KR100879223B1 - Enzyme-linked immunosorbent assay ELISA for the detection of insecticide bistrifluron residues - Google Patents

Abstract

According to the present invention, a hapten compound and protein of a bistrifluron immobilized on a solid support and an antibody produced from a complex comprising a sample containing an insecticide bistrifluron and a hapten compound and a protein of bistrifluron are bound by amide bonds. Contacting a complex bound by amide bonds and detecting binding of said antibody to bistrifluron immobilized on said solid support. According to the present invention, the insecticide bistrifluron can be detected quickly and quantitatively by using an antibody against a complex in which a specific hapten compound of bistrifluron and a protein are bound.

Description

Enzyme-linked immunosorbent assay (ELISA) for the detection of insecticide bistrifluron residues}

1 is a schematic diagram schematically showing the method of the present invention.

2 is a graph showing purification of antibodies against hapten I-KLH conjugates (I-KLH) using a Protein A column.

Figure 3 is the SDS-PAGE results of the isolated antibody.

4 shows indirect competitive ELISA results of polyclonal antibodies using I-BSA as the coating antigen and I-KLH as the antiserum.

5 shows indirect competitive ELISA results of polyclonal antibodies using various coating antigens.

6 shows the effect of various blocking reagents on indirect competitive ELISA.

7 and 8 show the effects of acetone and methanol on indirect competition ELISA, respectively.

9 shows the effect of pH on indirect competition ELISA.

10 shows the effect of ion concentration on indirect competitive ELISA.

11 shows the effect of Tween 20 on indirect competition ELISA.

12 shows standard curves under optimization conditions.

13 shows the results of cross-reactivity.

The present invention relates to an enzyme immunoassay for detecting pesticide bistrifluron residues.

Insecticide Bistrifluron is a low-toxic alternative to organophosphorus and carbamate insecticides and is effective against greenhouse dusts, lepidopteran pests and termites. Compared to the organophosphorus, which causes neurotransmission and kills pests, bistrifluron suppresses the biosynthesis of chitin, the main component of the epidermis, which does not exist in humans and animals but exists only in crustaceans and insects. It is a pesticide with a new mechanism of action that leads to. Therefore, it is a pesticide that has excellent effects on environmentally friendly and organophosphorus resistant pests, which are not toxic to humans, livestock and useful animals that do not have chitin.

Residual pesticides were mainly analyzed by gas chromatography (GC) or high performance liquid chromatography (HPLC), which have high sensitivity and accuracy. In addition, in the case of GC, analysis of thermally unstable substances is impossible, and in the case of HPLC, pesticides without chromophores are difficult to detect. In order to solve this problem, attempts to apply immunoassays, which are mainly used for the analysis of biological components or clinical diagnosis, began in the 1970s. Enzyme-linked Immunosorbent Assay (ELISA) is an enzyme- and antibody-related enzyme involved in the analysis process. The analysis of residual pesticides by this enzyme immunoassay is very sensitive and almost no pretreatment of the sample is required. The advantage is that multiple samples can be analyzed quickly and simultaneously, and at a lower cost.

Since ELISA (enzyme immunoassay) is based on the specific and high affinity binding between the antibody and the antigen, in order to develop an immunoassay, an antibody to an analyte must be produced and thus an appropriate antigen must be prepared. . However, since low-molecular substances, such as pesticides, cannot function as antigens themselves and thus cannot produce antibodies, they are similar in structure to pesticides, and have a group of atoms that can covalently bind proteins, that is, the synthesis of hapten. This was required. In addition, synthesis of hapten has been required for the preparation of coating antigens and enzyme tracers, which are competitors used in competitive immunoassays.

The ELISA of pesticides has been actively studied since the 1970s by the US FDA and other organizations around the world. Currently, ELISA assays for pesticides developed or developed in foreign countries include 7 fungicides, 30 pesticides, 28 pesticides and plants. There are about 80 kinds of growth regulators.

However, some universities and research institutes in Korea have been tested for research purposes, and there are about 7 kinds of pesticides produced by antisera, and they have not been commercialized. ELISA kits for the detection of pesticide residues in use are imported products. Therefore, in addition to the opening of the agricultural market internationally, the farming system tends to pursue technology-intensive, high quality, and high safety in domestic agriculture. There is a continuing need for ELISA using excellent antisera.

The inventors of the present invention, while studying the detection method for the insecticide bistrifluron based on the prior art as described above, by performing an ELISA using an antibody to a complex of a specific hapten compound and protein of bistrifluron, pesticide beast The present invention was completed, confirming that rifluron can be detected quickly and quantitatively.

Accordingly, an object of the present invention relates to a method for detecting bistrifluron in a sample using an antibody produced from a complex in which a hapten compound and a protein of insecticide bistrifluron are bound by amide bonds.

In order to achieve the object of the present invention, the present invention

The hapten compound and protein of bistrifluron immobilized on a solid support and the antibody produced from the sample containing the insecticide bistrifluron and the hapten compound and protein of the bistrifluron are bound by the amide bond. Contacting the bound complex by And detecting binding of the antibody to bistrifluron immobilized on the solid support.

1 is a schematic diagram schematically showing the method of the present invention. The complex in which the hapten compound of bistrifluron and the protein are bound by amide bond as a coating antigen is immobilized on the solid support by hydrophobic interaction through the protein. Washing and blocking using the protein used for complex formation, and a sample containing bistrifluron, for example, an antibody produced from a complex in which a hapten compound of bistrifluron and a protein are bound by an amide bond React with the complex immobilized on the solid support. The antibody binds competitively to bistrifluron and hapten in a complex immobilized on a solid support. If the amount of bistrifluron in the sample is large, the amount of antibody that binds to the hapten of the complex immobilized on the solid support is small, and the absorbance measured thereafter decreases. The amount of antibody that binds to the hapten increases, and the absorbance measured thereafter increases. Therefore, by measuring the amount of the antibody that binds to the hapten of the complex immobilized on the solid support, the amount of bistrifluron in the sample can be measured.

The antibody may be a mixture of an antibody against a hapten and an antibody against a carrier protein. If the carrier protein used for forming the antibody and the carrier protein immobilized on the solid support are the same, the antibody to the carrier protein is immobilized on the solid support. This will interfere with the analysis of bistrifluron in the sample to be analyzed. Therefore, the carrier protein immobilized on the solid support and the protein used for forming the antibody are preferably different from each other.

When the bistrifluron in the sample binds to the antibody against the complex, it is not fixed to the solid support and is removed by the washing liquid, whereas when the antibody binds to the hapten immobilized on the solid support, the solid is not removed by the washing liquid. Will remain on the support. When an enzyme-labeled secondary antibody that specifically binds to an antibody against bistrifluon is added, the secondary antibody specifically binds to an anti-bistrifluron antibody, where the enzyme degrades to develop a color reaction. The addition of a substrate that can cause a color reaction occurs, the absorbance is measured by using an absorbance measuring instrument. The enzyme may include horseradish peroxidase, alkaline phosphatase, urease, and the like, but is not limited thereto. Substrates can be variously selected depending on the enzyme used. For example, in the case of horseradish peroxidase, an o -phenylenediamine (OPD) substrate is used and the absorbance is measured at 492 nm.

Smaller molecules can also bind to antibodies, but they do not induce immune responses in vivo. Therefore, this material must be linked to a carrier of high molecular weight. Since most pesticides are not large enough to stimulate the immune system, antibodies to pesticides must be injected into experimental animals by linking them to a carrier of high molecular weight, such as a protein. Some pesticides have functional groups such as -COOH, -SH, -OH or -NH ₂ , which are good for binding to the carrier. However, most pesticides do not have such functional groups. A substance that can bind to proteins is called a hapten.

The hapten may comprise a carboxyl or amino group to form an amide bond with the protein. The carboxyl group of the hapten may form an amide bond with the amino group of the protein, and the amino group of the hapten may form an amide bond with the carboxyl group of the protein. The binding of the hapten to the carrier protein can be carried out by methods known to those skilled in the art. The method will of course depend on the functional groups present in the carrier protein. In the present invention, keyhole limpet hemocyanin (KLH) protein, bovine serum albumin (BSA) protein, and Ovalbumin (OVA) protein were combined with hapten. As a method of binding a protein and a hapten having a carboxyl group, an active ester method using carbodiimide and N-hydroxysuccinimide as described below was used.

As a method of binding a hapten having an amino group and a protein, a glutaryl dialdehyde method as described below can be used.

Preferably, the hapten compound of bistrifluron is a compound of formula

<Formula 1>

[In the meal,

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, carboxyl group, hydroxy group, alkyl group having 1 to 10 carbon atoms, halogen atom, cyano group, amino group, thiol group, aldehyde group, A nitro group, a carboxyalkyl group having 2 to 10 carbon atoms, a hydroxyalkyl group having 1 to 10 carbon atoms, or a haloalkyl group having 1 to 10 carbon atoms,

R ₈ represents an amino group or a carboxyl group,

X ₁ and X ₂ each independently represent a simple bond, an arylene group having 6 to 20 carbon atoms, or an alkylene group having 1 to 10 carbon atoms, at least one hydrogen atom present in the arylene group is substituted with a halogen atom,

m represents an integer of 0 to 3, n represents an integer of 1 to 3;

In the context of the present invention, the term "alkyl" includes straight or branched radicals of 1 to 10 carbon atoms, with preferred alkyl radicals being lower alkyl having 1 to 6 carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, iso-amyl, hexyl and the like. More preferred are lower alkyl radicals having 1 to 3 carbon atoms.

In the context of the present invention, the term "halogen" includes, but is not limited to, fluoride, chloride, bromide and iodide.

In the context of the present invention, the term "arylene" is a divalent form of aryl meaning a carbocyclic aromatic ring or ring system, for example phenylene, naphthylene, biphenylene, fluorenylene, indenylene, and the like. .

In the context of the present invention, the term "alkylene" is a divalent form of the "alkyl" group as defined above.

More preferably, the hapten compound of bistrifluron can be a compound of the following hapten I, II, III, IV or V:

<Hapten I>

<Hapten II>

<Hapten III>

<Hapten IV>

<Hapten V>

Considerations in selecting the hapten compound of the bistrifluron are as follows.

1) Since almost all functional groups in the analyte must be present in the hapten, the hapten must possess the characteristics of the analyte in terms of form and electron distribution.

2) If any atom or group in the analyte is to be substituted, it should be replaced by a similar substituent such as sulfur instead of chlorine.

3) The length of the linker or spacer arm of the hapten is preferably 4 to 6 carbon atoms. In addition, the spacer arm should not contain characteristic functional groups or large keys that the antibody can recognize.

4) It is not good to make a spacer arm adjacent to the position of the characteristic functional group or atom. This is because antibodies due to steric hindrance of spacer arms may not recognize characteristic structures or functional groups of hapten.

5) Immune triggered hapten and assay hapten (plate coated hapten or tracer hapten or analyte complex) should be different. If different, there is an advantage that can eliminate the bridging effects associated with the affinity of the antibody due to spacer cancer.

Considering the above, 2,6, -di can be used to recognize the 2-chloro-3,5-bistrifluoromethyl aniline structure, which is a characteristic structure of bistrifluron (pictured below) in a benzoyl phenyl urea insecticide. The hapten I to V were synthesized by designing a hapten with a spacer having COOH or NH ₂ , which is the functional group most introduced at the fluorobenzoic acid position (position a), and the structure of the synthesized hapten was obtained by using NMR and MS. It was confirmed and used as an immunogen and a coating antigen.

In the complex of the present invention, the protein may be bovine serum albumin (BSA), human serum albumin (HSA), fibrinogen, ovalbumin, thyroglobulin or keyhole limpet hemocyanin (KLH), but is not limited thereto. . Carrier proteins of the invention include molecules containing at least one T-cell epitope that can stimulate T-cells and then induce B-cells to produce antibodies against the entire hapten-carrier complex molecule. Epitopes determinants generally consist of chemically active surface groups of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. To be immunogenic it is believed that a protein or polypeptide must be able to stimulate T-cells. However, carrier proteins lacking T-cell epitopes can also have immunogenicity.

The antibody used in the method of the present invention may be a monoclonal antibody or a polyclonal antibody. Techniques for producing monoclonal antibodies are known in the art. Monoclonal antibodies are injected into mice with a composition comprising a hapten-carrier complex, followed by removal of serum samples to confirm antibody production, removal of the spleen to obtain B-lymphocytes, and fusion of these B-lymphocytes and myeloma cells Prepare hybridomas, clone these hybridomas, select positive clones to produce antibodies against hapten-carrier complexes, culture clones to produce antibodies to antigens, and antibody from hybridoma cultures It can be obtained by separating

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well established techniques. Such separation techniques include hydrophilic chromatography, protein-exclusion chromatography and ion exchange chromatography with Protein-A Sepharose. See, for example, pages 2.7.1–2.7.12 and 2.9.1–2.9.3 of Cogali. See also, "Purification of Immunoglobulin G (IgG)" by Baines et al. (METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104, The Humana Press, Inc. 1992).

Techniques for preparing polyclonal antibodies are also known in the art. Polyclonal antibodies are prepared according to known standard techniques. To prepare polyclonal antibodies, immunogenic materials are injected into animals and serum is collected which contains antibody mixtures for numerous epitopes of the injected immunogen. Suitable host mammals for antibody production include, but are not limited to, humans, mice, mice, rabbits, and goats.

In the method of the invention, bistrifluron in the sample can be detected quantitatively. Although the method of the present invention can be used to qualitatively detect bistrifluron in a sample, it is also possible to detect quantitatively. This is to obtain a standard curve indicating the absorbance according to the bistrifluron concentration using a standard material, and then to measure the absorbance by the same procedure using the sample, and then quantitatively obtain the concentration in the sample from the measured absorbance. .

In the method of the present invention, the solid support may be any one capable of fixing the carrier protein, and may be sheets, films, beads, pellets, disks, plates, rings, rods, nets, membranes, filters, trays, and microplates. , Stick, slide, or tube, but is not limited thereto.

Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1 Synthesis of Hapten

1) Synthesis of Hapten I

① Synthesis of I-2 (2,6-difluoro-3-iodobenzoic acid)

2,6-difluorobenzoic acid (I-1, 15.8 g) and iodine (12.7 g) were placed in a 250 mL three neck flask, and 30 mL of acetic acid was added to dissolve. The temperature was maintained at 85-90 ° C, and a mixture of 4.8 mL of nitric acid and 10.6 mL of sulfuric acid was slowly added using a dropping funnel. Sublimed iodine was dissolved into the reaction solution using a small amount of carbon tetrachloride. After heating under reflux for 6 hours, the solvent was concentrated under reduced pressure, and the residue was dissolved in 80 mL ethyl acetate, and the remaining iodine was removed with Na ₂ S ₂ O ₃ solution. The organic solvent layer was washed with distilled water and brine, the aqueous layer was reextracted with ethyl acetate, the organic solvent layers were combined, dehydrated with anhydrous Na ₂ SO ₄ and distilled under reduced pressure to obtain 25 g of the product.

NMR (CDCl ₃ ): 6.84 (1 H, m), 7.86 (1 H, m), 10.62 (1 H, br)

② Synthesis of I-3 (2,6-difluoro-3-iodobenzamide)

20 g of compound I-2 was added to excess thionylchloride and heated to reflux for 2 hours. After heating under reflux, the solvent was evaporated to dryness and a cold saturated ammonia solution was added to the residue in an ice bath. After shaking vigorously for 10 minutes, the resulting solid was filtered and washed with saturated ammonia solution to give 19.5 g of crude compound. This crude compound was recrystallized in ethanol / water = 8: 3 solution to give 16.1 g of a white desired compound.

NMR (CDCl ₃ ); 5.98 (2H, br), 6.82 (1H, m), 7.80 (1H, m)

③ Synthesis of I-4 (2,6-difluoro-3- [2- (ethoxycarbonyl) vinyl] benzamide)

11.3 g of I-3 compound was dissolved in 40 mL of acetonitrile, followed by stirring to reflux 2.8 g of dichloro bis (triphenylphosphine) palladium (II) catalyst, 44 mL of ethyl acrylate, and 8.1 g of triethylamine under nitrogen reflux. It was added under.

After the anhydrous calcium chloride trap was installed, the reaction mixture was heated to reflux overnight, the solvent was dried under reduced pressure, and the residue was separated by 5.3 g of the target compound using column chromatography.

NMR (CDCl ₃ ); δ 1.31 (t, 3H), 4.21 (q, 2H), 6.04 (s, 1H), 6.21 (s, 1H), 6.45 (d, 1H), 6.94 (m, 1H), 7.59 (m, 1H), 7.70 (d. 1 H)

GC-MS; m / z 255 (M &lt; + &gt;, 30), 210 (100), 191 (55), 119 (35).

④ Synthesis of I-5 (2,6-difluoro- [2- (ethoxycarbonyl) ethyl] benzamide)

After dissolving 1.3 g of I-4 in 130 mL of ethyl acetate, 0.26 g of platinum oxide catalyst was added and reduced while blowing hydrogen. When no more hydrogen was consumed, the reaction mixture was filtered using Celite and the solvent was dried under reduced pressure to recrystallize the resulting solid with an ethyl acetate / petroleum ether mixed solution to obtain 1.2 g of the target compound.

NMR (CDCl ₃ ); δ 1.19 (t, 3H), 2.56 (t, 2H), 2.93 (t, 2H), 4.09 (q, 2H), 5.97 (s, 1H), 6.02 (s, 1H), 6.85 (m, 1H), 7.35 (m. 1 H),

GC-MS; m / z 257 (M &lt; + &gt;, 10), 140 (100), 126 (65), 95 (90).

⑤ Synthesis of I-6 (2-chloro-3,5-bistrifluoromethyl aniline)

23 g of 3,5-bistrifluoromethyl aniline was dissolved in 100 mL carbon tetrachloride, and then 15 g of N-chlorosuccinimide was added and heated to reflux for 4 hours. After the reaction solution was cooled in an ice bath, the precipitated imide was removed, the organic layer was washed three times with 100 mL of distilled water, and the organic solvent layer was dehydrated with Na ₂ SO ₄ and dried under reduced pressure to obtain a crude extract. 20 mL of hexane was added to the crude extract, and recrystallized in a -20 ° C refrigerator to obtain 21 g of the target compound.

NMR (CDCl ₃ ); δ 4.53 (s, 2H), 7.12 (s, 1H), 7.22 (s, 1H).

GC-MS; m / z 263 (M +, 100), 265 (M + 2 +, 32), 244 (15), 208 (13)

⑥ of I-7 (1- [2-chloro-3,5-bistrifluoromethyl] -3- [2,6-difluoro-3- (2-ethoxycarbonylethyl) benzoyl] urea) synthesis

Compound I-5 was reacted with freshly distilled oxalyl chloride to prepare 2,6-difluoro- [2- (ethoxycarbonyl) ethyl] benzoyl isocyanate according to conventional methods. Next, a solution of crude isocyanate in carbon tetrachloride was added directly to a stirred solution of compound 5 in carbon tetrachloride. The mixture was stirred at rt for 6 h, the solvent was removed by distillation under reduced pressure and the residue was purified by column chromatography to give the product as a white solid.

NMR (CDCl ₃ ); δ 1.21 (t, 3H), 2.61 (t, 2H), 2.97 (t, 2H), 4.10 (q, 2H), 6.98 (m, 1H), 7.43 (m, 1H), 7.70 (s, 1H), 8.39 (s, 1 H), 8.88 (s, 1 H), 11.43 (s, 1 H)

⑦ Synthesis of I-8 (1- [2-chloro-3,5-bistrifluoromethyl] -3- [2,6-difluoro-3- (2-carboxyethyl) benzoyl] urea)

Hydrolysis of compound I-7 (0.3 g) was carried out in isopropyl alcohol (30 mL) with 2 equivalents of KOH. After 8 hours, the reaction mixture was extracted with ethyl acetate (50 mL). The aqueous phase was acidified to pH 2 with 6 M HCl and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were then dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography to give hapten I as a white solid (0.1 g):

NMR (CDCl ₃ ); δ 2.65 (t, 2H), 2.97 (t, 2H), 7.12 (m, 1H), 7.61 (m, 1H), 9.08 (s, 1H), 10.79 (s, 1H), 11.53 (s, 1H).

2) Synthesis of Hapten II

① Synthesis of II-2 (2,6-difluoro-3-nitrobenzoic acid)

3 ml of nitric acid and 12 ml of concentrated sulfuric acid were slowly added dropwise to 2,6-difluorobenzoic acid (I-1, 3.2 g) in an ice bath, followed by stirring for 2 hours. After the reaction was completed, water was added and extracted twice with 100 ml of ethyl acetate. The organic layer was dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and separated by column chromatography to obtain 3.1 g of the target compound. (Ethyl Acetate / Methanol = 2: 1)

GC-MS; m / z 203 (M +, 82), 186 (10), 173 (37), 153 (31), 140 (27), 125 (33), 112 (63), 101 (100), 63 (85)

② Synthesis of II-3 (2,6-difluoro-3-nitrobenzamide)

2.5 g of compound II-2 was added to excess thionylchloride and heated to reflux for 2 hours. After heating under reflux, the solvent was dried under reduced pressure, and a cold saturated ammonia solution was added to the residue in an ice bath. After shaking vigorously for 10 minutes, the resulting solid was filtered and the saturated ammonia solution was washed and separated by column chromatography to obtain 2.3 g of the target compound (ethyl acetate / hexane = 1: 2).

GC / MS; m / z 202 (M +, 90), 186 (100), 140 (87), 112 (67)

③ synthesis of II-4 (1- [2-chloro-3,5-bistrifluoromethyl] -3- [2,6-difluoro-3-nitrobenzoyl] urea)

1.0 g of compound II-3 was placed in a 50 ml flask, 30 ml of CCl ₄ was added thereto, and 2 ml of oxalyl chloride was added dropwise, followed by reflux for at least 12 hours. After the reaction was completed, the solvent and oxalyl chloride were blown off by distillation under reduced pressure, and redissolved in 10 ml of chloroform. 800 mg of compound I-6 was dissolved in 10 ml of chloroform and slowly added dropwise and stirred for 1 hour. After drying the solvent under reduced pressure, 653 mg of the target compound was obtained by column chromatography. (Ethyl acetate / hexane = 1: 7)

④ Synthesis of II-5 (1- [2-Chloro-3,5-bistrifluoromethyl] -3- [2,6-difluoro-3-aminobenzoyl] urea)

0.5 g of compound II-3 and 0.5 g of PtO ₂ were added to a 100 ml flask, dissolved in 30 ml of ethyl acetate, and hydrogen was bubbled into a balloon. If no more hydrogen was absorbed, the mixture was filtered through celite, distilled under reduced pressure, and separated by column chromatography. (Ethyl Acetate / Hexane = 1: 3)

LC-MS; m / z 462 ([M + H] +, 100), 464 (34), 426 (19), 263 (18), 173 (54), 156 (31)

3) Synthesis of Hapten III

① Synthesis of III-1

500 mg of hapten II and 370 mg of ethyl 4-chloro-4-oxo butyrate were dissolved in 20 ml of ethyl acetate, and 300 mg of K ₂ CO ₃ was added to 20 ml of water. After stirring for 4 hours, an ethyl acetate layer was taken, and dried under reduced pressure with anhydrous Na ₂ SO ₄ . Separation by column chromatography gave 570 mg of the target compound.

② Synthesis of Hapten III

500 mg of III-1 and 50 mg of LiOHH ₂ O were dissolved in 20 ml of isopropanol and stirred until the end of the reaction. After completion of the reaction, the mixture was adjusted to pH 2 with 1N HCl, and extracted twice with 50 ml of ethyl acetate. After extraction, the residue was separated by column chromatography to obtain 230 mg of the target compound.

LC-MS; m / z 562 ([M + H] +, 100), 544 (25), 462 (20), 263 (37)

4) Synthesis of Hapten IV

① Synthesis of IV-1

500 mg of I-6 and 370 mg of methyl 4-chloro-4-oxo butyrate were dissolved in 20 ml of ethyl acetate, and 300 mg of K ₂ CO ₃ was added to 20 ml of water. After stirring for 4 hours, an ethyl acetate layer was taken, and dried under reduced pressure with anhydrous Na ₂ SO ₄ . Separation by column chromatography gave 220 mg of the target compound.

NMR (CDCl ₃ ); δ 1.21 (t, 3H), 2.81 (m, 4H), 4.16 (q, 2H), 7.69 (s, 1H), 8.32 (s, 1H), 8.96 (s, 1H).

GC-MS; m / z 377 (M +, 3), 346 (10), 322 (5), 263 (8), 115 (100), 87 (19), 55 (48)

② Synthesis of Hapten IV

100 mg of IV-1 and 30 mg of LiOHH ₂ O were dissolved in 20 ml of isopropanol and stirred until the end of the reaction. After completion of the reaction, the mixture was adjusted to pH 2 with 1N HCl, and extracted twice with 50 ml of ethyl acetate. After extraction, the residue was separated by column chromatography to obtain 70 mg of the target compound.

NMR (CDCl ₃ ); δ 2.83 (m, 4H), 7.69 (s, 1H), 8.21 (s, 1H), 8.98 (s, 1H).

GC-MS (TMS) m / z 435 (M +, 0.3), 420 (10), 263 (100), 173 (34), 101 (90), 73 (84), 55 (54)

5) Synthesis of Hapten V

① Synthesis of V-1

500 mg of I-6 and 370 mg of methyl 4-chloro-4-oxo hexanoate were dissolved in 20 ml of ethyl acetate, and 300 mg of K ₂ CO ₃ was added to 20 ml of water. After stirring for 4 hours, an ethyl acetate layer was taken, and dried under reduced pressure with anhydrous Na ₂ SO ₄ . Separation by column chromatography gave 57 mg of the target compound.

NMR (CDCl ₃ ); 1.78 (m, 4H), 2.39 (t, 2H), 2.53 (t, 2H), 3.70 (s, 3H), 7.68 (s, 1H), 7.91 (s, 1H), 9.00 (s, 1H).

GC-MS; m / z 405 (M +, 3), 374 (12), 350 (8), 263 (14), 143 (55), 111 (100), 55 (83)

② Synthesis of Hapten V

50 mg of V-1 and 20 mg of LiOHH ₂ O were dissolved in 20 ml of isopropanol and stirred until the end of the reaction. After completion of the reaction, the mixture was adjusted to pH 2 with 1N HCl, and extracted twice with 50 ml of ethyl acetate. After extraction, the residue was separated by column chromatography to obtain 33 mg of the target compound.

NMR (CDCl ₃ ); δ 1.21 (t, 3H), 1.70 (d, 3H), 4.20 (q, 2H), 4.79 (q, 1H), 6.81-8.18 (m, 10H), 9.21 (s, 1H).

GC-MS (TMS); m / z 463 (M +, 0.3), 448 (13), 412 (8), 263 (7), 201 (43), 111 (100), 73 (42)

Example  2: Hapten - carrier  Protein binding

1) Protein binding with hapten I, III, IV, V

Hapten, including COOH functional groups, 1.1 equivalents of N-hydroxysuccinimide and 1.3 equivalents of DCC were dissolved in 1 ml of DMF and stirred overnight. The urea precipitate was removed by centrifugation, and slowly added dropwise to the protein (KLH, BSA, OVA) dissolved in pH9.0 buffer at 0.5m. After stirring for 12 hours, the PBS solution was dialyzed twice a day for three days.

2) protein binding with hapten II

10 mg of hapten II and 15 mg of BSA with NH ₂ functionality were dissolved in 2.5 ml PBS: dioxane = 2: 1 solution. 200ul of 25% glutaric dialdehyde was added to the solution, followed by stirring at 4 ° C for 5 hours. After the reaction solution was desalted using Sephadex G 25 (2 g), the PBS solution was dialyzed for 3 days while changing twice a day. Table 1 shows the amount of protein and hapten used for binding.

Table 1. Hapten-protein binding reaction

Hapten (mg) Protein (mg) Hapten (mg) Protein (mg) I-KLH 20 20 IV-BSA 16 50 I-BSA 20 20 V-BSA 17 50 II-BSA 10 15 I-OVA 5 50 III-BSA 23 50

3) Confirmation of hapten and protein binding

1.0 mL of 0.05M pH9.6 carbonate buffer was added to 1.0 mL of 0.5 mg / ml protein (BSA, KLH) and hapten-protein complex, followed by 1.0 mL of 0.1% TNBSA (trinitrobenzenesulfonic acid) solution. After reacting at 40 ° C. for 2 hours, 1.0 ml of 10% SDS (sodium dodecyl sulfate) solution was added, and 1 mL of 1M HCl solution was added to stop the reaction. The absorbance of the reaction solution was measured at 335 nm and the ratio of hapten bound to the protein was determined by the following equation.

Coupling density = (free protein absorbance-hapten complex protein absorbance) / (protein absorbance) * 100

Table 2. Hapten-protein binding concentrations

antigen Coupling Density (%) antigen Coupling Density (%) I-KLH 88.82 IV-BSA 81.75 I-BSA 98.15 V-BSA 79.34 II-BSA 73.22 I-OVA 34.31 III-BSA 75.56

Example  3: antiserum production and Titer  black

1) Production / Titer Test of Antisera

The amount of protein bound with hapten was quantified by Bradford method (Table 3) and used in the next experiment.

Table 3. Protein Concentration of Hapten-protein Complexes

antigen Protein (mg / ml) antigen Protein (mg / ml) I-KLH 1.9 IV-BSA 3.36 I-BSA 3.4 V-BSA 2.70 II-BSA 0.78 I-OVA 6.56 III-BSA 1.64

PBS solution containing 0.5 mg of hapten I-KLH conjugate (I-KLH) was mixed 1: 1 with Freund's complete adjuvant solution to prepare an emulsion, and then divided into 15 to 20 parts at the back of 2-4 kg rabbit. It was. Two rabbits were used for each antigen.

For boosting injections, PBS solution containing 0.2 mg of hapten I-KLH conjugate (I-KLH) is mixed 1: 1 with Freund's incomplete adjuvant to inject emulsion and then injected at two-week intervals in the same manner as the first injection. It was.

One week after the injection, blood was drawn from the ear vein to confirm antibody production and the antigen injected until sufficient titer appeared.

Blood was collected by heart puncture after the fifth injection.

2) Isolation of Antiserum

Collected blood was coagulated in the refrigerator for about one day, centrifuged at 3000rpm for 20 minutes to separate the serum and stored frozen at -70 ℃ until use.

3) Pure separation of antibodies

PIERCE's Protein A Immunoglobulin Purification Kit was used to purely isolate the antibody.

The kit was allowed to stabilize at room temperature and then equilibrated by flowing 5 ml of binding buffer. Serum diluted 1: 1 with binding buffer was loaded and washed with 15 ml of binding buffer. Eluted with 5 ml of elution buffer.

Each fraction was measured for absorbance at 280 nm and used for the next experiment by combining the E3-E5 moieties with absorbance. 2 is a graph showing purification of antibodies against hapten I-KLH conjugates (I-KLH) using a Protein A column.

SDS-PAGE of the combined fractions of the E3-E5 moiety revealed that the antibody was purely isolated by the bands of the light and heavy chains of the antibody after purification. Figure 3 is the SDS-PAGE results of the isolated antibody. 3A is for rabbit 1 of 2 rabbits, and B is for rabbit 2.

4) check board titration

In order to determine the dilution factor of the coating antigen and antiserum required for ELISA, it was measured by the following procedure by non-competitive indirect ELISA.

① 100 μl of the diluted antigen dissolved in 0.1 M carbonate-bicarbonate buffer (pH 9.6) (1 μg / ml, 0.5 μg / ml, 0.1 μg / ml, 50 ng / ml) was incubated in the wells of a microplate (4 ℃, 12 hours or more).

 ② washed 5 times with 100ul PBST (10mM phosphate water + 0.05% Tween 20).

③ Block by adding 100 μl of 1% BSA / PBS (37 ° C., 1 hour), and then antiserum diluted with PBS (1: 1,000, 1: 2,000, 1: 4,000, 1: 8,000, 1: 16,000, 1: 32,000, 1: 64,000, 1: 128,000) was added to the wells and incubated (37 ° C, 1 hour).

④ washed 5 times with PBST.

⑤ 100 μl of goat anti-rabbit IgG solution bound to horseradish peroxidase diluted with PBST (1: 10,000) was incubated (37 ° C., 1 hour).

⑥ Washed 5 times with PBST.

⑦ 100 μl of enzyme substrate o -phenylenediamine (OPD) dissolved in phosphate-citrate buffer (pH 5.0) was added and reacted at 37 ° C. for 30 minutes.

⑧ 50N of 4N H ₂ SO ₄ solution was added to stop the reaction, and the absorbance was measured at 492 nm.

Table 4 below shows the check board titration results.

Table 4. Check Board Titration Results

1-KLH I-BSA +++ II-BSA +++ III-BSA + IV-BSA + V-BSA +++ +++: O.D.> 1.0, ++: 1.0> O.D. > 0.5, +: 0.5> O.D. Coated antigen: 100 ng / well, antiserum diluted 1: 4,000 fold.

Example  4: Optimize method

1) Indirect Competition ELISA Act

In order to confirm the sensitivity and the standard linearity to bistrifluron, experiments were performed using the indirect competition ELISA method with the dilution factor of the coating antigen and antiserum identified in Example 3-4).

① 100 μl of diluted (I-BSA) coated antigen dissolved in 0.1 M carbonate-bicarbonate buffer (pH 9.6) was incubated in the wells of a microplate (4 ° C., 12 hours or more).

② washed 5 times with 100ul PBST (10mM phosphate + 0.05% Tween 20).

③ After blocking by adding 100 μl of 1% BSA / PBS (37 ° C., 1 hour), 50 μl of bistrifluon standard solution (100ppm ~ 1ppt) diluted in PBST and antiserum (1: 100) 50 diluted with PBS ΜL was added to the wells and incubated (37 ° C., 1 hour).

④ washed 5 times with PBST.

⑤ 100 ㎕l of a goat anti-rabbit IgG conjugated with horseradish peroxidase diluted with PBST (1: 10,000) was incubated (37 ° C., 1 hour).

⑥ Washed 5 times with PBST.

⑦ 100 μl of enzyme substrate OPD (0.4mg / ml) dissolved in phosphate-citrate buffer (pH 5.0) was added and reacted at 37 ° C. for 30 minutes.

⑧ 50N of 4N H ₂ SO ₄ solution was added to stop the reaction, and the absorbance was measured at 492 nm.

As a result of diluting bistrifluron in PBST and using I-BSA as a coating antigen, the IC ₅₀ was about 1.5 ppm with affinity for bistrifluron.

2) Indirect competition ELISA using hapten I-BSA complex as coating antigen

Indirect competitive ELISA was performed using a plate coated with I-BSA (2.5 ng), one polyclonal antibody, and three monoclonal antibodies. 4 shows indirect competitive ELISA results of polyclonal antibodies using I-BSA as the coating antigen and I-KLH as the antiserum. As a result of using one polyclonal antibody, the IC ₅₀ values of the polyclonal antibodies were 1.39 and 1.32 for rabbits 1 and 2, respectively. The reason for this low sensitivity is that the affinity between the antibody and the coated antigen is greater than the affinity between the antibody and the compound. In order to increase the sensitivity, a method of changing the coating antigen is mainly used to lower the affinity between the antibody and the coating antigen. Therefore, the experiment was performed by changing the type of coating antigen.

3) Indirect competition ELISA using various coating antigens

Indirect competition ELISA was performed using one polyclonal antibody and three monoclonal antibodies using a plate coated with II-BSA, III-BSA, V-BSA (50 ng). 5 shows indirect competitive ELISA results of polyclonal antibodies using various coating antigens. As a result of indirect competitive ELISA using various coating antigens, the IC ₅₀ values of polyclonal antibodies using II-BSA and V-BSA as coating antigens were 0.733 and 0.793, respectively. Therefore, the following experiment was performed using the V-BSA coated antigen showing a stable absorbance value of the two, the background value is low.

4) Influence of blocking reagent

In order to examine the effects of various blocking reagents, 1% Ovalbumin (OVA), fish gelatin, and skim milk powder were blocked on a plate coated with V-BSA (50 ng) with a blocking solution prepared in PBS solution and indirect competition ELISA was performed. It was. 6 shows the effect of various blocking reagents on indirect competitive ELISA. As a result, OVA showed the highest absorbance value, and skim milk powder showed the lowest absorbance. However, OVA showed high background absorbance. Therefore, in the present invention, fish gelatin was used as the blocking reagent.

5) Effect of solvent type and concentration

In order to investigate the effect of solvent type and concentration, each solvent concentration was 5% (5% organic solvent-) using methanol and acetone as a solvent for preparing non-strifluron standard solution on a plate coated with V-BSA (50 ng). Indirect competitive ELISA was performed using 10 mM PBS), 10%, and 25%. 7 and 8 show the effects of acetone and methanol on indirect competition ELISA, respectively. As can be seen in FIG. 7, as the acetone content was increased, the absorbance value decreased from 0.54 to 0.25 and the IC ₅₀ value decreased from 0.42 ppm to 0.04 ppm. As can be seen in FIG. 8, the absorbance value was almost unchanged as the methanol content was increased, and the IC ₅₀ value was not particularly inclined. Bistrifluron ELISA was found to be highly influenced by the solvent.

6) Effect of Buffer pH

Indirect competition ELISA was performed on a plate coated with V-BSA (50 ng) using a non-strifluron standard solution prepared with PBS solution at various pHs, namely pH5, 6, 7, 8, 9, 10. 9 shows the effect of pH on indirect competition ELISA. As a result, it was confirmed that bistrifluron ELISA was stable in a narrow pH range (pH7 to pH8).

7) Effect of buffer ion concentration

Indirect competition ELISA was performed on a plate coated with V-BSA (50 ng) using a bistrifluron standard solution prepared with PBS solution having a ionic strength of 5, 10, 20, 40 mM buffer. 10 shows the effect of ion concentration on indirect competitive ELISA. As a result, the absorbance value decreased with increasing ion concentration, and the IC ₅₀ value decreased but showed a stable value after 20 mM. Bistrifluron ELISA was found to be highly affected by ion concentration.

8) Impact of Detergent

To examine the effect of detergent on indirect competitive ELISA, non-strifluron standards in buffer prepared with various Tween 20 concentrations, ie 0.05, 0.1, 0.2, 0.4% PBS solution, on plates coated with V-BSA (50 ng) Indirect competitive ELISA was performed using the solution. 11 shows the effect of Tween 20 on indirect competition ELISA. As a result, it was confirmed that Tween 20 does not significantly affect bistrifluron ELISA.

9) Optimization of ELISA

The calibration curve for bistrifluron is established by optimizing the conditions specified in the above experiment. IC _50, which represents the sensitivity of ELISA, refers to the concentration of analyte that inhibits the maximum absorbance by 50%, and this value presented in the present invention is obtained by the following equation.

y = (AD) / [1+ (x / C) ^B ] + D

Where A is the maximum absorbance, B is the slope of the straight line, C is IC ₅₀ and D is the minimum absorbance.

A calibration curve was established by performing indirect competition ELISA using 25% acetone, pH7.5 containing 0.05% tween, and 20 mM phosphate buffered saline. 12 shows standard curves under optimization conditions. The IC ₅₀ was 82 ppb and the analytical range (IC ₂₀ IC ₈₀ ) was 1.0 ppb to 500 ppb. ELISA assay of the present invention was confirmed to show a high sensitivity and a wide range of analysis.

10) Cross Reactivity Studies

The optimized bistrifluron ELISA examined cross-reactivity for pesticides with a similar structure to bistrifluron and the same family of pesticides. The degree of cross reaction is expressed in% by the following equation.

Cross-reactivity (%) = (IC ₅₀ of the IC ₅₀ / Other compounds of the Beast ripple ruron) * 100

Interfering materials with high cross-reactivity exhibit higher values than actual concentrations. Therefore, in order to confirm cross-reactivity with benzoylphenyl urea insecticides, ELISA was performed under optimized conditions for 2-chloro-3,5-bistrifluoromethyl aniline (CBA), a metabolite of pseudo-system pesticides and bistrifluron. It was carried out to confirm the cross-reactivity. 13 shows the results of cross-reactivity. As a result, it was confirmed that there was no cross-reactivity with other pesticides, and thus the selectivity was high.

As described above, according to the present invention, the insecticide bistrifluron can be detected quickly and quantitatively by using an antibody against a complex of a specific hapten compound of bistrifluron and a protein.

Claims (9)

(a) an antibody produced from a complex comprising a pesticide bistrifluron and a hapten compound and protein of bistrifluron, and a hapten compound and protein of bistrifluron immobilized on a solid support Contacting the bound complex by amide bonds; And (b) detecting the binding of the antibody to the bistrifluron immobilized on the solid support using an enzyme labeled secondary antibody that specifically binds to the antibody. As a method, The protein is bovine serum albumin (BSA), human serum albumin (HSA), fibrinogen, ovalbumin, thyroglobulin or keyhole limpet hemocyanin (KLH), Method wherein the hapten compound of the bistrifluron is a compound of Formula 1 <Formula 1>

[In the meal, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, carboxyl group, hydroxy group, alkyl group having 1 to 10 carbon atoms, halogen atom, cyano group, amino group, thiol group, aldehyde group, A nitro group, a carboxyalkyl group having 2 to 10 carbon atoms, a hydroxyalkyl group having 1 to 10 carbon atoms, or a haloalkyl group having 1 to 10 carbon atoms, R ₈ represents an amino group or a carboxyl group, X ₁ and X ₂ each independently represent a simple bond, an arylene group having 6 to 20 carbon atoms, or an alkylene group having 1 to 10 carbon atoms, at least one hydrogen atom present in the arylene group is substituted with a halogen atom, m represents an integer of 0 to 3, n represents an integer of 1 to 3; delete delete The method of claim 1 wherein the hapten compound of bistrifluron is a compound of the following hapten I, II, III, IV or V: <Hapten I>

<Hapten II>

<Hapten III>

<Hapten IV>

<Hapten V>

delete The method of claim 1 wherein bistrifluron in the sample is detected quantitatively. The method of claim 1, wherein the protein immobilized on the solid support is different from the protein used to produce the antibody. The method of claim 1, wherein the solid support is a sheet, film, bead, pellet, disc, plate, ring, rod, net, membrane, filter, tray, microplate, stick, slide, or tube. delete

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