in the formula (1) can also be used as the labeling antigen.

In the step (d), the sample containing acetamiprid and the enzyme-bound hapten are brought into contact with the antibody-immobilized carrier, and the complex between the acetamiprid and the enzyme-bound hapten is formed by the competitive inhibition reaction between the acetamiprid and the enzyme-bound hapten. I do.
The sample containing acetamiprid 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 acetamiprid can be calculated from the calibration curve.

The monoclonal antibody 45D6-5 of the present invention
-1 and 45E10-1-1 are direct competitive ELISAs
About 0.2 ng / ml to about 50 ng / ml in the method,
It reacts with acetamiprid preferably in a concentration range of about 0.2 ng / ml to about 10 ng / ml (Example 6, FIG.
And FIG. 3).

Further, as described above, direct competition 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 Present Invention The direct competitive ELISA method or indirect competitive ELISA described above
By the method, the cross-reactivity of the monoclonal antibody of the present invention can be examined.

For example, monoclonal antibody 45D6-5
-1 and 45E10-1-1 show little reactivity with acetamiprid analogs (imidaproclide, nitenpyram) and have very high specificity for acetamiprid (Example 7, Table 2). Hereinafter, the present invention will be described specifically with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description in the present specification, and they are included in the technical scope of the present invention.

[0114]

EXAMPLES Example 1 Synthesis of Acetamiprid Hapten

[0115]

Embedded image

3- (5-formyl-2-pyridylthio)
Synthesis of propionic acid (1) In 80 ml of ethanol, 5.7 g (40 mmol) of 2-chloro-5-formylpyridine and 4.7 of thioglycolic acid were used.
g (44 mmol) and potassium carbonate 12.7 g (92
mmol) and the mixture was stirred at reflux for 2 hours. The reaction mixture was concentrated, 40 ml of water was added to the residue,
After adjusting the pH to 4 with 5N hydrochloric acid, the mixture was extracted twice with 70 ml of ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate, and concentrated. The residue was recrystallized from ligroin to obtain 5.5 g (yield 65%) of (1) as prism crystals. The melting point of this compound (1) is 85-
86 ° C.

3- (5-formyl-2-pyridylthio)
Synthesis of 2-trimethylsilylethyl propionate (2) 25 ml of dichloromethane and 5 ml of tetrahydrofuran
4.7 g (22.7 mmol) of 3- (5-formyl-2-pyridylthio) propionic acid (1), 5.3 g (45 mmol) of 2-trimethylsilylethanol and 4,
-Dimethylaminopyridine 0.2 g (1.6 mmol)
5.1 g of dicyclohexylcarbodiimide in a solution of
(25 mmol) was added at 10-15 ° C, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1) to obtain 6.4 g (yield 93%) of (2).

3- (5-hydroxymethyl-2-pyridi
2-trimethylsilylethyl luthio) propionate
Synthesis of (3) 3.1 g (10 mm) of 2-trimethylsilylethyl 3- (5-formyl-2-pyridylthio) propionate (2)
ol) in a solution of 30 ml of 1,4-dioxane and 0.4 ml of sodium borohydride dissolved in 4 ml of water.
g (10 mmol) was added at 10-15 ° C and stirred at room temperature for 1 hour. The reaction mixture was concentrated and the residue was treated with water 10
0 ml was added, and the mixture was extracted twice with 70 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 (n-hexane: ethyl acetate = 1: 1) to obtain 2.4.
g (yield 77%) of 3- (5-chloromethyl-2-pyridylthio) propio to give (3)
Synthesis of 2-trimethylsilylethyl 3- (5-hydroxymethyl-2-pyridylthio) propionate 2.2 g of 2- (3-hydroxymethyl-2-pyridylthio) propionate
(7.0 mmol) was dissolved in 7 ml of chloroform, and 1.3 g (11 mmol) of thionyl chloride was added to 1 ml of this solution.
The mixture was added at 0 to 15 ° C and stirred at room temperature for 1 hour. The reaction mixture was concentrated, and the residue was added with a 5% aqueous sodium hydrogen carbonate solution (25 m).
l and extracted twice with 70 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 (n-hexane: ethyl acetate = 5: 1) to obtain 1.8 g (yield 78%) of (4).

3- (5-methylaminomethyl-2-pyri)
2-trimethylsilylethyl propionate
Synthesis of (5) 10 g of acetonitrile was added to 2.0 g (25.8 mmol) of a 40% aqueous solution of methylamine in 20 ml of acetonitrile.
1.7 g of 2-trimethylsilylethyl 3- (5-chloromethyl-2-pyridylthio) propionate (4) in 1
(5.1 mmol) was added at 10-15 ° C. and stirred at room temperature for 2 hours. The reaction mixture was concentrated and the residue was 30 m
l of water was added and extracted with 100 ml of ethyl acetate. After washing the ethyl acetate layer with water, drying over anhydrous magnesium sulfate,
Concentrated. The residue was purified by silica gel chromatography (ethyl acetate: methanol = 1: 1, then methanol) to obtain 1.0 g (yield 60%) of (5).

3- [5- (N- (1-cyanoiminoethyl)
Ru) -N-methyl) aminomethyl-2-pyridylthio]
Synthesis of 2-trimethylsilylethyl propionate (6 ) 2.4 g (7.4 mmol) of 3- (5-methylaminomethyl-2-pyridylthio) propionate 2-trimethylsilylethyl (5) in 15 ml of ethanol and N-cyanoacetate A mixture of 1.0 g (7.8 mmol) of ethyl imidate was stirred at room temperature for 16 hours. The reaction mixture was concentrated, and the residue was purified by silica gel chromatography (ethyl acetate) to obtain 1.6 g (yield: 52%) of (6).

3- [5- (N- (1-cyanoiminoethyl)
Ru) -N-methyl) aminomethyl-2-pyridylthio]
Synthesis of propionic acid (7) 3- [5-
A solution of 1.4 g (3.4 mmol) of 2-trimethylsilylethyl (6) (N- (1-cyanoiminoethyl) -N-methyl) aminomethyl-2-pyridylthio] propionate in a solution of 1.0 g in n-hexane. 8.2 ml of a molar tetrabutylammonium fluoride solution (8.2 mmol
l) was added, and the mixture was stirred at room temperature for 5 minutes and concentrated. 40 ml of water was added to the residue, 1N hydrochloric acid was further added until the pH became 4, and then extracted twice with 50 ml of ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate, and concentrated. The residue solid was mixed with ethyl acetate: n-hexane = 1: 1.
Recrystallization from the solvent 1 gave 0.22 g (22% yield) of (7) as a white powder. The melting point of this compound (7) was 121-123 ° C.

[0122] ¹ H of the above acetate Tamil pre-de-hapten (7)
The physical property data (chemical shift δ) by NMR are shown below.

[0123]

Table 1 ¹ H-NMR (DMSO-D ₆ , 400 MHz) δ 2.43 (3H, d, CH ₃ ), 2.64 (2H, s, CH ₂ ), 3.03 (3H, d _{, CH 3), 3.29 (2H} , s, CH 2), 4.67 (2H, d, CH 2), 7.30 (1H, m, Pyr: H), 7.55 (1H, m, Pyr: H), 8.38 (1H, s, Pyr: H), 12.33 (1H, S, COOH) Example 2 Preparation of antigen for immunization and antigen for screening Acetamiprid as an antigen for immunization and an antigen for screening A conjugate of hapten and BSA) was made using the activated ester method.

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

On the other hand, 50 mg of BSA was added to 145 mM N
Dissolved in 5.0 ml of aCl-0.01M phosphate buffer (pH 7.2: hereinafter referred to as “PBS”), and added DMF 1.0.
A solution to which 05 ml was added was prepared, and 0.25 ml of the above-mentioned supernatant was added to this solution, followed by reaction at 4 ° C. for 16 hours. After the reaction, the mixture was dialyzed against distilled water at 4 ° C. to prepare a conjugate of acetamiprid and BSA (hereinafter, referred to as “acetamiprid hapten / BSA conjugate”). Hereinafter, it was used as an antigen for immunization.

Further, using the same method, a conjugate of acetamiprid hapten and RSA (hereinafter referred to as an “acetamiprid hapten / RSA conjugate”) and a conjugate of acetamiprid hapten and HRP (hereinafter “HRP
Conjugated acetamiprid hapten).

Example 3 Immunization Balb / c mice were used for immunization. 50 μg of the acetamiprid hapten / BSA conjugate prepared in Example 2 was dissolved in 100 μl of PBS as an antigen for immunization, mixed with an equal amount of complete Freund's adjuvant, and mixed with Balb / c.
The mice were inoculated subcutaneously. Further, four weeks later, the immunizing antigen prepared in the same manner as described above was boosted with Freund's incomplete adjuvant. In addition, 180 μl in the 6th week
The mice were boosted with 10 μg of the immunizing antigen dissolved in PBS from the tail vein of the mouse.

Example 4 Antiserum against acetamiprid
That immediately before inoculation into mice tail vein in reactive Example 3, the antiserum bled diluted prepared, indirect competitive detailed below ELI
Acetamiprid was measured by the SA method, and the antiserum was evaluated.

The acetamiprid hapten / RSA conjugate solution prepared in Example 2 in the same manner as the immunizing antigen (0.5 μg
/ Ml) was coated on a 96-well microplate in a volume 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. Next, a 5000-fold dilution of the antiserum and an equal amount of a 20% methanol solution containing various concentrations of acetamiprid were mixed, 50 μl of the mixture was added to each well, and reacted at room temperature for 1 hour.

After washing 5 times with PBS, a 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 reacted at room temperature for 1 hour. After washing 5 times with PBS, 2 mg / ml OP
A 0.1 M citrate-phosphate buffer (pH 5.0) containing D 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, 1 N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
One example of the result is shown in FIG. From FIG. 1, an inhibitory reaction was observed at about 10 ng / ml or more of acetamiprid, confirming that the antiserum used was reactive with acetamiprid.

Example 5 Preparation of Hybridomas Following Example 3, spleen cells from mice with high anti-acetamipride antibody activity in serum and myeloma cells (Sp
2 / 0-Ag14) was fused and cultured by the polyethylene glycol method according to the method of Shuji Yamashita et al. 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 using Block Ace diluted 10-fold was added in an amount of 50 μl / well, and the mixture was added at room temperature. Allowed to react for hours. 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 the color was developed at room temperature for 10 minutes.

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
Reactive cells (hybridomas) were selected. Next, the reactivity of each well with acetamiprid 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 (45D6
-5-1, 45E10-1-1) were cloned as cells producing anti-acetamipride antibodies. Among them, 45D6-5-1 was deposited on October 27, 1999 under the deposit number FERM P-17622, under the name of 1-3-1 Higashi, Tsukuba City, Ibaraki Pref. Deposited.

Example 6 A direct competition ELISA method
Measurement of Cetamiprid The hybridoma cells obtained in Example 5 (45D6-5
-1 and 45E10-1-1) were transplanted into the abdominal cavity of a mouse, and ascites obtained after 10 to 15 days were collected, and the monoclonal antibodies 45D6-5-1 and 45E10-1-1 were purified by affinity chromatography. did. (Hereinafter, monoclonal antibodies use the same names as the hybridomas that produce them.)
Using the -5-1 antibody or the 45E10-1-1 antibody, the amount of acetamiprid was measured by a direct competitive ELISA method.

The above 45D6-5-1 antibody solution (7 μg
/ Ml) or 45E10-1-1 antibody solution (5 μg / m
l) was placed in a 96-well microplate in an amount of 50 μl / well, and was allowed to stand still at 4 ° C. overnight for coating. Was prepared. An equal volume mixture of a 10% methanol solution containing acetamiprid at each concentration and a PBS solution containing the HRP-bound acetamiprid hapten prepared in Example 2 was added to each well in an amount of 50 μl, and reacted at 25 ° C. for 1.5 hours.

After the reaction, the plate was washed 5 times with PBS, and then 2 m
50 μl of a citrate-phosphate buffer (pH 5.0) containing g / ml OPD and 0.02% hydrogen peroxide was added to each well, and 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 results are shown in FIG. 2 and FIG. Using this direct competition ELISA method, the monoclonal antibodies 45D6-5-1 and 45E10-1-1 of the present invention could measure acetamiprid in the range of 0.2 ng / ml to 50 ng / ml.

Example 7 Cross-Reaction of Monoclonal Antibody
Using the indirect competitive ELISA method described in Example 4
The reactivity of the monoclonal antibodies 45D6-5-1 and 45E10-1-1 against various acetamiprid analogs was examined. Table 2 shows the results. From Table 2,
Monoclonal antibodies 45D6-5-1 and 45E10-
1-1 showed reactivity with acetamiprid,
It showed little reactivity with analogs.

[0140]

[Table 2]

[Brief description of the drawings]

FIG. 1 shows indirect competitive ELI using mouse antiserum.
4 shows the measurement of acetamiprid by SA.

FIG. 2 shows the monoclonal antibody 45D6 of the present invention.
The reactivity with acetamiprid by the direct competition ELISA method using -5-1 is shown.

FIG. 3 shows the monoclonal antibody 45E1 of the present invention.
The reactivity with acetamiprid by the direct competition ELISA method using 0-1-1 is shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/08 15/02 G01N 33/53 G C12P 21/08 33/577 B G01N 33 / 53 (C12P 21/08 33/577 C12R 1:91) // (C12P 21/08 C12N 5/00 B C12R 1:91) 15/00 C (72) Inventor Uchida Uchiaki Hamamatsucho, Minato-ku, Tokyo 1-27-14 Inside the Institute for Environmental Immunity Technology (72) Inventor Tetsuo Yamazaki 1-27-14, Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Shigeyuki Watanabe Tokyo 1-27-14 Hamamatsucho, Minato-ku F-term in the Institute for Environmental Immunity Technology (Reference) 4B024 AA11 BA43 DA02 GA05 HA15 4B064 AG27 CA10 CA19 CC24 DA13 4B065 AA92X AB05 AC14 CA25 CA46 4C055 AA01 BA02 BA33 CA02 CA28 CB02 DA01 4H045 AA11 AA20 AA30 BA51 BA72 DA76 DA86 EA50 FA72 GA26

Claims (13)

[Claims] (1) The following formula (1): [In the formula (1), A, is sulfur atom, oxygen atom, or -NH group; R ¹
And R ² are each an alkyl group having 1 to 3 carbon atoms which may be different from each other; and n is an integer of 1 to 5]. 2. The compound according to claim 1, wherein in the formula (1), R ¹ and R ² are both methyl groups. 3. In the formula (1), A is a sulfur atom.
The compound according to claim 1. 4. In the formula (1), n is 2.
4. The compound according to any one of claims 1 to 3. 5. A conjugate of the compound according to claim 1 with a polymer compound or a labeling substance. 6. An antigen is prepared by binding the compound according to any one of claims 1 to 4 to a polymer compound, and the antigen is used to obtain the following formula (2): ] [In the formula (2), L ¹ is a halogen compound selected from the group consisting of Cl, Br, and I; and R ¹ and R
² is an alkyl group having 1 to 3 carbon atoms, which may be different from each other.] An antibody which is reactive to a compound having a structure represented by the formula: A method for producing an antibody or a fragment thereof which is reactive with a compound having a structure to be obtained. 7. An antibody or a fragment thereof, which is produced by using the conjugate according to claim 5 as an antigen, and which is reactive with the compound of the formula (2). 8. The antibody or fragment according to claim 7, which is a monoclonal antibody. 9. The antibody or fragment according to claim 7, which is a monoclonal antibody 45D6-5-1 produced by the hybridoma deposited under accession number FERM P-17622. A hybridoma that produces the antibody or fragment according to any one of claims 7 to 9. 11. The hybridoma according to claim 10, which has been deposited under accession number FERM P-17622. 12. A method for immunochemically measuring a compound represented by the formula (2), comprising using the antibody or fragment according to any one of claims 7 to 9. 13. The method according to claim 1, further comprising:
The immunochemical measurement method according to claim 12, which comprises using the compound according to claim or the conjugate according to claim 5.