JP2000191624A - Hapten compound of carbaryl, antibody and determination thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound producible an antibody reacting with carbaryl or its fragment and useful for immunological determination, etc., by introducing a functional group having a specific structure and usable for a spacer arm of a carbaryl part and bonding to a high polymer. SOLUTION: This compound has a structure of formula I. The compound is obtained by, e.g. reacting 1-naphthol having a structure of formula II with a reagent generating phosgene, etc., in a solvent such as toluene, in the presence of a base such as sodium hydroxide at a temp. from -20 deg.C to the boiling point of the solvent for about 30 m-24 h, then reacting the resultant compound with 4-aminomethylcyclohexane carboxylic acid having a structure of formula III in a solvent in the presence of a base at a temp. from -20 deg.C to a boiling point of the solvent for about 30 m-40 h.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to 1-naphthyl-N
The present invention relates to a hapten compound, an antigen, an antibody and a fragment thereof of methyl carbamate (hereinafter referred to as “carbaryl” in the present specification).

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

[0003]

BACKGROUND OF THE INVENTION Carbaryl is represented by the following formula (3):

[0004]

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A carbamate insecticide having a structure represented by the following formula: Carbamate insecticides are a group of compounds having an insecticidal action, which do not contain Cl or P as a component of the compound and are composed of C, H, O and N. The main carbamate insecticides are roughly classified into three structural groups. First, naphthyl or substituted phenyl-N-methyl type, second, heterocyclic N, N-disubstituted type, and third, oxime type having an oxime (-C = NO-) bond in the molecule. Yes, carbaryl belongs to the first category.

[0006] Carbaryl is mainly used for controlling planthoppers and leafhoppers, and is also used as a preventive agent for pine weevil. It also acts as a plant growth regulator and is used as a fruit picker for apples. As an action characteristic, in addition to acting as a contact agent, it has a penetrative transfer property into a plant body, and when applied to the water surface, is absorbed from rice roots and stems, and migrates through the rice body to exhibit an insecticidal effect. At low temperatures, the insecticidal power decreases. It has a special effect on leafhoppers and aphids, and it is slightly slower than a marathon on leafhoppers, but has a longer residual effect. In addition, there are also spotted leafhoppers and brown planthoppers resistant to this drug. (Agrochemical Handbook pages 62-65 and 522, 1994 edition, Japan Plant Protection Association; "Residual Analysis Method for the Latest Pesticides", pp. 104-107, edited by the Research Group for Pesticide Residue Analysis Method, Chuo Law Publishing) .

[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 carbaryl, the residual standard value based on the Food Sanitation Law is, for example, rice, peach, summer tangerine, tangerine, Japanese pear, apple, oyster, grape, radish (leaves, roots), spinach, tea (more than 1 ppm), potato ( 0.1p
pm), etc. (“Recent analysis method for the latest pesticides”
Id.). Therefore, it is necessary to quickly and accurately measure the amount of carbaryl contained in crops, especially rice, in order to ensure safety with respect to the environment and foods.

Conventionally, for example, carbaryl in agricultural crops has been extracted from cereals, beans, seeds, seeds, tea, vegetables, fruits and the like, purified, and then analyzed by high performance liquid chromatography (HPLC). That is, for example, a method of extracting a sample with acetone, dissolving the sample in dichloromethane, distributing in hexane-acetonitrile, purifying by Florisil column chromatography, and measuring by HPLC is adopted. These methods have problems in that the preparation of the sample is complicated, requires a large amount of procedures and time, requires a high level of skill in the analysis, and requires high costs for measuring devices and equipment. The measurement of carbaryl requires the analysis of a large number of samples in a short period of time.
There is a need for new measurement methods that are also fast and economical.

An immunoassay is a method for detecting an antigen or an antibody based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen. Due to its excellent accuracy, simplicity, speed, and economy, In recent years, it has been attracting attention. In an immunoassay, a very wide variety of labels can be used as a detection method, for example,
Enzymes, radiotracers, chemiluminescent or fluorescent substances,
Metal atoms, sols, latexes and bacteriophages have been applied.

[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 molecules such as carbaryl 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] Despite the extremely high need for carbaryl, no hapten for producing such antibodies, as well as suitable antibodies, had been obtained prior to the present invention.

[0014]

An object of the present invention is to provide a novel antibody or a fragment thereof which reacts with carbaryl, 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 carbaryl.

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

Another object of the present invention is to provide a conjugate of a carbaryl hapten and a polymer compound.

Another object of the present invention is to provide a hybridoma that produces the antibody or a fragment thereof.

Another object of the present invention is to provide a method for immunologically measuring carbaryl, which comprises using a conjugate of the antibody or a fragment thereof and / or the carbaryl hapten and a polymer compound. .

[0020]

Means for Solving the Problems The present inventors have conducted intensive studies and as a result, have found that carbaryl or a carbaryl derivative in which a functional group usable for bonding to a polymer compound has been introduced into carbaryl or a portion thereof. As a result, an antibody having reactivity with the compound was successfully obtained, and the present invention was completed.

The carbaryl to be used in the present invention has the following formula (3):

[0022]

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

[0024]

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The compound having the structure represented by the following formula is used as a hapten for preparing an antibody. Also, the following equation (2):

[0026]

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In the formula (2), n is an integer of 1 to 10. A compound having a structure represented by the following formula can also be used as a hapten for preparing an antibody against carbaryl.

The present invention relates to the hapten compound, a conjugate of the hapten compound and a polymer compound, an antibody reacting with carbaryl, a method for producing the same, and a method for immunological measurement of carbaryl using the hapten compound or the antibody. Preparation of carbaryl hapten I. Synthesis of carbaryl hapten of formula (1) The carbaryl hapten represented by 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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1-naphthol having a structure represented by the following formula is reacted with a reagent for generating phosgene such as phosgene, trichloromethyl chloroformate (TCF) or triphosgene in a solvent in the presence of a base, and the following formula ( X2):

[0032]

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[In the formula (X2), L ¹ represents F, Cl, Br
Or a halogen atom selected from I] is obtained.

The reaction is carried out at a temperature of −20 ° C. to the boiling point of the solvent,
The reaction is preferably performed at 0 ° C. to 40 ° C. for 30 minutes to 24 hours, preferably 1 hour to 3 hours.

As a solvent, toluene, benzene, dioxane, tetrahydrofuran or the like can be used, and as a base, sodium hydroxide, sodium carbonate, potassium carbonate, potassium hydroxide, sodium methylate, sodium ethylate or the like can be used. A preferred base is sodium hydroxide.

Next, a compound of the formula (X2) is added to a compound of the following formula (X3) in a solvent in the presence of a base:

[0037]

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By reacting 4-aminomethylcyclohexanecarboxylic acid having a structure represented by the following formula, a compound represented by the formula (1) is obtained.

The reaction is carried out from -20 ° C to the boiling point of the solvent,
The reaction is preferably performed at 0 ° C. to 40 ° C. for 30 minutes to 40 hours, preferably for 10 hours to 20 hours.

As the solvent and the base, those similar to those used for the synthesis of the compound of the formula (X2) can be used.

II. Combination of carbaryl hapten of formula (2)
The carbaryl hapten represented by the formula (2) can also be produced according to a known method. Although not limited, for example, the following method can be used.

First, the following equation (X4):

[0043]

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A 1,5-dihydroxynaphthalene having a structure represented by the following formula (X5):

[0045]

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[In the formula (X5), L ² represents F, Cl, Br
Or a halogen atom selected from I; P is a protecting group for a carboxyl group; and n is as defined above], and a carboxyl group protected with a protecting group having a structure represented by the formula: By reacting a compound having the following formula (X6):

[0047]

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A compound having a structure represented by the formula (X6), wherein P and n are as defined above, is obtained.

The reaction is carried out, for example, in an appropriate organic solvent such as acetonitrile, dichloromethane, 1,2-dichloroethane, ethyl acetate, benzene, toluene, THF, diethyl ether, etc., in an inorganic base such as potassium carbonate, sodium carbonate, or triethylamine. -20 ° C to the boiling point of the solvent, preferably 0 ° C to 40 ° C, for 30 minutes to 30 ° C in the presence of an organic base such as diisopropylethylamine.
Time, preferably 3 to 10 hours.

Next, the compound of the formula (X6) is reacted with methyl isocyanate in an organic solvent to give a compound of the following formula (X7):

[0051]

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In the formula (X7), P and n are as defined above, to obtain a compound having a structure represented by the following formula:

The reaction is carried out from -20 ° C to the boiling point of the solvent,
The reaction is preferably performed at 0 ° C. to room temperature for 30 minutes to 24 hours, preferably 2 hours to 10 hours.

As the organic solvent, dichloromethane, benzene, dioxane, tetrahydrofuran and the like can be used.

Further, the compound of the formula (2) can be obtained by removing the protecting group of the carboxyl group represented by P from the compound of the formula (X7). 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 7) 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 (2) 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 7) 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 the formula (2) can be obtained by stirring and reacting for 1 hour to 2 hours.

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

Further, when P is a group containing a silyl atom, deprotection can also be carried out using a reagent that generates a fluorine anion such as tetra-n-butylammonium fluoride or pyridinium fluoride.

By subjecting the compound obtained by the above-mentioned production method to silica gel chromatography or recrystallization, if necessary, 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. Preparation of Conjugate of Carbaryl Hapten and Polymer Compound The carbaryl hapten synthesized as described above is bound to an appropriate polymer compound and then used as an antigen for immunization.

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

The bonding between the carbaryl hapten and the polymer compound can be performed, for example, by the activated ester method (AEKARU).
et al. : J. Agric. Food Chem.
42301-309 (1994)) or the mixed acid anhydride method (BF Erlanger et al .: JB).
iol. Chem. 234 1090-1094 (19
54)) 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 4 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 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.
The reaction time is 5 minutes to 24 hours, preferably 1 hour to 16 hours. The reaction product can be purified by dialysis, a desalting column or the like to obtain a conjugate of the carbaryl hapten and the polymer compound.

On the other hand, the mixed acid anhydride used in the mixed acid anhydride method is obtained by a reaction between a carboxylic acid and a halogen formate, and the resulting hapten-polymer compound is reacted with the polymer compound. A conjugate is produced. This reaction is performed in the presence of a basic compound. Examples of the basic compound include tributylamine, triethylamine, trimethylamine, N-methylformalin, pyridine, N, N-dimethylaniline, D
Organic bases such as BN, DBU, and DABCO; and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The reaction is usually carried out at a temperature between -20 ° C and 100 ° C, preferably between 0 ° C and 50 ° C
And the reaction time is 5 minutes to 10 hours, preferably 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually performed at a temperature of −20 ° C. to 150 ° C.
C., preferably at 0 ° C. to 100 ° 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 compounds 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.

In the same manner as described above, a product obtained by binding a labeling substance such as an enzyme to a carbaryl hapten can be used in an immunoassay. 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 carbaryl hapten and a polymer compound. For example, a carbaryl hapten-BSA conjugate is added to 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.

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.

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 carbaryl.

In the present invention, the antiserum obtained by using a conjugate of a carbaryl hapten and a polymer compound as an antigen for immunization can react with carbaryl at a concentration of at least about 100 ng / ml in an indirect competitive inhibition ELISA method described below ( Example 5, Table 1). Preparation of Monoclonal Antibody The monoclonal antibody of the present invention can be prepared by a known method using a conjugate of a carbaryl hapten and a polymer compound.

In producing a monoclonal antibody,
At least the following work steps are required. (A) Preparation of a conjugate of a carbaryl 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 (Syuji Yamashita et al., edited by the Japanese Society of Tissue Chemistry; interdisciplinary project, 1986).

Hereinafter, a method for preparing a monoclonal antibody against carbaryl 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, 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-Ag4-1 (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, X63.65.
3, myeloma cell line such as S194, or rat-derived cell line 210. RCY3. Ag 1.2.3. (Y3) (Na
cure, 277, 131-133, (1979)) and the like.

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

The cell fusion in the step (e) is carried out by a known method, for example, the method of Milstein et al. (Met
hods in Enzymology, 73, 3 (1
981)). Currently, the most commonly used method is polyethylene glycol (PE).
G). The PEG method is described in, for example, Cell Histochemistry, Shuji Yamashita et al. (Supra). As another fusion method, a method by electric treatment (electric fusion) can be adopted (Etsuko Okochi et al., Experimental Medicine 5.1315-19, 1987). Other methods can be appropriately adopted. In addition, the ratio of cells used may be the same as in a known method. For example, spleen cells may be used about 3 to 10 times as much as myeloma cells.

The selection of a hybridoma group in which spleen cells and myeloma cells are fused to obtain antibody secretion ability and proliferation ability is performed, for example, when a hypoxanthine guanine phosphoribosyltransferase-deficient strain is used as a myeloma cell line, 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 carbaryl is measured by the LISA method.

Further, cell cloning of a hybridoma that has been found by measurement to produce an antibody reactive with carbaryl 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-carbaryl 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-carbaryl monoclonal antibody. Further, dialysis, salting out with ammonium sulfate, gel filtration, lyophilization, etc. are performed to collect and purify the antibody fraction. By doing so, an anti-carbaryl 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-carbaryl 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 later. Measurement of carbaryl using an antibody As a method for measuring carbaryl using an antibody used in the present invention, radioisotope immunoassay (RIA), ELISA
Method A (Engval, E., Methods in E)
nzymol. , 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.

The measurement of carbaryl can be performed by the following procedure using, for example, an indirect competitive inhibition ELISA method among various ELISA methods.

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

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

(C) A sample and an antibody containing various concentrations of carbaryl are added thereto, and the antibody is allowed to react competitively with the immobilized antigen and carbaryl to form an immobilized antigen-antibody complex and carbaryl- Generate antibody conjugates.

(D) By measuring the amount of the immobilized antigen-antibody complex, the amount of carbaryl 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 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 can be selected from a wide range.
From μg / ml to about 100 μg / ml, preferably 0.1 μg / ml.
A range of from 05 μg / ml to 10 μg / ml is suitable. When a 96-well microtiter plate is used as a carrier, 50 μl at 300 μl / well or less is used.
1 / well to about 150 μl / well is desirable. Furthermore, there is no particular limitation on the incubation conditions,
Usually, overnight incubation at about 4 ° C. is suitable.

The antigen to be immobilized on a carrier includes:
Not only the conjugate of the carbaryl hapten with which the antibody was prepared and the polymer compound itself, but also other carbaryl analogs not included in the formula (1) can be used as the immobilized antigen.

In the blocking in the step (b), the carrier on which the antigen (the conjugate of the carbaryl hapten and the high molecular compound) is immobilized has a portion other than the carbaryl hapten portion to which the antibody to be added later can be adsorbed. May be
It is performed solely to prevent it. As a blocking agent, for example, BSA or skim milk solution can be used. Or, Block Ace ("Block-Ac
e ", manufactured by Dainippon Pharmaceutical Co., Ltd., code No. A commercially available blocking agent such as UK-25B) can also be used. Specifically, it is not limited,
For example, an appropriate amount of Block Ace diluted 4-fold with PBS is added to a portion where the antigen is immobilized, and the mixture is incubated at about 4 ° C. overnight, and then washed with a washing solution.
The washing solution is not particularly limited, and for example, PBS can be used.

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

At this time, as the antibody, an antibody against the carbaryl 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 dissolved in a buffer and added. Although not limited, the reaction 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 washing liquid, for example, PBS 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 anti-mouse-goat antibody conjugated with an enzyme (for example, peroxidase or alkaline phosphatase). 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 0.3 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. Also,
A labeled first antibody may be used, in which case a 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 carbaryl 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, for example, color is developed using p-nitrophenyl phosphate as a substrate, the enzyme reaction is stopped by adding 2N NaOH, and the absorbance at 415 nm is measured. The method is suitable.

With respect to the absorbance of the reaction solution to which carbaryl 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 carbaryl in a sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of a reaction solution to which a known concentration of carbaryl has been added.

Alternatively, the measurement of carbaryl can also be performed by, for example, 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) Blocking the surface of the carrier on which the antibody is not immobilized, with a protein unrelated to the antigen, for example, a protein.

(C) Separately from the above steps, a mixture is prepared by adding an enzyme-bound hapten obtained by binding a carbaryl hapten and an enzyme to a sample containing various concentrations of carbaryl.

(D) The mixture is reacted with the antibody-immobilized carrier. (E) By measuring the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of carbaryl 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), the carrier on which the antibody is immobilized may have a portion where carbaryl and enzyme-bound hapten in the sample to be added later are adsorbed independently of the antigen-antibody reaction. 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 preparation of the enzyme-bound hapten used in the step (c) is not particularly limited as long as it is a method of binding a carbaryl hapten to an enzyme, and may be carried out 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 carbaryl.

The hapten to be bound to a labeling substance such as an enzyme is not limited to the carbaryl hapten itself used for preparing the antibody, but also to the formula (1), as in the case of the immobilized antigen in the indirect competitive inhibition ELISA method. Other carbaryl analogs not included can also be used as haptens for coupling to enzymes.

In the step (d), the sample containing carbaryl and the enzyme-bound hapten are brought into contact with an antibody-immobilized carrier, and a competition inhibition reaction between carbaryl and the enzyme-bound hapten is carried out.
A complex of these and the solid-phased carrier is formed. The sample containing carbaryl is diluted with an appropriate buffer and used. 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), a chromogenic substrate solution which reacts with the enzyme of the enzyme-bound hapten is added in the same manner as in the indirect competitive inhibition ELISA method, and the amount of carbaryl is calculated from the calibration curve by measuring the absorbance. Can be.

The monoclonal antibody 29B1-1 of the present invention
-1 is about 0.01 in the direct competitive inhibition ELISA method.
ng / ml to 1000 ng / ml, preferably 0.1 ng / ml.
Carbaryl can be measured in the range of 1 ng / ml to 100 ng / ml (Example 7, FIG. 1). 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.

[0113]

EXAMPLES Example 1 Synthesis of carbaryl hapten-1

[0114]

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Synthesis of (2) 1-naphthol (1) (14.4 g, 100 mmol) was dissolved in a 2.5N aqueous sodium hydroxide solution to give 85%.
Heated at 0 ° C. for 1 hour. After returning to room temperature, 10.5 g (52.8 mmol) of trichloromethyl chloroformate (TCF)
50 ml of a toluene solution of l) was added dropwise over 30 minutes, and 1
Stirred for hours. The toluene layer was collected, concentrated and filtered through a short silica gel column. The filtrate was concentrated and then purified through a silica gel column (n-hexane: ethyl acetate = 19: 1).
6.0 g (29% yield) of (2) was obtained. Synthesis of (3) 1.88 g of 4-aminomethylcyclohexanecarboxylic acid
(12 mmol) in 6 m of 2N aqueous sodium hydroxide solution
and after cooling with ice, 3.0 g (14.5 mmol)
Then, 30 ml of a benzene solution of 1-naphthyloxycarbonyl chloride (2) was added dropwise, and 6 ml of a 2N aqueous sodium hydroxide solution was further added dropwise. Stir at room temperature for 15 hours,
After adjusting the pH of the reaction solution to 2 with concentrated hydrochloric acid while cooling with ice, the mixture was extracted with ethyl acetate. The ethyl acetate layer was dehydrated (anhydrous magnesium sulfate), filtered, concentrated and then purified by a silica gel column (n-hexane: ethyl acetate = 4: 1 → 1: 1) to obtain 0.31 g (yield 8%) of (3 ) Got.

¹ H-NM of the above carbaryl hapten-1
Physical property data (chemical shift) by R are shown below. ^{1 H-NMR (DMSO) 0.88-1.08} (2H, m), 1.20-1.5
4 (3H, duplicate) 1.72-2.00 (4H, duplicate), 2.03-2.2
1 (1H, m) 2.90-3.03 (2H, t), 7.20-7.3
1 (1H, d) 7.42-7.61 (2H, m), 7.74-8.1
1 (4H, m) Example 2 Synthesis of carbaryl hapten-2

[0117]

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Synthesis of (2) Sodium hydride (oil-based, 60%) 0.8 g (20 mm
ol) with N, N-dimethylaminoformamide (DM
F) Suspended in 30 ml, a solution of 3.2 g (20 mmol) of 1,5-dihydroxynaphthalene (1) in dimethyl sulfoxide (DMSO) (20 ml) was added dropwise at room temperature.
After stirring at room temperature for 20 minutes, 5 ml of a DMF solution of 3.6 g (20 ml) of tert-butyl 4-chlorobutyrate was added dropwise.
After heating and stirring at room temperature for 5 hours, the reaction solution was diluted with diethyl ether.
Partition extracted with water. Wash the collected diethyl ether layer with water,
After dehydration (anhydrous magnesium sulfate), filtration and concentration, silica gel chromatography (n-hexane: ethyl acetate =
4: 1 → 2: 1), 0.61 g as a brown solid
(Yield 10%) (2) was obtained. 0.61 g (2 m ) of the synthetic dihydroxynaphthalene derivative (2) of (3)
mol) was dissolved in 50 ml of dichloromethane, 0.14 g (2.5 mmol) of methyl isocyanate was added, and the mixture was stirred at room temperature for 4 hours. After concentrating the reaction solution, silica gel chromatography (n-hexane: ethyl acetate = 4: 1)
→ 1: 1) to obtain 0.67 g (yield 92%) of (3) as white crystals. 0.67 g (1.9 mmol) of synthetic hapten ester (3) of (4 )
Was dissolved in 60 ml of dichloromethane, 5 ml of trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 1 hour.
The reaction solution was concentrated under reduced pressure to obtain 0.53 g (yield 94%) of (4) as white crystals.

¹ H-NM of the above carbaryl hapten-2
Physical property data (chemical shift) by R are shown below. ¹ H-NMR (DMSO) 2.00-2.17 (m, 2H), 2.41-2.5
3 (m, 2H) 2.64-2.76 (d, 3H), 4.10-4.2
3 (t, 2H) 6.92-7.03 (d, 1H), 7.21-7.3
2 (d, 1H) 7.37-7.52 (duplicate, 2H), 7.80-7.9
0 (m, 1H) 7.97-8.08 (d, 1H) Example 3 Carbaryl hapten and polymer compound or
Preparation of a conjugate with a labeled compound A conjugate of carbaryl hapten and BSA was prepared as an immunogen and an antigen for screening using the activated ester method.

The carbaryl hapten prepared in Example 1
Was dissolved in 1.0 ml of DMF, and N-
0.3 mmol of hydroxysuccinimide and 0.3 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 Na
Cl-0.01 M phosphate buffer (pH 7.2: hereinafter referred to as “P
BS)) and dissolved in 5.0 ml and DMF 1.05 m
A solution to which l was added was prepared, and 0.25 ml of the above-mentioned supernatant was added to this solution and reacted at room temperature for 16 hours. After the reaction, carbaryl hapten dialyzed against distilled water at 4 ° C.
1 and BSA (hereinafter referred to as “carbaryl hapten 1
-BSA conjugate ") was used as the immunizing antigen.

Further, using the same method, a conjugate of carbaryl hapten-1 and RSA (hereinafter referred to as a “carbaryl hapten 1-RSA conjugate”) and a conjugate of carbaryl hapten-1 and HRP (hereinafter referred to as “conjugate”) , "Carbaryl hapten 1-HRP conjugate"). Example 4 Immunization Balb / c mice were used for immunization. 100 µg of the carbaryl hapten 1-BSA conjugate prepared in Example 3 was dissolved in 50 µl of PBS, mixed with an equal amount of complete Freund's adjuvant, and inoculated subcutaneously into Balb / c mice. Further, four weeks later, the immunizing antigen prepared in the same manner as described above was boosted with Freund's incomplete adjuvant. On the 6th week, 20 μg of the immunizing antigen dissolved in 180 μl of PBS was boosted through the tail vein of the mouse. Example 5 Reactivity of carbaryl with antiserum Immediately before inoculation into the mouse tail vein in Example 4, the antiserum collected was diluted and prepared for indirect competitive inhibition E as described in detail below.
Carbaryl was measured by the LISA method, and the antiserum was evaluated.

The carbaryl hapten-RSA conjugate solution (0.5 μg / ml) prepared in the same manner as the
A 96-well microplate was coated in an amount of μl / well (25 ng / 50 μl / well), and assayed by blocking with 4-fold diluted Block Ace (“Block Ace”, Snow Brand Milk Products Co., Ltd., code No. UK-25B). A plate for production was prepared. Antiserum 10
20% containing 000-fold diluted solution and various concentrations of carbaryl
An equal amount of the mixture was mixed with a methanol solution, 50 μl of the mixture was added to each well, and reacted at room temperature for 1 hour.

After washing five times with PBS, a peroxidase-conjugated goat anti-mouse IgG antibody (manufactured by Tago) diluted 2000-fold with 10-fold diluted Block Ace was added in an amount of 50 μl / well, and the mixture was added at room temperature for 1 hour. Allowed to react for hours.

After washing 5 times with PBS, 2 mg / ml
Of OPD and 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide in an amount of 50 μl / well, and reacted at room temperature for 10 minutes to develop color.

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

[0127]

[Table 1]

Table 1 shows that an inhibitory reaction was observed at 100 ng / ml of carbaryl, confirming that the antiserum used was reactive with carbaryl. Example 6 Preparation of Hybridoma Following Example 4, spleen cells of a mouse with high anti-carbaryl antibody activity in serum and myeloma cells (Sp2 /
0-Ag14) and the method of Yamashita Shuji et al. (Histocytochemistry:
Japan Society for Tissue and Cell Chemistry: Interdisciplinary project. (1986) according to the polyethylene glycol method and cultured.
A culture supernatant in which cell growth was observed was added in an amount of 50 μl / well to each of the coated and blocked plates in the same manner as in Example 5, and reacted at room temperature for 1 hour.

After washing five 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
0.1M containing OPD and 0.02% hydrogen peroxide
Citrate-phosphate buffer (pH 5.0) was added 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 carbaryl was examined by the indirect competitive inhibition ELISA described in Example 5, and cells producing the target antibody were cloned by the limiting dilution method. As a result, several hybridomas were cloned as cells producing anti-carbaryl antibodies. On December 17, 1998, 29B1-1-1 was
It was deposited under the deposit number FERM P-17093 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (1-1-3 Higashi, Tsukuba 305-0046, Ibaraki Prefecture). Example 7 Carbaryl by Direct Competition Inhibition ELISA
The hybridoma 29B1-1-1 obtained in Example 6 was transplanted into the abdominal cavity of a mouse, the ascites obtained 10 to 15 days later was collected, and the monoclonal antibody 29B1-1-1 was purified by affinity chromatography. . (Hereinafter, monoclonal antibodies use the same names as the hybridomas that produce them.) This 29B1-1-1
The amount of carbaryl was measured by direct competitive inhibition ELISA using the antibody.

The above 29B1-1-1 antibody solution (10 μl
g / ml) in a 96-well microplate in an amount of 50 μl / well, coating was allowed to stand at 4 ° C. overnight, and a 4-fold dilution of Block Ace (manufactured by Snow Brand Milk Products)
Was performed to prepare a plate for assay. An equal volume mixture of a 20% methanol solution containing each concentration of carbaryl and a PBS solution containing the carbaryl hapten-HRP conjugate prepared in Example 3 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 mg
50 μl of a citrate-phosphate buffer solution (pH 5.0) containing 0.02% / 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 result is shown in FIG. This direct competitive inhibition EL
Using the ISA method, the monoclonal antibody 29 of the present invention
B1-1-1 is carbaryl from 0.1 ng / ml to 1
It could be measured in the range of 00 ng / ml.

[Brief description of the drawings]

FIG. 1 shows the monoclonal antibody 29B1 of the present invention.
1 shows the measurement of carbaryl by the direct competitive inhibition ELISA method of 1-1.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12P 21/08 G01N 33/53 S G01N 33/53 J C12N 5/00 B 15/00 C // (C12N 5/10 (C12R 1:91) (C12P 21/08 C12R 1:91) (72) Inventor Masao Hayashi 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, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Naiji 1-27-114, Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72 ) Inventor Motoyuki Watanabe 1-27-14 Hamamatsucho, Minato-ku, Tokyo F-term in the Institute for Environmental Immunity Technology 4B024 AA05 AA07 AA11 BA53 BA61 GA03 HA15 4B064 AG26 AG27 CA10 CA20 CC24 DA10 DA11 DA13 4B065 AA92X AB05 AC14 BA08 CA24 CA25 CA41 CA46 CA47 4H006 AA01 AB20 RA24 4H045 AA11 AA20 BA10 BA30 CA40 DA75 DA76 DA86 EA50 FA72

Claims (11)

[Claims] (1) The following formula (1): A compound having a structure represented by the formula: 2. The following formula (2): [In the formula (2), n is an integer of 1 to 10]. 3. A conjugate of the compound according to claim 1 or 2 with a polymer compound or a labeling substance. 4. An antigen is prepared by binding the compound according to claim 1 or 2 with a polymer compound, and the antigen is used to obtain the following formula (3): A method for producing an antibody or a fragment thereof reactive to a compound having a structure represented by the formula (3), characterized by producing an antibody reactive to a compound having a structure represented by the formula: 5. An antibody or a fragment thereof, which is produced by using the conjugate according to claim 3 as an antigen, and which is reactive with the compound of the formula (3). 6. The antibody or fragment according to claim 5, which is a monoclonal antibody. 7. The antibody or fragment according to claim 5, which is a monoclonal antibody 29B1-1-1. A hybridoma that produces the antibody or fragment according to any one of claims 5 to 7. 9. The hybridoma according to claim 8, which has been deposited under accession number FERM P-17093. 10. A method for immunologically measuring a compound represented by the formula (3), comprising using the antibody or fragment according to any one of claims 5 to 7. 11. The immunological assay method according to claim 10, further comprising using the compound according to claim 1 or the conjugate according to claim 3.