JP2000236875A - Malathion haptenic compound, antibody and measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new malathion haptenic compound which consists of a malathion haptenic compound having a specific structure and is useful in the production of an antibody for measuring the residual amount of malathion of a dithio type low toxic oroganophosphorus insecticide by the immunological measurement method. SOLUTION: This malathion haptenic compound is a new haptenic compound of malathion [S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate] having the structure represented by formula I [X is NH or S; Z is NH or O; (k) is 0 or 1; (m) is an integer of 0-2; (n) is an integer of 1-20] and is useful as an antigen, or the like, for producing an antibody used in the immunological measurement, or the like, of the residual agrochemical concentration of malathion of a dithio type low toxic organophosphorus insecticide. This compound is obtained by reacting methanol with diphosphorus pentasulfide in an organic solvent followed by reacting a compound of formula II (L is a halogen) in the presence of a base to form the compound of formula III, reacting this product with a compound of formula IV (P is a protective group of carboxylic groups) in the presence of a lithium compound and then eliminating the protective group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a hapten compound of S-1,2-bis (ethoxycarbonyl) ethyl O, O-dimethylphosphorodithioate (hereinafter referred to as "malathion" in the present specification) and an antigen. , Antibodies and fragments thereof.

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

[0003]

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

[0004]

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A dithio-type low-toxic organophosphorus insecticide having a structure represented by the following formula: It is effective against sucking pests such as planthoppers, leafhoppers, aphids, thrips, and thrips. The main insecticidal mechanism of this drug is parathion, EP
It is in the inhibition of cholinesterase in the same way as the N agent. Insecticidal activity is generally weaker than them, but it is strong, selective and fast-acting depending on the type of insect. Although there is considerable osmotic migration, it is considered that the residual effect period is short because it is easily degraded in plant tissues and is also lost by volatilization and decomposition from the plant surface. Therefore, it can be used for fruit trees and vegetables up to several days before harvesting, but it is less effective unless used repeatedly for pests with a long outbreak or short generation.
It has little effect on soil pests and larvae. As for the chemical damage, it is necessary to pay particular attention to the seedlings of cucurbits and tomatoes because they are highly sensitive to malathion agents (Agrochemical Handbook pages 28-32 and 514, 1994 edition, Japan Plant Protection Association; "Recent Analytical Methods for the Latest Pesticides", pp. 384-386, edited by the Research Group for Pesticide Residue Analytical Methods, published by Chuo Hoshin).

[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 malathion, based on the Food Sanitation Law, the residual standard value is rice (0.1 ppm), cereals (2-8 ppm),
Beans, fruits, vegetables (0.5-8 ppm), flour (1.
2 ppm), oil seeds (0.1-8 ppm), nuts (8 ppm), hops (1 ppm), etc. ("Recent Analytical Methods for Pesticides" Same as above). Regarding water quality, the standard value for registration suspension for water pollution is 0.1 m.
g / l (Revised 3rd edition Agricultural Chemical Registration Suspension Standard Handbook pp. 874-876, September 25, 1998 Chemical Daily, Inc.) It is necessary to quickly and accurately determine the amount of malathion contained.

Conventionally, for example, malathion in agricultural crops has been extracted from samples of cereals, beans, seeds, seeds, hops, fruits, vegetables, etc., purified, and then analyzed by gas chromatography (GC). That is, for example, a method of extracting a sample with acetone, dissolving the sample in hexane, partitioning with hexane-acetonitrile as necessary, purifying by florisil column chromatography, and measuring by GC is adopted. These methods have problems in that the preparation of the sample is complicated, requires a large amount of procedures and time, requires a high level of skill in the analysis, and requires high costs for measuring devices and equipment.
In the measurement of malathion, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and a new measurement method which is not only accurate but also simple, quick and economical has been demanded.

[0008] The immunological measurement method is a method for detecting an antigen or an antibody based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen. Due to its excellent accuracy, simplicity, rapidity, and economy, In recent years, it has been attracting attention. In an immunoassay, a very wide variety of labels can be used as a detection method, for example,
Enzymes, radiotracers, chemiluminescent or fluorescent substances,
Metal atoms, sols, latexes and bacteriophages have been applied.

[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 molecule compounds such as malathion 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.

As described above, although malathion was very highly required, a hapten for producing such an antibody as well as a suitable antibody had not been obtained before the present invention. Was.

[0013]

An object of the present invention is to provide a novel antibody or a fragment thereof which reacts with malathion, and a method for producing the same. As used herein, the term “fragment” of an antibody means a part of an antibody capable of binding to an antigen, for example, a _Fab fragment or the like.

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

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

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

[0017] It is still another object of the present invention to provide a hybridoma that produces the antibody or a fragment thereof.

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

[0019]

Means for Solving the Problems As a result of intensive studies, the present inventors have used malathion haptens in which a functional group which can be used for bonding to a spacer arm and a polymer compound is introduced into malathion or a portion thereof. As a result, an antibody reactive with the compound was successfully obtained, and the present invention was completed.

The malathion which is an object of the present invention is represented by the following formula (2):

[0021]

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Is a compound represented by the formula: In the present invention, the stereoisomers of the compound are not particularly limited, and include all stereoisomers.

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

[0024]

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[In the formula (1), X is NH or S;
Z is NH or O; k is 0 or 1; m is 0
And n is an integer of 1 to 20], is used as a hapten for the production of an antibody.

Preferably, k is 0. Preferably,
m is 2. Preferably, n is an integer from 5 to 10.

More preferably, X is NH and k is 0
Where m is 2 and n is 5.

The present invention relates to the hapten compound, a conjugate of the hapten compound and a polymer compound, an antibody reacting with malathion, a method for producing the same, and a method for immunologically measuring malathion using the hapten compound or the antibody. Malathion hapten The malathion hapten represented by the formula (1) can be produced according to a known method. Although not limited, for example, the following method can be used. I. When X = S i) When k = 0 and Z = NH First, methanol is reacted with diphosphorus pentasulfide in an organic solvent. The reaction is carried out at 0 ° C. to the boiling point of the solvent, preferably at 40 ° C.
To 70 ° C. for 1 to 24 hours, preferably 2 to 5 hours, with stirring.

Examples of the organic solvent include toluene, acetonitrile, acetone, hexane, pentane, benzene, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, diglyme, N, N-dimethylformamide, hexamethylphosphoric triamide and the like. Or a mixed solvent thereof can be used.

Next, a base is added, and further, the following formula (Y1):

[0031]

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In the formula (Y1), L is a halogen atom selected from F, Cl, Br or I, and is reacted with an ester compound having a structure represented by the following formula (Y
2):

[0033]

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A compound having a structure represented by the following formula is obtained. As the base, for example, potassium carbonate, sodium carbonate,
Lithium carbonate, potassium bicarbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate, triethylamine,
N, N-dimethylaniline, lithium diisopropylamide, and the like can be used.

The reaction is carried out at 0 ° C. to the boiling point of the solvent, preferably at 40 ° C. to 70 ° C., for 1 hour to 24 hours, preferably for 1 hour to 3 hours.

Next, the compound of the formula (Y2) is converted to a compound of the following formula (Y) in an organic solvent in the presence of lithium bis (trimethylsilyl) amide, lithium diisopropylamide or the like.
3):

[0037]

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Wherein, in the formula (Y3), P is a protecting group for a carboxyl group; and L and m are as defined above, by reacting an ester compound having a structure represented by the following formula: Formula (Y4):

[0039]

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A compound having a structural formula represented by the following formula (Y4), wherein P and m are as defined above, is obtained.

As the organic solvent, tetrahydrofuran (THF), dioxane, diethyl ether, dimethoxyethane and the like can be used.

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, tert-butyldiphenylsilyl group,
Examples include a triethylsilyl group, a triisopropylsilyl group, a trimethylsilylethoxymethyl group, and the like. However,
Those which are more easily released from the carboxyl group than the ethyl group in the removal step by hydrolysis described below are preferable. For example, t-butyl group, p-methoxybenzyl group, 3,4-
A dimethoxybenzyl group is preferred.

The reaction is carried out at a temperature of −100 ° C. to the boiling point of the solvent, preferably at −80 ° C. to 30 ° C., for 1 hour to 20 hours, preferably 1 hour to 3 hours.

Next, the protecting group of the carboxyl group represented by P is removed from the compound of the formula (Y4) to give the following formula (Y5):

[0045]

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In the formula (Y5), m is as defined above to obtain a compound having a structure represented by the following formula:

The removal of the 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 (Y
The compound of 4) 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 and the like, and the mixture is stirred at 0 ° C. to the boiling point of the solvent, preferably at 0 ° C. to 50 ° C. for 5 minutes to 10 hours, preferably 1 hour to 5 hours to give a compound of the formula (Y5) Can be obtained.

In the case of alkali hydrolysis, the formula (Y
The compound of 4) is preferably methanol, ethanol,
Dissolve in an organic solvent such as THF and ethylene glycol, and then add sodium hydrogencarbonate, sodium carbonate, potassium carbonate, lithium hydroxide, sodium hydroxide or potassium hydroxide aqueous solution, etc., and bring the solvent to a boiling point of 0 ° C, preferably 0 ° C. The compound of formula (Y5) can be obtained by stirring and reacting at a temperature of from 0 ° C. to room temperature for 5 minutes to 10 hours, preferably 1 hour to 2 hours.

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

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

Next, the compound represented by the formula (Y) is preferably dissolved in an organic solvent such as THF, methanol, ethanol and ethylene glycol.
The compound of 5) is represented by the following formula (Y6):

[0053]

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By reacting a compound having a structure represented by the formula (Y6), P and n are as defined above, the following formula (Y7):

[0055]

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A compound having a structure represented by the formula (Y7), wherein P, m and n are as defined above, is obtained.

The reaction is carried out at a temperature of −20 ° C. to the boiling point of the solvent,
Preferably at minus 15 ° C to 30 ° C for 30 minutes to 2
It is carried out for 0 hour, preferably for 1 hour to 2 hours.

It is desirable that a base such as N-methylmorpholine and a haloformate such as isobutyl chloroformate be present in the reaction solution.

Further, by removing the protecting group of the carboxyl group represented by P from the compound of (Y7), a compound of the formula (1) (X = S, Z = NH and k = 0) can be obtained. it can. The removal of the protecting group for the carboxyl group can be performed by the same method as described above in the synthesis of the compound of the formula (Y5).

Ii) In the case where k = 0 and Z = 0, the compound of the formula (Y2) described above in i) is added to an organic solvent in the presence of lithium bis (trimethylsilyl) amide, lithium diisopropylamide, etc. Y
8):

[0061]

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[In the formula (Y8), L, P, m and n
Is as defined above], and a compound having a structure represented by the following formula (Y9):

[0063]

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In the formula (Y9), P, m and n are as defined above to obtain a compound having a structure represented by the formula:

As the organic solvent, those similar to those used for the synthesis of the compound of the formula (Y4) can be used. The reaction is carried out at a temperature of −100 ° C. to the boiling point of the solvent, preferably −80 ° C.
C. to 30.degree. C. for 1 hour to 20 hours, preferably 1 hour to 3 hours.

The compound of the formula (1) (X = S, Z = O and k = 0) can be obtained by removing the protecting group for the carboxyl group of P from the compound of the formula (Y9). Removal of the carboxyl-protecting group can be performed by the same method as described above.

Iii) In the case of k = 1 and Z = NH First, as in the synthesis of the compound of the formula (Y1), diphosphorus pentasulfide is reacted with methanol in an organic solvent. Next, a base is added, and further, the following formula (Y10):

[0068]

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In the formula (Y10), L, P and m are as defined above, and an ester compound having the structure represented by the following formula (Y11) is reacted:

[0070]

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A compound having a structure represented by the formula (Y11), wherein P and m are as defined above, is obtained.
The synthesis of the compound of the formula (Y11) can be carried out in the same manner as the synthesis of the compound of the formula (Y2).

Next, the compound of the formula (Y11) is converted into the following formula (Y12):

[0073]

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In the formula (Y12), L is as defined above, and an ester compound having a structure represented by the following formula (Y13) is reacted:

[0075]

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A compound having the structural formula represented by the formula (Y13), wherein P and m are as defined above, is obtained. The compound of the formula (Y13) can be synthesized in the same manner as in the synthesis of the compound of the formula (Y4).

Next, the protecting group of the carboxyl group represented by P is removed from the compound of the formula (Y13),
The following formula (Y14):

[0078]

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A compound having a structure represented by the formula (Y14), wherein m is as defined above, is obtained. Removal of the carboxyl-protecting group can be performed by the same method as described above.

Next, the compound of the formula (Y6) is reacted with the compound of the formula (Y14) to give a compound of the following formula (Y1)
5):

[0081]

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A compound having a structure represented by the formula (Y15), wherein P, m and n are as defined above, is obtained. The compound of the formula (Y15) can be synthesized in the same manner as in the synthesis of the compound of the formula (Y7).

Further, the compound of formula (1) (X = S, Z = NH and k = 1) can be obtained by removing the protecting group of the carboxyl group represented by P from the compound of (Y15). it can. Removal of the carboxyl-protecting group can be performed by the same method as described above. II. When X = NH When Z = O and k <m First, the following formula (Y16):

[0084]

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In the formula (Y16), k and m are as defined above.
H ₂ ) A protecting group is introduced into a carboxyl group near the _m side, and is represented by the following formula (Y17):

[0086]

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In the formula (Y17), P is a protecting group for a carboxyl group; and k and m are as defined above.

As the protecting group for the carboxyl group represented by P, a known protecting group can be used, and the protecting group can be introduced by a known method. For example, when a benzyl group is introduced as a protective group, benzyl alcohol is reacted in an organic solvent such as diethyl ether or water in the presence of a condensing agent such as sulfuric acid, and the benzyl group is converted to a compound (Y16) by a condensation reaction.
To the compound.

The reaction is carried out at 0 ° C. to the boiling point of the solvent, preferably at 20 ° C. to 30 ° C., for 1 hour to 40 hours, preferably 20 hours to 30 hours.

Next, a protecting group is introduced into the amino group of the compound of the formula (Y17) to give the following formula (Y18):

[0091]

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In the formula (Y18), a compound having a structure represented by the formula: P ′ is a protecting group for an amino group; and P, k and m are as defined above.

As the protecting group for the amino group represented by P ′, known groups can be used, and for example, a benzyloxycarbonyl group, a tert-butoxycarbonyl group and the like can be used. Introduction of a protecting group can also be performed using a known method.

For example, when a benzylcarbonyl group is introduced as a protecting group, benzyloxycarbonyl chloride is reacted in water in the presence of a base to convert the protecting group to (Y1
The compound of 7) is introduced. As the base, sodium hydrogen carbonate, potassium carbonate, sodium carbonate, lithium carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate,
Triethylamine, N, N-dimethylaniline and the like can be used. The reaction is carried out at 0 ° C. to the boiling point of the solvent, preferably 0 ° C. to 80 ° C., for 1 hour to 20 hours, preferably 1 hour to 3 hours.

Next, the compound of the formula (Y18) is reacted with a solution of hydrogen chloride in diethyl ether to give the compound of the following formula (Y1)
9):

[0096]

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A compound having a structure represented by the formula (Y19), wherein P, P ', k and m are as defined above, is synthesized.

The reaction is carried out in ethanol at 0 ° C. to the boiling point of the solvent, preferably at 40 ° C. to 60 ° C., for 1 hour to 20 hours, preferably for 1 hour to 3 hours.

Next, the protecting group for the carboxyl group represented by P and the protecting group for the amino group represented by P ′ are removed from the compound of the formula (Y19) to give the following formula (Y2)
0):

[0100]

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In the formula (Y20), k and m are as defined above, to obtain a compound having a structure represented by the following formula:
The removal of the protecting group can be performed by a known method as described above for the removal of the protecting group of the carboxyl group.

For example, when P and / or P 'is a benzyl group, the removal can be carried out by hydrogenolysis with hydrogen.

Next, the protecting group for the amino group is introduced again into the compound of the formula (Y20), and the compound of the following formula (Y21):

[0104]

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In the formula (Y21), a compound having a structure represented by the formula: P ′, k and m are as defined above is obtained.

Next, the compound of the formula (Y21) is added to the compound of the following formula (Y22) in an organic solvent:

[0107]

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By reacting a compound having a structure represented by the formula (Y22), P and n are as defined above, the following formula (Y23):

[0109]

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In the formula (Y23), P, P ′, k, m and n are as defined above, to obtain a compound having a structure represented by the formula:

The reaction is carried out in tetrahydrofuran (THF),
In organic solvents such as dioxane, diethyl ether, dimethoxyethane, dichloromethane, and 1,2-dichloroethane, the temperature is from 0 ° C to the boiling point of the solvent, preferably from 20 ° C to 30 ° C.
And 1 hour to 40 hours, preferably 20 hours to 30 hours
Do time.

The reaction is preferably carried out using a base catalyst such as N, N-dimethylaminopyridine (DMAP), an acid catalyst such as camphorsulfonic acid, N, N-dicyclohexylcarbodiimide (DCC), N, N-diisopropylcarbodiimide. And the like in the presence of a condensing agent.

Next, the protecting group for the amino group represented by P ′ is removed from the compound of the formula (Y23) to give the following formula (Y24):

[0114]

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A compound having a structure represented by the formula (Y24), wherein P, k, m and n are as defined above, is obtained. The removal of the protecting group for the amino group can be carried out by the same method as described above for the synthesis of the compound of the formula (Y20).

Next, the compound of the formula (Y24) is added to the compound of the following formula (Y25):

[0117]

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[0118] In the formula (Y25), L is as defined above, and reacted with a halogenated dimethyl thiophosphate having a structure represented by the following formula in an organic solvent in the presence of a base:
The following formula (Y26):

[0119]

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A compound having a structure represented by the formula (Y26), wherein P, k, m and n are as defined above, is obtained.

Examples of the organic solvent for synthesizing the compound of the formula (Y26) include acetonitrile, acetone, hexane, pentane, benzene, toluene, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, diglyme, N, N-dimethylformamide, hexamethylphosphoric triamide, etc., or a mixed solution 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, lithium diisopropyl Amides and the like can be used.

The reaction is carried out at 0 ° C. to the boiling point of the solvent, preferably at 40 ° C. to 60 ° C., for 1 hour to 10 hours, preferably for 1 hour to 2 hours.

Next, the protecting group of the carboxyl group represented by P is removed from the compound of the formula (Y26), whereby
The compound of formula (1) (when X = NH and Z = O) can be obtained.

The removal of the carboxyl-protecting group can be carried out by a known method as described above. 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. Preparation of Conjugate of Malathion Hapten and Polymer Compound The malathion hapten synthesized as described above is bound to an appropriate polymer compound and then used as an antigen for immunization or immobilization.

Examples of preferable 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, but KLH
And BSA are preferred.

The binding between the malathion hapten and the polymer compound can be performed, for example, by the activated ester method (AEKARU).
et al. : J. Agric. Food Chem.
42301-309 (1994)) or the mixed acid anhydride method (BF Erlanger et al .: JB).
iol. Chem. 234 1090-1094 (19
54)) and the like.

The activated ester method can be generally carried out as follows. First, a hapten compound is dissolved in an organic solvent and reacted with N-hydroxysuccinimide in the presence of a coupling agent to form an N-hydroxysuccinimide activated ester.

As the coupling agent, an ordinary coupling agent commonly used 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 (D
MF), dioxane and the like can be used. The molar ratio of the hapten compound to N-hydroxysuccinimide used in the reaction is preferably from 1:10 to 10: 1, more preferably from 1: 1 to 1:10, most preferably 1: 1. The reaction temperature is from 0 ° C to 100 ° C, preferably from 5 ° C to 50 ° C.
C., more preferably 22 ° C. to 27 ° C .;
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.
The reaction time is 5 minutes to 24 hours, preferably 1 hour to 16 hours. The reaction product can be purified by dialysis, a desalting column or the like to obtain a conjugate of malathion hapten and a polymer compound.

On the other hand, the mixed 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 obtained by reacting this with a polymer compound. Manufactured. 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, N-methylformalin, pyridine, N, N-dimethylaniline, DBN, DBU, DA
Organic bases such as BCO, potassium carbonate, sodium carbonate,
Inorganic bases such as potassium bicarbonate and sodium bicarbonate are exemplified. The reaction is usually carried out at a temperature of −20 ° C. to 15 ° C.
The reaction is carried out at 0 ° C., preferably at 0 ° C. to 100 ° C., and the reaction time is 5 minutes to 10 hours, preferably 5 minutes to 2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually carried out at a temperature of −20 ° C. to 100 ° C., preferably 0 ° C.
To 50 ° 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,
Specifically, ethers such as dioxane, diethyl ether, tetrahydrofuran and dimethoxyethane, halogenated hydrocarbons such as dichloromethane, chloroform and dichloroethane, aromatic hydrocarbons such as benzene, toluene and xylene, methyl acetate, ethyl acetate and the like Esters,
Aprotic polar solvents such as N, N-dimethylformamide, dimethylsulfoxide, hexamethylphosphoric triamide and the like; Examples of the haloformate used in the mixed acid anhydride method include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, and isobutyl chloroformate. The proportions of the hapten, the haloformate and the polymer compound used in the method can be appropriately selected from a wide range.

[0132] In the same manner as described above, a substance obtained by binding a labeling substance such as an enzyme to a malathion hapten can be used in an immunoassay. As a labeling substance, horseradish peroxidase (hereinafter “HR”)
P "), enzymes such as alkaline phosphatase, fluorescent substances such as fluorescein isocyanate and rhodamine, radioactive substances such as ³² P and ¹²⁵ I, and chemiluminescent substances. Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be prepared by a conventional method using a conjugate of malathion hapten and a polymer compound. For example, a malathion hapten-KLH conjugate is added to a sodium phosphate buffer (hereinafter, referred to as a sodium phosphate buffer)
It can be obtained by immunizing an animal with a solution dissolved in an adjuvant such as Freund's complete adjuvant or incomplete adjuvant, or alum. As the animal to be immunized, any of those commonly used in the art can be used, and examples thereof include mice, rats, rabbits, goats, and horses.

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

Blood is collected from the immunized animal, and the serum separated therefrom can be used to evaluate the presence of a polyclonal antibody that reacts with malathion. Preparation of Monoclonal Antibody The monoclonal antibody of the present invention can be prepared by a known method using a conjugate of malathion hapten and a polymer compound.

In producing a monoclonal antibody,
At least the following work steps are required. (A) Preparation of a conjugate of malathion hapten used as an antigen for immunization and a polymer compound (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 producing a monoclonal antibody against malathion 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 carried out by substantially the same method as described for the polyclonal antibody.

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

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

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

The cell fusion in the step (e) 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.

[0142] 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 malathion is measured by the LISA method.

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

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

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

The culture supernatant or ascites fluid thus obtained can be used as an anti-malathion 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-malathion monoclonal antibody can be obtained. Further, when purification is necessary, ion exchange column chromatography, affinity chromatography, high performance liquid chromatography (HPL)
It can be carried out by combining commonly used methods such as C).

The anti-malathion 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 Malathion by Antibody As a method of measuring malathion by an antibody used in the present invention, radioisotope immunoassay (RIA), ELISA
Method A (Engval, E., Methods in E)
nzymol. , 70, 419-439 (198).
0)), various methods generally used for detection of antigens such as a fluorescent antibody method, a plaque method, a spot method, an agglutination method, and Ouchterlony (“Hybridoma method and monoclonal antibody”, published by R & D Planning, Inc. Pages 30-53, March 5, 1982
Days). ELIS from the viewpoint of sensitivity, simplicity, etc.
Method A is widely used.

[0149] Malathion can be measured by the following procedure using, for example, an indirect competitive inhibition ELISA method among various ELISA methods.

(A) First, a conjugate of malathion 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 protein unrelated to the antigen, for example, a protein.

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

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

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

To immobilize the solid phase antigen on a carrier,
For example, a buffer containing an antigen may be placed on a carrier and incubated. As the buffer, a known buffer can be used, and examples thereof include a phosphate buffer. The concentration of the antigen in the buffer 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 immobilization antigen to be immobilized on the carrier includes not only the conjugate of the malathion hapten for which the antibody was prepared and the polymer compound itself, but also other hapten represented by the formula (1). A conjugate with a polymer compound can also be used as an immobilized antigen. For example, in formula (1), a compound in which X, Z, k, m or n is different from the antigen for preparing an antibody can be used as the immobilized antigen. Further, other malathion-like compounds not included in the formula (1) can be used as the immobilized antigen.

In the blocking in the step (b), an antibody to be added later besides the malathion hapten portion can be adsorbed on a carrier on which an antigen for immobilization (malathion hapten and a polymer compound) is immobilized. Parts may be present and are used solely to prevent them. As a blocking agent, for example, BSA or skim milk solution can be used. Or, Block Ace ("Block-
Ace ", manufactured by Dainippon Pharmaceutical Co., Ltd., code No. UK-25
A commercially available blocking agent such as B) can also be used. Specifically, although not limited, for example, a buffer solution containing a blocking agent [eg, 1% BSA and 60 mM
85 mM borate buffer (pH
8.0)], and at about 4 ° C. and room temperature, for 1 hour to 5 hours.
After incubation for a period of time, washing is performed with a washing solution. The washing solution is not particularly limited, but for example, an 85 mM borate buffer (pH 8.0) to which 60 mM NaCl is added can be used.

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

At this time, as the antibody, an antibody against malathion 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 25 ° C. to 37 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the first antibody that has not bound to the immobilized antigen. As the washing solution, for example, 85 mM borate buffer (pH 8.0) to which 60 mM NaCl is added can be used.

Next, a second antibody is added. For example, when a mouse monoclonal antibody is used as the first antibody, it is appropriate to use an anti-mouse-goat antibody conjugated with an enzyme (for example, peroxidase or alkaline phosphatase). About 500-fold to about 1-fold for the first antibody bound to the carrier
It is desirable to react the second antibody diluted so as to have a final absorbance of 0000 times, preferably 4 or less, more preferably 0.5 to 3.0. Buffer is used for dilution. Without limitation, the reaction is carried out at room temperature for about 1 hour,
After the reaction, the plate is washed 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 malathion can be calculated from the calibration curve by measuring the absorbance.

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

With respect to the absorbance of the reaction solution to which malathion was not added, the rate of decrease in the absorbance of the solution in which malathion was added and reacted with the antibody was calculated as the inhibition rate. The concentration of malathion in a sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of a reaction solution to which malathion of a known concentration has been added.

Alternatively, the measurement of malathion 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) Blocking the surface of the carrier on which the antibody is not immobilized, with a protein unrelated to the antigen, for example, a protein.

(C) A sample containing malathion at various concentrations and an enzyme-bound hapten obtained by binding a malathion hapten 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 malathion in the sample is determined from a previously prepared calibration curve.

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

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

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

The hapten to be bound to a labeling substance such as an enzyme is not limited to the malathion hapten itself used for preparing the antibody, but also to the formula (1), as in the case of the immobilized antigen in the indirect competitive inhibition ELISA method. It is also possible to use a conjugate of another represented hapten and a polymer compound as an antigen for labeling competition. For example, a compound in which X, Z, k, m, or n in formula (1) is different from the antigen for preparing an antibody can be used as the antigen for labeling competition. Further, other malathion-like compounds not included in the formula (1) can also be used as antigens for labeling competition.

In the step (c), a sample containing malathion and an enzyme-bound hapten are brought into contact with an antibody-immobilized carrier, and a competition inhibition reaction between malathion and the enzyme-bound hapten is carried out.
A complex of these and the solid-phased carrier is formed. The sample containing malathion 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. The washing solution is, for example, 85 mM containing 60 mM NaCl.
A borate buffer (pH 8.0) can be used.

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

For example, the monoclonal antibody ML of the present invention
Direct competitive inhibition E using T2-23 and MLT40-4
In the LISA method, 5 ng / ml to 1000 ng / ml,
Malathion can be measured preferably in the range of 10 ng / ml to 300 ng / ml, and with MLT45-3, in the direct competitive inhibition ELISA method, malathion can be measured in the range of 50 ng / ml to 5000 ng / ml, preferably 100 ng / ml to 1000 ng / ml. Example 13, FIG. 1).

Further, as described above, the direct competitive inhibition EL
In the ISA method, a hapten different from that for antibody production can be used as a hapten for labeling competition. For example, the monoclonal antibody MLT2-23 of the present invention is a malathion hapten-
5, but when malathion hapten-6 was used as the hapten for labeling competition, 4 ng /
When malathion hapten-4 is used in a concentration range of 100 ng / ml to 100 ng / ml, 10 ng / ml to 1000 n is used.
Reacts with malathion in a concentration range of g / ml. Monoclonal antibody MLT40-4 is also an antibody obtained using malathion hapten-5, but 40 ng when malathion hapten-6 is used as a hapten for labeling competition.
When malathion hapten-4 is used in a concentration range of 10 ng / ml to 300 ng / ml to 300 ng / ml.
Reacts with malathion in a concentration range of ng / ml (Example 14, FIG. 2).

As described above, the monoclonal antibody of the present invention exhibits a unique reactivity in the direct competitive inhibition ELISA method in combination with the hapten for labeling competition. Particularly, when the monoclonal antibody MLT2-23 and malathion hapten-6 / HRP are combined, the reactivity with malathion is highest. Cross Reactivity of Antibodies of the Invention Direct Competition Inhibition ELISA or Indirect Competition Inhibition E
The cross-reactivity of the monoclonal antibody of the present invention can be examined by the LISA method.

For example, MLT2-23 shows 1.6% cross-reactivity with malathion, a metabolite of malathion, but all other compounds only cross-react with less than 1%, indicating high reactivity with malathion. It has specificity (Example 15, Table 28). 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.

[0179]

EXAMPLES Example 1 Synthesis of Malathion Hapten-1

[0180]

Embedded image

Synthesis of (2) 22.2 g (100 mmol) of diphosphorus pentasulfide was dissolved in 100 ml of a toluene solution, and methanol (16 g, 500 g) was dissolved.
mmol) was added dropwise at 50 ° C. and stirred for 3.5 hours. The pH of this solution was adjusted to 6 with a 2N aqueous sodium hydroxide solution while cooling with ice. 34 g (200 mmol) of ethyl bromoacetate was added dropwise at room temperature and stirred at 60 ° C. for 30 minutes and at 70 ° C. for 30 minutes. The toluene layer was collected, dehydrated (anhydrous magnesium sulfate), filtered and concentrated, and then subjected to a silica gel column (n-hexane: ethyl acetate = 49: 1 → 19:
Purification (1 → 9: 1 → 2: 1) afforded 22.4 g (46% yield) of (2) as a clear yellow oil.

[0182]

TABLE 1 _{^{1 H-NMR (CDCl 3)}} 1.21-1.34 (3H, t), 3.53-3.65 (2H, d) 4.13-4.26 (2H, q) ( Synthesis of 3) 1.0 M lithium bis (trimethylsilyl) amide
(LiHMDS: lithium hexamethyldisilazane)
31 ml was cooled to −70 ° C. or less with dry ice-acetone, and 7.5 g (30 mmol) of (2) was added dropwise under a nitrogen stream. After stirring for 1 hour, 6.0 g (30 mm
ol) in THF solution of tert-butyl bromoacetate 5m
1 was added dropwise. After dropping, remove the Dewar bath and remove
Stirred for hours. The reaction solution was concentrated under reduced pressure and subjected to silica gel chromatography (n-hexane: ethyl acetate = 19:
1 → 9: 1), 7.1 g as a clear oil
(Yield 66%) (3) was obtained.

[0183]

¹ H-NMR (CDCl ₃ ) 1.21-1.33 (3H, t), 1.37-1.49 (9H, s) 2.75-3.00 (2H, m), 3.70-3.87 (6H, m) 3.99-4.27 (3H, m) Synthesis of (4) 7.0 g (19 mmol) of (3) was dissolved in 100 ml of dichloromethane, and trifluoroacetic acid was dissolved. (TFA) 5
The mixture was stirred at room temperature for 1 hour, and 5 ml of TFA was further added and stirred for 1.5 hours. The reaction solution was concentrated under reduced pressure, and purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1 → 2: 1 → 1: 1) to obtain 5.06 g (yield 86%) of a transparent oil as a transparent oil (yield: 86%). 4) was obtained.

[0184]

Table 3 ¹ H-NMR (CDCl ₃ ) 1.23-1.35 (3H, t), 2.90-3.18 (2H, m) 3.76-3.87 (6H, d), 4.06-4.29 (3H, m) Synthesis of (5) 2.46 g (8.1 mmol) of (4) was dissolved in 30 ml of tetrahydrofuran (THF), and the solution was cooled to 0 with salt-ice. 10 ml of a THF solution of 0.82 g (8.1 mmol) of N-methylmorpholine (NMM) and then 1.1 g (8.1 mmol) of isobutyl formate in THF
10 ml of the solution was added dropwise. 1.5g after stirring for 10 minutes
(8.1 mmol) of tert- 6-aminohexanoic acid
10 ml of a THF solution of butyl was added dropwise. After stirring under ice-cooling for 5 minutes, the mixture was stirred at 4 ° C. for 20 minutes and further at room temperature for 10 minutes.
After evaporating the solvent, the residue was redissolved with dichloromethane,
After washing with water, dehydration (anhydrous magnesium sulfate), filtration and concentration, crude purification was performed by silica gel chromatography (n-hexane: ethyl acetate = 2: 1 → 1: 1). Synthesis of (6) The crude product containing (5) was dissolved in 100 ml of dichloromethane, 10 ml of TFA was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and silica gel chromatography (n-hexane: ethyl acetate = 2: 1 → 1: 1 →
(Ethyl acetate) to give 1.94 as a clear oil.
g (57% of the total yield from (4)) was obtained.

[0185]

TABLE 4 _{^{1 H-NMR (CDCl 3)}} 1.14-1.71 (9H, overlapping), 2.25-2.41 (2H, t) 2.61-2.94 (2H, m), 3.12-3.31 (2H, m) 3.67-3.86 ( 6H, m), 4.03-4.27 (3H, overlapping) 5.59-5.65 (1H, br) carried Example 2 Synthesis of Malathion Hapten-2

[0186]

Embedded image

Synthesis of (2) 22.2 g (100 mmol) of diphosphorus pentasulfide was dissolved in 100 ml of a toluene solution, and methanol (16 g, 500 g) was dissolved.
mmol) was added dropwise at 50 ° C. and stirred for 3.5 hours. The pH of this solution was adjusted to 6 with a 2N aqueous sodium hydroxide solution while cooling with ice. Tert-butyl bromoacetate 35
g (179 mmol) was added dropwise at room temperature, and the mixture was stirred at 60 ° C. for 1 hour. The toluene layer was collected, dehydrated (anhydrous magnesium sulfate), filtered, concentrated, and then purified by a silica gel column (n-hexane: ethyl acetate = 49: 1 → 9: 1).
18.7 g (34% yield) of (2) was obtained as a yellow transparent oil.

[0188]

Table 5 Synthesis of ¹ H-NMR (CDCl ₃ ) 1.48 (9H, s), 3.41-3.57 (2H, d) 3.68-3.87 (6H, d) (3) 1.0 M lithium bis (trimethylsilyl) amide 1
9 ml was cooled to −70 ° C. or less with dry ice-acetone, and 5.0 g (18 mmol) of (2) was added dropwise under a nitrogen stream. After stirring for 1 hour, 3.1 g (18 mm
ol) in 5 ml of a THF solution of ethyl bromoacetate. After completion of the dropping, the Dewar bath was removed and the mixture was stirred for 1 hour. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, and washed with water. The concentrate is subjected to silica gel chromatography (n
-Hexane: ethyl acetate = 19: 1 → 9: 1) gave 3.5 g (53% yield) of (3) as a clear oil.

[0189]

TABLE 6 _{^{1 H-NMR (CDCl 3)}} 1.20-1.30 (3H, t), 1.48 (9H, s) 2.79-3.05 (2H, m), 3.73- 3.87 (6H, m) 3.97-4.23 (3H, m) Synthesis of (4) 3.4 g (9.5 mmol) of (3) was dissolved in 100 ml of dichloromethane, and trifluoroacetic acid (TFA) was dissolved. )
5 ml was added and the mixture was stirred at room temperature for 3 hours.
Was added and stirred for 40 minutes. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1 → 2: 1) to obtain 2.2 g (yield 76%) of (4) as a transparent oil. Was.

[0190]

Table 7 ¹ H-NMR (CDCl ₃ ) 1.20-1.31 (3H, t), 2.84-3.11 (2H, m) 3.72-3.88 (6H, m), 4.06-4.27 (3H, m) synthesis 1.5g of (5 mmol) of (5) (4) was dissolved in tetrahydrofuran (THF) 30 ml, the solution salt - 0.67 g while cooling with ice (6.6 mmol) of N-methylmorpholine (NMM) in 5 ml of THF and then 0.9
g (6.6 mmol) of isobutyl formate in THF 5
ml was added dropwise. After stirring for 20 minutes, 1.1 g (5.9
5 mmol of tert-butyl 6-aminohexanoate in THF was added dropwise. After stirring for 30 minutes under ice-cooling, the mixture was further stirred at room temperature for 15 minutes. After the solvent was distilled off, the residue was redissolved in dichloromethane, washed with water, dehydrated (anhydrous magnesium sulfate), filtered, concentrated and purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1) to give a transparent oil. 390 mg (16% yield) of (5) were obtained.

[0191]

Table 8 ¹ H-NMR (CDCl ₃ ) 1.20-1.67 (18H, m), 2.16-2.27 (2H, t) 2.81-3.15 (2H, m), 3.20-3.31 (2H, m) 3.71-3.83 (6H, m), 3.99-4.19 (3H, m) 6.49-6.58 (1H, m) ( Synthesis of 6) 390 mg (830 μmol) of (5) was dissolved in 50 ml of dichloromethane, 5 ml of TFA was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and silica gel chromatography (n-hexane: ethyl acetate = 2:
Purification by (1 → 1: 1 → ethyl acetate) yielded 260 mg (76% yield) of (6) as a clear oil.

[0192]

Table 9 ¹ H-NMR (CDCl ₃ ) 1.22-1.74 (9H, m), 2.32-2.43 (2H, t) 2.82-3.14 (2H, m), 3.23-3.33 (2H, m) 3.73-3.84 (6H, m), 3.99-4.20 (3H, m) 6.56-6.77 (1H, m), 8.69-9.25 (1H, br) Example 3 Synthesis of Malathion Hapten-3

[0193]

Embedded image

Synthesis of (2) 22.2 g (100 mmol) of diphosphorus pentasulfide was dissolved in 100 ml of a toluene solution, and methanol (16 g, 500 g) was dissolved.
mmol) was added dropwise at 50 ° C. and stirred for 3.5 hours. The pH of this solution was adjusted to 6 with a 2N aqueous sodium hydroxide solution while cooling with ice. 34 g (200 mmol) of ethyl bromoacetate was added dropwise at room temperature and stirred at 60 ° C. for 30 minutes and at 70 ° C. for 30 minutes. The toluene layer was collected, dehydrated (anhydrous magnesium sulfate), filtered and concentrated, and then subjected to a silica gel column (n-hexane: ethyl acetate = 49: 1 → 19:
Purification (1 → 9: 1 → 2: 1) afforded 22.4 g (46% yield) of (2) as a clear yellow oil.

[0195]

Table 10 ¹ H-NMR (CDCl ₃ ) 1.21-1.34 (3H, t), 3.53-3.65 (2H, d) 4.13-4.26 (2H, q) ( dry ice lithium bis (trimethylsilyl) amide 5.3ml synthesis 1.0M 3) - minus 70 ° C. with acetone
The mixture was cooled to 1.3 g (5.3 mmo) under a nitrogen stream.
1) (2) was added dropwise. After stirring for 1 hour, 2.0 g
2 ml of a THF solution of (5.3 mmol) tert-butyl 11- (bromoacetoxy) undecanoate was added dropwise. After completion of the dropping, the Dewar bath was removed and the mixture was stirred for 1 hour. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, and washed with water. The concentrate is subjected to silica gel chromatography (n
-Hexane: ethyl acetate = 19: 1 → 9: 1) to give 1.3 g (yield 45%) as a yellow transparent oil.
(3) was obtained.

[0196]

[Table 11] ¹ H-NMR (CDCl ₃ ) 1.17-1.47 (28H, m), 2.11-2.23 (2H, t) 2.82-3.11 (2H, m), 3.71-3.86 (6H, m) 4.00-4.27 ( 5H, m) (4) synthesis 1.3g of (2.4 mmol) of (3) was dissolved in dichloromethane 50ml of tri 5 ml of fluoroacetic acid was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1 → 1: 1) to obtain 1.0 g (88% yield) of (4) as a yellow transparent oil. I got

[0197]

Table ^{1 1} H-NMR (CDCl ₃ ) 1.17 to 1.40 (15H, m), 1.50 to 1.70 (4H, m) 2.24-2.40 (2H, t), 2.79-3.10 (2H, m) 3.68-3.86 (6H, m), 3.99-4.26 (5H, m) Example 4 Synthesis of Malathion Hapten-4

[0198]

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Synthesis of (2) 22.2 g (100 mmol) of diphosphorus pentasulfide was dissolved in 100 ml of a toluene solution, and methanol (16 g, 500 g) was dissolved.
mmol) was added dropwise at 50 ° C. and stirred for 3.5 hours. The pH of this solution was adjusted to 6 with a 2N aqueous sodium hydroxide solution while cooling with ice. 34 g (200 mmol) of ethyl bromoacetate was added dropwise at room temperature and stirred at 60 ° C. for 30 minutes and at 70 ° C. for 30 minutes. The toluene layer was collected, dehydrated (anhydrous magnesium sulfate), filtered and concentrated, and then subjected to a silica gel column (n-hexane: ethyl acetate = 49: 1 → 19:
Purification (1 → 9: 1 → 2: 1) afforded 22.4 g (46% yield) of (2) as a clear yellow oil.

[0200]

Table 13 ¹ H-NMR (CDCl ₃ ) 1.21-1.34 (3H, t), 3.53-3.65 (2H, d) 4.13-4.26 (2H, q) ( Synthesis of 3) 1.0 M lithium bis (trimethylsilyl) amide 8
The mixture was cooled to −70 ° C. or lower with dry ice-acetone, and 1.96 g (8 mmol) of (2) was added dropwise under a nitrogen stream. After stirring for 1 hour, 5 ml of a THF solution of 2.35 g (8 mmol) of tert-butyl 6- (bromoacetoxy) hexanoate was added dropwise. After completion of the dropping, the Dewar bath was removed and the mixture was stirred for 1 hour. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, and washed with water. The concentrate was roughly purified by silica gel chromatography (n-hexane: ethyl acetate = 19: 1 → 9: 1). Synthesis of (4) The crude product containing (3) was dissolved in 50 ml of dichloromethane, 5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure and silica gel chromatography (n-hexane: ethyl acetate = 4: 1 →
Purification in 1: 1) yielded 0.54 g as a clear oil.
(4) of (total yield from (2) 20%) was obtained.

[0201]

Table ^{1 1} H-NMR (CDCl ₃ ) 1.20-1.50 (5H, overlap), 1.58-1.76 (4H, m) 2.31-2.43 (2H, t), 2.82-3.11 (2H, m) 3.71-3.88 (6H, m), 4.03-4.27 (5H, duplicate) Example 5 Synthesis of Malathion Hapten-5

[0202]

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[0203] After addition of synthetic sulfate 10ml of (2) in diethyl ether 100ml, was added benzyl alcohol 100ml with stirring. After distilling off diethyl ether, 13.4 g (100 m
l) L-Aspartic acid was added in small portions. 1 at room temperature
After stirring for 200 days, ethanol 200 ml and pyridine 50 ml
Was added and left in the refrigerator overnight. The precipitated crystals were collected by filtration, washed with ether and water, and dried, to give 11 g.
(Yield 49%) (2) was obtained. Synthesis of (3) 11 g (49 mmol) of (2) was added to 600 ml of water and dissolved at 80 ° C. When heating was stopped and cooled to 60 ° C., 10.5 g (125 mmol) of sodium hydrogen carbonate and 12 g (70 mmol) of benzyloxycarbonyl chloride (Z-Cl) were added, and the mixture was stirred for 3 hours. After washing with diethyl ether, the mixture was adjusted to pH 1 with concentrated hydrochloric acid while cooling with ice, and extracted with ethyl acetate. After dehydration (anhydrous sodium sulfate), filtration and concentration, purification by a silica gel column (n-hexane: ethyl acetate = 9: 1 → 1: 2) gave 4.5 g (yield 25%) of (3) as white crystals. Obtained.

[0204]

Table 15 ¹ H-NMR (CDCl ₃ ) 2.80-3.17 (2H, m), 4.54-4.76 (1H, m) 5.11 (4H, s), 5.74- 5.89 (1H, d) 7.31 (10H, s), 9.38-10.02 (1H, br) Synthesis of (4) 4.5 g (12.6 mmol) of (3) in ethanol 1
The mixture was dissolved in 50 ml, and 15 ml of a 1.0 M solution of hydrogen chloride in diethyl ether was added, followed by reflux for 30 minutes. Further, 15 ml of the solution was added, and the mixture was refluxed for 30 minutes.
Was added and refluxed for 30 minutes. The reaction solution was filtered and concentrated, and then purified by a silica gel column (n-hexane: ethyl acetate = 4: 1 → 2: 1) to obtain 1.8 g (yield 37%) of (4) as a transparent oil. .

[0205]

Table 16 ¹ H-NMR (CDCl ₃ ) 1.13-1.27 (3H, t), 2.83-3.14 (2H, m) 4.08-4.22 (2H, m), 4.58-4.69 (1H, m) 5.11 (4H, s), 5.69-5.81 (1H, d) 7.34 (10H, s) Synthesis of 8.6 g of ( 5) 22 mmol) of (4) in ethanol 200
Then, 2.0 g of 5% palladium carbon was added, and the mixture was stirred at room temperature for 1 hour under a hydrogen atmosphere. The reaction solution was filtered and concentrated to obtain (5). Synthesis of (6) 4.2 g of crude carboxylic acid (5) was added to 300 ml of water and dissolved at 80 ° C. When heating was stopped and cooled to 60 ° C., 0.88 g (10.5 g) of sodium hydrogencarbonate was added.
3.0 g (17.6 mmol) after the addition of
Of Z-Cl was added. 1.3 g (15.5 mmo)
1) Sodium bicarbonate was added and stirred for 3 hours. After washing with diethyl ether, pH was adjusted with concentrated hydrochloric acid while cooling with ice.
Was adjusted to 2 and extracted with ethyl acetate. After dehydration (anhydrous sodium sulfate), filtration and concentration, the silica gel column (n-
Purification with hexane: ethyl acetate = 1: 1 → 1: 2) gave 1.63 g (total yield 50% from (4)) of (6) as white crystals.

[0206]

Table 17 ¹ H-NMR (CDCl ₃ ) 1.11-1.30 (3H, t), 2.74-3.12 (2H, m) 4.06-4.25 (2H, m), 4.55-4.70 (1H, m) 5.10 (2H, s), 5.84-5.99 (1H, d) 7.33 (5H, s), 8.49-9.26 ( 1H, br) Synthesis of (7) 1.63 g (5.5 mmol) of (6), 224 mg
(1.8 mmol) N, N-dimethylaminopyridine (DMAP), 426 mg (1.8 mmol) camphorsulfonic acid (CSA), 1.4 g (7.4 mmol)
l) Tert-butyl 6-hydroxyhexanoate was added to 50 ml of tetrahydrofuran (THF). 1.7
g (8.3 mmol) of N, N-dicyclohexylcarbodiimide (DCC) in 10 ml of THF was added dropwise. After stirring at room temperature for 1 day, the reaction solution was filtered and concentrated, and then silica gel column (n-hexane: ethyl acetate = 4: 1 → 2:
Purification in 1) gave 2.16 g (84% yield) of (7) as a clear oil.

[0207]

[Table 18]¹ H-NMR (CDCl_Three1.20-1.46 (14H, duplication), 1.51-1.69 (4H, m) 2.15-2.26 (2H, t), 2.77-3.07 (2H, m) 4.02-4.26 (4H, m), 4.57-4.67 (1H, m) 5.12 (2H, s), 5.73-5.82 (1H, d) 7.37 (5H, s)Synthesis of (8) 2.16 g (4.6 mmol) of (7) in ethanol 2
Dissolve in 00ml and 300mg 5% palladium carbon
Was added and the mixture was stirred at room temperature for 1 hour under a hydrogen atmosphere. Reaction liquid
Was filtered and concentrated to obtain (8). Without purification
Used for reaction. Synthesis of (9) Crude amine (8) and 0.8 g (5 mmol) of chlorothio
Dimethyl phosphate, 0.7 g (5 mmol) of potassium carbonate
Was added to 80 ml of acetonitrile and stirred at 50 ° C for 1 hour.
Stirred. After filtration and concentration, a silica gel column (n-hexa
And purified with ethyl acetate = 4: 1).
(9) of (total yield 100% from (7)) was obtained.

[0208]

Table 19 ¹ H-NMR (CDCl ₃ ) 1.23-1.49 (14H, duplication), 1.54-1.71 (4H, m) 2.15-2.27 (2H, t), 2.71-3.00 (2H, m) 3.63-3.75 (6H, m), 4.00-4.11 (5H, t) 4.17-4.34 (3H, duplicate) ( Synthesis of 10) 1.71 g (4.6 mmol) of (9) was dissolved in 100 ml of dichloromethane, and trifluoroacetic acid (TF
A) 5 ml was added and the mixture was stirred at room temperature for 1 hour. Further TFA
5 ml was added and stirred for 1 hour. The reaction solution is concentrated under reduced pressure and subjected to silica gel chromatography (n-hexane:
Purification with ethyl acetate = 4: 1 → 1: 1) gave 1.44 g (99% yield) of (10) as a clear oil.

[0209]

Table 20 ¹ H-NMR (CDCl ₃ ) 1.22-1.33 (3H, t), 1.35-1.49 (2H, m) 1.59-1.75 (4H, m), 2.31-2.42 (2H, t) 2.71-3.00 ( 2H, m), 3.62-3.76 (6H, m) 4.01-4.34 (5H, overlapping) carried Example 6 Synthesis of Malathion Hapten-6

[0210]

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Synthesis of (2) 58.8 g (400 mmol) of L-glutamic acid and 4
8 g (440 mmol) of benzyl alcohol to 60%
In addition to sulfuric acid (39.2 g of concentrated sulfuric acid + 26 ml of water), 70
Stir at 45 ° C. for 45 minutes. After concentration at 70 ° C. for 3 hours, 67.
The mixture was poured into 200 ml of cold water containing 2 g (800 mmol) of sodium hydrogen carbonate with stirring, 150 ml of cold water was added, and the mixture was allowed to stand. The precipitated crystals were separated by filtration and washed with water. The crystals were recrystallized from water to give 35 as white crystals.
g (37% yield) of (2) was obtained. Synthesis of (3) 4.74 g (20 mmol) of (2) was added to 300 ml of water and dissolved at 80 ° C. Stop heating and 60 ℃
When cooled down, 4.2 g (50 mmol) of sodium hydrogen carbonate and 4.1 g (24 mmol) of benzyloxycarbonyl chloride (Z-Cl) were added, and the mixture was stirred for 3 hours. After washing with diethyl ether, the mixture was adjusted to pH 1 with concentrated hydrochloric acid while cooling with ice, and extracted with ethyl acetate. After dehydration (anhydrous sodium sulfate), filtration and concentration, purification by a silica gel column (n-hexane: ethyl acetate = 1: 2)
6.4 g (yield 86%) of (3) was obtained as white crystals.

[0212]

Table 21 ¹ H-NMR (CDCl ₃ ) 1.93-2.37 (2H, m), 2.37-2.61 (2H, m) 4.30-4.50 (1H, m), 5.10 (4H, s) 5.40-5.53 (1H, d), 7.33 (10H, s) Synthesis of (4) 6.0 g (16 mmol) of (3) was treated with ethanol 150
Then, 1 ml of a 1.0 M hydrogen chloride diethyl ether solution was added, and the mixture was refluxed for 30 minutes. 3m of the solution
After 1 minute was added and refluxed for 30 minutes, 3 ml of the solution was added three times and refluxed for 1 hour. The reaction solution was filtered and concentrated, and then concentrated on a silica gel column (n-hexane: ethyl acetate = 9: 1 →
4: 1 → 2: 1) to give a clear oil.
2 g (yield 65%) of (4) was obtained.

[0213]

Table 22 ¹ H-NMR (CDCl ₃ ) 1.15-1.33 (3H, q), 1.87-2.32 (2H, m) 2.33-2.58 (2H, m), 4.07-4.25 (2H, m) 4.32-4.48 (1H, m), 5.10 (4H, s) 5.32-5.45 (1H, d), 7.34 ( 10H, s) Synthesis of (5) 16 g (40 mmol) of (4) was treated with 250 ml of ethanol.
Then, 2.5 g of 5% palladium carbon was added, and the mixture was stirred at room temperature for 1 hour under a hydrogen atmosphere. The reaction solution was filtered and concentrated to obtain (5). 8.6 g of crude yield (crude yield 1
22%). Synthesis of (6) 8.6 g of crude carboxylic acid (5) was added to 300 ml of water and dissolved at 80 ° C. When heating was stopped and the temperature was cooled to 60 ° C., 8.4 g (100 mmol) of sodium hydrogen carbonate and 8.2 g (48 mmol) of Z-Cl were added, and the mixture was stirred for 3 hours. After washing with diethyl ether, the mixture was adjusted to pH 1 with concentrated hydrochloric acid while cooling with ice, and extracted with ethyl acetate. After dehydration (anhydrous sodium sulfate), filtration and concentration, a silica gel column (n-hexane: ethyl acetate = 1: 1 →
1: 2), 5.4 g as white crystals
(6) of (Total yield 43% from (4)) was obtained.

[0214]

Table 23 ¹ H-NMR (CDCl ₃ ) 1.17-1.35 (3H, q), 1.88-2.30 (2H, m) 2.33-2.57 (2H, m), 4.06-4.26 (2H, m) 4.34-4.49 (1H, m), 5.11 (2H, s) 5.38-5.51 (1H, d), 7.34 ( 5H, s) Synthesis of (7) 1.0 g (3.2 mmol) of (6), 130 mg
(1.1 mmol) N, N-dimethylaminopyridine (DMAP), 250 mg (1.1 mmol) camphorsulfonic acid (CSA), 600 mg (3.2 mmol)
l) Tert-butyl 6-hydroxyhexanoate was added to 15 ml of tetrahydrofuran (THF). 1.0
g (4.9 mmol) of N, N-dicyclohexylcarbodiimide (DCC) in 15 ml of THF was added dropwise. After stirring at room temperature for 20 hours, the reaction solution was filtered and concentrated, and then silica gel column (n-hexane: ethyl acetate = 4: 1 →
2: 1), 940 mg as a clear oil
(7) of (yield 60%) was obtained.

[0215]

Table 24 ¹ H-NMR (CDCl ₃ ) 1.21-1.47 (14H, overlap), 1.52-1.70 (4H, m) 1.90-2.06 (1H, m), 2.16-2.27 (3H, m) 2.34-2.47 (2H, m), 4.00-4.10 (2H, t) 4.14-4.26 (2H, q), 4.33-4.44 (1H, m) 5.11 (2H, s), 5.37-5.46 (1H, d) 7.36 (5H, s) Synthesis of 5.9 g of ( 8) 12.3 mmol) of (7) in ethanol 1
Dissolve in 50 ml, 5% palladium carbon 600mg
Was added and stirred at room temperature for 30 minutes under a hydrogen atmosphere. The reaction solution was filtered and concentrated to obtain (8). This was used for the next reaction without purification. Synthesis of (9) Crude amine (8) and 2.0 g (12, 4 mmol) of dimethyl chlorothiophosphate, 1.7 g (12.3 mmol)
Of potassium carbonate in 100 ml of acetonitrile
Stirred at 0 ° C. for 1 hour. After filtration and concentration, the residue was purified by a silica gel column (n-hexane: ethyl acetate = 4: 1) to obtain 4.7 g (total yield 100% from (7)) of (9).

[0216]

Table 25 ¹ H-NMR (CDCl ₃ ) 1.21-1.47 (14H, overlap), 1.54-1.70 (4H, m) 1.83-2.15 (2H, m), 2.18-2.26 (2H, t) 2.37-2.48 (2H, m), 3.60-3.73 (6H, m) 3.93-4.11 (3H, duplication), 4.15-4.27 (2H, q) Synthesis of (10) 4.7 g (12.3 mmol) of (9) was dissolved in 80 ml of dichloromethane, and trifluoroacetic acid (TFA) was dissolved.
5 ml was added and the mixture was stirred at room temperature for 1 hour. 5 ml of TFA was added, and the mixture was further stirred for 1 hour. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1 → 1: 1) to obtain 3.5 g (87% yield) of (10) as a transparent oil. Was.

[0219]

Table 26 ¹ H-NMR (CDCl ₃ ) 1.24 to 1.36 (3H, t), 1.36-1.50 (2H, m) 1.60-1.74 (4H, m), 1.83-2.20 (2H, m) 2.34-2.50 ( 4H, overlapping), 3.61-3.76 (6H, m) 3.94-4.28 (5H, m) carried Example 7 Preparation of Antigen for Immunization and Antigen for Screening Malathion haptens-1 to 6 prepared in Examples 1 to 6 were used as haptens, and 3.6 μmol of each was DMS.
O was dissolved in 100 μl. To these solutions, a solution of N-hydroxysuccinimide (16 μmol) dissolved in 10 μl of DMSO was added. Next, a solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5.6 μmol) dissolved in 20 μl of DMSO was added, and the mixture was reacted at room temperature for 1.5 hours.
A solution prepared by dissolving 10 mg each of BSA or KLH in 500 μl of 85 mM borate buffer (pH 8.0) was added to these reaction solutions, and the reaction was performed again at room temperature for 1.5 hours. After completion of the reaction, Dulbecco's phosphate buffer (hereinafter referred to as
Dialysed with “PBS (−)”), a conjugate of malathion hapten-1 to 6 and KLH (hereinafter, referred to as “malathion hapten-1 to 6 / KLH”), malathion hapten-1 to 6 and BSA And a conjugate with
"Malathion hapten-1 to 6 / BSA")
Was prepared respectively. Example 8 Immunization Balb / c mice (7 weeks old, female) were used for immunization. 100 µg of malathion hapten-1 to 6 / KLH prepared in Example 7 was dissolved in 50 µl of PBS (-). This was emulsified and mixed with an equal volume of Freund's complete adjuvant, and then inoculated intraperitoneally into mice. One month later,
A booster immunization was performed with a quarter of the initial immunization amount, and 5 weeks later, the same immunization as the booster immunization was finally performed. Example 9 Preparation of Hybridoma Cell fusion for obtaining a hybridoma producing a monoclonal antibody was performed using the spleen cells of a mouse 3 days after the final immunization in Example 8.

From the excised spleen, spleen cells were taken out in DMEM and washed three times with DMEM. After washing, the cells were mixed with mouse myeloma cells P3-X63-Ag8.653 at a cell ratio of 5: 1 (spleen cells: myeloma cells), and centrifuged (1,200 rpm, 5 minutes). The cell sediment was collected. To this cell sediment, 50% polyethylene glycol (molecular weight: 150
0) 1 ml was added to fuse the cells. DMEM 10m
1 was gradually added, and 1 ml of fetal bovine serum (hereinafter, referred to as “FBS”) was further added to stop the cell fusion. The fused cells were prepared by adding 10% FBS, hypoxanthine (100 μM), aminopterin (0.4 μM) to DMEM.
μM) and thymidine (16 μM), and suspended in a HAT medium, and dispensed at 2 × 10 ⁵ cells / well into each well of a 96-well microplate. After culturing at 37 ° C. in the presence of 5% carbon dioxide for 10 to 14 days, colonies of hybridomas were formed in each well. Example 10 Preparation of Monoclonal Antibody Among the wells in which colonies were formed in Example 9, those producing a monoclonal antibody that reacts with malathion in their culture supernatant were subjected to indirect competitive inhibition E as follows.
Screening was performed using the LISA method.

First, malathion hapten-1 to 6 / BSA (4 μg / ml) dissolved in PBS (−) was added to 96
100 wells per well of a microtiter plate
The mixture was added at μl / well, and allowed to stand at 4 ° C. overnight to immobilize the solid phase. Next, 300 μl / well of blocking buffer (85% with 1% BSA and 60 mM NaCl) was added.
mM borate buffer (pH 8.0)) and blocked for 1 hour at room temperature. Add diluent A (150m
85 mM borate buffer (pH
The malathion solution serially diluted to an appropriate concentration in 8.0)) was added at 50 μl / well. Then, immediately dilute the culture supernatant to the appropriate concentration with the same diluent, and add 50
The mixture was added at μl / well, mixed, and reacted at room temperature for 1 hour. Appropriate concentration refers to this EL without malathion added.
This is the antibody concentration at which the absorbance of the ISA method can be の of that at the time of antibody saturation.

Wash solution (8 with 60 mM NaCl added)
After washing three times with 5 mM borate buffer (pH 8.0), a diluent B for secondary antibody (0.3% BSA and 60 mM
85 mM borate buffer (pH
8.0)) HRP-conjugated anti-mouse I diluted 1000-fold
A gG antibody (manufactured by Capel) was added at 100 μl / well, and reacted at room temperature for 1 hour. After washing 3 times again, HRP
Color was developed for 10 minutes with a substrate solution (0.1 M sodium acetate buffer (pH 5.5) supplemented with 100 μg / ml 3,3′5,5′-tetramethylbenzidine and 0.006% hydrogen peroxide), After stopping the reaction with 1N sulfuric acid, 450 nm
Was measured for absorbance.

After qualitatively screening the reactivity of each well, the hybridomas in the wells that showed reactivity with malathion were cloned by the limiting dilution method. In this manner, three monoclonal antibody-producing cells (MLT2-2) that produce antibodies reactive to malathion were prepared.
3, MLT40-4, MLT45-3). All of these three strains are hybridomas obtained using malathion hapten-5 / BSA as an antigen.

Further, these monoclonal antibody-producing cells were cultured in DMEM supplemented with 10% of FBS, and the resulting culture supernatants were used as monoclonal antibodies. Since then
Each monoclonal antibody has the same name as the monoclonal antibody-producing cells that produce them.

The subclasses of the prepared monoclonal antibodies are shown in Table 27 below.

[0224]

Table 27: Subclass of the prepared monoclonal antibody Monoclonal antibody Subclass MLT2-23 IgG2a MLT40-4 IgG1 MLT45-3 IgG1 Of the obtained monoclonal antibodies, MLT2-23 was deposited on January 22, 1999, and deposited under FERM. P-171
At 57, the Institute of Biotechnology, Institute of Industrial Science (¥ 305-00
46 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture). Example 11 Purification of Monoclonal Antibody Each monoclonal antibody obtained in Example 10 was purified. First, Balb / c mice were inoculated with each monoclonal antibody-producing cell intraperitoneally to obtain ascites. MLT2-
The ascites from the 23-producing cells had 35% saturation, and the MLT40-4 producing cells and the MLT45-3
Ammonium sulfate was added to the ascites from the producing cells so as to be 33% saturated, and each was stirred at 4 ° C. and then centrifuged to obtain a precipitate. After these precipitates were solubilized with PBS (-), they were further dialyzed against PBS (-) for 2 nights to obtain purified antibodies. The purity of these antibodies was at least 90% or more as a result of measurement by SDS-polyacrylamide gel electrophoresis. Example 12 Conjugate of Malathion Hapten and HRP
Each of the prepared malathion haptens-4 to 6 was 1.25 μmo
was dissolved in 50 μl of DMSO. To these solutions, N
-Hydroxysuccinimide (5.5 μmol).
Solution in 4 μl of DMSO, then 1-ethyl-
3- (3-dimethylaminopropyl) carbodiimide.
A solution of hydrochloride (1.9 μmol) dissolved in 6.8 μl of DMSO was added, and the mixture was reacted at room temperature for 1 hour. 40 μl of 1 M sodium bicarbonate was added to these reaction solutions,
Further, a solution in which HRP (5 mg) was dissolved in 250 μl of distilled water was added, and reacted at room temperature for 3 hours. The low-molecular-weight compound was removed from the obtained reaction solution using a gel filtration column (Sephadex G-25), and malathion hapten-4 was removed.
-6 and HRP (hereinafter referred to as "malathion hapten-4 to 6 / HRP"). Example 13 Malachi in Direct Competition Inhibition ELISA
Reactivity of Monoclonal Antibody to On The reactivity of the obtained monoclonal antibody to malathion was examined using a direct competitive inhibition ELISA method.

MLT2-23 purified in Example 11
LT40-4 has a concentration of 3 μg / ml, and MLT45-3
Was dissolved in PBS (−) at a concentration of 5 μg / ml. These were added to each well of a 96-well microtiter plate at 100 μl / well, and allowed to stand at 4 ° C. overnight for immobilization. Then, it was replaced with 300 μl / well of a blocking buffer, and blocked at room temperature for 1 hour. On the other hand, diluent C (0.3% BSA and 150 mM
100 mM phosphate buffer containing NaCl (pH
7.0)), and malathion diluted serially to an appropriate concentration, and malathion hapten, also diluted to an appropriate concentration.
A mixture of 5 / HRP was prepared. The appropriate concentration means that the absorbance of this ELISA method under the condition where malathion is not added is HR
It is the concentration of the HRP-bound hapten that can show half of the P-bound hapten saturation. These were added at 100 μl / well to each of the previously blocked wells, and reacted at room temperature for 1 hour. After washing three times with the washing solution, color was developed in the same manner as in the indirect competitive inhibition ELISA shown in Example 10,
The absorbance at 450 nm was measured.

The results are shown in FIG. Here, the inhibition rate was calculated by the following equation.

[0227]

Embedded image

As shown in FIG. 1, the monoclonal antibodies MLT2-23 and MLT40-4 were 10 ng / ml.
To 300 ng / ml, monoclonal antibody MLT45
-3 reacted with malathion in a concentration range of 100 ng / ml to 1000 ng / ml. Therefore, it was revealed that the monoclonal antibodies MLT2-23 and MLT40-4 show high reactivity to malathion. Example 14 Direct competition using a heterologous hapten
Monoclonal antibody MLT in inhibition ELISA
Response of 2-23 and MLT40-4 to malathion
For the monoclonal antibodies MLT2-23 and MLT40-4 showing high reactivity to malathion in Example 13, changes in reactivity to malathion were examined by changing the HRP-binding hapten used in the direct competitive inhibition ELISA method. Specifically, the same procedure as in Example 13 was carried out except that malathion hapten-6 / HRP or malathion hapten-4 / HRP was used instead of malathion hapten-5 / HRP as the HRP-bound hapten.

The results are shown in FIG. As shown in FIG. 2, the reactivity of each antibody to malathion varied depending on the type of HRP-binding hapten used. That is, the monoclonal antibody MLT2-23 is different from the result of Example 13 in the concentration range of 4 ng / ml to 100 ng / ml when malathion hapten-6 / HRP is used, and 10 ng when malathion hapten-4 / HRP is used. /
Reacted with malathion in a concentration range from ml to 1000 ng / ml. On the other hand, monoclonal antibody MLT40-4
Is 40 when malathion hapten-6 / HRP is used.
10 ng / m when malathion hapten-4 / HRP is used in a concentration range of ng / ml to 1000 ng / ml.
It reacted with malathion in a concentration range of 1 to 300 ng / ml.

As described above, the monoclonal antibody MLT2-
23 and MLT40-4 all showed unique reactivity in combination with the hapten for labeling competition. In particular, when the monoclonal antibody MLT2-23 and malathion hapten-6 / HRP were combined, it was revealed that the reactivity with malathion was the highest. Example 15 Cross-over of monoclonal antibody MLT2-23
Reactivity From the results of Example 14, cross-reactivity with malathion metabolites and related compounds was examined using a combination of the monoclonal antibody MLT2-23 and malathion hapten-6 / HRP that showed the highest reactivity to malathion.

The results are shown in Table 28 below.

[0232]

[Table 28]

In Table 28, IC50 indicates the reaction
% The concentration of the compound that inhibits, and the cross-reactivity is the IC50 value of malathion / IC50 value of each compound x 100. From Table 28, it can be seen that the monoclonal antibody MLT2-23
Is 1.6% with malaoxon, a metabolite of malathion
Despite showing cross-reactivity, all compounds cross-reacted less than 1% with all other compounds, revealing high specificity to malathion.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows monoclonal antibodies MLT2-23 and MLT40- of the present invention obtained by direct competitive inhibition ELISA.
4 shows the reactivity of MLT4 and MLT45-3 to malathion.

FIG. 2 shows a heterologous direct competitive inhibition ELISA
The monoclonal antibody MLT2-23 of the present invention by the method A
And MLT40-4 shows reactivity to malathion.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12P 21/08 G01N 33/53 G 4H050 G01N 33/53 33/531 A 33/531 33/577 B 33/577 A01N 57 / 12Z // A01N 57/12 C12N 5/00 B (C12P 21/08 C12R 1:91) (72) Inventor Shunichi Takewaki 1-27-14 Hamamatsucho, Minato-ku, Tokyo Research on environmental immunity technology Institution (72) Inventor Shiro Miyake 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental Immunity Technology (72) Inventor Yuki 1-27-14 Hamamatsucho, Minato-ku, Tokyo Co., Ltd. Within the Institute for Environmental Immunity Technology (72) Inventor Katsuya Oide 1-27-14 Hamamatsucho, Minato-ku, Tokyo F-term within the Institute for Environmental Immunity Technology 4B024 AA11 BA53 GA03 4B064 AG26 AG27 CA10 CA20 CC24 DA13 4B065 AA90X AA91X AB02 AB05 AC14 BA08 CA25 CA46 4H011 AC01 BB17 4H045 AA11 BA10 CA40 DA75 DA76 EA50 FA72 FA74 4H050 AA01 AA03 AB20

Claims (11)

[Claims] (1) The following formula (1): [In the formula (1), X is NH or S; Z is NH or O; k is 0 or 1; m is an integer of 0 to 2; Which is an integer]. 2. In the formula (1), X is NH, Z is O, k is 0, m is 2, and n is 5
The compound of claim 1, wherein 3. A conjugate of the compound according to claim 1 or 2 with a polymer compound or a labeling substance. 4. An antigen is prepared by binding the compound according to claim 1 or 2 with a polymer compound, and the antigen is used to obtain the following formula (2): A method for producing an antibody or a fragment thereof reactive to a compound having a structure represented by the formula (2), characterized by producing an antibody reactive to a compound having a structure represented by the formula: 5. An antibody or a fragment thereof, which is produced by using the conjugate according to claim 3 as an antigen, and which is reactive with the compound of the formula (2). 6. The antibody or fragment thereof according to claim 5, which is a monoclonal antibody. 7. The antibody or fragment according to claim 5, which is the monoclonal antibody MLT2-23 or a fragment thereof. A hybridoma that produces the antibody or fragment according to any one of claims 5 to 7. 9. The hybridoma according to claim 8, which has been deposited under accession number FERM P-17157. 10. An immunoassay for a compound represented by the formula (2), comprising using the antibody or fragment according to any one of claims 5 to 7. 11. The method according to claim 11, further comprising using the compound according to claim 1 or 2 or the conjugate according to claim 3.
The immunological measurement method according to claim 10.