KR100644205B1 - Haptens and antibodies for the class-specific determination of organophosphorus pesticides and the immunoassay method using the same - Google Patents

Abstract

Haptens and antibodies for class-specific determination of organophosphorus pesticides and the immunoassay method by using the same haptens and antibodies are provided to improve determination accuracy and efficiency, so that remained pesticides are efficiently analyzed. The hapten having the formula and its N-succinimidyl ester derivative are provided, wherein R1 is CH3, CH2CH3 or OCH3, and R2 is CO2H or CONH(CH2)2CO2H. The monoclonal antibody for class-specific determination of organophosphorus pesticides having an O,O-diethyl group is obtained by conjugating the hapten with a carrier protein, wherein the organophosphorus pesticides having the O,O-diethyl group are phosphorothioates or phosphorodithioates, wherein the monoclonal antibody is obtained by immunizing a mouse with the conjugate of hapten and carrier protein, extracting the spleen from the immunized mouse, hybridizing the spleen cells with myeloma cells, and cloning the hybrids.

Description

Haptens and antibodies for the class-specific determination of organophosphorus pesticides and the immunoassay method using the same}

The present invention relates to a hapten (hapten) and antibodies for the total detection of organophosphorus pesticides and an immunoassay method using the same.

More specifically, the present invention provides a monoclonal antibody obtained from an hapten which can collectively detect an organophosphorus pesticide having a common O, O-diethyl group and an immunogen conjugated with a carrier protein to the hapten. It relates to a direct and indirect competition immunoassay using the same.

Pesticides have resulted in improved agricultural productivity and improved quality of agricultural products, but contamination of food and natural environment due to misuse of pesticides is a serious threat to our health. As a result, there is a need for a new way of understanding pesticides' risks and methods for efficient analysis of residual pesticides.

Conventional pesticide analysis methods have mainly used gas chromatography (GC) and high performance liquid chromatography (HPLC). However, GC and HPLC have the advantage of being very sensitive, accurate, and capable of analyzing multiple components at the same time. However, GC and HPLC require a pretreatment process before analysis, which requires a long time and requires professional personnel and expensive equipment.

In order to solve these shortcomings, the development of more efficient, simple and economical pesticide assays has been actively attempted. Among the new pesticide assays, the most anticipated pesticide detection method is immunoassay. Immunoassays have high sensitivity and specificity due to the high affinity and specificity of the antibodies, and thus, the predominant process is rapidly increasing and the cost is low. Among immunoassays, an enzyme-linked immunosorbent assay (ELISA), which immobilizes an antigen or an antibody on a solid surface and quantifies a substance by measuring an antigen-antibody reaction by an enzyme reaction, is a bio-related substance. It has been widely used for the analysis of pesticides and has been used for the analysis of pesticides in the 80s (Vanderlaan et al., 1991).

Antibodies used in immunoassays are polyclonal antibodies (pAbs) and monoclonal antibodies (monoclonal antibodies, mAbs). Polyclonal antibodies refer to a collection of various antibodies produced from various B-cell clones. The method of obtaining antibodies is relatively simple since they are generated by an immune response occurring spontaneously in the animal body against the injected antigen. However, polyclonal antibodies are obtained from the sera of immunized animals and vary in sensitivity and specificity depending on the type of animal and the time of blood collection in the immunization process, and have a disadvantage in that immunization takes a long time. Monoclonal antibodies are antibodies produced from single B-cell clones, which have the same affinity and specificity, which increases their use in agrochemical immunoassays due to the constant cross-reactivity to substances and the availability of permanent antibodies. It is becoming.

Another approach to improving the efficiency of pesticide analysis has been a method called class-specific detection of pesticides. If the total amount of each class can be identified prior to analysis of the individual pesticides, it is possible to determine whether the analysis of the individual pesticides of the class is necessary, and thus it is possible to exclude a large number of samples that are not necessary for analysis. This approach has been tried in several ways, one of which is an enzyme assay based on the principle that cholinesterase is inhibited by organophosphorus and carbamate pesticides and the degree of inhibition depends on the concentration of the pesticide (Mendoza et al., 1968). Guilbault et al., 1970; Alfthan et al., 1989; Argauer & Brown, 1994; Evtyugin et al., 1997). However, the enzyme analysis method has a problem that the quantitative analysis is impossible because the degree of inhibition varies greatly depending on the pesticide.

For the overall analysis of organophosphorus pesticides, a method using an immunoassay (immunoassay) has been attempted. There are hundreds of organophosphorus pesticides, but they have a common structure of (EtO) ₂ P (= S)-, such as parathion, diazinon, and disulfone, that is , phosphorothioate and phosphate having O, O- diethyl groups in common. A group of porodithioates and a group having a structure of (MeO) ₂ P (= S)-in common, such as phenytrothion, fenthion, and dimethoate, that is, phosphorothioate having O, O- dimethyl group in common And phosphorodithioates account for the majority.

Figure 1. Two major classes of organophosphorus pesticides.

Immunoassay is a method of quantifying analyte by measuring the reaction (binding) between an antibody and an antigen (analyte) by obtaining an antibody of an analyte. Therefore, if an antibody to these common structures can be obtained, each class can be analyzed collectively. Immunoassay can be established. In other words, if a compound having only a common structure is synthesized and an antibody is obtained therefrom, the antibody will show affinity for the entire pesticide belonging to the class, and thus the antibody can be used for the overall analysis of the class.

In order to establish an immunoassay based on this principle, an antibody must be obtained by first synthesizing a substance having these common structures and injecting it into an animal. However, in the case of a substance having a low molecular weight, immunization is not possible as it is, so the structure is similar, and a substance having an atomic group that can be covalently bonded to a protein, hapten, should be synthesized by binding to a protein and obtaining an antibody using this as an immunogen. In the case of pesticides, the common structures are small in mass, so hapten must be synthesized and attached to proteins and used as immunogens.

Detected immunoassays by class of organophosphorus pesticides using polyclonal and monoclonal antibodies have already been tried (Sudi & Heeschen, 1988; Banks et al., 1998; Johnson et al., 1998; Alcocer et al., 2000; Jang et al., 2002), Reference may be made to References 1 to 5 in this regard. However, the overall analysis of these organophosphorus pesticides immunoassays have been developed by obtaining antibodies against the common structure of organophosphorus pesticides. It is hard to see that. For example, the immunoassay developed by Jang et al. (2002) showed a much smaller variation in the response of antibodies to organophosphorus pesticides than other immunoassays, but an exceptionally low response to diazinon was observed. . This may be because diazinone has a fairly large isopropyl group at the meta position of the benzene ring, unlike other organophosphorus pesticides.

Reference 1; Sudi J & Heeschen W. Studies on the development of an immunoassay for the group-specific detection of the diethyl ester of phosphates, thiophosphates, dithiophosphates and phosphonates. Kiel. Milchwirtsch. Forschungsber. 1988, 40, 179-203.]

Reference 2; Banks JN, Chaudhry MQ, Matthews WA, Haverly M, Watkins T, Northway BJ. Production and chracterisation of polyclonal antibodies to the common moiety of some organophosphorus pesticides and development of a generic type ELISA. Food and Agricultural Immunology. 1998, 10, 349-361.]

Reference 3; Johnson JC, Van Emon JM, Pullman DR, Keeper KR. Development and evaluation of antisera for detection of the O, O-diethyl phosphorothionate and phosphorothionothiolate organophosphorus pesticides by immunoassay. J Agric. Food Chem. 1998, 46, 3116-3123.]

Reference 4; Alcocer MJC, Dillon PP, Manning BM, Doyen C, Lee HA, Daly SJ, O'Kennedy R, Morgan, MRA. Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody. J. Agric. Food Chem. 2000, 48, 2228-2233.]

Reference 5; Jang MS, Lee SJ, Xue XP, Kwon HM, Rha CS, Lee YT, Chung TW. Production and characterization of monoclonal antibodies to a generic hapten for class-specific determination pesticides. Bull. Kor Chem. Soc. 2002, 23, 1116-1120.]

The present inventors have attempted to develop a total analysis method of organophosphorus pesticides having a common O , O -diethyl group using indirect competition and direct competition immunoassay in order to solve these shortcomings and develop more efficient pesticide analysis method. Theoretically, if an antibody against the structure of (EtO) ₂ P (= S)-can be obtained, an immunoassay using this antibody can provide a comprehensive analysis of this class of pesticides. Since this common structure is small in mass and cannot itself become an immunogen, it is impossible to produce antibodies. Therefore, in order to obtain an antibody against this structure, first, a hapten must be synthesized with a substance having an atomic group covalently bonded to the protein.

The inventors of the present study have found that, when an hapten having the following structural formula is used as an immunogen, the affinity for an antibody to the structure of (EtO) ₂ P (= S)-can be enhanced.

or

Where

R ₁ is CH ₃ , CH ₂ CH ₃ or OCH ₃ ;

R ₂ is CO ₂ H or CONH (CH ₂ ) ₂ CO ₂ H.

Examples of immunogen haptens according to the present invention include the following (Figure 2).

Figure 2. Structure of Immunogen Hapten

In addition to the immunogen hapten mentioned above, the following substances (Fig. 3) were used as coating antigens or enzyme tracer haptens used as competitors in immunoassays.

Figure 3. Structure of hapten for coating antigen and enzyme tracer

Among these haptens, hapten I and J have been reported for synthesis (Jang et al., 2002), and hapten J was synthesized by a new method. The remaining hapten was used synthetically.

Synthesis of hapten for the preparation of immunogens, coated antigens and enzyme tracers

A) Synthesis of Hapten A

The hapten A can be synthesized by the following process (Figure 4).

Figure 4. Synthesis process of hapten A.

B) synthesis of hapten B

The hapten B can be synthesized by the following process.

Figure 5. Synthesis process of hapten B.

C) synthesis of hapten C and D

The hapten C and D can be synthesized by the following process.

    Figure 6. Synthesis process of hapten C and D.

D) Synthesis of Hapten E

The hapten E can be synthesized by the following process.

Figure 7. Synthesis process of hapten E.

E) synthesis of hapten F

The hapten F can be synthesized by the following process.

Figure 8. Synthesis of Hapten F

F) synthesis of hapten G and H;

The hapten G and H can be synthesized by the following process.

Figure 9. Synthesis process of hapten G and H

G) synthesis of hapten J, K, L, M, N

The hapten J, K, L, M, N can be synthesized by the following process.

Figure 10. Synthesis of hapten J, K, L, M, N.

Hapten-protein binding

As a protein for hapten-protein binding, KLH (Kalbiochem Co., Ltd .; hemocyanin of giant keyhole limpet) and OVA (Sigma Co .; Ovalbumin) were used. KLH conjugate was used as an immunogen, and OVA conjugate was used as a coating antigen. . Sigma's horseradish peroxidase (HRP) was used as the enzyme of the enzyme tracer. The conjugation method uses carbodiimide and N-hydroxysucciniimide to make each hapten into a highly active N-succinimidyl ester type, which is then converted into a protein. So-called active ester method was used.

10 mg of KLH was dissolved in 1 mL of 0.2 M borate buffer (pH 8.7), followed by vigorous stirring dropping 0.1 mL of N, N-dimethylformamide (DMF). A solution of DMF (0.15 mL) in which the active ester (3.1 mg) of hapten A was dissolved therein was slowly dropped over 10 minutes, followed by stirring at room temperature for 1 hour and overnight at 4 ° C. The hapten A-OVA conjugate was made in the same way using OVA (20 mg, 0.00044 mmol) and the active ester of hapten A (11 mg, 0.028 mmol). The binding process with other hapten proteins was also the same. The hapten A-HRP conjugate was made in the same manner using peroxidase (1 mg, 0.00002 mmol) and the active ester of hapten A (0.182 mg, 0.0004 mmol). The binding process between the other haptens and the enzyme was the same.

For hapten-protein binding unbound hapten was removed by gel filtration (Separdex G25, 2 g) with PBS (phosphate-buffered saline; 10 mM, pH 7.4) as eluant. The solution obtained from the column was considered to have a hapten-protein conjugate present for a solution having a value of at least 0.1 by measuring absorbance at 278 nm, and the solution was dialyzed (distilled water, 4 ° C., overnight) and then lyophilized to -80. Stored at ℃. For hapten-enzyme binding, the supernatant was dialyzed for 24 hours (10 mM PBS, pH 7.4, 4 ° C., overnight) to remove unbound hapten. Enzyme tracer solution was stored at -80 ° C.

Measurement of titers of immunization and antiserum

The immunogen was mixed with an adjuvant and emulsified and injected intravenously into the mouse, followed by additional immunization with the immunogen dissolved in PBS every two weeks, and the antiserum titer was determined by indirect non-competitive immunoassay to confirm the antibody production. Measured. Splenocytes and myeloma cells (SP2 / O) of mice exhibiting high titers to the immunogen were fused and seeded into wells. In order to confirm the production of antibodies in the wells dispensed, a non-competitive indirect immunoassay using homologous coated antigens as a culture supernatant was performed. Culture supernatants of selected wells were taken to perform indirect competitive immunoassays using homologous coated antigens to select wells that were inhibited by at least 50% by pesticides. Finally, hybridomas were obtained through three rounds of cloning. The hybridomas were propagated and transplanted into the abdominal cavity of mice to obtain antibodies from ascites.

Indirect competitive immunoassay development

Eight monoclonal antibodies and three pesticides, chlorpyrephos, diazinon, and parathion, were used to determine the optimal dilution factor by performing indirect competition assays with different dilutions of antibody and coated antigen. An essay is conducted under the optimized conditions to establish a calibration curve for each pesticide. The IC ₅₀ value is obtained from the calibration curve, which represents the concentration of the analyte that inhibits the maximum absorbance by 50%.

The process of an indirect competition essay consists of the following steps:

a) immobilizing a coating antigen having hapten for phosphorothioate and phosphorodithioate organophosphorus pesticides having an O, O- diethyl group on a solid support.

b) contacting an antibody and a sample against phosphorothioate and phosphorodithioate organophosphorus pesticides having an O, O- diethyl group with a coating antigen.

c) quantifying the concentration of pesticide by quantifying the antibody bound to the coating antigen.

The method of quantifying the antibody bound to the coated antigen is performed by using an antibody labeled with an enzyme, a fluorescent atomic group, a radioisotope, or a colloidal gold, or by adding a labeled anti-mouse secondary antibody and measuring the signal by the label. It is possible by doing.

As a method for quantifying the product of the enzymatic reaction can be used a method such as absorption, fluorescence, light emission according to the optical properties of the product. Using primary antibodies with fluorescent atomic groups, radioactive isotopes, or colloidal gold eliminates the need for an anti-mouse antibody step, reducing the essay process.

Development of Direct Competition Immunoassay

Optimizing each dilution factor by measuring affinity for all kinds of enzyme tracers for antibodies and two-dimensional screening with different dilutions of antibody and enzyme tracers for affinity enzyme tracers. Calibration curves are obtained as in the case of an indirect competition essay.

The process of a direct competition essay consists of the following steps:

a) Immobilizing a monoclonal antibody obtained by using a conjugated protein conjugated to hapten for phosphorothioate and phosphorothiothioate organophosphorus pesticides having an O, O- diethyl group as an immunogen to a solid support.

b) contacting the conjugate with the antibody conjugated with the hapten for the phosphorothioate and phosphorodithioate organophosphorus pesticides having an O, O- diethyl group to the enzyme and the sample.

c) Quantifying the pesticide concentration by quantifying the hapten-label conjugate bound to the antibody.

As a method for quantifying a hapten-label conjugate bound to an antibody, it is possible to measure a signal by a label using an enzyme, a fluorescent atomic group, a radioisotope, colloidal gold, or the like as a label.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Example

Synthesis of succinimidyl esters of hapten and hapten

The chemical shift values of the NMR spectra described in the following hapten synthesis process are based on tetramethylsilane (TMS) as an internal standard, and the coupling constant values are expressed in Hz. Among them, d, t, q, m, ar means double, triple, quadruple, multiple and aromatic protons, respectively.

Example 1; Synthesis of Compound 1

1.79 g (10.8 mmol) of 4-methoxy-2-methyl benzoic acid (benzoic acid) mixes 4.9 g of red phosphorus and 26 mL of acetic anhydride / hydriodic acid 1: 1 After the solution was added, the mixture was stirred under reflux for 3 hours. The reaction solution was filtered through Celite, extracted with ethyl acetate (50 mL × 3) and washed with saturated brine. After evaporation of the solvent under reduced pressure, the residue was subjected to column chromatography (silica gel, hexane / ethyl acetate / acetic acid 200: 100: 8, Rf 0.42) to obtain 1.4 g (yield 85%) of product. 1 H NMR (CDCl 3): δ 8.00 (1H, d, J = 9.3, ar), 6.72 (1H, s, ar), 6.71 (1H, m, ar), 2.60 (3H, s, CH 3).

Example 2; Synthesis of Compound 2

3.5 mL of sulfuric acid / methanol (1:10) mixed solution was added to 1.35 g (8.9 mmol) of compound 1, followed by stirring under reflux at 80 ° C. for 24 hours. After evaporating the solvent under reduced pressure, the residue was subjected to column chromatography (silica gel, hexane / ethyl acetate 2: 1, Rf 0.54) to give 1.16 g (yield 79%) of the product. 1 H NMR (CDCl ₃ ): δ 7.84 (1H, d, J = 9.6, ar), 7.69 (1H, s, ar), 7.67 (1H, d, J = 9.2, ar), 3.85 (3H, s, OCH ₃ ), 2.58 (3H, s, CH ₃ ).

Example 3; Synthesis of Compound 3

0.26 g (1.57 mmol) of Compound 2 and 0.5 g (2.65 mmol) of diethylchlorothiophosphate were mixed, and 5 g of K ₂ CO ₃ was added thereto, and the mixture was stirred under reflux at 80 ° C. for 12 hours. The reaction solution was filtered using Celite, and then the solvent was evaporated under reduced pressure, and the residue was subjected to column chromatography (silica gel, hexane / ethyl acetate 5: 1, Rf 0.48) to give 362 mg (yield 72%) of the product. 1 H NMR (CDCl ₃ ): δ 7.93 (1H, d, J = 9.5, ar), 7.07 (1H, d, J = 8.8, ar), 7.06 (1H, s, ar), 4.25 (4H, q × d , J = 7.0 & 10.0, CH ₃ CH ₂ O), 3.87 (3H, s, OCH ₃ ), 2.60 (3H, s, CH ₃ ), 1.38 (6H, t, J = 7.1, CH ₃ CH ₂ O) .

Example 4; Synthesis of Hapten A

318 mg (1 mmol) of Compound 3 was dissolved in ethanol (60 mL) and then hydrolyzed by addition of 1 M KOH (25 mL) at room temperature for 30 minutes. 1 N HCl (30 mL) was added to the reaction solution, and the product was extracted with ethyl acetate (50 mL). The extract was washed twice with 1 N HCl, dehydrated with MgSO 4 and the solvent was evaporated and column chromatography (silica gel, hexane / ethyl acetate / acetic acid 200: 100: 8, Rf 0.57) gave 70 mg (yield 23%) of product. . 1 H NMR (CDCl 3): δ 8.07 (1H, d, J = 8.3, ar), 7.10 (1H, d, J = 9.8, ar), 7.09 (1H, s, ar), 4.26 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 2.65 (3H, s, CH ₃ ), 1.38 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 5; Synthesis of Compound 4

Compound 4 was synthesized in the same manner as in Example 2, except that 4.04 g (26.2 mmol) of 2,4 dihydroxy benzoic acid and 11 mL of sulfuric acid / methanol (1:10) mixed solution were used. Yield 61%. TLC: hexanes / ethyl acetate 2: 1, Rf 0.52. 1 H NMR (CDCl ₃ ): δ 7.72 (1H, d, J = 8.6, ar), 6.41 (1H, s, ar), 6.39 (1H, d, J = 8.6, ar), 3.91 (3H, s, CO ₂ CH ₃ ).

Example 6; Synthesis of Compound 5

Compound 5 was synthesized with some modifications to the reported synthesis method (Masada & Oishi, 1978). 1.34 g (7.98 mmol) of Compound 4 and 4.37 g (31.9 mmol) of t-butyl bromide were mixed, 10 g of K ₂ CO ₃ was added thereto, and the mixture was stirred at 70 ° C. for 48 hours. The reaction solution was filtered through celite, and the solvent was evaporated under reduced pressure, and the residue was then subjected to column chromatography (silica gel, hexane / ethyl acetate 8: 1, Rf 0.64) to give 262 mg (yield 15%) of the product. 1 H NMR (CDCl ₃ ): δ 7.69 (1H, d, J = 8.9, ar), 6.69 (1H, s, ar), 6.46 (1H, d, J = 8.7, ar), 3.89 (3H, s, CO ₂ CH ₃ ). 1.41 (9H, s, CH ₃ of t-butyl group).

Example 7; Synthesis of Compound 6

76 mg (0.34 mmol) of Compound 5 was dissolved in acetone, and then 172 mg (1.36 mmol) of dimethyl sulfate was added thereto, 1 g of K ₂ CO ₃ was added thereto, and the mixture was stirred at 60 ° C. for 12 hours. The reaction solution was filtered using Celite, and the solvent was evaporated under reduced pressure, and the residue was then subjected to column chromatography (silica gel, hexane / ethyl acetate 4: 1, Rf 0.52) to obtain 78 mg (yield 97%) of the product. 1 H NMR (CDCl ₃ ): δ 7.75 (1H, d, J = 8.6, ar), 6.59 (1H, d × d, J = 8.6 & 2.2, ar), 6.53 (1H, d, J = 2.1, ar) , 3.85 (3H, s, CO ₂ CH ₃ ). 1.40 (CH ₃ of 9H, s, t-butyl group)

Example 8; Synthesis of Compound 7

68 mg (0.288 mmol) of compound were dissolved in 0.2 mL of trifluoroacetic acid and stirred for 10 minutes. After evaporating the solvent under reduced pressure, the residue was subjected to column chromatography (silica gel, hexane / ethyl acetate 1: 1, Rf 0.42) to give 51 mg (yield 97%) of product. 1 H NMR (CDCl ₃ ) δ 7.77 (1H, d, J = 8.7, ar), 6.43 (1H, s, ar), 6.42 (1H, d × d, J = 8.1 & 2.3, ar), 3.84 (3H, s, CO ₂ CH ₃ ). 3.79 (3H, s, OCH ₃ ).

Example 9; Synthesis of Compound 8

A compound 8 was synthesized in the same manner as in Example 3, except that 51 mg (0.28 mmol) of Compound 7 and 64 mg (0.34 mmol) of diethylchlorothiophosphate were used. Yield 56%. TLC: hexanes / ethyl acetate 4: 1, Rf 0.53. 1 H NMR (CDCl ₃ ): δ 7.80 (1H, d, J = 9.5, ar), 6.82 (1H, m, ar), 6.73 (1H, m, ar), 4.25 (4H, q × d, J = 7.1 & 9.9, CH ₃ CH ₂ O), 3.87 (3H, s, CO ₂ CH ₃ ), 3.85 (3H, s, OCH ₃ ), 1.34 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 10; Synthesis of Hapten B

The hapten B was synthesized in the same manner as in Example 4, except that 51 mg (0.153 mmol) of Compound 8 was dissolved in ethanol (10 mL) and then 1 M KOH (10 mL) was added. Yield 71%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.46. 1 H NMR (CDCl ₃ ): δ 8.15 (1H, d, J = 9.2, ar), 6.94 (1H, m, ar), 6.93 (1H, m, ar), 4.26 (4H, q × d, J = 7.1 & 10.1, CH ₃ CH ₂ O), 4.05 (3H, s, OCH ₃ ), 1.35 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 11; Synthesis of Compound 9

In a solution of 130 mg (0.324 mmol) of N-succinimidyl ester of hapten A (Example 35) in methanol (0.2 mL), KOH (25 mg) and β-alanine ethyl ester (76 mg, 0.628 mmol) were added. The dissolved methanol solution (0.5 mL) was dropped and stirred at room temperature for 2 hours. After filtering the reaction solution, the solvent was evaporated under reduced pressure, and the residue was subjected to column chromatography (silica gel, hexane / ethyl acetate / acetic acid 15: 15: 1, Rf 0.67) to give 88 mg (yield 62%) of the product. 1 H NMR (CDCl ₃ ): δ 7.32 (1H, d, J = 9.0, ar), 7.07 (1H, d, J = 7.8, ar), 7.00 (1H, t, J = 1.9, ar), 4.11-4.28 (4H, q × d, J = 7.0 & 10.0, CH ₃ CH ₂ O), 4.06 (2H, q × d, J = 7.2, CH ₃ CH ₂ OCO), 3.67 (2H, q, J = 6.0, NHCH ₂ ), 2.62 (2H, t, J = 6.0 CH ₂ CO ₂ ), 2.41 (3H, s, CH ₃ ), 1.35 (6H, t, J = 7.1, CH ₃ CH ₂ O), 1.25 (3H, t , J = 7.1, CH ₃ CH ₂ OCO).

Example 12; Synthesis of Hapten C

A hapten C was synthesized in the same manner as in Example 4, except that 78 mg (0.178 mmol) of Compound 9 was dissolved in ethanol (12 mL) and then 1 M KOH (5 mL) was added. Yield was 52%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.45. 1 H NMR (CDCl ₃ ): δ 7.32 (1H, d, J = 8.9, ar), 7.08 (1H, d, J = 7.8, ar), 7.00 (1H, m, ar), 6.32 (1H, m, NH ), 4.16 to 4.27 (4H, q × d, J = 7.0 & 10.0, CH ₃ CH ₂ O), 3.68 (2H, q, J = 6.0, NHCH ₂ ), 2.79 (2H, t, J = 5.8, CH ₂ CO ₂ ), 2.41 (3H, s, CH ₃ ), 1.35 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 13; Synthesis of Compound 10

The same procedure as in Example 11 was carried out except that 90 mg (0.216 mmol) of N-succinimidyl ester of hapten B (Example 36) and β-alanine ethyl ester (51 mg, 0.436 mmol) were used. 10 was synthesized. Yield 53%. TLC: hexanes / ethyl acetate / acetic acid 15: 15: 1, Rf 0.51. 1 H NMR (CDCl ₃ ): δ 8.29 (1H, m, NH), 8.16 (1H, d, J = 8.5, ar), 6.87 (1H, m, ar), 6.84 (1H, m, ar), 4.15- 4.28 (4H, qxd, J = 7.0 & 10.0, CH ₃ CH ₂ O), 3.94 (3H, s, OCH ₃ ), 3.70 (2H, q, J = 6.0, NHCH ₂ ), 2.63 (2H, t , J = 6.0, CH ₂ CO ₂ ), 1.34 (9H, t, J = 6.9, CH ₃ CH ₂ O).

Example 14; Synthesis of Hapten D

A hapten D was synthesized in the same manner as in Example 4, except that 44 mg (0.097 mmol) of Compound 10 was dissolved in ethanol (10 mL) and then 1 M KOH (5 mL) was added. Yield was 48%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.44. 1 H NMR (CDCl ₃ ): δ 8.37 (1H, m, NH), 8.15 (1H, d, J = 8.6, ar), 6.87 (1H, d × t, J = 8.7 & 2.0, ar), 6.83 (1H , m, ar), 4.16 to 4.27 (4H, q × d, J = 7.1 & 9.9, CH ₃ CH ₂ O), 3.90 (3H, s, OCH ₃ ), 3.70 (2H, q, J = 5.9, NHCH ₂ ), 2.69 (2H, t, J = 5.9, CH ₂ CO ₂ ), 1.34 (6H, t × d, J = 7.0 & 0.7, CH ₃ CH ₂ O).

Example 15; Synthesis of Compound 11

Except 300 mg (1.8 mmol) methyl 3-hydroxy-5-methyl benzoic acid, 406 mg (2.16 mmol) diethylchlorothiophosphate, 8 g K ₂ CO ₃ , 10 mL butanone were used Compound 11 was synthesized in the same manner as in Example 3. Yield was 93% (TLC, silica gel, hexanes / ethyl acetate 5: 1, Rf 0.49). 1 H NMR (CDCl ₃ ) δ 7.70 (1H, s, ar), 7.63 (1H, d, ar, J = 1.2), 7.20 (1H, d, ar, J = 0.9), 4.25 (4H, d × q, CH ₃ CH ₂ , J = 9.9 × 7.1), 3.91 (3H, s, CO ₂ CH ₃ ), 2.40 (3H, s, CH ₃ ), 1.38 (6H, t, CH ₃ CH ₂ , J = 7.1)

Example 16; Synthesis of Hapten E

The hapten E was synthesized in the same manner as in Example 4 except that 530 mg (1.67 mmol) of Compound 11 was dissolved in ethanol (84 mL) and then 1 M KOH (33 mL) was added. The yield was 60% (TLC, silica gel, hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.50). 1 H NMR (CDCl ₃ ) δ 7.74 (1H, s, ar), 7.68 (1H, s, ar), 7.23 (1H, s, ar), 4.25 (4H, d × q, CH ₃ CH ₂ , J = 9.9 7.9), 2.40 (3H, s, CH ₃ ), 1.38 (6H, t, CH ₃ CH ₂ , J = 7.1)

Example 17; Synthesis of Compound 12

Except for using 230 mg (1.26 mmol) methyl 3-hydroxy-5-methoxybenzoic acid, 284 mg (1.51 mmol) diethylchlorothiophosphate, 5 g K ₂ CO ₃ , 7 mL butanone Compound 12 was synthesized in the same manner as in Example 3. Yield was 84% (TLC, silica gel, hexane / ethyl acetate 5: 1, Rf 0.43). 1 H NMR (CDCl ₃ ): δ 7.44 (1H, m, ar), 7.41 (1H, m, ar), 6.96 (1H, m, ar), 4.25 (4H, d × q, CH ₃ CH ₂ , J = 10.0 × 7.1), 3.91 (3H, s, CO ₂ CH ₃ ), 3.85 (3H, s, OCH ₃ ), 1.38 (6H, d × t, CH ₃ CH ₂ , J = 0.8 × 7.1)

Example 18; Synthesis of Hapten F

The hapten F was synthesized in the same manner as in Example 4 except that 355 mg (1.06 mmol) of Compound 12 was dissolved in ethanol (30 mL) and then 1 M KOH (21 mL) was added. Yield was 55%. (TLC, silica gel, hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.42). 1 H NMR (CDCl ₃ ): δ 7.51 (1H, s, ar), 7.47 (1H, s, ar), 7.01 (1H, m, ar), 4.26 (4H, d × q, CH ₃ CH ₂ , J = 10.0 × 7.1), 3.86 (3H, s, CH ₃ ), 1.38 (6H, d × t, CH ₃ CH ₂ , J = 0.8 × 7.1)

Example 19; Synthesis of Compound 13

Add THF (2 mL) to hapten E (72 mg, 0.24 mmol), β-alanine ethyl ester (76 mg, 0.49 mmol), DMAP (4 mg, 0.033 mmol), and dissolve in THF solution (0.3 mL) with stirring. DCC (61 mg, 0.30 mmol) was added dropwise. After reacting for 1 hour, the urea was removed by filtration . The solvent was volatilized and then prep. TLC (silica gel, hexane / ethyl acetate 200: 100, Rf 0.50) gave 55 mg (yield 57%) of product. 1 H NMR (CDCl ₃ ) δ 7.39-7.37 (2H, s, ar), 7.14 (1H, s, ar), 6.84 (1H, s, NH), 4.25 (4H, d × q, CH ₃ CH ₂ , J = 9.9 × 7.1), 4.18 (2H, q, CH ₃ CH ₂ , J = 7.2), 3.72 (2H, q, NHCH ₂ , J = 6.0), 2.64 (2H, t, CH ₂ , J = 5.9), 2.39 (3H, s, CH ₃ ), 1.38 (6H, d × t, CH ₃ CH ₂ , J = 0.9 × 7.1), 1.28 (2H, t, J = 7.2).

Example 20; Synthesis of Hapten G

The hapten G was synthesized in the same manner as in Example 4 except that 55 mg (0.14 mmol) of Compound 13 was dissolved in ethanol (10 mL) and then 1 M KOH (6 mL) was added. Yield was 60%. (TLC, silica gel, hexanes / ethyl acetate / acetic acid, 100: 200: 8 Rf 0.48) 1H NMR (CDCl ₃ ) δ 7.34 (1H, s, ar), 7.07 (2H, s, ar), 4.19 (4H, d Xq, CH ₃ CH ₂ , J = 9.9 × 7.1), 3.62 (2H, q, NHCH ₂ , J = 5.7), 2.62 (2H, t, CH ₂ , J = 5.9), 2.29 (3H, s, CH ₃ ), 1.32 (6H, t, CH ₃ CH ₂ , J = 7.1).

Example 21; Synthesis of Compound 14

THF (1 mL) using hapten F (18 mg, 0.056 mmol), β-alanine ethyl ester (17 mg, 0.11 mmol), DMAP (1 mg, 0.008 mmol), DCC (14 mg, 0.067 mmol) Except for the synthesis of Compound 14 in the same manner as in Example 19. Yield 63%. Rf 0.45 (silica gel, hexane / ethyl acetate 200: 100) 1 H NMR (CDCl ₃ ) δ 7.39-7.37 (2H, s, ar), 7.14 (1H, s, ar), 6.77 (1H, m, NH), 4.25 (4H, m, CH ₃ CH ₂ ), 4.14 (2H, q, CH ₃ CH ₂ , J = 7.2), 3.82 (3H, s, OCH ₃ ), 3.69 (2H, q, NHCH ₂ , J = 6.0) , 2.62 (2H, t, CH ₂ , J = 5.2), 1.36 (6H, t, CH ₃ CH ₂ , J = 7.1), 1.26 (2H, t, J = 6.7).

Example 22; Synthesis of Hapten H

A hapten H was synthesized in the same manner as in Example 4, except that 15 mg (0.036 mmol) of Compound 14 was dissolved in ethanol (5 mL) and then 1 M KOH (2 mL) was added. Yield 71%. (TLC, silica gel, hexane / ethyl acetate / acetic acid, 100: 200: 8 Rf 0.43) 1 H NMR (CDCl ₃ ) δ 7.18 (1H, s, ar), 7.14 (1H, s, ar), 6.87 (1H, s , ar), 6.76 (1H, m, NH), 4.22 (4H, d × q, CH ₃ CH ₂ , J = 10.0 × 7.1), 3.81 (3H, s, OCH ₃ ), 3.70 (2H, q, NHCH ₂ , J = 5.9), 2.70 (2H, t, CH ₂ , J = 5.9), 1.35 (6H, t, CH ₃ CH ₂ , J = 7.1).

Example 23; Synthesis of Compound 15

Compound 15 was synthesized in the same manner as in Example 2, except that 0.865 g (5.5 mmol) of 4-fluoro-3-hydroxy benzoic acid and 5 mL of sulfuric acid / methanol (1:10) mixed solution were used. It was. Yield 63%. TLC: hexanes / ethyl acetate 2: 1, Rf 0.45. 1 H NMR (CDCl ₃ ): δ 7.68 (1H, d × d, J = 8.5 & 2.1, ar), 7.56 (1H, m, ar), 7.09 (1H, t, J = 9.3, ar), 3.88 (3H , s, CO ₂ CH ₃ ).

Example 24; Synthesis of Compound 16

Compound 16 was prepared in the same manner as in Example 2 except for using 774 mg (3.9 mmol) of 4-hydroxy-2,6-dimethoxy benzoic acid in a solution of 8.5 mL of sulfuric acid / methanol (1:48). Synthesized. Yield was 50%. (TLC, silica gel, hexanes / ethyl acetate 3: 1, Rf 0.22). 1 H NMR (CDCl ₃ ) δ 7.33 (2H, s, ar), 5.92 (1H, s, OH), 3.95 (6H, s, (OCH ₃ ) ₂ ), 3.90 (3H, s, CO ₂ OCH ₃ )

Example 25; Synthesis of Compound 17

Compound 17 was synthesized in the same manner as in Example 3, except that 0.27 g (1.59 mmol) of Compound 17 and 0.603 g (3.2 mmol) of diethylchlorothiophosphate were added. Yield was 69%. TLC: hexanes / ethyl acetate 4: 1, Rf 0.49. 1 H NMR (CDCl ₃ ): δ 7.94 (1H, d × t, J = 7.6 & 1.9, ar), 7.86 (1H, m, ar), 7.18 (1H, t, J = 9.0, ar), 4.19-4.32 (4H, qxd, J = 7.1 & 9.9, CH ₃ CH ₂ O), 3.89 (3H, s, CO ₂ CH ₃ ), 1.35 (6H, t, J = 7.0, CH ₃ CH ₂ ).

Example 26; Synthesis of Compound 18

Compound 18 was synthesized in the same manner as in Example 3, except that 0.401 g (2.64 mmol) of the compound 18 and 0.6 g (3.18 mmol) of diethylchlorothiophosphate were added. Yield was 95%. TLC: hexanes / ethyl acetate 8: 1, Rf 0.55. 1 H NMR (CDCl ₃ ): δ 7.86 (1H, d, J = 6.6, ar), 7.80 (1H, s, ar), 7.40 (1H, m, ar), 7.38 (1H, m, ar), 4.17- 4.28 (4H, qxd, J = 7.1 & 10.0, CH ₃ CH ₂ O), 3.90 (3H, s, CO ₂ CH ₃ ), 1.35 (6H, t, J = 7.0, CH ₃ CH ₂ O).

Example 27; Synthesis of Compound 19

A compound 19 was synthesized in the same manner as in Example 3, except that 0.147 g (1.57 mmol) of the compound 19 and 0.2 g (1.06 mmol) of diethylchlorothiophosphate were added. The yield was 33%. TLC hexanes / ethyl acetate 4: 1, Rf 0.67. 1 H NMR (CDCl ₃ ): δ 8.04 (2H, d, J = 8.7 ar), 7.23 (2H, d × d, J = 8.5 & 1.5, ar), 4.19-4.30 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 3.90 (3H, s, CO ₂ CH ₃ ), 1.37 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 28; Synthesis of Compound 20

A compound 20 was synthesized in the same manner as in Example 3, except that 0.53 g (2.94 mmol) of the compound 20 and 0.72 g (3.82 mmol) of the diethylchlorothiophosphate were added. Yield was 79%. TLC: hexanes / ethyl acetate 4: 1, Rf 0.47. 1 H NMR (CDCl ₃ ): δ 7.13 (2H, d, J = 8.6, ar), 7.07 (2H, d × d, J = 8.7 & 1.4, ar), 4.14-4.26 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 3.64 (3H, s, CO ₂ CH ₃ ), 2.90 (2H, t, J = 7.7, (CH ₂ CH ₂ CO ₂ ), 2.59 (2H, t, J = 7.7, CH ₂ CO ₂ ) 1.33 (6H, t × d, J = 7.1 & 0.7, CH ₃ CH ₂ O).

Example 29; Synthesis of Compound 21

The same procedure as in Example 3 was carried out except that 414 mg (1.95 mmol) of Compound 16 and 439 mg (2.34 mmol) of diethylchlorothiophosphate were mixed, followed by addition of 10 g of K2CO3 and 15 mL of butanone. Compound 21 was synthesized. Yield was 65%. (TLC, silica gel, hexanes / ethyl acetate 5: 1, Rf 0.30). 1 H NMR (CDCl ₃ ) δ 7.33 (2H, s, ar), 4.33 (4H, d × q, CH ₃ CH ₂ , J = 8.9 × 7.1), 3.91 (3H, s, (OCH ₃ ) ₂ ), 3.90 (3H, s, CO ₂ CH ₃ ), 1.41 (6H, d × t, CH ₃ CH ₂ , J = 0.8 × 7.1)

Example 30; Synthesis of hapten J

The hapten J was synthesized in the same manner as in Example 4 except that 283 mg (0.88 mmol) of Compound 17 was used. Yield was 99%. TLC hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.49. 1 H NMR (CDCl ₃ ): δ 8.01 (1H, d × t, J = 7.5 & 1.9, ar), 7.93 (1H, m, ar), 7.22 (1H, t, J = 9.1, ar), 4.22-4.33 (4H, qxd, J = 7.1 & 9.9, CH ₃ CH ₂ O), 1.38 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 31; Synthesis of hapten K

The hapten K was synthesized in the same manner as in Example 4, except that 304 mg (1 mmol) of Compound 18 was used. Yield was 79%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.59. 1 H NMR (CDCl ₃ ): δ 7.94 (1H, m, ar), 7.90 (1H, s, ar), 7.45 (1H, m, ar), 7.43 (1H, m, ar), 4.19-4.30 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 1.36 (6H, t, J = 7.1, CH ₃ CH ₂ O)

Example 32; Synthesis of hapten L

A hapten L was synthesized in the same manner as in Example 4, except that 97 mg (0.32 mmol) of Compound 19 was dissolved in ethanol (12 mL) and then 1 M KOH (10 mL) was added. Yield was 82%: TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.56. 1 H NMR (CDCl ₃ ): δ 8.12 (2H, d, J = 8.5, ar), 7.23 (2H, d × d, J = 8.7 & 1.4, ar), 4.17 to 4.33 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 1.39 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 33; Synthesis of hapten M

The hapten M was synthesized in the same manner as in Example 4, except that 351 mg (1.06 mmol) of Compound 20 was used. Yield was 98%: TLC (hexane / ethyl acetate / acetic acid 15: 15: 1, Rf 0.58); 1 H NMR (CDCl ₃ ) δ 7.13 (2H, d, J = 8.5, ar), 7.06 (2H, d × d, J = 8.4 & 1.0, ar), 4.14-4.26 (4H, q × d, J = 7.1 9.8, CH ₃ CH ₂ O), 2.87 (2H, t, J = 7.8, CH ₂ CH ₂ CO ₂ ), 2.58 (2H, t, J = 7.7, CH ₂ CO ₂ ), 1.33 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 34; Synthesis of hapten N

The hapten N was synthesized in the same manner as in Example 4 except that 460 mg (1.26 mmol) of Compound 21 was dissolved in ethanol (50 mL) and then 1 M KOH (27 mL) was added. Yield was 62%: (TLC, silica gel, hexane / ethyl acetate / acetic acid 200: 100: 8, Rf 0.39). 1 H NMR (CDCl ₃ ) δ 7.37 (2H, s, ar), 4.33 (4H, d × q, CH ₃ CH ₂ , J = 8.9 × 7.0), 3.90 (6H, s, (OCH ₃ ) 2), 1.42 (6H, d × t, CH ₃ CH ₂ , J = 0.6 × 7.0)

Example 35; Active ester synthesis of hapten A

N-hydroxysuccinimide (22 mg, 0.19 mmol) dissolved in dichloromethane (10 mL) and then hapten A (52 mg, 0.17 mmol) and 4-dimethylaminopyridine (DMAP, 2) dissolved in a small amount of dichloromethane. mg, 0.017 mmol) and N, N-dicyclohexylcarbodiimide (DCC, 39 mg, 0.19 mmol) were added sequentially. The reaction solution was stirred at room temperature for 4 hours and then filtered to remove dicyclohexylurea. After evaporation of the solvent, the product was separated by column chromatography (silica gel, hexane / ethyl acetate / acetic acid, 200: 100: 8, Rf 0.44) to give 44 mg (64% yield) of the product. 1 H NMR (CDCl 3): δ 8.15 (1H, d, J = 9.4, ar), 7.15 (1H, d, J = 7.5, ar), 7.14 (1H, s, ar), 4.26 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 2.91 (4H, s, succinyl), 2.62 (3H, s, CH ₃ ), 1.38 (6H, t, J = 7.0, CH ₃ CH ₂ O).

Example 36; Active ester synthesis of hapten B

N-hydroxysuccinimide (10.3 mg, 0.09 mmol) dissolved in dichloromethane (3 mL), hapten B (25 mg, 0.075 mmol) and DMAP (1 mg, 0.008 mmol) dissolved in a small amount of dichloromethane, and An active ester of hapten B was synthesized in the same manner as in Example 35 except that DCC (18 mg, 0.087 mmol) was added. The yield was 43%. TLC: hexanes / ethyl acetate / acetic acid, 200: 100: 8, Rf 0.49. 1 H NMR (CDCl ₃ ): δ 8.05 (1H, d, J = 8.5, ar), 6.94 (1H, m, ar), 6.88 (1H, m, ar), 4.24 (4H, q × d, J = 7.1 & 10.1, CH ₃ CH ₂ O), 3.90 (3H, s, OCH ₃ ), 2.87 (4H, s, succinyl), 1.36 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 37; Active ester synthesis of hapten C

N-hydroxysuccinimide (6.8 mg, 0.059 mmol) dissolved in dichloromethane (2 mL), hapten C (20 mg, 0.049 mmol) and DMAP (0.6 mg, 0.0049 mmol) dissolved in a small amount of dichloromethane; An active ester of hapten C was synthesized in the same manner as in Example 35 except that DCC (12 mg, 0.059 mmol) was added. The yield was 49%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.47. 1 H NMR (CDCl ₃ ): δ 7.36 (1H, d, J = 9.1, ar), 7.01 (1H, m, ar), 7.00 (1H, m, ar), 6.50 (1H, m, NH), 4.15- 4.27 (4H, qxd, J = 7.1 & 9.9, CH ₃ CH ₂ O), 3.80 (2H, q, J = 5.7, NHCH ₂ ), 2.95 (2H, t, J = 6.0, CH ₂ CO ₂ ) , 2.83 (4H, s, succinyl), 2.42 (3H, s, CH ₃ ), 1.34 (6H, t × d, J = 7.1 & 0.6, CH ₃ CH ₂ O).

Example 38; Active ester synthesis of hapten D

N-hydroxysuccinimide (4.1 mg, 0.036 mmol) dissolved in dichloromethane (2 mL), followed by hapten D (13 mg, 0.03 mmol) and DMAP (0.4 mg, 0.008 mmol) dissolved in a small amount of dichloromethane. An active ester of hapten D was synthesized in the same manner as in Example 35, except that DCC (7.4 mg, 0.036 mmol) was added. Yield was 48%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.47. 1 H NMR (CDCl ₃ ): δ 8.24 (1H, m, NH), 8.15 (1H, d, J = 8.5, ar), 6.88 (1H, d × t, J = 8.6 & 2.0, ar), 6.84 (1H , m, ar), 4.16 to 4.28 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 3.91 (3H, s, OCH ₃ ), 3.82 (2H, q, J = 6.0, NHCH ₂ ), 2.93 (2H, t, J = 5.9, CH ₂ CO ₂ ), 2.83 (4H, s, succinyl), 1.34 (6H, t, J = 7.0, CH ₃ CH ₂ O).

Example 39; Active ester synthesis of hapten E

N-hydroxysuccinimide (15 mg, 0.13 mmol) dissolved in dichloromethane (3 mL), hapten E (34 mg, 0.11 mmol) and DMAP (1.3 mg, 0.011 mmol) dissolved in a small amount of dichloromethane; An active ester of hapten E was synthesized in the same manner as in Example 35 except that DCC (27 mg, 0.132 mmol) was added. Yield was 45%. TLC (silica gel, hexane / ethyl acetate / acetic acid, 200: 100: 8 Rf 0.37) 1 H NMR (CDCl ₃ ) δ 7.80 (1H, s, ar), 7.72 (1H, d, ar, J = 1.5), 7.33 ( 1H, m, ar, J = 0.73), 4.25 (4H, d × q, CH ₃ CH ₂ , J = 10.0 × 7.1), 2.92 (4H, s, succinyl), 2.43 (3H, s, CH ₃ ) , 1.38 (6H, d × t, CH ₃ CH ₂ , J = 0.7 × 7.1)

Example 40; Active ester synthesis of hapten F

N-hydroxysuccinimide (19 mg, 0.16 mmol) dissolved in dichloromethane (4 mL), hapten F (42 mg, 0.14 mmol) and DMAP (2 mg, 0.014 mmol) dissolved in a small amount of dichloromethane; An active ester of hapten F was synthesized in the same manner as in Example 35 except that DCC (33 mg, 0.16 mmol) was added. The yield was 40%. TLC (silica gel, hexane / ethyl acetate / acetic acid, 200: 100: 8 Rf 0.34) 1 H NMR (CDCl ₃ ) δ 7.53 (1H, m, ar), 7.47 (1H, m, ar), 7.09 (1H, m, ar), 4.25 (4H, d × q, CH ₃ CH ₂ , J = 10.0 × 7.1), 3.86 (3H, s, OCH ₃ ), 2.91 (4H, s, succinyl), 1.38 (6H, d × t , CH ₃ CH ₂ , J = 0.8 × 7.1)

Example 41; Active ester synthesis of hapten G

N-hydroxysuccinimide (12 mg, 0.102 mmol) in dichloromethane (3 mL), hapten G (32 mg, 0.085 mmol) and DMAP (1 mg, 0.0085 mmol) dissolved in a small amount of dichloromethane, and An active ester of hapten G was synthesized in the same manner as in Example 35 except that DCC (21 mg, 0.102 mmol) was added. The yield was 33%. Rf 0.60 (silica gel, hexane / ethyl acetate / acetic acid, 100: 200: 8) 1H NMR (CDCl ₃ ) δ 7.44 (1H, s, ar), 7.38 (1H, s, ar), 7.11 (1H, s, ar ), 4.22 (4H, d × q, CH ₃ CH ₂ , J = 9.9 × 7.1), 3.85 (2H, q, NHCH ₂ , J = 6.1), 2.91 (2H, t, CH ₂ , J = 5.9), 2.85 (4H, s, succinyl), 2.36 (3H, s, CH ₃ ), 1.34 (6H, t, CH ₃ CH ₂ , J = 6.8).

Example 42; Active ester synthesis of hapten H

N-hydroxysuccinimide (3.2 mg, 0.028 mmol) dissolved in dichloromethane (2 mL), hapten H (9 mg, 0.023 mmol) and DMAP (1 mg, 0.008 mmol) dissolved in a small amount of dichloromethane, and An active ester of hapten H was synthesized in the same manner as in Example 35 except that DCC (6 mg, 0.028 mmol) was added. Yield was 45%. Rf 0.57 (silica gel, hexane / ethyl acetate / acetic acid, 100: 200: 8) 1H NMR (CDCl ₃ ) δ 7.23 (1H, s, ar), 7.21 (1H, s, ar), 7.04 (1H, s, ar ), 6.91 (1H, NH, m), 4.26 (4H, d × q, CH ₃ CH ₂ , J = 9.9 × 7.1), 3.89 (2H, q, NHCH ₂ , J = 5.9), 3.86 (3H, s , OCH ₃ ), 2.95 (2H, t, CH ₂ , J = 5.9), 2.89 (4H, s, succinyl), 1.39 (6H, t, CH ₃ CH ₂ , J = 7.1).

Example 43; Active ester synthesis of hapten J

N-hydroxysuccinimide (69 mg, 0.6 mmol) dissolved in dichloromethane (5 mL), hapten J (154 mg, 0.5 mmol) and DMAP (6 mg, 0.05 mmol) dissolved in a small amount of dichloromethane; An active ester of hapten J was synthesized in the same manner as in Example 35 except that DCC (124 mg, 0.6 mmol) was added. Yield was 99%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.35. 1 H NMR (CDCl ₃ ): δ 8.04 (1H, d × t, J = 7.4 & 2.0, ar), 7.99 (1H, m, ar), 7.27 (1H, t, J = 8.8, ar), 4.21-4.32 (4H, q × d, J = 7.1 & 9.9, CH ₃ CH ₂ O), 2.89 (4H, s, succinyl), 1.37 (6H, t × d, J = 7.1 & 0.7, CH ₃ CH ₂ O) .

Example 44; Active ester synthesis of hapten K

N -hydroxysuccinimide (44 mg, 0.38 mmol) dissolved in dichloromethane (5 mL), hapten K (101 mg, 0.35 mmol) and DMAP (4.6 mg, 0.038 mmol) dissolved in a small amount of dichloromethane; An active ester of hapten K was synthesized in the same manner as in Example 35 except that DCC (79 mg, 0.38 mmol) was added. Yield was 81%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.4. 1 H NMR (CDCl ₃ ): δ 8.15 (2H, d, J = 8.5, ar), 7.32 (2H, d × d, J = 8.7 & 1.4, ar), 4.24 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 2.91 (4H, s, succinyl), 1.39 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 45; Active ester synthesis of hapten L

N-hydroxysuccinimide (32 mg, 0.28 mmol) dissolved in dichloromethane (5 mL), hapten L (73 mg, 0.25 mmol), DMAP (3 mg, 0.025 mmol) dissolved in a small amount of dichloromethane, and An active ester of hapten L was synthesized in the same manner as in Example 35 except that DCC (58 mg, 0.28 mmol) was added. Yield was 81%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.44. 1 H NMR (CDCl ₃ ): δ 7.95 (1H, m, ar), 7.89 (1H, s, ar), 7.45 (1H, m, ar), 7.45 (1H, m, ar), 4.17-4.28 (4H, q × d, J = 7.1 & 10.0, CH ₃ CH ₂ O), 2.89 (4H, s, succinyl), 1.35 (6H, t, J = 7.1, CH ₃ CH ₂ O).

Example 46; Active ester synthesis of hapten M

N-hydroxysuccinimide (63.3 mg, 0.55 mmol) dissolved in dichloromethane (5 mL) and then hapten M (159 mg, 0.5 mmol) and DMAP (6.7 mg, 0.008 mmol) dissolved in a small amount of dichloromethane. An active ester of hapten M was synthesized in the same manner as in Example 35, except that DCC (113 mg, 0.55 mmol) was added. Yield was 79%. TLC: hexanes / ethyl acetate / acetic acid 200: 100: 8, Rf 0.32. 1 H NMR (CDCl ₃ ): δ 7.13 (2H, d, J = 8.6, ar), 7.06 (2H, d × d, J = 8.6 & 1.4, ar), 4.11-4.21 (4H, q × d, J = 7.1 & 9.8, CH ₃ CH ₂ O), 2.97 (2H, t, J = 7.4, (CH ₂ CH ₂ CO ₂ ), 2.86 (4H, s, succinyl), 2.84 (2H, m, CH ₂ CO ₂ 1.29 (6H, t, J = 7.0, CH ₃ CH ₂ O).

Example 47; Active ester synthesis of hapten N

N-hydroxysuccinimide (15 mg, 0.13 mmol) dissolved in dichloromethane (3 mL), hapten N (37 mg, 0.11 mmol) and DMAP (1.3 mg, 0.011 mmol) dissolved in a small amount of dichloromethane, and An active ester of hapten N was synthesized in the same manner as in Example 35 except that DCC (26 mg, 0.13 mol) was added. Yield was 59%. 1 H NMR (CDCl ₃ ) δ 7.40 (2H, s, ar), 4.33 (4H, d × q, CH ₃ CH ₂ , J = 9.0 × 7.1), 3.91 (6H, s, (OCH ₃ ) ₂ ), 2.92 (4H, s, succinyl), 1.42 (6H, d × t, CH ₃ CH ₂ , J = 0.9 × 7.1)

Example 48; Detection of Organophosphorus Pesticides by Indirect Competition Immunoassay

First, organophosphorus pesticides were prepared using 10 mg / mL stock solution using methanol as a solvent, followed by dilution stepwise with 10% methanol-PBS to make standard solutions of various concentrations. 100 μl of the coated antigen dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6) was added to a 96 well immunoplate, incubated at 4 ° C. for 14 hours, and then washed 5 times with PBST. 50 μl of pesticide standard solution is added, and then 50 μl of the antibody diluted with PBS is added and reacted at 25 ° C. for 1 hour. After washing 5 times with PBST, HRP-coupled goat anti-mouse IgG was diluted 1: 3000 times with PBST, added 100 μl / well, and reacted at 25 ° C. for 1 hour. After washing 5 times with PBST, 100 μl / well of tetramethylbenzidine (3 ', 3,5', 5-tetramethylbenzidine, TMB) solution is added and reacted at 25 ° C. At the appropriate time, add 50 μl / well of 2 MH ₂ SO ₄ to stop the reaction and measure the absorbance at 450-650 nm.

The calibration curve of the indirect competitive immunoassay was fitted by a 4-parameter logistic equation.

Since eight monoclonal antibodies and 11 coated antigens were used, a total of 88 immunoassays could be established. For each immunoassay, the optimal dilution factor was determined by indirect competition immunoassay using three pesticides, chlorpyrephos, diazinon, and parathion, with different dilutions of antibody and coated antigen. The IC ₅₀ value was obtained by performing the calibration curve under the immunoassay under optimal conditions, which means the concentration of the analyte that inhibits the maximum absorbance by 50% and is obtained by the four-parameter logistic equation.

Among the eight monoclonal antibodies, antibodies produced from hapten H did not show affinity for most pesticides. Therefore, immunoassay was performed on 20 pesticides using various coated antigens for the remaining seven antibodies. As a result, the IC ₅₀ of each pesticide is shown in Tables 1-7. Pesticides 1 to 11 are phosphorothioate or phosphorodithioate pesticides with O, O -diethyl groups, pesticides 12 to 18 are other classes of organophosphorus pesticides and pesticides 19 and 20 are carbamate These are pesticides. In the table below, ni indicates that the IC50 value is 50000 ng / mL or more, indicating no inhibition.

Example 49; Detection of Organophosphorus Pesticides by Direct Competition Immunoassay

Direct competitive immunoassays were established using antibodies selected from indirect competitive immunoassays. The process of detecting organophosphorus pesticides by direct competitive immunoassay is as follows.

100 μl / well of Gout anti-mouse antibody dissolved in 50 mM carbonate-bicarbonate buffer (pH 9.6) was added to 96 well immunoplate and incubated at 4 ° C. for 16 hours. Then wash 5 times with PBST. 100 μl / well of antibody diluted with PBS is added and reacted at 25 ° C. for 1 hour. After washing 5 times with PBST, 50 µl of a pesticide standard solution dissolved in 10% methanol-PBS (10 mM, pH 7.4) and 50 µl of enzyme tracer diluted with PBS were added to the wells, and then reacted at 25 ° C for 1 hour. After washing 5 times with PBST, TMB solution was added to 100 μl / well and reacted at 25 ° C. After the appropriate time, 50 μl / well of 2 MH ₂ SO ₄ is added to stop the reaction and the absorbance is measured at 450-650 nm.

The affinity was measured for all kinds of enzyme tracers for antibodies, and the dilution folds of the affinity enzyme tracers were changed by two-dimensional screening with different dilutions of antibody and enzyme tracers. Calibration curves were obtained in the same way as indirect competitive immunoassays.

The affinity between each antibody and 14 enzyme tracers (hapten A to N) was measured. Calibration curves were obtained by direct competitive immunoassay for 18 pesticides using enzyme tracers with relatively good coloration, and IC ₅₀ values of the pesticides are shown in Tables 8 to 13. In the table below, ni indicates that the IC50 value is 50000 ng / mL or more, indicating no inhibition.

Comparative example

In order to compare the performance of the indirect competitive immunoassay and the conventional immunoassay according to the present invention, the IC ₅₀ values of the pesticides by each immunoassay are shown in Table 14. In the table below, ni indicates that the IC50 value is 50000 ng / mL or more, indicating no inhibition.

Table 1; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides in an indirect competitive immunoassay using antibodies (C5) obtained from hapten A and various coated antigens.

Table 2; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides in an indirect competitive immunoassay using antibodies (H10) from hapten B and various coated antigens.

Table 3; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides in indirect competitive immunoassays using antibodies (3E) obtained from hapten C and various coated antigens.

Table 4; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides in an indirect competitive immunoassay using antibodies (10F) from hapten D and various coated antigens.

Table 5; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides from indirect competitive immunoassays using antibodies (E6) from hapten E and various coated antigens.

Table 6; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides from indirect competitive immunoassays using antibodies (H6) from hapten F and various coated antigens.

Table 7; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides in an indirect competitive immunoassay using antibodies (B11) obtained from hapten G and various coated antigens.

Table 8; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides in direct competitive immunoassays using antibodies (C5) obtained from hapten A and various enzyme tracers.

Table 9; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides of direct competition ELISA using antibody (H10) obtained from hapten B and various enzyme tracers.

Table 10; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides of direct competitive ELISA using antibodies (3E) obtained from hapten C and various enzyme tracers.

Table 11; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides of direct competitive ELISA using antibodies (10F) obtained from hapten D and various enzyme tracers.

Table 12; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides of direct competitive ELISA using antibodies (E6) obtained from hapten E and various enzyme tracers.

Table 13; IC ₅₀ values (ng / mL) for organophosphorus and carbamate pesticides of direct competitive ELISA using antibody (B11) obtained from hapten G and various enzyme tracers.

Table 14; Comparison of performance (IC ₅₀ value, ng / mL) of immunoassay and conventional immunoassay according to the present invention

Comparing IC ₅₀ values of each pesticide by each immunoassay, several important characteristics of the immunoassay according to the present invention can be seen.

First, the immunoassay according to the present invention is much more sensitive to phosphorothioate and phosphorodithioate-type organophosphate pesticides having O, O- diethyl groups than conventional assays of the same purpose.

Second, the immunoassay according to the present invention is much less sensitive to phosphorothioate and phosphorodithioate-based organophosphorus pesticides having O, O- diethyl groups than conventional assays of the same purpose. will be.

Third, the immunoassay according to the present invention shows similar sensitivity to phosphorothioate pesticides for EPN, which is a phosphonothioate pesticide, so that EPN can be detected.

Fourth, the immunoassay according to the present invention has much higher selectivity since it shows little response to the oxon and other class of pesticides of organophosphorus pesticides, unlike conventional assays of the same purpose. The only exceptional pesticide is phenytrothion, an O, O- dimethyl phosphorothioate family.

As can be seen from the above, the immunoassay according to the present invention has a very high and similar response to phosphorothioate and phosphorodithioate having O, O- diethyl group which are most used among organophosphorus pesticides. Indicates. In terms of sensitivity and selectivity, it is far superior to conventional immunoassays developed so far. These facts are derived from the very high specificity for the (CH ₃ CH ₂ O) ₂ P (= S) -structure of the antibody according to the invention, wherein the (CH ₃ CH ₂ O) ₂ P (= S)-moiety is epitope. It is an important structure for forming an epitope.

From the above results, the immunoassay according to the present invention has a high sensitivity and specificity for the (CH ₃ CH ₂ O) ₂ P (= S)-structure, and therefore , an organophosphorus pesticide having an O, O- diethyl group, particularly preferably Phosphorothioate or phosphorodithioate can be regarded as a suitable method for analyzing the organophosphorus pesticides as a whole.

Monoclonal antibodies according to the present invention are much more sensitive than other antibodies of the same purpose for phosphorothioate and phosphorodithioate having O, O- diethyl groups, which are the most widely used organophosphorus pesticides. High and small variation in response

Therefore, by using the hapten and monoclonal antibodies according to the present invention, it is possible to more effectively perform a total analysis of organophosphorus pesticides such as phosphorothioate or phosphorodithioate having an O, O-diethyl group in common.

Claims (7)

Hapten and its N-succinimidyl ester derivatives having the structure:

or

Where R ₁ is CH ₃ , CH ₂ CH ₃ , or OCH ₃ ; R ₂ is CO ₂ H or CONH (CH ₂ ) ₂ CO ₂ H. A monoclonal antibody for the total analysis of an organophosphorus pesticide having an O , O -diethyl group, which is obtained from an immunogen conjugated to a carrier protein according to claim 1. The monoclonal antibody according to claim 2, wherein the organophosphorus pesticide having an O , O -diethyl group is phosphorothioate or phosphorodithioate. A method of immunizing a mouse with an immunogen conjugated with a carrier protein to a hapten of claim 1, extracting the spleen of the immunized mouse, fusing the cells with myeloma cells, and cloning to obtain a monoclonal antibody. Indirect competitive immunoassay for the total detection of phosphorothioate and phosphorodithioate organophosphorus pesticides having O, O- diethyl groups , comprising the following steps: a) immobilizing a coating antigen having hapten for phosphorothioate and phosphorodithioate organophosphorus pesticides having an O, O- diethyl group on a solid support; b) contacting an antibody and a sample against phosphorothioate and phosphorodithioate organophosphorus pesticides having an O, O- diethyl group with a coating antigen;   c) Pesticides can be determined by quantifying the antibody bound to the coated antigen by measuring the signal from the label using an antibody labeled with a fluorescent atom group, a radioisotope, or a colloidal gold, or by adding an anti-mouse secondary antibody with such a label. Quantifying the concentration. Direct competition immunoassay for the total detection of organophosphorus pesticides with O , O -diethyl groups, comprising the following steps: a) immobilizing a monoclonal antibody obtained by using a conjugate attached with a protein to hapten for phosphorothioate and phosphorothiothioate organophosphorus pesticides having an O, O- diethyl group as an immunogen to a solid support; b) contacting the antibody with a conjugate labeled with hapten for the phosphorothioate and phosphorodithioate-containing organophosphorus pesticides having an O, O- diethyl group and the antibody;   c) Quantifying the pesticide concentration by quantifying the hapten-label conjugate bound to the antibody by measuring the signal from the label using enzymes, fluorescent atomic groups, radioisotopes, colloidal gold, etc. as the label. Hapten and its N-succinimidyl derivatives having the following structure: