Hapten and complete antigen of imazethapyr as well as preparation method and application of hapten and complete antigen

Abstract

The invention discloses a hapten and a complete antigen of imazethapyr as well as a preparation method and application of the hapten and the complete antigen. The hapten of imazethapyr is an imazethapyr hapten A or an imazethapyr hapten B1 or an imazethapyr hapten B2; the imazethapyr complete antigen is an imazethapyr complete antigen C1 or an imazethapyr complete antigen C2; the hapten disclosed by the invention can be conveniently and quickly obtained, and is low in synthesis cost and good in effect; the antiserum obtained through complete antigen immunization is good in titer and high in specificity, can be effectively used for fusion of hybridoma cells and preparation of an imazethapyr antibody, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural detection, in particular to a hapten and a complete antigen of imidacloprid and a preparation method and application thereof. Background Art

[0002] Imazethapyr, English name is Imazethapyr, chemical name is 2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid, trade name is: Imazethapyr, CAS registration number: 81335-77-5, molecular formula: C 15 H 19 N3O3, relative molecular weight: 289.33.

[0003] With the gradual improvement of people's living standards and health awareness, food safety issues from farmland to table have become one of the main focuses of daily life, and food safety detection technology has therefore received great attention. Pesticides and veterinary drugs are complex and diverse, and agricultural products are diverse. Research and development of rapid detection technology for agricultural product pesticide and veterinary drug residues with strong specificity, high sensitivity, rapidity, convenience, accuracy and stability has become a current research hotspot and key direction for pesticide and veterinary residue detection. As the source of livestock products, forage pesticide residues are highly concealed. When people pay attention to food safety issues, they only focus on herbivorous livestock such as meat, eggs, and milk, and agricultural products such as poultry, but usually ignore the safety of forage products.

[0004] Imidazolin is a highly effective, low-toxic pyrazolone herbicide. This type of herbicide has high activity, low dosage, broad weed control spectrum, strong selectivity and is easy to use. It occupies an important position in the world herbicide market. It is mainly used to remove malignant weeds in crops. In the large-scale cultivation of alfalfa in China, imazethapyr is a widely used herbicide. Its structure is stable and it is not easy to volatilize and photolyze in the soil. It has a long residual effect. Its residue in the soil has phytotoxicity problems to certain subsequent crops and non-target plants.

[0005] At present, the detection of imazethapyr is mainly based on large-scale instrument methods, which have high accuracy and sensitivity, but its extraction, purification and detection processes are cumbersome, require high professional level of operators, and are expensive, so it is not suitable for popularization and application in grassroots regulatory departments. Compared with instrumental analysis methods, immunoassay has the advantages of being fast, simple, real-time, easy to conduct on-site detection, simple sample pretreatment, high sensitivity, strong selectivity, suitable for high-throughput analysis, and can significantly reduce the cost of detection.

[0006] However, currently, there is a lack of hapten and complete antigen and immunoassay data for imidacloprid.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for preparing imazethapyr hapten and complete antigen and its application, which can be used to establish imazethapyr monoclonal antibody and enzyme-linked immunosorbent assay technology, develop a rapid detection method for imazethapyr residues in environment-alfalfa-livestock products, realize the monitoring of imazethapyr in the whole process of environment-forage-livestock products, ensure the quality and safety of agricultural and livestock products from the source, and ensure the health and safety of consumers; the hapten provided by the present invention can be obtained conveniently and quickly, with low synthesis cost and good effect; and the antiserum obtained by immunization with the complete antigen of the present invention has good titer and high specificity, and the host animal spleen cells obtained by immunization can be effectively used for the fusion of hybridoma cells and the preparation of anti-imazethapyr antibodies, and has broad application prospects as follows: A hapten of imidazole acetylcholine, wherein the hapten of imidazole acetylcholine is: imidazole acetylcholine hapten A, imidazole acetylcholine hapten B1, or imidazole acetylcholine hapten B2; The imidazolin hapten A is: 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamide)methyl)benzoic acid; The molecular structure of the imidazolin hapten A is: ; The imidazolin hapten B1 is: 2,5-dioxopyrrolidin-1-yl 2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-5-methylnicotinate; The molecular structure of the imidazolin hapten B1 is: ; The imidazolin hapten B2 is: 2,5-dioxopyrrolidin-1-yl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamide)methyl)benzoate; The molecular structure of the imidazolin hapten B2 is: .

[0009] Preferably, the preparation method of the imidazolin hapten A is as follows: S1, weigh 2.89 g (10 mmol) A0 (imidazole nicotinic acid), 1.58 g benzylamine hydrochloride (11 mmol), 2.88 g EDC hydrochloride (15 mmol), 2.3 g 1-hydroxybenzotriazole (15 mmol) and 2.63 g triethylamine (26 mmol), add 100 mL dichloromethane, and react at room temperature for 24 hours; after the reaction, evaporate the solvent, add water to redissolve and extract, dry over anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 2.5 g intermediate product A1; S2. Take 1.0 g of the intermediate product A1, add 10 mL of tetrahydrofuran, and stir to dissolve; take another 0.2 g of sodium hydroxide and 10 mL of water, stir to dissolve; mix the two solutions, and react at room temperature for about 3 hours; after the raw material is completely converted, add dilute hydrochloric acid to adjust the pH to 3-4; separate and purify to obtain 0.46 g of the final product, imidazole ethyl nicotinic acid hapten A.

[0010] Preferably, A0 is imidazolin; The molecular structure of A0 is: ; Preferably, the intermediate product A1 is: methyl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamido)methyl)benzoate; The molecular structure of the intermediate product A1 is: .

[0011] Preferably, the preparation method of the imidazolin hapten B1 is as follows: Weigh 0.0225 mmol A0, 0.045 mmol N-hydroxysuccinimide (NHS) and 0.045 mmol dicyclohexylcarbodiimide (DCC), dissolve in 1 mL anhydrous N,N-dimethylformamide (DMF), stir and react at room temperature 25°C for 4 hours, centrifuge the reaction solution at 6000 r / min for 5 minutes, take the supernatant, and obtain the supernatant containing imidazolyl acetylcholine hapten B1; The A0 is imidazolin; The molecular structure of A0 is: .

[0012] Preferably, the preparation method of the imidazolin hapten B2 is as follows: Weigh 0.0225 mmol imidazole acetylcholine hapten A, 0.045 mmol N-hydroxysuccinimide (NHS) and 0.045 mmol dicyclohexylcarbodiimide (DCC), dissolve them in 1 mL of anhydrous N,N-dimethylformamide (DMF), stir and react at room temperature 25°C for 4 hours, centrifuge the reaction solution at 6000 r / min for 5 minutes, take the supernatant for use, and obtain imidazole acetylcholine hapten B2.

[0013] On the other hand, the present invention provides a complete imidazole antigen prepared from an imidazole hapten, wherein the complete imidazole antigen is the complete imidazole antigen C1 or the complete imidazole antigen C2; The chemical structural formula of the imidazolin complete antigen C1 is: ; The chemical structural formula of the imidazolin complete antigen C2 is: .

[0014] Preferably, the preparation method of the imidazolin complete antigen C1 is as follows: Adding acetonicotinic acid hapten B1 to the carrier protein OVA solution, the feeding molar ratio of acetonicotinic acid hapten B1 to the carrier protein OVA is 40:1, stirring at 25°C, and obtaining a reaction solution after 8 hours; The obtained reaction solution was dialyzed for three days with a PBS solution having a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution was diluted to a 1 mg / mL solution to obtain the imidazolin complete antigen C1; i.e., the conjugate of the imidazolin hapten (B1) and OVA; The carrier protein OVA is ovalbumin; The carrier protein OVA solution is prepared by dissolving 20 mg of OVA in 4 mL of phosphate buffered saline (PBS) with a pH value of 7.4 and mixing the mixture.

[0015] Preferably, the preparation method of the imidazolin complete antigen C2 is as follows: Slowly drop imidazole acetyl nicotinic acid hapten A into the carrier protein BSA solution, the feeding molar ratio of imidazole acetyl nicotinic acid hapten A to the carrier protein BSA is 40:1, stirring at 25° C., and obtaining a reaction solution after 8 hours; The obtained reaction solution was dialyzed for three days with a phosphate buffer solution having a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution was diluted to a 1 mg / mL solution, quickly frozen in liquid nitrogen, and stored at -20°C for later use, thereby obtaining the imidazole acetylcholine complete antigen C2, i.e., the conjugate of the imidazole acetylcholine hapten A and BSA; The carrier protein BSA is bovine serum albumin; The carrier protein BSA solution is prepared by dissolving 20 mg of the carrier protein BSA in 5 mL of phosphate buffer (PBS) having a pH value of 7.4 and mixing the mixture.

[0016] Furthermore, the present invention provides an application of a complete imidazolin antigen for preparing antibodies.

[0017] On the other hand, the present invention provides a method for preparing antibodies against imidazole acetylcholine, which comprises: immunizing a host animal with a complete antigen as described in any of the above items, isolating spleen cells from the host animal, fusing them with SP2 / 0 tumor cells, and screening monoclonal cell lines capable of secreting anti-imidazole acetylcholine antibodies to prepare anti-imidazole acetylcholine antibodies; or extracting mRNA transcribed from B lymphocytes in the blood of the host animal, reverse transcribing it into cDNA, and screening antibodies through a phage or yeast antibody expression library constructed by bioengineering technology.

[0018] Monoclonal hybridoma cell lines are easy to prepare for those skilled in the art. For example, spleen cells are obtained after immunizing a host animal with the above-mentioned complete antigen, and they are fused with SP2 / 0 tumor cells in vitro. Single hybridomas that can secrete anti-imidazole acetylcholine antibodies are screened through selective culture medium to obtain hybridoma cell lines. Anti-imidazole acetylcholine antibodies can also be obtained by means of genetic engineering technology, etc. These technologies are easy to implement for those skilled in the art. Based on this, no matter what method is used to prepare anti-imidazole acetylcholine antibodies, as long as the hapten or complete antigen provided by the present invention is used, it belongs to the protection scope of the present invention.

[0019] The antibody or antiserum prepared by immunizing a host animal with the complete antigen provided by the present invention can specifically bind to imidazolin and has a good immune effect.

[0020] Preferably, in some embodiments of the present invention, the antibody is anti-imidazole serum.

[0021] Based on the hapten or complete antigen disclosed in the present invention, those skilled in the art can easily think of using a suitable antibody preparation method to prepare an anti-imidazole acetylcholine antibody, no matter what kind of animal is used for immunization, no matter how the immunization conditions or parameters are set or changed, as long as the hapten or complete antigen provided by the present invention is used, it falls within the protection scope of the present invention.

[0022] In another aspect, the present invention provides an antibody against imazethapyr, which is prepared by the method described above.

[0023] In another aspect, the present invention provides a reagent or kit for detecting imazethapyr, which comprises the antibody as described above.

[0024] In another aspect, the present invention provides a method for detecting imazethapyr, using the antibody as described above, or the reagent or kit as described above for detection.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for preparing a hapten and a complete antigen of imazethapyr and their application, which can be used to establish an imazethapyr monoclonal antibody and an enzyme-linked immunosorbent assay technology, develop a rapid detection method for imazethapyr residues in the environment-alfalfa-livestock product, and realize the monitoring of imazethapyr in the whole process of environment-forage-livestock product, thereby ensuring the quality and safety of agricultural and livestock products from the source and the health and safety of consumers; the hapten designed by the present invention can be obtained conveniently and quickly, with low synthesis cost and good effect; the complete antigen prepared by using the hapten exhibits excellent antiserum titer and high specificity in the immunization process, which not only greatly promotes the fusion process of hybridoma cells, but also opens up a new way for the preparation of anti-imazethapyr antibodies, indicating that it has extremely broad application potential and market prospects.

[0026] The antiserum obtained by immunization with the complete antigen of the present invention has good titer and high specificity, and the spleen cells of the immunized host animals can be effectively used for the fusion of hybridoma cells and the preparation of anti-imidazole acetylcholine antibodies.

[0027] 2. The complete antigen prepared by the hapten structure designed and synthesized by the present invention and its coupling technology with the carrier protein has high immunogenicity and specificity, and can stimulate the body to produce high-titer specific antibodies, thereby achieving high-sensitivity detection of imidazole acetylcholine residues.

[0028] 3. The present invention is not only suitable for the detection of imidacloprid residues in farmland environments, but can also be applied to residue monitoring in forage and livestock products, thereby ensuring the quality and safety of agricultural products from the source and providing strong protection for the health of consumers.

[0029] 4. Compared with the traditional large-scale instrument detection method, the detection method provided by the present invention is more environmentally friendly, reduces the use of toxic and harmful chemical reagents and the discharge of waste, and is in line with the current green and sustainable development concept.

[0030] 5. The method for preparing the imidazole acetylcholine hapten and complete antigen provided by the present invention avoids the cumbersome steps and high costs of traditional large-scale instrument detection methods, and provides a high-throughput, rapid screening detection method for grassroots regulatory departments, which helps to reduce detection costs and improve detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a synthetic route of imidazolin hapten A; Figure 2It is a synthetic route of imidazolin hapten B1; Figure 3 It is a synthetic route of imidazolin hapten B2; Figure 4 It is a synthetic route of the imidazolin complete antigen C1; Figure 5 It is a synthetic route of the imidazolin complete antigen C2; Figure 6 For intermediate A1 1 H NMR test results; Figure 7 Imidazole acetylcholine hapten A 1 H NMR test results; Figure 8 To measure the high-resolution mass spectrometry results of imidazolin hapten A; Fig. 9 This is the MALDI-TOF-MS test result of OVA standard; Fig.10 This is the MALDI-TOF-MS test result of the imidazolin complete antigen C1; Fig.11 This is the MALDI-TOF-MS test result of BSA standard; Fig.12 This is the MALDI-TOF-MS detection result of the complete antigen C2 of imidacloprid. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0033] Sources of some materials in the following examples: imidazolin (A0) and benzylamine hydrochloride were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; EDC hydrochloride, o-phenylenediamine (OPD), sodium carbonate, sodium chloride, sodium dihydrogen phosphate dodecahydrate, gelatin, citric acid monohydrate, Tween-20, and concentrated sulfuric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; 1-Hydroxybenzotriazole (HOBt), triethylamine, dichloromethane, ethyl acetate, anhydrous sodium sulfate, tetrahydrofuran, sodium hydroxide, and hydrochloric acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Freund's complete adjuvant, Freund's incomplete adjuvant, bovine serum albumin, and ovalbumin were purchased from Merck; Goat anti-mouse IgG-HRP was purchased from Jackson Company.

[0034] Example 1 Synthesis of Imidazole Acetyl Hapten A Imidazole nicotinic acid hapten A: 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamido)methyl)benzoic acid; Synthesis route see Figure 1 , weigh 2.89 g (10 mmol) of imidazolyl nicotinic acid, 1.58 g of benzylamine hydrochloride (11 mmol), 2.88 g of EDC hydrochloride (15 mmol), 2.3 g of 1-hydroxybenzotriazole (15 mmol) and 2.63 g of triethylamine (26 mmol), add 100 mL of dichloromethane, and react at room temperature for 24 hours; after the reaction, evaporate the solvent, add water to dissolve it again, extract it with water, dry it with anhydrous sodium sulfate, and separate and purify it by column chromatography to obtain 2.5 g of intermediate product A1 methyl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) nicotinamide) methyl) benzoate, the molecular structure of which is as follows: ; Figure 6 For intermediate A1 1 H NMR test results, from Figure 6 It can be seen that: 1 H NMR (300 MHz, CDCl3) δ12.06 (s, 1H), 9.15 (s, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.55 (d, J = 2.2 Hz, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 4.72 (ddd, J = 38.7, 15.2, 5.3 Hz, 2H), 3.92 (s, 3H), 1.32 (dd, J = 16.2, 8.6 Hz, 6H), 0.82 (dd, J = 16.7, 6.8 Hz, 6H). 1 H NMR ( Figure 6 ) confirmed that the structure of the intermediate product (A1) was correct.

[0035] Take 1.0 g of intermediate product A1, add 10 mL of tetrahydrofuran (THF), stir to dissolve, take 0.2 g of sodium hydroxide and 10 mL of water, stir to dissolve, mix the two solutions, and react at room temperature for about 3 hours; after the raw material is completely converted, add dilute hydrochloric acid to adjust the pH to acidic; separate and purify to obtain the final product imidazolyl nicotinic acid hapten A 0.46 g, the molecular structure of which is as follows: .

[0036] Figure 7 Imidazole acetylcholine hapten A 1 H NMR test results, from Figure 7 It can be seen that: 1 H NMR (400 MHz, CDCl3) δ 11.98 (s, 1H), 8.68 (d, J = 1.7 Hz, 1H), 8.57 (d, J = 1.8 Hz, 1H), 8.06 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 4.74 (ddd, J = 46.2, 15.3, 5.2 Hz, 2H), 2.81 (q, J = 7.6 Hz, 2H), 1.32 (dd, J = 14.0, 6.4 Hz, 6H), 0.84 (dd, J = 25.5, 6.8 Hz, 6H). HRMS (ESI) Calculated for C 23 H 25 N4O4 [MH] - 421.1881, found 421.1880. 1 H NMR ( Figure 7 ) and high-resolution mass spectrometry confirmation ( Figure 8 ), the final product hapten 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamide)methyl)benzoic acid (A) has an accurate structure.

[0037] Example 2 Synthesis of Imidazolin Hapten B1 Imidazole nicotinic acid hapten B1: 2,5-dioxopyrrolidin-1-yl 2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-5-methylnicotinate (B1), the structure is as follows: .

[0038] Synthesis route see Figure 2 , weigh 0.0225 mmol of imidazolin A0, N-hydroxysuccinimide (NHS, 0.045 mmol) and dicyclohexylcarbodiimide (DCC, 0.045 mmol) and dissolve them in 1 mL of anhydrous N,N-dimethylformamide (DMF). After stirring and reacting at room temperature (25°C) for 4 hours, the reaction solution was centrifuged at 6000 r / min for 5 minutes, and the supernatant was taken for later use to obtain imidazolin hapten B1. The reaction product can be directly used for subsequent carrier protein coupling.

[0039] Example 3 Synthesis of Imidazolin Hapten B2 Imidazole nicotinic acid hapten B2: 2,5-dioxopyrrolidin-1-yl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) nicotinamide) methyl) benzoate (B2), 0.0225 mmol of formula (A) prepared in Example 1, N-hydroxysuccinimide (NHS, 0.045 mmol) and dicyclohexylcarbodiimide (DCC, 0.045 mmol) were dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF), stirred at room temperature 25°C for 4 hours, and the reaction solution was heated at 6000 The mixture was centrifuged at 400 rpm for 5 minutes, and the supernatant was taken for use to obtain imidazolyl nicotinic acid hapten B2: 2,5-dioxopyrrolidin-1-yl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamide)methyl)benzoate. The synthetic route is shown in Figure 3 The reaction product can be directly used for subsequent carrier protein coupling. The molecular structure is as follows: .

[0040] Example 4 Synthesis of Imidazolinyl Acetate Complete Antigen C1 Imidazole complete antigen C1: .

[0041] Synthesis method: The supernatant containing the imidazole acetyl nicotinic acid hapten B1 obtained in Example 2 is slowly added dropwise to a carrier protein OVA (ovalbumin) solution (the carrier protein solution is obtained by dissolving 20 mg of OVA in 4 mL of phosphate buffer (PBS) with a pH value of 7.4 and mixing evenly), the feed molar ratio of the compound of formula B to the carrier protein is 40:1, and the mixture is stirred at 25°C. After 8 hours, the obtained reaction solution is dialyzed for three days with a PBS solution with a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution is diluted to a 1 mg / mL solution; the role of dialysis is to remove unreacted 4-(4-(methoxycarbonyl)-1,3-dimethyl-1H-pyrazole-5-sulfonamido)-4-oxobutanoic acid or other small molecules to obtain a compound shown in formula C1, i.e., a conjugate of imidazole acetyl nicotinic acid hapten (B1) and OVA. The synthesis route is shown in Figure 4 .

[0042] The results of OVA standard and imidazolin complete antigen C1 obtained by MALDI-TOF-MS are as follows Fig. 9 Fig.10 As shown in the figure, it can be seen that the single-charged ion peaks of OVA standard and complete antigen C1 are 44330.380 and 45615.103 respectively; the coupling ratio of carrier protein to hapten = (complete antigen C1 molecular weight - carrier protein OVA molecular weight) / imidazole acetylcholine molecular weight = (45615.103-44330.38) / 289.14; the coupling ratio of complete antigen C1 to imidazole acetylcholine calculated by the formula is 1:4.

[0043] Example 5 Synthesis of Imidazolinyl Acetate Complete Antigen C2 Imidacloprid complete antigen C2: .

[0044] The supernatant containing imidazole acetylcholine hapten (A) obtained in Example 2 was slowly added dropwise to a carrier protein BSA (bovine serum albumin) solution (the carrier protein solution was obtained by dissolving 20 mg BSA in 5 mL phosphate buffer (PBS) with a pH value of 7.4 and mixing evenly), and the molar ratio of the compound of formula A to the carrier protein was 40:1. The mixture was stirred at 25°C. After 8 hours, the obtained reaction solution was dialyzed for three days with a PBS solution with a pH value of 7.4 and a concentration of 0.01 mol / L. The dialyzed reaction product solution was diluted to a 1 mg / mL solution, quickly frozen with liquid nitrogen, and then stored at -20°C for later use to obtain a compound shown in formula C2, i.e., a conjugate of imidazole acetylcholine hapten A and BSA. The synthesis route is shown in Figure 5 .

[0045] The results of BSA standard and imidazolin complete antigen C2 obtained by MALDI-TOF-MS are as follows Fig.11 , Fig.12 As shown in the figure, the single-charged ion peaks of BSA standard and complete antigen C2 are 66416.533 and 69536.310 respectively; the coupling ratio of carrier protein to hapten = (complete antigen C2 molecular weight - carrier protein BAS molecular weight) / hapten A molecular weight = (69536.130-66416.533) / 422.20; the coupling ratio of complete antigen C2 to imidazolinone hapten A calculated by the formula is 1:7.

[0046] Example 6 Application of Imidazole Complete Antigen 1. Preparation of antibodies using the complete imipenem antigen C2 of Example 5 (1) 8-10 week old Balb / C mice were used as experimental animals (8-week old Balb / c mice weigh approximately 23-25 ​​g).

[0047] (2) Primary immunization: The diluted imidazolin complete antigen C2 antigen solution (concentration of 1 mg / mL) obtained in Example 6 was added with an equal volume of Freund's complete adjuvant. After sufficient stirring and emulsification, the emulsified antigen was injected into the abdominal cavity and back subcutaneously of mice. The total injection dose was 0.1 mg of emulsified antigen per mouse.

[0048] (3) Booster immunization: 2 weeks after the first immunization, take 1 mL of the diluted imidazolin complete antigen C2 antigen solution, then add Freund's incomplete adjuvant, stir thoroughly to emulsify, and then inject the emulsified complete antigen into the abdominal cavity and back subcutaneous tissue of the mouse, with a total injection dose of 0.1 mg of emulsified antigen per mouse; booster immunization is performed every 14 days. Starting from the fourth booster immunization, blood is collected from the mouse eye sockets on the 3rd to 5th day after each immunization to determine the antibody titer; the blood is allowed to stand at room temperature for 1 hour, then allowed to stand in a refrigerator at 25°C for 2 hours, and then centrifuged in a centrifuge at 4°C and 10,000 r / min for 5 minutes to separate the antiserum, thus obtaining imidazolin antiserum, which is used in the following experiments: 2. Detection of Antibody Titer in Antiserum The various buffers used in the following experiments are as follows: (1) Coating buffer (pH 9.6, 0.05 M carbonate buffer): Weigh 1.5 g sodium carbonate and 2.94 g sodium bicarbonate, and dilute to 1000 mL with ultrapure water.

[0049] (2) Phosphate buffer (0.01 M, pH 7.4): Weigh 0.2 g of sodium dihydrogen phosphate, 8 g of sodium chloride, and 2.92 g of disodium hydrogen phosphate dodecahydrate, and dilute to 1000 mL with ultrapure water.

[0050] (3) Washing buffer: Add 0.1% by volume of Tween-20 to the prepared phosphate buffer solution.

[0051] (4) Sample diluent: Add 1% by volume of Tween-20 and 1 g of gelatin (melted in a microwave oven) to the prepared phosphate buffer solution.

[0052] (5) Substrate buffer (pH 5.5): Weigh 9.22 g of disodium hydrogen phosphate dodecahydrate, 2.55 g of citric acid monohydrate, and 0.5 mL of Tween-20, and dilute to 1 L with ultrapure water.

[0053] (6) Stop solution (2M H2SO4): Take 445.6 mL of distilled water and add 54.4 mL of 98% (v / v) concentrated sulfuric acid dropwise while stirring.

[0054] 1. Inhibition experiment of antibodies in antiserum 1. Preparation of Imidazobactam Complete Antigen C1 Coating Antigen Solution The imidazolin complete antigen C1 prepared in Example 5 was diluted in a gradient manner at 1:500, 1:1000, 1:2000, and 1:4000 with a coating buffer to obtain coating antigen solutions of imidazolin complete antigen C1 of different concentrations.

[0055] 2. Preparation of imidazolin standard solution (1) Weigh 10 mg of imidazolin standard and dissolve it in 10 mL of methanol to obtain a 1 mg / mL imidazolin standard solution.

[0056] (2) Use sample diluent to prepare the 1 mg / mL imidazolin standard solution in (1) to a final concentration of 1000 ng / mL imidazolin standard solution.

[0057] 3. Preparation of Imidazole Antiserum / Antibody Dilution Solution The imidazolin antiserum prepared in the above step was diluted with the sample diluent in a gradient manner of 1:250, 1:500, 1:1000, and 1:2000 to obtain an imidazolin antiserum dilution.

[0058] 4. Checkerboard experiment of antigen and antiserum Coating: Add 100 μL of the imidazolin complete antigen C1 coating antigen solution prepared in step 1 to each well of a 96-well ELISA plate, incubate at 37°C for 3 hours, wash three times with sample washing solution, and spin dry.

[0059] Competition: Add 50 μL of sample diluent to each well of zero well; add 50 μL of imidazolin standard solution prepared in step 2 to each well of inhibition well. Add the imidazolin antiserum dilution obtained in step 3 to the ELISA plate (50 μL / well), incubate at 37°C for 30 min, wash the plate 3 times, and spin dry.

[0060] Add enzyme-labeled secondary antibody: dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 1000 times, the diluent is 0.1 M, pH 9.6 sample washing solution, add 100 μL to each well, incubate at 37°C for 30 min, wash the plate 3 times, and spin dry.

[0061] Color development: Prepare the color development substrate before use. Add 15-20 mg OPD to every 10 mL substrate buffer. After dissolving the weighed OPD in the substrate buffer, add 4 μL 30% hydrogen peroxide to every 10 mL. Add 100 μL to each well and develop the color for 15 minutes at room temperature away from light.

[0062] Stop: Add 50 μL of 2 M sulfuric acid stop buffer to each well and measure the OD value of each well at 492 nm using a microplate reader.

[0063] The calculation formula of inhibition rate: inhibition rate = (A0-A 50 ) / A0×100%, A0 is the OD value of the control well; A 50 is the OD value of the inhibition well.

[0064] After the fourth and fifth immunizations, the antiserum titer results are shown in Table 1.

[0065] Table 1 Serum titer detection of imidazolin-resistant mice

[0066] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0067] The results in Table 1 show that after the fifth immunization, when the coating antigen dilution was 1:500 and the antiserum dilution was 1:4000, the antiserum inhibition rate was the best, which was 67.46%, indicating that the imidazole complete antigen C2 prepared in Example 6 above can be used as an immunogen to prepare anti-imidazole antibodies.

[0068] Example 7 Preparation of antibodies using imidazopyrine complete antigen Immunize a host animal with imidazolin complete antigen C2 or imidazolin complete antigen C1, separate spleen cells from the host animal, fuse them with SP2 / 0 tumor cells, screen monoclonal cell lines that can secrete anti-imidazolin antibodies, and use them to prepare anti-imidazolin antibodies; or extract mRNA transcribed from B lymphocytes in the blood of the host animal, reverse transcribe it into cDNA, and use phage or yeast antibody expression libraries constructed by bioengineering technology to screen antibodies.

[0069] Preparation method of monoclonal hybridoma cell line: spleen cells are obtained after immunizing the host animal with the above-mentioned complete antigen, and they are fused with SP2 / 0 tumor cells in vitro. Single hybridomas that can secrete anti-imidazole nicotinic acid antibodies are screened through selective culture medium to obtain hybridoma cell lines.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hapten of imidacloprid, characterized in that: The hapten of imidazole acetylcholine is: imidazole acetylcholine hapten A or imidazole acetylcholine hapten B1 or imidazole acetylcholine hapten B2; The imidazolin hapten A is: 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamide)methyl)benzoic acid; The molecular structure of the imidazolin hapten A is: ； The imidazolin hapten B1 is: 2,5-dioxopyrrolidin-1-yl 2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-5-methylnicotinate; The molecular structure of the imidazolin hapten B1 is: ； The imidazolin hapten B2 is: 2,5-dioxopyrrolidin-1-yl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamide)methyl)benzoate; The molecular structure of the imidazolin hapten B2 is: 。 2. The hapten of imidazolin according to claim 1, characterized in that: The preparation method of the imidazolin hapten A is as follows: S1, weigh 2.89 g A0, 1.58 g benzylamine hydrochloride, 2.88 g EDC hydrochloride, 2.3 g 1-hydroxybenzotriazole and 2.63 g triethylamine, add 100 mL dichloromethane, and react at room temperature for 24 hours; after the reaction, evaporate the solvent, add water to redissolve, extract with ethyl acetate, dry with anhydrous sodium sulfate, and separate and purify by column chromatography to obtain 2.5 g intermediate product A1; S2. Take 1.0 g of the intermediate product A1, add 10 mL of tetrahydrofuran, and stir to dissolve; take another 0.2 g of sodium hydroxide and 10 mL of water, stir to dissolve; mix the two solutions and react at room temperature for about 3 hours; after the raw material is completely converted, add dilute hydrochloric acid to adjust the pH to 3-4; separate and purify to obtain 0.46 g of imidazole acetylcholine hapten A.

3. The hapten of imidazolin as claimed in claim 2, characterized in that: The A0 is imidazolin; The molecular structure of A0 is: 。 4. The hapten of imidazolin as claimed in claim 3, characterized in that: The intermediate product A1 is: methyl 4-((5-ethyl-2-(5-isopropyl-5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)nicotinamido)methyl)benzoate; The molecular structure of the intermediate product A1 is: 。 5. The hapten of imidazolin as claimed in claim 1, characterized in that: The preparation method of the imidazolin hapten B1 is as follows: Weigh 0.0225 mmol A0, 0.045 mmol N-hydroxysuccinimide and 0.045 mmol dicyclohexylcarbodiimide and dissolve them in 1 mL of anhydrous N,N-dimethylformamide. Stir and react at room temperature (25°C) for 4 hours. Centrifuge the reaction solution at 6000 r / min for 5 minutes and take the supernatant to obtain the supernatant containing imidazolyl nicotinic acid hapten B1. The A0 is imidazolin; The molecular structure of A0 is: 。 6. The hapten of imidazolin as claimed in claim 2, characterized in that: The preparation method of the imidazolin hapten B2 is as follows: Weigh 0.0225 mmol of imidazole acetylcholine hapten A, 0.045 mmol of N-hydroxysuccinimide and 0.045 mmol of dicyclohexylcarbodiimide, dissolve them in 1 mL of anhydrous N,N-dimethylformamide, stir and react at room temperature 25°C for 4 hours, centrifuge the reaction solution at 6000 r / min for 5 minutes, take the supernatant for use, and obtain imidazole acetylcholine hapten B2.

7. The imidazolin complete antigen prepared by using the imidazolin hapten according to claim 1, characterized in that: The imidazole complete antigen is imidazole complete antigen C1 or imidazole complete antigen C2; The chemical structural formula of the imidazolin complete antigen C1 is: ； The chemical structural formula of the imidazolin complete antigen C2 is: 。 8. The imidazolin complete antigen according to claim 7, characterized in that The preparation method of the imidazolin complete antigen C1 is as follows: Adding acetonicotinic acid hapten B1 to the carrier protein OVA solution, the feeding molar ratio of acetonicotinic acid hapten B1 to the carrier protein OVA is 40:1, stirring at 25°C, and obtaining a reaction solution after 8 hours; The obtained reaction solution was dialyzed for three days with a PBS solution having a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution was diluted to a 1 mg / mL solution to obtain the imidazolin complete antigen C1; The carrier protein OVA is ovalbumin; The carrier protein OVA solution is prepared by dissolving 20 mg of OVA in 4 mL of phosphate buffer having a pH value of 7.4 and mixing evenly.

9. The imidazolin complete antigen according to claim 7, characterized in that The preparation method of the imidazolin complete antigen C2 is as follows: Slowly drop imidazole acetyl nicotinic acid hapten A into the carrier protein BSA solution, the feeding molar ratio of imidazole acetyl nicotinic acid hapten A to the carrier protein BSA is 40:1, stirring at 25° C., and obtaining a reaction solution after 8 hours; The obtained reaction solution was dialyzed for three days with a phosphate buffer solution having a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution was diluted to a 1 mg / mL solution, and after quick freezing with liquid nitrogen, it was stored at -20°C for later use, thereby obtaining the imidazolin complete antigen C2; The carrier protein BSA is bovine serum albumin; The carrier protein BSA solution is prepared by dissolving 20 mg of the carrier protein BSA in 5 mL of a phosphate buffer solution having a pH value of 7.4 and mixing them evenly.

10. The use of the imidazolin complete antigen according to any one of claims 7 to 9, characterized in that: Used to prepare antibodies.

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