JPH11140100A - Hapten compound of isoxathion, antibody and assay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound, a hapten compound of O,O- diethyl-O-(5-henyl-3-isoxazolyl)phosphorothioate (isoxathion), usable for e.g. an antigen for preparing antibody to isoxathion. SOLUTION: This compound is a new hapten compound of O,O-diethyl-O-5- phenyl-3-isoxazolyl)phosphorothioate (isoxathion) of formula II, which has a structure shown by formula I (R is a 1-4C alkyl; (n) is an integer of 1-10), being useful for e.g. an antigen for preparing antibody to isoxathion as an organophosphorus-based insecticide. This new compound is obtained by reaction between a dichlorothiophosphoric ester and an aminocarboxylic ester to form a chlorothiophosphoric acid derivative of formula III (Z is a protective group for carboxyl group) which, in turn, is reacted with 5-phenyl-3-isoxazolone in an organic solvent in the presence of a base. An antigen for preparing the antibody is obtained by coupling the compound of formula I with a polymeric compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a hapten compound of O, O-diethyl-O- (5-phenyl-3-isoxazolyl) phosphorothioate (hereinafter referred to as "isoxathione"), an antigen, an antibody and a hapten compound. Regarding that fragment.

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

[0003]

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

Embedded image An organophosphorus insecticide having a structure represented by
Acts as a poison and has a long lasting effect ("Recent Analytical Methods for Pesticides", pp. 376-377, edited by the Research Group for Pesticide Residue Analytical Methods; Chugoku-Horoki Publishing; Pesticide Handbook, p. 19, 1994 Edition, Japanese Plants Epidemics Association). Isoxathion is widely used for controlling pests of vegetables, fruit trees, and flowers, but is also used for controlling pests such as scarab beetles because it is relatively stable in soil. It is widely used to control soil pests such as beetles, scarab beetles, grasshopper weevil and grasshopper.

[0004] In recent years, great social interest has been given to the effects of pesticide residues in the environment, such as water, soil and air, and in foods on human health. Therefore, it is necessary to quickly and accurately measure the amount of isoxathion remaining in isoxathion, which is a widely used agricultural chemical, in order to ensure the safety of the environment and foods.

[0005] At present, no standards for pesticide residues based on the Food Sanitation Law have been set for isoxathion. However, since it is a widely used pesticide such as this, the analysis of residues in crops is important.

[0006] Various guidelines for water quality of isoxathion are determined and monitored. For example, it is listed as a required monitoring item of the water quality environmental standard based on the Basic Environmental Law and a monitored item of the tap water quality standard based on the Water Supply Law.
/ L or less. In the "Provisional Guidance Guidelines for Prevention of Water Pollution Caused by Pesticides Used at Golf Courses" (1990) by the Environment Agency, the provisional guidance values for golf course pesticide drainage are set at 0.08 mg / l. (Journal of Japan Society on Water Environment Vol.16 No.4 (19
93) p. 13-21, Industry and Environment Vol. 25
No. 7 (1996) p. 26-30).

Conventionally, for example, a test on the water quality of isoxathion has been performed by preparing a sample solution by solid phase extraction or solvent extraction and then performing measurement based on GC-MS or GC. For example, a sample is developed and extracted with acetone in a solid phase extraction column, and then subjected to gas chromatography (G
C) Measure the concentration of isoxathion using a mass spectrometer or a gas chromatograph equipped with a phosphorus / nitrogen specific thermionic detector (FTD or NPD) or a phosphorus / sulfur specific flame photometric detector (FPD). can do. These methods have the problems that the preparation of the sample is complicated, requires a great deal of labor and time, has a high level of skill in analysis, and requires high costs for measuring devices and equipment. In the measurement of isoxathion, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and a new measurement method having not only accuracy but also simplicity, speed, and economy has been demanded.

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

[0009] Among the immunoassays, 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 isoxathion 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.

[0012] Despite the great need for isoxathion, not only suitable antibodies,
Haptens for producing antibodies have not been obtained before the present invention.

[0013]

DISCLOSURE OF THE INVENTION The present invention provides a novel antibody or a fragment thereof that reacts with isoxathion,
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.

[0014] In one aspect, the present invention provides a monoclonal antibody reactive to isoxathione.

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

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

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

Another object of the present invention is to provide a method for immunologically measuring isoxathion, which comprises using a conjugate of the antibody or its fragment and / or the isoxathion derivative with a polymer compound. .

[0019]

Means for Solving the Problems As a result of intensive studies, the present inventors have introduced a functional group which can be used for bonding to a spacer arm and a polymer compound to isoxathion.
By using a derivative of isoxathion as a hapten, an antibody reactive with the compound was successfully obtained, and the present invention was completed.

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

Embedded image It is a compound represented by these.

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

Embedded image [Wherein, in the formula (1), R is an optionally branched alkyl group having 1 to 4 carbon atoms (preferably an ethyl group); and n is an integer of 1-10]. Is used as a hapten for antibody production. In the present invention, the stereoisomers of the compound are not particularly limited, and include all stereoisomers.

The present invention relates to the hapten compound, a conjugate of the hapten compound and a polymer compound, an antibody reacting with isoxathion, a method for preparing the same, and a method for immunologically measuring isoxathion using the hapten compound or the antibody.

Preparation of Derivative of Isoxathion The isoxathion derivative represented by the formula (1) can be prepared according to a known method. For example, but not limited to, the methods described below.

First, the following equation (X1):

Embedded image [In the formula (X1), R is as defined above] and a dichlorothiophosphate represented by the following formula (X
2):

Embedded image [In the formula (X2), Z is a protecting group for a carboxyl group,
n is as described above] with an ester represented by the following formula (X)
3):

Embedded image [In the formula (X3), R, Z and n are the same as those described above].

The ester compound represented by the formula (X2) is
Known methods, for example, Skerritt et al.
Agric. Food Chem. 1992, 40, 1
466-1470), and can be easily synthesized.

In the synthesis of the formula (X3), the organic solvent includes
For example, acetonitrile, acetone, hexane, pentane, benzene, toluene, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, diglyme,
N, N-dimethylformamide, hexamethylphosphoric triamide and the like, or a mixed solvent thereof can be used. As the base, for example, potassium carbonate, sodium carbonate, lithium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate, triethylamine, N, N-dimethylaniline, hydroxide sodium,
Lithium diisopropylamide or the like can be used. The reaction is carried out at a temperature of from −10 ° C. to the boiling point of the solvent, preferably 20 ° C.-30 ° C., for 10-50 hours, preferably 20-30 hours. Dichlorothiophosphate ether and the amino ester of the formula (X2) are in a molar ratio of 2.0: 1.
0-1.0: 2.0, preferably 1.2: 1.0-1.
Used in a ratio of 0: 1.2.

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

Next, the chlorothiophosphoric acid derivative of the formula (X3) and the following formula (X4):

Embedded image Is reacted in an organic solvent in the presence of a base to give the following formula (X5):

Embedded image [In the formula (X5), R, Z and n are the same as those described above] to obtain an ester compound of an isoxathion derivative.

The organic solvent and the base are represented by the formula (X)
The same compounds as in the case of synthesizing the compound of 3) can be used. The reaction is carried out at a temperature between -10 ° C and the boiling point of the solvent, preferably at 60 ° C to 85 ° C for 0.5 to 5 hours, preferably 2-3 hours. The chlorothiophosphoric acid derivative of the formula (X3) and the compound of the formula (X4) are in a molar ratio,
2.0: 1.0-1.0: 2.0, preferably 1.2: 1.0
Used in the ratio -1.0: 1.2.

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

That is, in the case of acid hydrolysis, the formula (X)
The ester compound 5) is preferably dissolved in an organic solvent such as benzene, dichloromethane, 1,2-dichloroethane and the like, and then hydrochloric acid, sulfuric acid, boron trifluoride, trifluoroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid At 0 ° C. to the boiling point of the solvent, preferably 0 ° C. to room temperature, for 5 minutes to 10 hours, preferably 1-
The carboxylic acid compound of the formula (1) can be obtained by stirring and reacting for 5 hours.

In the case of alkali hydrolysis, the formula (X
The ester compound of 5) is preferably dissolved in an organic solvent such as methanol, ethanol, tetrahydrofuran or ethylene glycol, and then an aqueous solution of sodium hydroxide or potassium hydroxide is added thereto. The isoxathione derivative carboxylic acid compound of the formula (1) can be obtained by stirring and reacting at a temperature of from 5 ° C. to 5 hours, preferably 1-2 hours.

Further, when Z is a benzyl group, the removal can also be carried out by a hydrogenolysis reaction using a catalyst such as palladium carbon or a Birch reduction using liquid ammonium-sodium.

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

The compound obtained by the above production method is
By performing a silica gel chromatography or a recrystallization operation as needed, a purified product with higher purity can be obtained.

The preparation of the antigen and antibody of the present invention and the method for immunological measurement will be described below. 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.

The isoxathione derivative and the polymer compound
Preparation of Conjugate The isoxathion derivative synthesized as described above is bound to an appropriate polymer compound and then used as an antigen for immunization.

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

The binding between the isoxathion derivative and the polymer compound can be performed by, for example, the activated ester method (AE KAR).
U et al. : J. Agric. Food C
hem. 42 301-309 (1994)) or the mixed acid anhydride method (BF Erlanger e).
t al. : J. Biol. Chem. 234 10
90-1094 (1954)).

The activated ester method can be generally carried out as follows. First, a hapten compound is dissolved in an organic solvent and reacted with N-hydroxysuccinimide in the presence of a coupling agent to produce an N-hydroxysuccinimide 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 N, N-dimethylformamide (hereinafter, referred to as “DMF”), dimethyl sulfoxide (DM
SO), dioxane and the like can be used. The molar ratio of the hapten compound and N-hydroxysuccinimide used in the reaction is preferably 1: 10-10: 1, more preferably
1: 1-1: 10, most preferably 1: 1. The reaction temperature is 0 ° C-100 ° C, preferably 5 ° C-50 ° C, more preferably 22 ° C-27 ° C, and the reaction time is 5 minutes-24.
Time, preferably 30 minutes-6 hours, more preferably 1-
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 supernatant is added to the solution of the polymer compound and reacted. For example, when the polymer compound has a free amino group, the amide is located between the amino group and the carboxyl group of the hapten compound. A bond is created. The reaction temperature is 0 ° C-60 ° C, preferably 5 ° C-40 ° C.
C., more preferably 22C to 27C, and the reaction time is 5 minutes to 24 hours, preferably 1 to 16 hours. The reaction product can be purified by dialysis, a desalting column, or the like to obtain a conjugate of the isoxathion derivative and the polymer compound.

On the other hand, the mixed acid anhydride used in the mixed acid anhydride method is obtained by reacting a carboxylic acid with a haloformate, and by reacting this with a polymer, the desired hapten-polymer compound is obtained. A conjugate is produced. This reaction is performed in the presence of a basic compound.
Examples of the basic compound include N-methylmorpholine,
Triethylamine, trimethylamine, pyridine, N,
Organic bases such as N-dimethylaniline, DBN, DBU, and DABCO; and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The reaction is usually performed at minus 20 ° C to 100 ° C, preferably at 0 ° C to 50 ° C, and the reaction time is 5 minutes.
10 hours, preferably 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually performed by minus 2
The reaction is carried out at 0 ° C to 150 ° C, preferably at 0 ° C to 100 ° C, and the reaction time is 5 minutes to 10 hours, preferably 5 minutes to 5 minutes.
Time. 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, dioxane,
Ethers such as diethyl ether, tetrahydrofuran, dimethoxyethane, dichloromethane, chloroform,
Halogenated hydrocarbons such as dichloroethane, benzene,
Examples thereof include aromatic hydrocarbons such as toluene and xylene, esters such as methyl acetate and ethyl acetate, and aprotic polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide, and hexamethylphosphoric triamide.
Examples of the haloformate used in the mixed acid anhydride method include isobutyl chloroformate, methyl chloroformate, methyl bromoformate, ethyl chloroformate, and ethyl bromoformate. 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 an isoxathion derivative is used.
It can be used in immunoassays. As a labeling substance, horseradish peroxidase (hereinafter, “HR”)
P ") and enzymes such as alkaline phosphatase, coloring substances such as fluorescein isothiocyanate and rhodamine, and radioactive substances such as ³² P and ¹²⁵ I.

Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of an isoxathion derivative and a polymer compound. For example, an isoxathion derivative-BSA conjugate is added to a phosphate buffer (hereinafter referred to as “PB
S ") and immunizing the animals with adjuvants such as Freund's complete 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. Dosing 1
It can be performed multiple times or at appropriate intervals, preferably 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 isoxathion.

The antiserum of the mouse immunized with the conjugate of the isoxathion derivative of the present invention and the polymer compound was used in an indirect competitive inhibition ELISA method described below, wherein the amount of isoxathione was about 0.01-1.0 μg / dose. It can be measured in the range of ml (Example 4).

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

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

(A) Preparation of conjugate of isoxathione derivative used as antigen for immunization and polymer compound (b) Immunization of animal (c) Collection of blood, assay, and preparation of antibody-producing cells (d) Myeloma Preparation of cells (e) Cell fusion between antibody-producing cells and myeloma cells and selective culture of hybridomas (f) Screening and cloning of hybridomas producing the desired antibody (g) Culture of hybridomas or hybridomas to animals Preparation of Monoclonal Antibody by Transplantation (h) Measurement of Reactivity of Prepared Monoclonal Antibody, etc. Conventional methods for producing a hybridoma producing a monoclonal antibody include, for example, Hybridoma Technologies, Cold Spring Harbor Laboratory (Cold
Spring Harbor Laborator
y), 1980 edition), and Cell Histochemistry (Syuji Yamashita et al., edited by the Japanese Society for Tissue and Cell Chemistry; interdisciplinary project, 1986).

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

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

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

The myeloma cells that can be used in the step (d) include, for example, P3 / X63-Ag8 (X63) (Nat) of a myeloma cell line derived from Balb / c mouse.
ure, 256 , 495-497 (1975)),
P3 / X63-Ag8. U1 (P3U1) (Curre
nt Topics. in Microbiology
and Immunology, 81 1-7
(1987)), P3 / NSI-1-Ag4-1 (NS
-1) (Eur. J. Immunol., 6 , 511).
-519 (1976)), Sp2 / 0-Ag14 (S
p2 / 0) (Nature 276 , 269-270)
(1978)), FO (J. Immuno. Me)
th. , 35 , 1-21 (1980)), MPC
Myeloma cell lines such as -11, X63.653, S194, or rat-derived 210. RCY3. Ag1.
2.3. (Y3) (Nature 277 , 131-
133, (1979)) and the like.

The above cell line is subcultured in Dulbecco's modified Eagle's medium (DMEM) or Iscove's modified Dulbecco's medium (IMDM) containing fetal bovine serum, and the number of cells is 1 × 10 ⁶ or more 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.
methods in Enzymology, 73 ,
3 (1981)). At present, the method most commonly used is a method using polyethylene glycol (PEG), in which the fusion operation is also easy. P
The EG method is described in, for example, Cell Histochemistry, Shuji Yamashita et al. (Supra). As another fusion method, a method by electric treatment (electric fusion) or the like can be appropriately adopted (Etsuko Okochi et al., Experimental Medicine 5.1315).
-19, 1987). Also, the ratio of cells used may be the same as in the known method. For example, spleen cells may be used about 3 to 10 times as much as myeloma cells. Selection of a hybridoma group in which spleen cells and myeloma cells are fused to obtain antibody-producing ability and proliferation ability is performed, for example, when a hypoxanthine guanine phosphoribosyltransferase-deficient strain is used as a myeloma cell line, for example, when the above-mentioned DMEM or IMEM is used.
It can be carried out by using a HAT medium prepared by adding hypoxanthine / aminopterin / thymidine to MDM.

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 isoxathion is measured by the LISA method. Further, cell cloning of a hybridoma that has been found by measurement to produce an antibody that reacts with isoxathion 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 the wells in which the antibody titer has been recognized, cell cloning is repeated 1-4 times, for example, by the limiting dilution method, and those with a stable antibody titer are selected as anti-isoxathione monoclonal antibody-producing hybridoma strains.

The culture medium for culturing hybridomas includes:
For example, DMEM or IMDM containing fetal bovine serum 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, Ba
lb / c) or hybridomas can be grown in the abdominal 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-isoxathione 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-isoxathione monoclonal antibody can be obtained. If further purification is required, the operation of collecting the antibody fraction using a commonly used method such as ion exchange column chromatography, affinity chromatography, open column chromatography, high performance liquid chromatography (HPLC), etc. is performed. It can be carried out once or more than once.

The anti-isoxathione 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 Isoxathion with Antibody As a method for measuring isoxathion with an antibody used in the present invention, a radioisotope immunoassay (RIA),
ELISA method (Engval, E., Meth. E)
nsymol. , 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 an Ouchterlony method (“Hybridoma method and monoclonal antibody”, published by R & D Planning Inc.) Pp. 30-53, March 5, 1982
Days). ELIS from the viewpoint of sensitivity, simplicity, etc.
Method A is widely used.

The measurement of isoxathion 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 an isoxathion derivative 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 isoxathion are added thereto, and the antibody is allowed to react competitively with the immobilized antigen and free isoxathion to form an immobilized antigen-antibody complex and a free isoxathion-antibody complex. Generate body. (D) By measuring the amount of the immobilized antigen-antibody complex, the amount of free isoxathion 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. Known buffers 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.
About 100 μg / ml, preferably 0.05-10 μg
/ Ml is suitable. When a 96-well microtiter plate is used as a carrier, 300 μm
1 / well or less, preferably about 50-150 μl / well. Furthermore, the conditions for incubation are not particularly limited, but usually incubation at about 4 ° C. overnight is suitable.

As the antigen to be immobilized on the carrier,
Not only the conjugate of the isoxathione derivative and the polymer compound used for preparing the antibody itself, but also the conjugate of another derivative represented by the formula (1) and the polymer compound can be used. Further, other isoxathione-like compounds not included in the formula (1) can also be used as the immobilized antigen.

In the blocking in the step (b), there may be a portion to which the antibody to be added later can be adsorbed in addition to the isoxathione derivative portion on the carrier having the antigen-bound conjugate immobilized thereon. Done on purpose.
As a blocking agent, for example, BSA or skim milk solution can be used. Alternatively, Block Ace (“Bl
ock Ace ", manufactured by Snow Brand Milk Products Co., Ltd., code No. UK-
A commercially available blocking agent such as 25B) can also be used. Specifically, for example, but not limited to, 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 buffer. It is done by doing. The washing liquid is not particularly limited, but for example, PBS is suitable.

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

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

The first antibody is added after being dissolved in a buffer. Although not limited, the reaction may be performed at about 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 is preferable.

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 antibody-goat antibody conjugated with an enzyme (eg, peroxidase or alkaline phosphatase). It is desirable that the first antibody bound to the carrier is reacted with the second antibody diluted so that the final absorbance is 4 or less, 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. 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 enzyme of the second antibody bound to the carrier is added, and the amount of isoxathion can be calculated from the calibration curve by measuring the absorbance.

When peroxidase is used as the enzyme that binds to the second antibody, a chromogenic substrate solution containing, for example, hydrogen peroxide and orthophenylenediamine (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. If OPD is used, measure the absorbance at 490 nm. On the other hand, when alkaline phosphatase is used as the enzyme that binds to the second antibody, for example, p-
The color is developed using nitrophenyl phosphate as a substrate and 2N N
A method of stopping the enzyme reaction by adding aOH and measuring the absorbance at 415 nm is suitable.

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

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

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

(C) A sample containing various concentrations of isoxathion and an enzyme-bound hapten in which an isoxathion derivative is bound to an enzyme are reacted with an antibody immobilized on a carrier.

(D) By measuring the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of isoxathion 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. Alternatively, a product obtained by binding an antibody to an amino group-bonded microtiter plate using a chemical bonding method can be used.

In the blocking in the step (b), there is a case where a portion of the carrier on which the antibody is immobilized is present where the isoxathion and the enzyme-bound hapten in the sample to be added later are adsorbed independently of the antigen-antibody reaction. , To prevent it. As the blocking agent and the method therefor, those similar to the indirect competitive inhibition ELISA method described above can be employed.

The preparation of the enzyme-bound hapten used in the step (c) may be carried out by any method without particular limitation as long as it is a method of binding an isoxathion derivative to an enzyme. For example, the activated ester method described above can be employed. The prepared enzyme-linked hapten is mixed with a sample containing isoxathion.

As the hapten to be bound to the enzyme, not only the isoxathione derivative itself used for preparing the antibody but also other derivatives represented by the formula (1) can be used. For example, compounds having different numbers of R and n in the formula (1) can be used for antibody production and as a label-competitive compound, respectively. Furthermore, other isoxathione-like compounds not included in the formula (1) can also be used as haptens to be bound to the enzyme. In step (c), a sample containing isoxathion and an enzyme-bound hapten are brought into contact with the antibody-immobilized carrier, and a complex between the isoxathion and the enzyme-bound hapten is formed by a competitive inhibition reaction between the isoxathion and the enzyme-bound hapten. The sample containing isoxathion is diluted with an appropriate buffer before use. Without limitation, the reaction is performed, for example, at room temperature for about 1 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. The cleaning liquid is, for example, PB
S can be adopted.

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

The monoclonal antibody 15A18- of the present invention
1-1 is about 0.0 in direct competitive inhibition ELISA.
1-10 ng / ml, preferably 0.1-10 ng / m
1 can be measured for isoxathion (Example 7 and FIG. 3).

Cross-Reactivity of the Antibody of the Present Invention The cross-reactivity of the monoclonal antibody of the present invention can be examined by the above-described direct competitive inhibition ELISA or indirect competitive inhibition ELISA. For example, 15A18-
1-1 showed almost no reactivity with other similar organic phosphorus compounds in the indirect competitive inhibition ELISA method (Example 6 and FIG. 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.

[0089]

EXAMPLES Example 1 Synthesis of Isoxathion Derivative

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1.8 g of ethyl dichlorothiophosphate (1)
(10 mmol) and 1.6 g (10 mmol) of tert-butyl 4-aminobutyrate in 150 ml of acetonitrile
And 4.3 g (30 mmol) of potassium carbonate was added thereto, followed by stirring at room temperature for 24 hours. The reaction solution was filtered through celite, concentrated, and then purified by a silica gel column (n-hexane: ethyl acetate = 4: 1) to give 1.
6 g (yield 59%) of (2) was obtained. (2) 2.7 g
(10 mmol) and 1.6 g (10 mmol) of 5-phenyl-3-isoxazolone in 100 ml of acetonitrile
, 4.3 g (30 mmol) of potassium carbonate was added, and the mixture was stirred at 85 ° C. for 3.5 hours. The reaction solution was filtered through Celite, concentrated, and purified by a silica gel column (n-hexane: ethyl acetate = 9: 1) to obtain 0.9 g (yield 23%) of (3) as a yellow transparent liquid. .

Hapten ester (3) 0.9 g (2.3
mmol) was dissolved in 50 ml of dichloromethane, 5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2.5 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1) to obtain 0.42 g (yield 54%) of (4) as white crystals.

¹ H-NMR (CDCl ₃ ) 1.05-1.55 (t, 3H), 1.68-2.08
(M, 2H), 2.23-2.60 (t, 2H), 2.
85-3.42 (m, 2H), 4.00-4.42
(Q, 2H), 6.58 (s, 1H), 7.33-7.
55 (m, 3H), 7.60-7.92 (m, 2H)

Example 2 Isoxathione Derivative and Polymer
Preparation of a conjugate with a compound or a labeling substance As an immunogen and an antigen for screening, a conjugate of an isoxathion derivative and BSA was prepared by the activated ester method.

The isoxathion derivative (0.05 mmol) prepared in Example 1 was dissolved in DMF (0.25 ml), and N-
Hydroxysuccinimide (0.05 mmol) and dicyclohexylcarbodiimide (0.05 mmol) 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, 25 mg of BSA was added to 145 mM Na
Cl-0.01 M phosphate buffer (pH 7.2: hereinafter referred to as “P
BS)) dissolved in 2.5 ml and DMF 0.5 ml
Was added thereto, and 0.125 ml of the above supernatant was added to this solution, followed by reaction at room temperature for 16 hours.
After the reaction, an isoxathion derivative-BSA conjugate dialyzed against distilled water at 4 ° C was used as an antigen for immunization.

Further, using the same method, isoxathion derivative-RSA conjugate, isoxathion derivative-HRP
Conjugates were also made.

Example 3 Immunization 100 μg of the immunizing antigen prepared in Example 2 was added to PBS 10
It was dissolved in 0 µl, mixed with an equal volume of Freund's complete adjuvant, and inoculated subcutaneously into Balb / c mice. Two weeks later, an incomplete immunizing antigen was further boosted with Freund's incomplete adjuvant. Four weeks later, 50 μg of the immunizing antigen dissolved in 250 μl of PBS was boosted into the tail vein of the mouse.

Example 4 Isoxathione by antiserum
Immediately before inoculation into the tail vein of the mouse in Reactive Example 3, the antiserum collected was diluted and prepared for indirect competitive inhibition E described in detail below.
Isoxathion was measured by the LISA method, and the antiserum was evaluated.

A solution of isoxathion derivative-RSA conjugate (0.5 μg / ml) prepared in the same manner as the immunizing antigen was coated on a 96-well plate at a volume of 50 μl / well and diluted 4-fold with Block Ace (“Block Ac”).
e ", manufactured by Snow Brand Milk Products Co., Ltd., code No. The plate was blocked with UK-25B) to prepare an assay plate. An equal amount of a 4000-fold diluted antiserum and a 20% methanol solution containing various concentrations of isoxathion were mixed with each other.
μl was placed in a well 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 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
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.
One example of the result is shown in FIG. From FIG. 1, it was confirmed that isoxathion was measured in the range of 0.01 to 1.0 μg / ml by using mouse antiserum (polyclonal antibody).

Example 5 Preparation of Hybridomas Following Example 3, spleen cells from mice having high anti-isoxathione 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. The culture supernatant in which cell growth was observed was added to the coated and blocked plates in the same manner as in Example 4 in an amount of 50 μl / well, and reacted at room temperature for 1 hour.

After washing 5 times with PBS, a peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Tago) diluted 2000-fold with 10-fold diluted Block Ace was added in an amount of 50 μl / well, and incubated at room temperature for 1 hour. Reacted.

After washing 5 times with PBS, 0.1 M citrate-phosphate buffer (pH 5.0) containing 2 mg / ml OPD and 0.02% hydrogen peroxide was added in an amount of 50 μl / well. The reaction was allowed to proceed 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.
Reactive cells (hybridomas) were selected. Next, the reactivity of each well with isoxathion was examined by the indirect competitive inhibition ELISA method described in Example 4, and cells producing the desired antibody were cloned by the limiting dilution method. As a result, several hybridomas were cloned as cells producing anti-isoxathione antibodies. 15A18-1-1 of which was purchased on November 6, 1997.
On the day, it was deposited under the deposit number FERM P-16504 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture 305).

Example 6 Cross-Reaction of Monoclonal Antibodies
For monoclonal antibodies 15A18-1-1 derived from sexual evaluation cloned hybridomas 15A18-1-1, an organophosphorus compound in an indirect competitive inhibition ELISA method using the culture supernatant triazophos, pyraclofos,
The reactivity to pyridafenthion and sulfotep was examined. The result is shown in FIG. From FIG. 2, 15A18
-1-1 showed reactivity with isoxathione, but hardly showed reactivity with the other four organophosphorus compounds.

Example 7 Direct competitive inhibition ELISA
Measurement of isoxathione Hybridoma 15A18-1-1 obtained in Example 5
Was transplanted into the abdominal cavity of a mouse, the ascites obtained 10 to 15 days later was collected, and the monoclonal antibody 15A18-1-1 was purified by affinity chromatography using Protein G (manufactured by Pharmacia). This 15A1
The amount of isoxathion was measured by the direct competitive inhibition ELISA method using the 8-1-1 antibody.

The 15A18-1-1 antibody solution (3 μl
g / ml) in a 96-well plate in an amount of 50 μl / well, coating was allowed to stand at 4 ° C. overnight, and blocking was further performed with 4-fold diluted Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.), for assay. Was prepared. 50 μl of a PBS solution containing each concentration of isoxathion and the HRP-bound isoxathion derivative prepared in Example 2 was added to each well, and reacted at 25 ° C. for 1 hour.

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

Next, 50 μl of 1N sulfuric acid was added to each well to stop the color reaction, and the absorbance at 490 nm was measured. The result is shown in FIG. This direct competitive inhibition EL
Using the ISA method, the monoclonal antibody 15 of the present invention
A18-1-1 has an amount of isoxathion of 0.10-10.
It could be measured in the range of ng / ml.

[Brief description of the drawings]

FIG. 1 shows the reactivity of isoxathion with mouse antiserum.

FIG. 2 shows the monoclonal antibody 15A1 of the present invention.
8 shows the reactivity with an organic phosphorus compound by an indirect competitive inhibition ELISA method using 8-1-1.

FIG. 3 shows the monoclonal antibody 15A1 of the present invention.
The reactivity with isoxathion by the direct competitive inhibition ELISA method using 8-1-1 is shown.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12P 21/08 C12N 15/00 C (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91) (72) Invention Person Atsushi Fujii 1-27-14, Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology, Inc. (72) Inventor Naoki 1-27-14, Hamamatsucho, Minato-ku, Tokyo Environmental Immunity Technology, Inc. Inside the laboratory (72) Inventor Shigeyuki Watanabe Inside the Institute for Environmental Immunity Technology, 1-27-14 Hamamatsucho, Minato-ku, Tokyo

Claims (9)

[Claims] (1) The following formula (1): [In the formula (1), R is an optionally branched alkyl group having 1 to 4 carbon atoms; and n is an integer of 1-10.]
A compound having a structure represented by the formula: 2. A conjugate of the compound according to claim 1 with a polymer compound or a labeling substance. 3. An antigen is prepared by binding the compound according to claim 1 with a polymer compound, and the antigen is used to obtain the following formula (2): A method for producing an antibody or a fragment thereof reactive to a compound of the formula (2), characterized by producing an antibody reactive to a compound having a structure represented by the formula: 4. An antibody or a fragment thereof, which is produced by using the conjugate according to claim 2 as an antigen, and which is reactive with the compound of the formula (2). 5. The antibody or fragment thereof according to claim 4, which is a monoclonal antibody. 6. The antibody or fragment thereof according to claim 4, which is 15A18-1-1. 7. A hybridoma producing the antibody according to any one of claims 4 to 6. 8. The hybridoma according to claim 7, which has been deposited under accession number FERM P-16504. 9. A method for immunologically measuring a compound of the formula (2), comprising using the antibody or fragment according to any one of claims 4 to 6.