A myclobutanil hapten, a complete antigen and a method for detecting myclobutanil

Abstract

The application discloses a myclobutanil hapten, a complete antigen and a method for detecting myclobutanil, and relates to the technical field of myclobutanil detection. The hapten provided by the application can be conveniently and quickly obtained, has low synthesis cost and good effect; the complete antigen prepared based on the hapten has good titer, high specificity and the spleen cells of the host animal obtained through immunization can be effectively used for fusion of hybridoma cells and preparation of anti-myclobutanil antibodies, so that the application has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of cyproconazole detection technology, and more specifically, to a cyproconazole hapten, a complete antigen, and a method for detecting cyproconazole. Background Technology

[0002] Myclobutanil, also known as 2-(4-chlorophenyl)-2-(1H,1,2,4-triazol-1-methyl)hexanoic acid, is a commercially available product. Its CAS Registry Number is 88671-89-0. The molecular formula is C15H17ClN4, and the relative molecular weight is 288.78. Its chemical structure is as follows: .

[0003] Cyazofamid is a systemic triazole fungicide with both protective and curative activities. It has a long residual effect, is safe for crops, and has a certain growth-stimulating effect. It can be used to control *Hylocereus* fungi on grapes, as well as *Smutella smut* and *Ustilago maydis* on wheat. It also has good control effects on powdery mildew, rust, black spot, gray spot, brown spot, and smut, and can be used for seed treatment. However, according to literature reports, long-term application of cyazofamid to crops can lead to residue accumulation. Large-scale application may pose a potential threat to soil microorganisms and soil nitrogen transformation, and may induce unique neurotoxicity syndromes in mammals. Therefore, conducting cyazofamid residue analysis in food is of significant practical importance for protecting consumer health.

[0004] Currently, the main analytical methods for cyproconazole are chromatography and chromatography-mass spectrometry (GC-MS). However, these instrumental methods require expensive equipment, complex sample preparation, are time-consuming, and costly, and require specialized technicians to operate, making them unsuitable for rapid on-site detection of pesticide residues. Compared to instrumental methods, cyproconazole immunoassay has a simpler sample preparation method, is easier for operators to perform on-site, and can significantly reduce detection costs. However, it currently lacks haptens and complete antigens for cyproconazole, and accurate immunoassay methods are also lacking.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a hapten, a complete antigen, and a method for detecting hapten to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a cyazofamid hapten having the following structure: .

[0008] The hapten provided by this invention can be obtained conveniently and quickly, with low synthesis cost and good effect; the complete antigen prepared based on it has good antiserum titer and high specificity after immunization, and the host animal spleen cells obtained by immunization can be effectively used for hybridoma cell fusion and preparation of anti-cyproconazole antibodies, which has broad application prospects.

[0009] Secondly, this invention also provides a method for preparing cyproconazole hapten, which is obtained from cyproconazole as a raw material through a chemical reaction, and its synthetic route is as follows: ; Wherein, X-Phos represents 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene, Pd(oAc)2 represents palladium acetate, NaH represents sodium hydride, DMF represents N,N-dimethylformamide, TFA represents trifluoroacetic acid, DCM represents dichloromethane, THF represents tetrahydrofuran, LiOH represents lithium hydroxide, and MeOH represents methanol.

[0010] In the above synthetic route, numbers 3, 4, 5, and 6 represent compounds 3, 4, 5, and 6, respectively. TL-1 is a cyazofamid hapten, and SM1 is cyazofamid.

[0011] The above-mentioned methods are simple and easy to implement, and the product yield is high.

[0012] In a preferred embodiment of the present invention, the synthesis of the cyazofamid hapten includes the following steps: (1) The cyproconazole, tert-butyl carbamate, cesium carbonate, X-Phos and palladium acetate were mixed in an organic solvent and reacted; after extraction and separation, compound 3 was obtained; The molar ratio of cyproconazole, tert-butyl carbamate, cesium carbonate, X-Phos, and palladium acetate is 1:1-1.5:2-3:0.1-0.2:0.03-0.1. In one optional embodiment, the organic solvent is 1,4-dioxane, and the reaction is carried out in a nitrogen atmosphere at a reaction temperature of 100-160°C for 10-20 hours. (2) Mix a DMF solution containing 0.03 mol of compound 3 with 0.045-0.06 mol of sodium hydride and 0.045 mol of compound 4 and react for 15-20 h; extract and separate to obtain compound 5; (3) Mix the dichloromethane solution containing compound 5 with trifluoroacetic acid, react for 10-20 h, and then concentrate, extract and separate to obtain compound 6; (4) Compound 6 was mixed with methanol, tetrahydrofuran and lithium hydroxide and reacted for 10-20 h. After removing methanol and tetrahydrofuran, water was added to the reaction mixture and extracted with an organic solvent. Then the mixture was washed, dried and concentrated. In an optional embodiment, the extraction and separation in step (1) includes: extracting the reactants with water and ethyl acetate at least twice, collecting the organic phase, washing it with water and / or brine, drying and concentrating it using at least one of anhydrous sodium sulfate, anhydrous magnesium sulfate, and anhydrous calcium sulfate, and separating the concentrated product by silica gel column chromatography to obtain compound 3. The reaction time is 10-20 h. For example, the reaction time is 15 h, 16 h, 17 h, 18 h, 19 h, or 20 h.

[0013] The brine is a saturated brine, i.e., a saturated NaCl aqueous solution. In one embodiment, the collected organic phase is washed repeatedly with water and then with saturated brine, for example, washed three times with water and once with brine.

[0014] The mobile phase used in silica gel column chromatography was petroleum ether (PE): ethyl acetate (EtOAc) = 3:1, until the mobile phase was 100% ethyl acetate.

[0015] In an optional embodiment, in step (2), compound 3 is dissolved in 80-120 mL of DMF, and 0.045-0.06 mol of sodium hydride is added. After stirring in an ice bath for 20-30 minutes, compound 4 is added. After reacting for 15-20 h, water and ethyl acetate are added for extraction at least twice. The organic phase is collected, washed with water and / or brine, dried and concentrated with anhydrous sodium sulfate, and the concentrated product is separated by silica gel column chromatography to obtain compound 5. The reaction time is, for example, 15 h, 16 h, 17 h, 18 h, 19 h or 20 h.

[0016] The mobile phase used in silica gel column chromatography was petroleum ether (PE): ethyl acetate (EtOAc) = 3:1, until the mobile phase was 100% ethyl acetate.

[0017] In an optional embodiment, in step (3), compound 5 is reacted with 100 mL of dichloromethane and 30 mL of trichloroacetic acid; 50-200 mL of saturated sodium bicarbonate is added to the concentrated product, followed by extraction with dichloromethane at least twice. The organic phase is collected, washed with water and / or brine, dried and concentrated with anhydrous sodium sulfate, and the concentrated product is separated by silica gel column chromatography to obtain compound 6. The reaction time is, for example, 10-14 h, 15 h, 16 h, 17 h, 18 h, 19 h, or 20 h.

[0018] The mobile phase used in silica gel column chromatography was petroleum ether (PE): ethyl acetate (EtOAc) = 1:1, until the mobile phase was 100% ethyl acetate.

[0019] The brine is a saturated brine, i.e., a saturated NaCl aqueous solution. In one embodiment, the collected organic phase is washed repeatedly with water and then with saturated brine, for example, washed three times with water and twice with brine.

[0020] In an optional embodiment, in step (4), compound 6 is mixed with 30 mL of methanol, 30 mL of tetrahydrofuran and 50 mL of lithium hydroxide solution and reacted. After the reaction is completed, methanol and tetrahydrofuran are removed under vacuum. Then water is added to the reaction mixture and extracted with ethyl acetate at least twice. The organic phase is collected, washed with water and / or brine, dried and concentrated with anhydrous sodium sulfate, and the concentrated product is added with ethyl acetate and stirred for 0.5-1 h. The precipitate is collected by filtration and dried under vacuum to obtain cyproconazole hapten.

[0021] The reaction time of compound 6 with methanol, tetrahydrofuran and lithium hydroxide is 10-15 h, 12-16 h or 17-20 h.

[0022] The brine is a saturated brine, i.e., a saturated NaCl aqueous solution. In one embodiment, the collected organic phase is washed repeatedly with water and then with saturated brine, for example, washed three times with water and twice with brine.

[0023] Thirdly, the present invention provides a complete antigen comprising a carrier protein and a cyazofamid hapten coupled to the carrier protein or a cyazofamid hapten prepared by the above preparation method.

[0024] In a preferred embodiment of the present invention, the carrier protein includes, but is not limited to, any one or more of bovine serum albumin, human serum albumin, ovalbumin, diphtheria toxoid, the non-toxic mutant of diphtheria toxoid CRM197, tetanus toxoid, and keyhole hemocyanin to form a fusion protein.

[0025] In other embodiments, the carrier protein includes, but is not limited to, proteins expressed by bacteria.

[0026] Fourthly, the present invention provides the use of cyproconazole hapten or cyproconazole hapten prepared by the above-described preparation method in the preparation of a complete antigen, wherein the complete antigen includes a carrier protein and a cyproconazole hapten coupled to the carrier protein.

[0027] In one alternative embodiment, when preparing the complete antigen, the molar ratio of the hapten to the carrier protein is 5-60:1, preferably 30:1.

[0028] In one optional embodiment, when preparing the complete antigen, the hapten is mixed with the carrier protein, the reaction temperature is 0-50°C, more preferably 4°C, the reaction time is 8-36 hours, more preferably 12 hours, and the pH is 5-9, more preferably 7.4.

[0029] Fifthly, the present invention provides the application of cyproconazole hapten, cyproconazole hapten prepared by the above-described preparation method, or the above-described complete antigen in the preparation of anti-cyproconazole polyclonal antibodies or monoclonal antibodies. This application includes, but is not limited to, preparing the cyproconazole hapten as a complete antigen, isolating the corresponding anti-cyproconazole polyclonal antibodies from the blood of a host animal through immunization; or isolating spleen cells from the host animal, fusing them with SP2 / 0 tumor cells, screening for monoclonal cell lines capable of secreting anti-cyproconazole antibodies, and using these cells to prepare anti-cyproconazole antibodies; or extracting mRNA transcribed from B lymphocytes in the blood of the host animal, reverse transing it into cDNA, and screening for anti-cyproconazole antibodies using a phage or yeast antibody expression library constructed using bioengineering technology.

[0030] Monoclonal hybridoma cell lines are easily prepared by those skilled in the art. For example, spleen cells can be obtained by immunizing host animals with the above-mentioned complete antigen, and then fused with SP2 / 0 tumor cells in vitro. Single hybridomas that can secrete anti-cyproconazole antibodies can be screened through selective culture medium to obtain hybridoma cell lines. Anti-cyproconazole antibodies can also be obtained through genetic engineering techniques, which are easily implemented by those skilled in the art. Therefore, regardless of the method used to prepare anti-cyproconazole antibodies, as long as the hapten or complete antigen provided by this invention is used, it falls within the protection scope of this invention.

[0031] Antibodies or antiserum prepared by immunizing host animals with the complete antigen provided by the present invention can specifically bind to cyazofamid and have a good immunizing effect.

[0032] Optionally, in some embodiments of the present invention, the antibody is serum resistant to cyproconazole.

[0033] Based on the hapten or complete antigen disclosed in this invention, those skilled in the art will readily conceive of using suitable antibody preparation methods to prepare antibodies against cyazofamid. Regardless of the type of animal used for immunization, or how the immunization conditions or parameters are set or changed, as long as the hapten or complete antigen provided by this invention is used, it falls within the protection scope of this invention.

[0034] In a sixth aspect, the present invention provides an anti-cyproconazole polyclonal antibody, which is obtained by immunizing a host animal with the above-mentioned complete antigen and isolating the anti-cyproconazole polyclonal antibody from the blood of the host animal.

[0035] The aforementioned anti-cyproconazole polyclonal antibody is anti-cyproconazole serum.

[0036] Seventhly, this invention provides a hybridoma cell capable of secreting monoclonal antibodies against cyazofamid, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 45730 and original accession number JJZ3E10. The deposit date is October 13, 2023. The suggested classification name is mouse hybridoma cell line, and the cell is identified as viable. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0037] Eighthly, the present invention provides an anti-cyproconazole monoclonal antibody, which is obtained by secretion from the above-mentioned hybridoma cells.

[0038] In a ninth aspect, the present invention provides the application of cyproconazole hapten, cyproconazole hapten prepared by the above preparation method, complete antigen, anti-cyproconazole polyclonal antibody, or the above-mentioned anti-cyproconazole monoclonal antibody in cyproconazole immunoassay.

[0039] The aforementioned immunoassay for cyazofamid includes, but is not limited to, immunoassay for cyazofamid using kits or test strips. The aforementioned applications include, but are not limited to, analysis of cyazofamid residues in food, pharmaceuticals, soil, and air.

[0040] In a preferred embodiment of the present invention, the kit includes a carrier, and the carrier carries an antibody or a cyazofamid complete antigen. In a preferred embodiment of the present invention, the carrier is nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, or porous materials.

[0041] The carrier is linked to the antibody or complete antigen via coupling and / or physical connection. Physical connection includes, but is not limited to, electrostatic adsorption.

[0042] In other embodiments, the porous material is selected from ELISA plates or other solid supports.

[0043] For example, activated magnetic beads can be incubated with antibodies to coat the beads with antibodies. Alternatively, antibodies can be conjugated to agarose gel microspheres or silica gel microspheres to prepare corresponding immunoaffinity adsorption materials.

[0044] For example, activated magnetic beads can be incubated with complete antigens to coat the beads with the complete antigens. Alternatively, complete antigens can be coupled to agarose gel microspheres or silica gel microspheres to prepare corresponding immunoaffinity adsorption materials.

[0045] In one alternative embodiment, the nanoparticles are selected from organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0046] Magnetic beads, also known as magnetic microspheres, are composite microspheres with specific magnetic properties formed by combining organic polymers and inorganic magnetic nanoparticles through appropriate methods. Magnetic beads include, but are not limited to, nano-magnetic beads and micron-magnetic microspheres. In one optional embodiment, magnetic beads include, but are not limited to: carboxyl magnetic beads, amino magnetic beads, oleylamine-modified magnetic beads, silanol magnetic beads, sulfonic acid-based magnetic microspheres, thiol-based magnetic microspheres, PEG-modified magnetic beads, unmodified magnetite beads, monodisperse silica-coated magnetic beads, epoxy-based magnetic beads, monodisperse mesoporous silica-coated magnetic beads, gold-coated magnetic nanoparticles, streptavidin-modified magnetic beads, polylysine-modified magnetic beads, nickel magnetic beads, magnetic polystyrene microspheres, and silica magnetic microspheres.

[0047] Latex microspheres are spherical polymer particles in the colloidal size range formed from amorphous polymers (usually polystyrene), such as particles with a diameter of less than 100 nm, or particles with a particle size of 0.3-0.5 µm, or particles with a particle size of more than 1 µm.

[0048] Latex microspheres include, but are not limited to, latex microspheres with at least one functional group selected from hydroxyl, carboxyl, or Toysal on their surface.

[0049] In one alternative embodiment, the kit further includes a buffer solution, a positive control, and a washing solution.

[0050] In a preferred embodiment of the present invention, the antibodies (monoclonal and / or polyclonal antibodies) or complete antigens are further labeled with detectable markers.

[0051] In a preferred embodiment of the present invention, the detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and colloids.

[0052] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0053] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0054] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0055] In one alternative embodiment, the chemiluminescent reagent is selected from at least one of acridine ester, luminol, luciferin, luteolin, ruthenium bipyridine, dioxane, rofen, isoluminol, and peroxyoxalate.

[0056] Colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0057] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0058] In a tenth aspect, the present invention provides a reagent or kit for detecting cyazofamid, comprising at least one of the above-mentioned anti-cyazofamid polyclonal antibody, the above-mentioned anti-cyazofamid monoclonal antibody, and the above-mentioned complete antigen.

[0059] In one alternative embodiment, the kit includes an ELISA plate, the microwells of which are coated with cyproconazole complete antigen.

[0060] This test reagent or kit has the technical advantage of high specificity for the detection of cyazofamid.

[0061] In one aspect, the present invention provides a method for detecting cyazofamid, using the above-mentioned complete antigen, the above-mentioned anti-cyazofamid polyclonal antibody, the above-mentioned anti-cyazofamid monoclonal antibody, or the above-mentioned reagents or kits for detection.

[0062] The present invention has the following beneficial effects: The hapten provided by this invention can be obtained conveniently and quickly, with low synthesis cost and good effect; the complete antigen prepared based on it has good antiserum titer and high specificity after immunization, and the host animal spleen cells obtained by immunization can be effectively used for hybridoma cell fusion and preparation of anti-cyproconazole antibodies, which has broad application prospects. Attached Figure Description

[0063] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0064] Figure 1 This is a schematic diagram of the synthesis pathway of cyproconazole hapten. Figure 2 The high-performance liquid chromatogram of the cyazofamid hapten; Figure 3 The 1H NMR spectrum of the cyazofamid hapten; Figure 4 MADLI-TOF-MS spectrum of cyazofamid complete antigen C1; Figure 5 MADLI-TOF-MS spectrum of cyazofamid complete antigen C2; Figure 6 The MADLI-TOF-MS spectrum of BSA; Figure 7 MADLI-TOF-MS spectrum of OVA; Figure 8 Standard curve for detecting cyazofamid using monoclonal antibody 3E 10. Detailed Implementation

[0065] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0066] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0068] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0069] Example 1 This embodiment provides a method for preparing cyazofamid hapten; the synthetic route is as follows: Figure 1 As shown. The specific preparation method includes the following steps: (1) Weigh 13 g of 2-(4-chlorophenyl)2-(1H-1,2,4-triazol-1-methyl)hexanonitrile, 6.3 g of tert-butyl carbamate, 29.3 g of cesium carbonate, 2.15 g of 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene), and 310 mg of palladium acetate and dissolve them in 150 mL of 1,4-dioxane. Stir the mixture at 110 °C for 16 h under nitrogen atmosphere. After the reaction is complete, cool the reactants to room temperature, add 250 mL of water and 100 mL of ethyl acetate to extract the organic phase three times, and collect the organic phase. Wash the organic phase three times with 100 mL of water and once with 100 mL of brine, and finally dry and concentrate it with anhydrous sodium sulfate. The concentrated product was separated by silica gel column chromatography (elution buffer was a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1, gradient elution, and elution with 100% volume of ethyl acetate as the eluent) to obtain compound 3 (14.5 g, recovery rate 89%).

[0070] (2) 11.1 g of compound 3 was weighed and dissolved in 100 mL of dimethylformamide at 0℃. 1.8 g of sodium hydride was added, and the mixture was stirred at 0℃ for 30 minutes. Then, 7.5 g of compound 4 was added, and the mixture was stirred at room temperature for 16 h. After the reaction was completed, 400 mL of water and 100 mL of ethyl acetate were added to extract the product three times, and the organic phase was collected. The organic phase was washed three times with 120 mL of water and twice with 150 mL of brine. Finally, the product was dried over anhydrous sodium sulfate and concentrated to obtain 21.5 g of concentrated product. The concentrated product was separated by silica gel column chromatography (using a 3:1 volume ratio of petroleum ether and ethyl acetate as the eluent, gradient elution, and elution with 100% volume of ethyl acetate as the eluent) to obtain compound 5 (15.2 g, recovery rate 100%).

[0071] (3) 15.0 g of compound 5 was weighed and dissolved in 100 mL of dichloromethane. 30 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 16 h before concentration. 200 mL of saturated sodium bicarbonate was added to the concentrated product, followed by extraction three times with 100 mL of dichloromethane. The organic phase was collected. The organic phase was washed three times with 120 mL of water and twice with 150 mL of brine. Finally, it was dried over anhydrous sodium sulfate and concentrated to obtain 12.2 g of concentrated product. The concentrated product was separated by silica gel column chromatography (eluting solution was a 1:1 mixture of petroleum ether and ethyl acetate) to obtain compound 6 (6.2 g, recovery rate 53%).

[0072] (4) 6.2 g of compound 6 was weighed and dissolved in 30 mL of methanol and 30 mL of tetrahydrofuran. 50 mL of lithium hydroxide solution was added, and the mixture was stirred at room temperature for 16 h. Methanol and tetrahydrofuran were then removed under vacuum. 50 mL of water was added to the reaction mixture, and the pH was adjusted to 3 with 1 M hydrochloric acid solution. Then, 100 mL of ethyl acetate was added to extract twice, and the organic phase was collected. The organic phase was washed three times with 120 mL of water and twice with 150 mL of brine. Finally, it was dried and concentrated with anhydrous sodium sulfate to obtain 7.2 g of concentrated product. 25 mL of ethyl acetate was added to the concentrated product, and the mixture was stirred at room temperature for 1 h. The precipitate was collected by filtration and dried under vacuum to finally obtain the cyazofamid hapten (4.60 g, recovery rate 81%).

[0073] High performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) were used to analyze the hydrogen spectroscopy. 1 The hapten for cyazofamid was identified by 1H NMR. The identification results by high-performance liquid chromatography were as follows: Figure 2 As shown, only one chromatographic peak is displayed.

[0074] reference to the proton NMR spectrum Figure 3 As shown: 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 0.81 (t, J = 6.4 Hz, 3H), 1.04–1.07 (m, 1H), 1.25–1.26 (m, 3H), 1.91–2.03 (m, 2H), 3.08 (s, 2H), 4.65–4.768 (m, 2H), 6.58 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.95 (s, 1H), 8.110 (s, 1H). Based on the ¹H NMR results, the molecular structure of the cyproconazole hapten 2-(4-(2-cyano-1-(1H-1,2,4-triazol-1-yl)hexane-2-yl)phenylamino)acetic acid is correct, indicating successful synthesis.

[0075] Example 2 Synthetic cyazofamid complete antigen C1: The cyazofamid complete antigen C1 is prepared by conjugating the cyazofamid hapten prepared in Example 1 with the carrier protein OVA (oval albumin).

[0076] The specific method includes: slowly adding the supernatant of the cyproconazole-activated hapten dropwise to the carrier protein OVA solution (the solution is obtained by dissolving 20 mg of OVA in phosphate-buffered saline (PBS) with a pH of 7.4 and mixing well, with a concentration of 10 mg / mL). The molar ratio of hapten to carrier protein is 30:1. After stirring overnight at 4°C, the resulting reaction solution is dialyzed 6 times with PBS solution with a pH of 7.4 and a concentration of 0.01 mol / L. The completely dialyzed reaction product solution is diluted to a 4 mg / mL solution and stored at -20°C for later use.

[0077] MADLI-TOF-MS spectrum of cyazofamid complete antigen C1 (refer to) Figure 4 As shown, the MADLI-TOF-MS spectrum of OVA is referenced. Figure 7 As shown, the single-charged ion peaks for OVA and complete antigen C1 are 44536.325 and 47620.036, respectively. The coupling ratio of the carrier protein to the hapten is calculated as (molecular weight of complete antigen - molecular weight of carrier protein) / molecular weight of hapten. The coupling ratio of complete antigen C1 is calculated to be 1:9.4 using this formula.

[0078] Example 3 In this embodiment, the complete antigen C2 of cyproconazole was synthesized: the complete antigen C2 of cyproconazole was prepared by coupling the cyproconazole hapten prepared in Example 1 with the carrier protein BSA.

[0079] The supernatant of the hapten activated by cyazofamid was slowly added dropwise to the carrier protein solution (the carrier protein solution was prepared by dissolving 20 mg BSA in phosphate buffer (PBS) at pH 7.4, with a concentration of 10 mg / mL). The molar ratio of hapten to carrier protein was 30:1. After stirring overnight at 4°C, the resulting reaction solution was dialyzed six times with PBS solution at pH 7.4 and a concentration of 0.01 mol / L. The completely dialyzed reaction product solution was diluted to a 4 mg / mL solution and stored at -20°C for later use.

[0080] MADLI-TOF-MS spectrum of cyazofamid complete antigen C2 (refer to) Figure 5 As shown, the MADLI-TOF-MS spectrum of BSA is referenced. Figure 6 As shown, the single-charged ion peaks of BSA and complete antigen C2 are 67457.779 and 75575.306, respectively. The coupling ratio of carrier protein to hapten = (molecular weight of complete antigen - molecular weight of carrier protein) / molecular weight of hapten. The coupling ratio of complete antigen C2 calculated by the formula is 1:24.8.

[0081] Example 4 This embodiment provides a polyclonal antibody against cyazofamid and its preparation method.

[0082] 1. Animal immunization Balb / C mice aged 6-8 weeks (weighing approximately 23-25g) were used as experimental animals.

[0083] Basic immunization: The diluted cyazofamid complete antigen C2 solution (concentration of 1 mg / mL) was filtered through a sterile filter and an equal volume of Freund's complete adjuvant was added. The mixture was then thoroughly emulsified using a magnetic stirrer until it did not diffuse when dropped into water. Balb / c mice were injected intraperitoneally and subcutaneously at multiple sites on the back with the emulsified complete antigen at a dose of 0.1 mg emulsified antigen per mouse.

[0084] Boosting immunization: Two weeks after the primary immunization, take 1 mL of the diluted cyazofamid complete antigen C2 solution and add 1 mL of Freund's incomplete adjuvant. Emulsify thoroughly with a magnetic stirrer until it does not diffuse when dropped into water. Administer the emulsified antigen to Balb / c mice via multiple intraperitoneal and subcutaneous injections on the back at a dose of 0.1 mg emulsified antigen per mouse.

[0085] Booster immunizations were administered every 14 days, starting with the third booster immunization. Three days after each immunization, blood was collected from the orbital cavity of mice to determine antibody titers. The coating antigen was 1 mg / mL cyazofamid complete antigen C1, diluted four times: 1000, 2000, 4000, and 8000 times. Once the titer reached or exceeded 1:8000 (titer defined as the serum dilution factor when the zero-well colorimetric value is 1), the eyeballs were enucleated to collect blood. The blood was allowed to stand at room temperature for 1 hour, then incubated at 4°C for 2 hours. Finally, the blood was centrifuged at 4°C, 10000 r / min for 5 minutes to separate the antiserum, yielding the cyazofamid polyclonal antibody.

[0086] 2. Detection of antibody titer in antiserum The buffer solutions used in the following experiments are as follows: (1) Coating buffer (pH 9.6, 0.05 M carbonate buffer): Weigh 1.5 g of Na2CO3 and 2.94 g of NaHCO3, and bring the volume to 1000 mL with ultrapure water; (2) Phosphate buffer (0.01 M, pH 7.4): Weigh 0.2 g of KH2PO4, 8 g of NaCl, and 2.92 g of Na2HPO4·12H2O, and bring the volume to 1000 mL with ultrapure water; (3) Washing buffer: Weigh 0.2 g of KH2PO4, 8 g of NaCl, 2.92 g of Na2HPO4·12H2O, 1 mL of Tween-20, and bring the volume up to 1000 mL with ultrapure water; (4) Sample dilution solution: Weigh 0.2 g of KH2PO4, 8 g of NaCl, 2.92 g of Na2HPO4·12H2O, 1 g of gelatin (melted by microwave heating), and make up to 1000 mL with ultrapure water; (5) Substrate buffer (pH 5.5): Weigh 9.22 g of Na2HPO4·12H2O, 2.55 g of citric acid monohydrate, measure 0.5 mL of Tween-20, and bring the volume up to 1 L with ultrapure water; (6) Termination solution (2M H2SO4): Measure 445.6 mL of distilled water, add 54.4 mL of 98% (volume / volume) concentrated sulfuric acid dropwise and stir.

[0087] Antiserum and antibody inhibition experiments: (1) Preparation of C1 coating antigen solution of cyproconazole complete antigen: After thawing the cyproconazole complete antigen prepared above and diluted to 1 mg / mL, it was serially diluted with coating buffer at 1:1000, 1:2000, 1:4000 and 1:8000 to obtain coating antigen solutions of different concentrations of cyproconazole complete antigen.

[0088] (2) Preparation of cyproconazole standard solution: Weigh 10 mg of cyproconazole standard sample and dissolve it completely in 10 mL of methanol to obtain 1 mg / mL cyproconazole standard solution. Use sample diluent to prepare 1 mg / mL cyproconazole standard solution to a final concentration of 1000 ng / mL.

[0089] (3) Preparation of cyproconazole antiserum / antibody dilution: The cyproconazole antiserum was serially diluted with the sample dilution at 1:4000, 1:8000, 1:16000 and 1:32000 to obtain the cyproconazole antiserum dilution.

[0090] (4) Coating: Add 100 μL of cyproconazole complete antigen C1 coating antigen solution to each well of a 96-well microplate, incubate at 37°C for 3 hours, wash 3 times with sample washing solution, and spin dry.

[0091] (5) Competition: Add 50 μL of sample diluent to each well in the zero well; add 50 μL of the prepared 1000 ng / mL cyazofamid standard solution to each well in the inhibition well. Add cyazofamid antiserum diluent to the ELISA plate (50 μL / well), incubate at 37℃ for 30 min, wash the plate 3 times, and spin dry.

[0092] (6) Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 1000 times with sample diluent, add 100 μL to each well, incubate at 37℃ for 30 min, wash the plate 3 times, and spin dry.

[0093] (7) Color development: Prepare the color development substrate fresh for use. Add 15-20 mg OPD to every 10 mL of substrate buffer. After dissolving the weighed OPD in the substrate buffer, add 4 μL of 30% hydrogen peroxide to every 10 mL. Add 100 μL to each well and develop the color at room temperature in the dark for 15 min.

[0094] (8) Termination: Add 50 μL of 2 M sulfuric acid termination buffer to each well and measure the OD value of each well at 492 nm using an ELISA reader.

[0095] The formula for calculating the inhibition rate is: Inhibition rate = (A0 - A) / ... 50 () / A0×100%), where A0 is the OD value of the reference well; A 50 To suppress the pore OD value.

[0096] The results are shown in Table 1.

[0097] Table 1. Serum titer detection in mice treated with cyazofamid.

[0098] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0099] The results in Table 1 show that the inhibition rate was best at 46.95% when the coating antigen was diluted to 1:8000 and the serum was diluted to 1:32000. The prepared cyazofamid complete antigen C2 can be used as an immunogen to prepare antibodies for detecting cyazofamid.

[0100] Example 5 This embodiment provides a cyazofamid monoclonal antibody JJZ3E10 and its preparation method.

[0101] Mice with high serum titers and good specificity were screened for immunization by intraperitoneal injection of 100 μg immunogen C2 three days before cell fusion. Mouse spleen cells were fused with Sp2 / 0 cells in logarithmic growth phase using the PEG method to prepare hybridoma cells. These cells were cultured in HAT medium, and positive cell pores were detected using indirect ELISA. Further, the inhibitory effect of positive cell pores was determined using indirect competitive ELISA. Subcloning of positive cell pores with the best inhibition was performed using limiting dilution. Finally, the hybridoma cell line JJZ3E10 was obtained, with the preservation number CGMCC NO: 45730.

[0102] Titer assay for monoclonal antibodies: The antibody titer was detected using icELISA, following the method described in Example 4 for detecting antibody titer in antiserum. Specific results are shown in Table 2.

[0103] Table 2. Titer assay of monoclonal antibody JJZ3E10 (inhibition by 1000 ng / mL cyproconazole standard)

[0104] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0105] Table 2 shows that the highest inhibition rate and best inhibitory effect against cyazofamid were observed when the coating antigen dilution was 1:64000 and the antibody dilution was 1:4000. This indicates that the monoclonal antibody JJZ3E10 can effectively inhibit cyazofamid.

[0106] Example 6 This embodiment establishes a standard curve for the detection of cyazofamid, and the method for establishing the curve is as follows: The cyazofamid standard solution prepared in Example 4 was diluted with sample diluent to the following different concentrations: 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL.

[0107] (1) Coating of antigen: The prepared cyazofamid complete antigen C1 was diluted at 1:32000 and added to the microplate, 100 μL per well, and placed in a humidified chamber at 37°C overnight; the solution in the microplate was poured off, the plate was washed 3 times with washing solution, and then dried. (2) Dilute the cyproconazole standard solution in Example 4 and the monoclonal antibody prepared in Example 5 with sample diluent. Add 50 μL of the above diluted cyproconazole standard solution of different concentrations and 50 μL of antibody to the plate in sequence, and incubate at 37°C for 30 min. Pour off the solution in the ELISA plate, wash the plate 3 times with washing buffer, and spin dry. (3) Add 100 μL of goat anti-mouse enzyme-labeled antibody diluted 1:1000 to the experimental wells and control wells, incubate at 37℃ for 30 min; pour off the solution in the enzyme-labeled plate, wash the plate 3 times with washing buffer, and spin dry; (4) Color development: Prepare the color development substrate fresh for use. Add 15-20 mg OPD to every 10 mL of substrate buffer. After dissolving the weighed OPD in the substrate buffer, add 4 μL of 30% hydrogen peroxide to every 10 mL. Add 100 μL to each well and develop the color at room temperature in the dark for 15 min.

[0108] (5) Termination: Add 50 μL of 2 M sulfuric acid termination buffer to each well and measure the OD value of each well at 492 nm using an ELISA reader.

[0109] (6) Plot the standard curve: Plot the standard curve using the different concentrations (ng / mL) of the cyazofamid standard solution as the X-axis and the ratio of absorbance values ​​(B / B0, where B is the average absorbance value of the cyazofamid standard solution and B0 is the average absorbance value of the control well) as the Y-axis.

[0110] The experiment was repeated three times, and the average of the three results was taken to obtain the standard curve of cyazofamid as shown below. Figure 8 As shown.

[0111] The standard curve equation is y = A² + (A₁ - A₂) / (1 + (x / x₀)^p), where A₁ = 0.99783, A₂ = 0.07412, x₀ = 228.48187, p = 1.45052, and the correlation coefficient R of the standard curve is... 2 >0.99 (R²≈0.99435). Where IC 50 The value was 254.44 ng / mL.

[0112]

[0113] Example 7 This experimental example demonstrates the specificity of the cyazofamid monoclonal antibody JJZ3E10 provided in Example 5.

[0114] The coating antigen / antibody dilution factor is 32 × 10⁻⁶. 3 / 4×10 3 The monoclonal antibody JJZ3E 10 was subjected to cross-reactivity experiments with the tebuconazole, tebuconazole, triazole, and triazophos, which are structural analogs of cyproconazole. The results showed that the absorbance values ​​in each well of the ELISA plate corresponding to each structural analog did not differ significantly with the dilution concentration of the standard. The specific results are shown in Table 3.

[0115] This result indicates that the monoclonal antibody JJZ3E 10 has a very low cross-reactivity rate with the four structural analogs of cyazofamid, suggesting that the antibody prepared using the cyazofamid complete antigen C2 has good specificity for cyazofamid.

[0116] Cross-reactivity rate (%) = (Cyazofamid IC50) 50 ) / (analog IC 50 ) × 100%.

[0117] Table 3. Specificity detection of antibody JJZ3E 10

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cyazofamid hapten, characterized in that, It has the following structure: 。 2. The method for preparing the cyazofamid hapten as described in claim 1, characterized in that, The cyproconazole hapten is obtained from cyproconazole as a raw material through a chemical reaction, and its synthetic route is as follows: ； In this synthetic route, SM1 represents cyproconazole, X-Phos represents 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene, Pd(oAc)2 represents palladium acetate, NaH represents sodium hydride, DMF represents N,N-dimethylformamide, TFA represents trifluoroacetic acid, DCM represents dichloromethane, THF represents tetrahydrofuran, LiOH represents lithium hydroxide, MeOH represents methanol, and TL-1 is cyproconazole hapten. The numbers 3, 4, 5, and 6 in the synthetic route represent compounds 3, 4, 5, and 6, respectively.

3. The method for preparing the cyazofamid hapten according to claim 2, characterized in that, The synthesis of cyazofamid hapten includes the following steps: (1) The cyproconazole, tert-butyl carbamate, cesium carbonate, X-Phos and palladium acetate were mixed in an organic solvent and reacted; after extraction and separation, compound 3 was obtained; The molar ratio of the mixture of cyproconazole, tert-butyl carbamate, cesium carbonate, X-Phos, and palladium acetate is 1:1-1.5:2-3:0.1-0.2:0.03-0.

1. (2) Mix a DMF solution containing 0.03 mol of compound 3 with 0.045-0.06 mol of sodium hydride and 0.045 mol of compound 4 and react for 15-20 h; Compound 5 was obtained through extraction and separation; (3) Mix the dichloromethane solution containing compound 5 with trifluoroacetic acid, react for 10-20 h, and then concentrate, extract and separate to obtain compound 6; (4) The compound 6 was mixed with methanol, tetrahydrofuran and lithium hydroxide and reacted for 10-20 h. After removing methanol and tetrahydrofuran, water was added to the reaction mixture and extracted with an organic solvent. Then the mixture was washed, dried and concentrated.

4. The method for preparing the cyazofamid hapten according to claim 3, characterized in that, In step (1), the organic solvent is 1,4-dioxane, and the reaction is carried out in a nitrogen atmosphere at a reaction temperature of 100-160℃ for 10-20 hours.

5. The method for preparing the cyazofamid hapten according to claim 3, characterized in that, The reaction time in step (1) is 10-20 hours; The extraction and separation process includes: extracting the reactants with water and ethyl acetate at least twice, collecting the organic phase and washing it with water and / or brine, drying and concentrating it using at least one of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium sulfate, and separating the concentrated product by silica gel column chromatography to obtain compound 3.

6. The method for preparing the cyazofamid hapten according to claim 3, characterized in that, In step (2), compound 3 is dissolved in 80-120 mL of DMF, 0.045-0.06 mol of sodium hydride is added, and the mixture is stirred in an ice bath for 20-30 minutes before adding compound 4. After reacting for 15-20 h, water and ethyl acetate are added for extraction at least twice. The organic phase is collected, washed with water and / or brine, dried and concentrated with anhydrous sodium sulfate, and the concentrated product is separated by silica gel column chromatography to obtain compound 5.

7. The method for preparing the cyazofamid hapten according to claim 3, characterized in that, In step (3), compound 5 is mixed with 100 mL of dichloromethane and 30 mL of trichloroacetic acid and reacted; 50-200 mL of saturated sodium bicarbonate is added to the concentrated product obtained by concentration, and then dichloromethane is added to extract at least twice. The organic phase is collected, washed with water and / or brine, dried and concentrated with anhydrous sodium sulfate, and the concentrated product is separated by silica gel column chromatography to obtain compound 6.

8. The method for preparing the cyazofamid hapten according to claim 3, characterized in that, In step (4), compound 6 is mixed with 30 mL of methanol, 30 mL of tetrahydrofuran and 50 mL of lithium hydroxide solution and reacted. After the reaction is completed, methanol and tetrahydrofuran are removed under vacuum. Then water is added to the reaction mixture and extracted with ethyl acetate at least twice. The organic phase is collected, washed with water and / or brine, dried and concentrated with anhydrous sodium sulfate, and the concentrated product is added with ethyl acetate and stirred for 0.5-1 h. The precipitate is collected by filtration and dried under vacuum to obtain cyproconazole hapten.

9. A complete antigen, characterized in that, It includes a carrier protein and a cyazofamid hapten of claim 1 or a cyazofamid hapten prepared by the preparation method of any one of claims 2-8, wherein the carrier protein is selected from bovine serum albumin or ovalbumin.

10. The use of the cyproconazole hapten as described in claim 1 or the cyproconazole hapten prepared by the preparation method according to any one of claims 2-8 in the preparation of a complete antigen, characterized in that, The complete antigen includes a carrier protein and a cyazofamid hapten coupled to the carrier protein.

11. The application according to claim 10, characterized in that, When preparing the complete antigen, the molar ratio of the hapten to the carrier protein is 5-60:

1.

12. The application according to claim 11, characterized in that, When preparing the complete antigen, the molar ratio of the hapten to the carrier protein is 30:

1.

13. The application according to claim 10, characterized in that, When preparing the complete antigen, the hapten is mixed with the carrier protein, and the reaction temperature is 0-50℃, the reaction time is 8-36 hours, and the pH is 5-9.

14. The application according to claim 13, characterized in that, When preparing the complete antigen, the hapten is mixed with the carrier protein, and the reaction temperature is 4°C.

15. The application according to claim 13, characterized in that, When preparing the complete antigen, the hapten is mixed with the carrier protein, and the reaction time is 12 hours and the reaction pH is 7.

4.

16. The use of the cyproconazole hapten as described in claim 1, the cyproconazole hapten prepared by the preparation method according to any one of claims 2-8, or the complete antigen as described in claim 9 in the preparation of anti-cyproconazole polyclonal antibodies or monoclonal antibodies.

17. A polyclonal antibody against cyproconazole, characterized in that, It involves immunizing a host animal with the complete antigen as described in claim 9, and then isolating the anti-nitrazole polyclonal antibody from the blood of the host animal.

18. The anti-cyproconazole polyclonal antibody according to claim 17, characterized in that, The anti-nitrile polyclonal antibody is anti-nitrile polyclonal serum.

19. A hybridoma cell capable of secreting monoclonal antibodies against cyproconazole, characterized in that, It is prepared from the cyazofamid hapten described in claim 1, and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCCNO:45730.

20. A monoclonal antibody against cyproconazole, characterized in that, It is obtained by secretion from the hybridoma cells as described in claim 19.

21. The application of the cyproconazole hapten as described in claim 1, the cyproconazole hapten prepared by the preparation method according to any one of claims 2-8, the complete antigen as described in claim 9, the anti-cyproconazole polyclonal antibody as described in any one of claims 17-18, or the anti-cyproconazole monoclonal antibody as described in claim 19 in the immunoassay of cyproconazole, wherein the application is for the purpose of non-disease diagnosis or treatment.

22. A reagent or kit for detecting cyazofamid, characterized in that, It contains at least one of the following: the anti-cyproconazole polyclonal antibody according to any one of claims 17-18, the anti-cyproconazole monoclonal antibody according to claim 19, and the complete antigen according to claim 9.

23. A method for detecting cyazofamid, characterized in that, The detection is performed using the complete antigen of claim 9, the anti-cyproconazole polyclonal antibody of any one of claims 17-18, the anti-cyproconazole monoclonal antibody of claim 20, or the reagent or kit of claim 22, wherein the method is for the purpose of non-disease diagnosis or treatment.

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