JP2001233884A - Hapten compound of ethylthiometon, antibody and method for measuring the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new antibody reacting with ethylthiometon and analogous compounds thereof or fragments thereof and a method for preparation thereof, because the antibody and a hapten for preparing such antibody have not been obtained, though need of ethylthiometon was remarkably high. SOLUTION: This hapten compound of the present invention has a structure obtained by covalently bonding a spacer arm and a functional group for bonding to an analogous compound of ethylthiometon.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to O, O-diethyl-S-2- (ethylthio) ethyl phosphorodithioate (generally referred to as "ethylthiomethone" or "disulfoton"). And hapten compounds, antigens, antibodies and fragments thereof, which are referred to as “ethylthiomethone” in the above.

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

[0003]

2. Description of the Related Art Ethyl thiomethone has the following formula (4):

[0004]

Embedded image

An organic phosphorus compound having a structure represented by the following formula: Ethyl thiometon is a permeable organophosphorus insecticide and is used for controlling pests such as vegetables and rice. Specifically, it is effective against absorptive insect pests such as aphids and spider mites, and is absorbed from the roots of plants by soil treatment to prevent damage by germinated crop pests. It has a long aftereffect and is said to last for 6 to 8 weeks. It is effective to use top dressing while growing radish and Chinese cabbage. In addition, in the case of rice, application to the seedlings in the nursery box before rice planting can control planthoppers and leafhoppers in the early stage of Honda, but be aware that some populations of the leafhopper leafhopper have developed resistance (pesticides) Handbook page 2-3
Pp. 37-38 and 604, 1998 Edition, Japan Plant Protection Association.

[0006] In recent years, there has been a great social interest in pesticide residues in environments such as soil, water, and the atmosphere, and in residues of post-harvest pesticides in imported agricultural products, which have recently increased in particular. Regarding ethylthiomethone, the registration standard for pesticide registration is 0.07 ppm for rice, 0.05 ppm for fruits, 0.1 ppm for vegetables, 0.1 ppm for potatoes, 0.0 ppm for beans.
It is specified as 5 ppm, etc. (Latest Handbook of Regulations and Standards for Pesticides, 1995 edition, page 104, Japan Plant Protection Association). Therefore, in order to ensure safety with respect to the environment and food, it is necessary to measure the amount of ethylthiomethone contained therein quickly and accurately.

[0007] Conventionally, for example, ethyl thiometon in agricultural crops has been extracted from rice, fruits, vegetables, potatoes, beans, etc., purified, and analyzed by gas chromatography (GC). That is, for example, a method of extracting a sample with acetone, diverting to dichloromethane, distributing hexane-acetonitrile, concentrating the acetonitrile layer to dryness, dissolving in dichloromethane and acetone, and measuring by GC is adopted. ("Recent Analytical Methods for Pesticides", page 234-
P. 235, issued by Chuo Hokyou Shuppan, published April 1, 1995)). These methods have problems in that the preparation of the sample is complicated, requires a large amount of procedures and time, requires a high level of skill in the analysis, and requires high costs for measuring devices and equipment. In the measurement of ethylthiomethone, 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 the accuracy but also the simplicity, the quickness, and the economy is demanded.

[0008] The immunochemical measurement method is a method of detecting an antigen or an antibody based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen.
In recent years, it has attracted attention due to its economic efficiency. In immunochemical assay methods, a wide variety of labels have been applied as detection methods, for example, enzymes, radiotracers, chemiluminescent or fluorescent substances, metal atoms, sols, latexes and bacteriophages.

[0009] Among the immunochemical assay methods, 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, low molecular weight compounds such as ethyl thiometone usually cannot elicit an immune response when inoculated into animals. These molecules only act as a group of epitopes by binding to immunogenic macromolecules (proteins, polysaccharides, etc.).
An immune response is elicited in the presence of a cell receptor, resulting in the production of antibodies by a panel of B lymphocytes. Molecules that produce immunogenicity only when bound to a polymer compound in this way are collectively referred to as “haptens”.

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

[0012] Despite the extremely high need for ethyl thiomethone, not only antibodies but also haptens for producing such antibodies had not been obtained prior to the present invention.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antibody or a fragment thereof which reacts with ethylthiomethone and its analogs, and a method for producing the same. As used herein, the term “fragment” refers to a part of an antibody capable of binding to an antigen, for example, F
_ab fragment etc.

[0014] In one aspect, the present invention provides a monoclonal antibody reactive to ethylthiomethone. Another object of the present invention is to provide a hapten compound (ethylthiomethone hapten) which constitutes an antigen for producing a novel antibody reactive with ethylthiomethone.

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

[0016] The present invention further provides a method for immunochemically measuring ethylthiomethone, which comprises using a conjugate of the above-mentioned antibody or fragment thereof and / or the above-mentioned ethylthiomethone hapten with a polymer compound or a labeling substance. Aim.

[0017]

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

The ethylthiomethone analog compound to be used in the present invention is represented by the following formula (3):

[0019]

Embedded image

[In the formula (3), A ₁ is S or O;
A ₂ is S, O or NH; A ₃ is S, S = O or O = S = O; R ₁ , R ₂ and R ₄ are each independently selected, branched R ₃ is an optionally branched alkylene group having 1 to 4 carbon atoms; and R ₃ is an alkylene group having 1 to 4 carbon atoms which may be branched. ]
Is a compound having a structure represented by

A particularly preferred target compound, ethylthiomethone, has the following formula (4):

[0022]

Embedded image

A compound having a structure represented by the following formula: In the present specification, “ethylthiomethone” means ethylthiomethone represented by the formula (4) and / or an ethylthiomethone analog compound represented by the formula (3) depending on the context.

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

[0025]

Embedded image

[In the formula (1), A ₁ is S or O;
A ₂ is S, O or NH; A ₃ is S, S ＝ O or O ＝ S ＝ O; R ₁ and R ₂ are each independently selected R ₃ is an optionally branched alkylene group having 1 to 4 carbon atoms; and n is 1 to 4
It is an integer of 10. Alternatively, the following equation (2):

[0027]

Embedded image

[In the formula (2), A ₁ is S or O;
A ₂ is S, O or NH; A ₃ is S, S ＝ O or O ＝ S ＝ O; A ₄ is O or NH; R ₁ and R ₄ are each independently R 1 is an optionally branched alkyl group having 1 to 4 carbon atoms;
₃ is an optionally branched alkylene group having 1 to 4 carbon atoms; and m is an integer of 1-10. ]
Is used as a hapten for preparing an antibody.

In the formula (1), preferably, A ₁ and A ₃ are S
A ₂ is NH, R ₁ and R ₂ are ethyl groups, and R ₃ is an ethylene group. Preferably, n is 5.

In the formula (2), preferably, A ₁ and A ₃ are S
Wherein A ₂ and A ₄ are NH, R ₁ and R ₄ are ethyl groups, and R ₃ is an ethylene group. Preferably, m is 3.

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

Preparation of Ethylthiomethone Hapten The ethylthiomethone hapten represented by the formula (1) or (2) can be produced according to a known method. Although not limited, for example, the following method can be used.

I) Ethylthiomethone hapten of formula (1)
General production method First, the following formula (X1):

[0034]

Embedded image

A compound having the structure of formula (X1), wherein A ₂ , A ₃ and R ₃ are as defined above, is reacted with an organic solvent in the presence of a base in the following formula (X2):

[0036]

Embedded image

[In the formula (X2), P is a protecting group for a carboxyl group; L is a halogen atom selected from F, Cl, Br and I; and n is as defined above. Is reacted with a compound having a structure represented by the following formula (X3):

[0038]

Embedded image

An ester compound having a structure represented by the formula (X3), wherein A ₂ , A ₃ , R ₃ , P and n are as defined above, is obtained. Examples of the organic solvent for synthesizing the compound of the formula (X3) include ethanol, methanol, 1-propanol, 2-propanol, tetrahydrofuran and the like. Examples of the base include sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, sodium methylate, sodium ethylate, triethylamine, N, N-dimethylaniline,
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 at 20 ° C. to 80 ° C., for 30 minutes to 10 hours,
Preferably, it is performed for 30 minutes to 2 hours. The protecting group for the carboxyl group represented by P may be a known group, and specific examples include a methyl group, an ethyl group, a tert-butyl group, a benzyl group, a p-methoxybenzyl group, and a 3,4-dimethoxybenzyl group. Group, trichloroethyl group, trimethylsilyl group, tert-butyldimethylsilyl group, ter
t-butyldiphenylsilyl group, triethylsilyl group,
And a triisopropylsilyl group and a trimethylsilylethoxymethyl group.

Next, the compound of the formula (X3) is converted into the following formula (X4):

[0042]

Embedded image

In the formula (X4), A ₁ , R ₁ , R ₂ and L are as defined above, and a halogenated phosphoric acid ester having a structure represented by the following formula: And reacting under the following formula (X5):

[0044]

Embedded image

In the formula (X5), A ₁ , A ₂ , A ₃ , R ₁ , R
₂ , R ₃ , P and n are as defined above] to obtain a compound having a structure represented by the following formula:

Examples of the organic solvent for the synthesis of the compound of the formula (X5) include 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, the same bases as those usable for the synthesis of the compound of the above formula (3) can be used.

The reaction is carried out at a temperature of from −10 ° C. to the boiling point of the solvent,
The reaction is preferably performed at 20 ° C. to 80 ° C. for 30 minutes to 5 hours, preferably 30 minutes to 2 hours. Further, the compound of the formula (1) can be obtained by removing the protecting group for the carboxyl group represented by P from the compound of the formula (X5). The removal of the carboxyl protecting group can be carried out by a known method such as acid hydrolysis and alkali hydrolysis.

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

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

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

II) Ethylthiomethone hapte of formula (2)
General method for manufacturing a down First, the compound of formula (X1) described above, the following formula (X
6):

[0052]

Embedded image

In the formula (X6), R ₄ and L are as defined above. A compound having a structure represented by the following formula (X7) is reacted in an organic solvent in the presence of a base. ):

[0054]

Embedded image

A compound having a structure represented by the formula (X7), wherein A ₂ , A ₃ , R ₃ and R ₄ are as defined above, is obtained. The reaction is carried out at a temperature of -10 ° C to the boiling point of the solvent, preferably 20 ° C to 80 ° C, for 30 minutes to 10 hours, preferably 30 minutes to 2 hours. As the organic solvent and the base for synthesizing the compound of the formula (X7), the same organic solvents and bases that can be used for the synthesis of the compound of the formula (X3) described above can be used.

Next, the compound of the formula (X7) is added to the compound of the following formula (X8):

[0057]

Embedded image

Wherein, in the formula (X8), A ₁ , R ₁ and L are as defined above, with a halogenated phosphoric acid ester having a structure represented by the following formula: Then, the following formula (X9):

[0059]

Embedded image

In the formula (X9), A ₁ , A ₂ , A ₃ , R ₁ , R
₃ , R ₄ and L are as defined above] to obtain a compound having a structure represented by the following formula:

The reaction is carried out at a temperature of from −10 ° C. to the boiling point of the solvent,
The reaction is preferably performed at 0 ° C. to 30 ° C. for 30 minutes to 30 hours, preferably 30 minutes to 24 hours. As the organic solvent and the base for the synthesis of the compound of the formula (X9), the same organic solvents and bases as those usable for the synthesis of the compound of the formula (X5) described above can be used.

Next, the compound of the formula (X9) is added to the compound of the following formula (X10):

[0063]

Embedded image

The compound having the structure represented by the formula (X10), wherein A ₄ , P and m are as defined above is reacted to give the following formula (X11):

[0065]

Embedded image

In the formula (X11), A ₁ , A ₂ , A ₃ , A ₄ ,
R ₁ , R ₃ , R ₄ , P and m are as defined above]
A compound having a structure represented by the following formula is obtained.

The reaction is carried out at a temperature of from −10 ° C. to the boiling point of the solvent,
Preferably at 30 ° C. to 80 ° C., for 30 minutes to 24 hours,
Preferably, it is performed for 30 minutes to 2 hours. As the organic solvent and the base for synthesizing the compound of the formula (X11), the same organic solvents and bases that can be used for the synthesis of the compound of the formula (X5) described above can be used.

Further, by removing the carboxyl protecting group represented by P from the compound of the formula (X11),
The compound of the formula (2) can be obtained. The removal of the protecting group for the carboxyl group can be carried out by a known method such as acid hydrolysis and alkali hydrolysis described above for the synthesis of the compound of the formula (1). By subjecting the compound obtained by the above-mentioned production method to silica gel chromatography or recrystallization operation or the like as necessary, a purified product with higher purity can be obtained.

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

Ethylthiometon hapten and high molecular compound
Preparation of Conjugate with Ethylthiometon Hapten synthesized as described above is bound to an appropriate polymer compound and then used as an antigen for immunization or immobilization.

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

The coupling between the ethyl thiometon hapten and the polymer compound can be performed, for example, by the activated ester method (AEKA).
RU et al. : J. Agric. Food Ch
em. 42 301-309 (1994)), or the mixed acid anhydride method (BF Erlanger et a).
l. : J. Biol. Chem. 234 1090-1
094 (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 form an N-hydroxysuccinimide activated ester.

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

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

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

Further, a substance in which a labeling substance such as an enzyme is bound to an ethylthiometon hapten by the same method as described above can be used in an immunochemical measurement method. The labeling substance, (hereinafter referred to "HRP") horseradish peroxidase, an enzyme such as alkaline phosphatase, fluorescein isothiocyanate, a fluorescent substance such as rhodamine, radioactive substances such as ³² P, ¹²⁵ I, and the like chemiluminescent substance.

Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of ethylthiomethone hapten and a polymer compound. For example, a conjugate of ethyl thiometon hapten and KLH is dissolved in a sodium phosphate buffer (hereinafter, referred to as “PBS”), mixed with an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or alum. It can be obtained by immunizing an animal as an antigen for use. As the animal to be immunized, any of those commonly used in the art can be used, and examples thereof include mice, rats, rabbits, goats, and horses. However, humans are not included.

The method of administration 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 serum separated therefrom can be used to evaluate the presence of a polyclonal antibody that reacts with ethylthiomethone. In the present invention, an antiserum obtained by using a conjugate of ethylthiometon hapten and a polymer compound as an antigen for immunization is measured for the amount of ethylthiometone in the range of 0.01 μg / ml to 100 μg / ml by an indirect competitive ELISA method described later. (Example 5, FIG. 1).

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

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

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

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

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

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

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

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

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

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

The culture supernatant or ascites fluid obtained as described above can be used as an anti-ethylthiomethone 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-ethylthiometon monoclonal antibody can be obtained. Further, when purification is necessary, it can be carried out by combining commonly used methods such as ion exchange column chromatography, affinity chromatography, and high performance liquid chromatography (HPLC).

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

Measurement of Ethylthiomethone by Antibody As a method for measuring ethylthiomethone by the antibody used in the present invention, radioisotope immunoassay (RIA), EIA
LISA method (Engval, E., Methods)
in Enzymol. , 70, 419-439 (19
80)), 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 ethyl thiomethone was performed by various ELISAs.
For example, among the methods A, the indirect competitive ELISA method can be used in the following procedure. (A) First, a conjugate of ethylthiomethone hapten, which is an antigen for immobilization, and a polymer compound is immobilized on a carrier.

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

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

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

The antigen to be immobilized on the carrier includes:
Not only the conjugate of the ethylthiometon hapten for which the antibody was prepared and the polymer compound itself, but also the conjugate of the other hapten represented by the formula (1) or (2) and the polymer compound is used as the immobilized antigen. It is also possible. For example, a compound in which any one of A ₁ -A ₄ , R ₁ -R ₄ , m or n in formula (1) or (2) is different from that used for antibody production can be used as the immobilized antigen. . Further, equation (1)
Alternatively, another ethylthiomethone analog compound not included in (2) can be used as the immobilized antigen.

In the blocking in the step (b), in the carrier on which the antigen (the complex of the ethyl thiometone hapten and the polymer compound) is immobilized, there is a portion other than the ethyl thiometon hapten portion to which the antibody to be added later can be adsorbed. May be done solely to prevent it. As a blocking agent, for example, BSA or skim milk solution can be used. Alternatively, block ace ("Bloc
k-Ace ", manufactured by Snow Brand Milk Products Co., Ltd., code No. UK-25
A commercially available blocking agent such as B) can also be used. Specifically, for example, but not limited to, a buffer solution containing a blocking agent at the portion where the antigen is immobilized [eg, 1% BSA and 60 mM NaC
85 mM borate buffer (pH 8.0)
, And incubated at about 4 ° C for 1 to 5 hours, followed by washing with a washing solution. The washing solution is not particularly limited.
Can be used.

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

At this time, as the antibody, an antibody against the ethylthiometon 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 is carried out at 10 to 40 ° C,
Preferably, the heat treatment may be performed at about 25 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the first antibody that has not bound to the immobilized antigen. As the cleaning liquid, for example, PB
S can be used.

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

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

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

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

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

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

(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 ethylthiomethone in the sample is determined from a previously prepared calibration curve.

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

In the blocking in the step (b), the carrier on which the antibody is immobilized may have a portion where ethylthiomethone and the 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 ELISA method described above can be used.

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

The hapten to be bound to a labeling substance such as an enzyme is not limited to the ethylthiomethone hapten itself used for preparing the antibody, but also to the formula (1) or ( It is also possible to use a conjugate of another hapten represented by 2) and a polymer compound as a labeling antigen. For example, equation (1)
Alternatively, a compound in which any one of A ₁ -A ₄ , R ₁ -R ₄ , m or n in (2) is different from that for preparing an antibody can be used as a labeling antigen. Further, other ethylthiomethone analogs not included in the formula (1) or (2) can be used as the labeling antigen.

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

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

The monoclonal antibody 44K7-1 of the present invention
-1 is about 0.2 ng / in the direct competition ELISA method.
ml to about 50 ng / ml, preferably about 0.2 ng / ml.
Reacts with ethylthiomethone in a concentration range from ml to about 6 ng / ml (Example 7, FIG. 2).

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

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

For example, the monoclonal antibody 44K7-1
-1 shows little reactivity with ethylthiomethone analogs and has very high specificity for ethylthiomethone (Example 8, Table 7). 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.

[0122]

EXAMPLES Example 1 Ethylthiomethone Hapten-1
Synthesis

[0123]

Embedded image

Synthesis of Compound (2) 0.23 g (3 mmol) of Compound (1) was added to 50 ml of ethanol, and 0.12 g of sodium hydroxide (3 m
(mol) was stirred at room temperature for 30 minutes. 0.77 g (3 mmol) of tert-butyl 6-bromohexanoate
Of ethanol solution was added dropwise. After stirring at 50 ° C. for 30 minutes, the mixture was concentrated and purified by a silica gel column (ethyl acetate → chloroform: methanol = 9: 1) to obtain 0.6 g (yield 81%) of compound (2) as a colorless oil. .

The physical data (chemical shift δ) of the compound (2) by ¹ H-NMR are shown below.

[0126]

Table 1 ¹ H-NMR (CDCl ₃ ) 1.33-1.49 (2H, m), 1.53-1.6
8 (4H, m) 2.13-2.27 (2H, t), 2.46-2.5
5 (2H, t) 2.57-2.65 (2H, t), 2.83-2.9
Synthesis of 0 (2H, m) compound (3) 3.8 g (15 mmol) of compound (2) and 3.0 g (15 mmol) of diethyl chlorophosphate were dissolved in 150 ml of acetonitrile, and 2.1 g of potassium carbonate (15 m
mol) was added and the mixture was stirred at 50 ° C for 1 hour. The reaction mixture was filtered through celite, concentrated and purified by a silica gel column (n-hexane: ethyl acetate = 49: 1 → 19: 1 → 9: 1) to give 2.2 g (yield 36%) of the compound as a colorless oil. (3) was obtained.

The physical data (chemical shift δ) of the compound (3) by ¹ H-NMR are shown below.

[0128]

Table 2 ¹ H-NMR (CDCl ₃ ) 1.27-1.38 (6H, t), 1.38-1.52
(11H, duplication) 1.52-1.68 (4H, m), 2.17-2.24
(2H, t) 2.46-2.53 (2H, t), 2.58-2.67
(2H, t) 3.04-3.19 (2H, m), 3.37-3.51
(1H, m) 3.97-4.13 (4H, m) Synthesis of Compound (4) 2.2 g (5.5 mmol) of Compound (3) was obtained in 100 m.
of trifluoroacetic acid (T
FA) 5 ml was added, and the mixture was stirred at room temperature for 1 hour.
After adding 5 ml, the mixture was stirred for 1 hour. The reaction solution is concentrated and concentrated on a silica gel column (n-hexane: ethyl acetate = 4: 1 →
1: 1) to give 1.55 g (82% yield) of compound (4) as a colorless oil.

The above ethylthiomethone hapten-1 (4)
Physical property data according to ¹ H-NMR of the (chemical shift [delta])
Is shown below.

[0130]

Table 3 ¹ H-NMR (CDCl ₃ ) 1.26-1.35 (6H, t), 1.39-1.51
(2H, m) 1.53-1.72 (4H, m), 2.30-2.40
(2H, t) 2.44-2.55 (2H, t), 2.60-2.67
(2H, t) 3.04-3.19 (2H, m), 3.26-3.61
(1H, br) 3.96-4.12 (4H, m) Example 2 Synthesis of ethylthiomethone hapten-2

[0131]

Embedded image

Synthesis of compound (5) 5.4 g (70 mmol) of compound (1) was added to 50 ml of ethanol, and 2.8 g of sodium hydroxide (70 m
(mol) was stirred at room temperature for 30 minutes. 13 g (83 mmol) of ethyl iodide was added dropwise, the mixture was stirred at 50 ° C. for 30 minutes, concentrated, and crudely purified by a silica gel column (ethyl acetate → chloroform: methanol = 9: 1).

Synthesis of Compound (6) A crude product containing 6.0 g (57 mmol at maximum) of Compound (5) and 10.2 g (57 mmol) of ethyl dichlorophosphate were obtained.
l) was added to 100 ml of benzene and potassium carbonate was added.
9 g (57 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was filtered through celite, concentrated, and purified by a silica gel column (n-hexane: ethyl acetate = 49: 1 → 9: 1) as a colorless oil (0.5 g, 3% in total yield from compound (1)). ) Compound (6) was obtained.

The physical properties data (chemical shift δ) of the above compound (6) by ¹ H-NMR are shown below.

[0135]

Table 4 ¹ H-NMR (CDCl ₃ ) 1.21-1.32 (3H, t), 1.33-1.45
(3H, t) 1.50-1.60 (2H, q), 1.69-1.78
(2H, t) 3.20-3.37 (2H, m), 4.09-4.36
(3H, Duplicate) Synthesis of Compound (7) 0.5 g (2 mmol) of compound (6), 0.32 g (2 mmol) of tert-butyl 4-aminobutyrate, and 0.28 g (2 mmol) of potassium carbonate were added to acetonitrile 10
The mixture was added to 0 ml and stirred at 50 ° C. for 30 minutes. The reaction mixture was filtered, concentrated, and purified by a silica gel column (n-hexane: ethyl acetate = 19: 1 → 4: 1) to give 0.54 g (yield 73%) of compound (7) as a colorless oil. Obtained.

The physical data (chemical shift δ) of the above compound (7) by ¹ H-NMR are shown below.

[0137]

Table 5 ¹ H-NMR (CDCl ₃ ) 1.21-1.33 (6H, overlapping), 1.45 (9H,
s) 1.71-1.85 (2H, m), 2.23-2.3.
2 (2H, t) 2.49-2.60 (2H, q), 2.67-2.8
0 (3H, duplicate) 2.90-3.01 (2H, m), 3.06-3.2
0 (2H, m) 3.97-4.14 (2H, m) Synthesis of Compound (8) 540 mg (1.46 mmol) of Compound (7) was added to 10
It was dissolved in 0 ml of dichloromethane, added with 10 ml of TFA, and stirred at room temperature for 1 hour. The reaction solution is concentrated, and silica gel column (ethyl acetate: hexane = 4: 1 → 1: 1)
250 mg (55% yield) as a colorless oil
Compound (8) was obtained. The physical property data (chemical shift δ) of the above ethylthiometon hapten-2 (8) by ¹ H-NMR is shown below.

[0138]

Table 6 ¹ H-NMR (CDCl ₃ ) 1.19-1.37 (6H, duplicated), 1.77-1.9
0 (2H, m) 2.37-2.49 (2H, t), 2.49-2.5
9 (2H, q) 2.61-2.72 (2H, t), 2.92-3.1
8 (4H, m) 3.99-4.17 (2H, m) Example 3 Preparation of Immunization Antigen and Screening Antigen Activated ester of conjugate of ethylthiometon hapten and BSA as immunization antigen and screening antigen It was produced using the method.

0.2 mmol of ethylthiomethone hapten-1 prepared in Example 1 was dissolved in 1.0 ml of DMF, and 0.2 mmol of N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide were dissolved in 1.0 mmol of DMF. Was added and stirred at room temperature for 3.5 hours. After the reaction, centrifuge at 10,000 rpm for 15 minutes,
Separated into supernatant and precipitate. This supernatant is hereinafter referred to as “Solution A”.

On the other hand, 50 mg of BSA was added to 145 mM N
Dissolved in 5.0 ml of aCl-0.01M phosphate buffer (pH 7.2: hereinafter referred to as “PBS”), and added DMF 1.0.
A solution to which 05 ml was added was prepared, and 0.25 ml of solution A was added to this solution, followed by reaction at 4 ° C. for 16 hours. After the reaction, a conjugate of ethylthiomethone hapten-1 and BSA (hereinafter, referred to as “ethylthiomethone hapten-1 / BSA”) obtained by dialysis at 4 ° C. against distilled water was used as an antigen for immunization.

Further, using the same method, a conjugate of ethyl thiometone hapten-1 and RSA (hereinafter referred to as “ethyl thiometone hapten-1 / RSA”) and a conjugate of ethyl thiometon hapten-1 and HRP (hereinafter referred to as “ethyl thiometone hapten-1 / RSA”) "Ethylthiometon hapten-1 / HRP") was also prepared.

Example 4 Immunization Balb / c mice were used for immunization. 50 μg of ethylthiomethone hapten-1 / BSA prepared in Example 3 was dissolved in 100 μl of PBS, mixed with an equal amount of complete Freund's adjuvant, and inoculated subcutaneously into Balb / c mice. Four weeks later, 50 µg of the antigen for immunization and Freund's incomplete adjuvant were mixed in the same manner as described above, and the mice were boosted subcutaneously. Two weeks later, PBS1
10 μg of the immunizing antigen dissolved in 80 μl was boosted through the tail vein of the mouse.

Example 5 Antiserum against ethylthiomethone
Immediately before inoculation into mice tail vein in reactive Example 4, diluted prepare antiserum bled, indirect competitive ELI detailed below
Ethyl thiomethone was measured by the SA method, and the antiserum was evaluated.

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

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

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
One example of the result is shown in FIG. From FIG. 1, it was confirmed that the amount of ethylthiomethone can be measured in the range of 0.01 to 100 μg / ml by using mouse antiserum (polyclonal antibody).

Example 6 Preparation of Hybridomas Following Example 4, spleen cells from mice with high serum anti-ethylthiometone antibody activity and myeloma cells (S
p2 / 0-Ag14) and the method of Shuji Yamashita et al. (Histocytochemistry: Japan Society for Histocellular Chemistry: Interdisciplinary Planning, 1986)
And fused by the polyethylene glycol method according to the above. 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 added at room temperature.
Allowed to react for hours.

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 incubated at room temperature for 1 hour. Reacted. After washing 5 times with PBS, 2 mg / ml OPD
And 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide in an amount of 50 μl / well, and a color reaction was performed at room temperature for 10 minutes.

Next, 1N sulfuric acid was added in an amount of 50 μl / well to stop the reaction, and the absorbance at 490 nm was measured.
Reactive cells (hybridomas) were selected. Next, the reactivity of each well with ethylthiomethone was determined in Example 5.
Were examined by the indirect competitive ELISA method described in (1) above, and cells producing the desired antibody were cloned by the limiting dilution method. As a result, several strains of hybridomas were cloned as cells producing anti-ethylthiometon antibodies. Of these, 44K7-1-1 was transferred on February 15, 2000.
On the day, it was deposited under the deposit number FERM P-17732 with the Institute of Biotechnology, Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki 305-0046, Japan).

Example 7 By Direct Competition Inhibition ELISA
Measurement of Ethylthiometon Hybridoma 44K7-1-1 obtained in Example 6 was transplanted into the peritoneal cavity of a mouse, and ascites obtained 10 to 15 days later was collected, and a monoclonal antibody was fractionated by ammonium sulfate fractionation. (Hereinafter, monoclonal antibodies use the same names as the hybridomas that produce them.) Using this 44K7-1-1 antibody, the amount of ethylthiomethone was measured by a direct competitive ELISA method.

The above 44K7-1-1 antibody solution (5 μg
/ Ml) in a 96-well microplate in a volume of 50 μl / well, and left to stand overnight at 4 ° C. for coating.
Furthermore, blocking was performed with 4-fold dilution of Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) to prepare an assay plate.
A 10% methanol solution containing various concentrations of ethyl thiometone and the ethyl thiometone hapten prepared in Example 3
50 μl of an equal volume mixture with a PBS solution containing 1 / HRP
Each well was placed in each well and reacted at 25 ° C. for 1.5 hours.

After the reaction, the plate was washed 5 times with PBS, and then 2 mg
0.1 g / ml OPD and 0.02% hydrogen peroxide.
50 μl of 1 M citrate-phosphate buffer (pH 5.0)
Each well was placed in 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. As shown in FIG.
In the ISA method, the monoclonal antibody 44K of the present invention
It was confirmed that 7-1-1 can measure ethylthiomethone in the range of 0.2 ng / ml to 50 ng / ml.

Example 8 Cross-Reaction of Monoclonal Antibody
For monoclonal antibodies 44K7-1-1 derived from the obtained hybridomas 44K7-1-1 Evaluation Example 6 of sex, by indirect competitive ELISA method described in Example 5 using the culture supernatant, three The cross-reactivity to ethylthiomethone analogs was studied. Table 7 shows the results.

[0155]

[Table 7]

In Table 7, the cross-reactivity rate (%)
Meton's IC₅₀/ IC of target compound ₅₀) × 100 (%)
Is defined by Cross-reaction between ethylthiomethone and related compounds.
The more divergent the value of the response is, the more specific the ethylthiomethone is
Antibody that reacts specifically. Also, IC₅₀(Pmol / m
l) is a reaction without adding a compound such as ethylthiomethone.
Reaction solution that reduces the absorbance by 50% relative to the absorbance of the solution
Shows the concentration of the target compound in the liquid.

As shown in Table 7, the monoclonal antibody 44K7-
1-1 showed reactivity with ethylthiomethone, but showed a low cross-reactivity with ethylthiomethone analogs and showed little reactivity.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the relationship between the ethylthiomethone hapten of the present invention and
Indirect competitive ELISA of antisera obtained using 1 / BSA
The reactivity with ethylthiomethone according to Method A is shown.

FIG. 2 shows the monoclonal antibody 44K7 of the present invention.
The reactivity with ethylthiometon is shown by the direct competitive ELISA method using -1-1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/08 G01N 33/577 B G01N 33/53 G01N 33/563 33/577 C12R 1:91) // G01N 33/563 C12N 5/00 B (C12N 5/10 15/00 C C12R 1:91) C12R 1:91) (72) Inventor Tetsuo Yamazaki 1-27-14 Hamamatsucho, Minato-ku, Tokyo, Japan Co., Ltd. Inside the Institute for Environmental Immunity Technology (72) Insider of Inventor Uchida 1-27-114 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Yoshio Kamata 1-27, Hamamatsucho, Minato-ku, Tokyo No. 14 Inside the Institute of Environmental Immunity Technology (72) Inventor Shigeyuki Watanabe 1-27-14 Hamamatsucho, Minato-ku, Tokyo F-term inside the Institute of Environmental Immunity Technology 4B024 AA11 BA41 GA03 GA09 GA27 HA03 4B064 AG27 CA10 CA20 CC01 CC24 CE04 DA13 4B065 AA91X AB05 AC14 AC15 BA08 BA24 BC01 BD14 BD50 CA25 CA46 4H045 AA11 AA20 AA30 DA76 EA50 FA72 GA06 4H050 AB81 AC60 BA92 BB21 BE12 WA15 WA27 WA28

Claims (14)

[Claims] (1) The following formula (1): [Equation (1), A ₁ is S or O; A ₂ is S, O or NH; A ₃ is S, S = O or O = S = O; R ₁ and R ₂ is an optionally branched alkyl group having 1 to 4 carbon atoms, which may be independently selected;
₃ is an optionally branched alkylene group having 1 to 4 carbon atoms; and n is an integer of 1-10. ]
Alternatively, the following formula (2): [In formula (2), A ₁ is S or O; A ₂ is S, O or NH; A ₃ is S, S = O or O = S = O; A ₄ is ,
R ₁ and R ₄ are an optionally branched alkyl group having 1 to 4 carbon atoms, each of which may be independently selected; R ₃ is an optionally branched carbon atom; M is an alkylene group of 1 to 4;
It is an integer of 10. ] The compound which has a structure represented by these. 2. In the formula (1), A ₁ and A ₃ are S and A ₂
Is NH, R ₁ and R ₂ are ethyl groups, and R
The compound according to claim 1, wherein ₃ is an ethylene group. 3. In the formula (2), A ₁ and A ₃ are S and A ₂
And A ₄ is NH, R ₁ and R ₄ are ethyl groups,
The compound according to claim 1, wherein R ₃ is an ethylene group. 4. The method according to claim 1, wherein n is 5 in the formula (1).
The compound according to the above. 5. The method according to claim 1, wherein m is 3 in the formula (2).
The compound according to the above. 6. A conjugate of the compound according to claim 1 with a polymer compound or a labeling substance. 7. An antigen is prepared by binding the compound according to claim 1 to a high molecular compound, and the antigen is used to obtain the following formula (3): Wherein (3), A ₁ is S or O; A ₂ is S, O or NH; A ₃ is S, S = O or O = S = O; R _1, R
₂ and R ₄ are each independently selected an optionally branched alkyl group having 1 to 4 carbon atoms; and R ₃ is an optionally branched alkyl group having 1 to 4 carbon atoms.
Is an alkylene group. A method for producing an antibody or a fragment thereof reactive to the compound represented by the formula (3), characterized by producing an antibody reactive to the compound having the structure represented by the formula: [8] An antibody or a fragment thereof, which is produced by using the conjugate according to [6] as an antigen, and which is reactive with the compound represented by the formula (3). 9. The antibody or fragment according to claim 8, which is a monoclonal antibody. 10. The monoclonal antibody 44K7-1-1 produced from a hybridoma deposited under accession number FERM P-17732.
2. The antibody or fragment according to item 1. 11. A hybridoma producing the antibody according to any one of claims 8 to 10. 12. The hybridoma according to claim 11, which has been deposited under accession number FERM P-17732. 13. Use of the antibody or fragment according to any one of claims 8 to 10,
A method for immunochemical measurement of a compound represented by the formula (3). 14. The method according to claim 13, further comprising using the compound according to any one of claims 1 to 5 or the conjugate according to claim 6.