A conazole hapten, antigen, antibody, and methods of making and using the same

Abstract

The present application relates to the field of immunoassay technology, and provides a myclobutanil hapten, antigen, antibody and preparation method and application. The present application introduces an active group at the meta position of the chlorine atom on the benzene ring of myclobutanil. Compared with the existing hapten, the hapten provided by the present application completely simulates the structure of myclobutanil, and enhances the immunogenicity of the antigen. The antigen provided by the present application has strong immunogenicity after being coupled with a carrier protein. After injection immunization of mice, the titer of the produced antibody is 5.0x10 4 , the half-inhibitory concentration (IC 50 ) is about 50 ppb, and the minimum detection limit of colloidal gold for myclobutanil in dry tobacco leaf samples is about 2.5 μg / g.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, and particularly relates to a myclobutanil hapten, antigen, antibody and preparation method and application. BACKGROUND

[0002] Myclobutanil is a kind of protective and therapeutic activity of endosporic triazole fungicide. It mainly inhibits the biosynthesis of ergosterol of pathogenic fungi, and has good control effect on ascomycetes and basidiomycetes. The fungicide has long persistence, is safe to crops, has certain growth stimulating effect, and has the characteristics of strong endosporicity, high efficacy and safety to crops.

[0003] At present, the common detection methods of myclobutanil in food and agricultural products include liquid chromatography, liquid chromatography-mass spectrometry and immunoassay method. The immunoassay method mainly includes competitive enzyme-linked immunosorbent assay (ELISA) and colloidal gold method. Although the liquid chromatography and liquid chromatography-mass spectrometry have accurate detection results, they need expensive instrument equipment and the operation is complex, and are mainly used in professional laboratories. Compared with the above two methods, the ELISA kit method or colloidal gold method is simple and rapid in operation, does not need professional operation, only needs to be equipped with basic instrument equipment, and can be widely applied to raw material safety control of food processing enterprises and on-site supervision of industrial and commercial law enforcement departments. It is a very popular food safety rapid screening method.

[0004] However, the current immunoassay method for myclobutanil generally has the problems of insufficient antibody detection sensitivity and low titer. SUMMARY

[0005] Therefore, the present application provides a myclobutanil hapten, antigen, antibody and preparation method and application. The antigen obtained by coupling the myclobutanil hapten provided by the present application with a carrier protein can produce antibodies with high titer and high sensitivity after immunization of a host animal, thereby providing high-quality raw materials for rapid detection of myclobutanil by ELISA kit method and colloidal gold method.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] A myclobutanil hapten has the structure shown in formula I:

[0008]

[0009] The present application also provides a preparation method of the myclobutanil hapten described in the above-mentioned scheme, comprising the following steps:

[0010] The myclobutanil hapten is obtained by mixing myclobutanil, aluminum chloride, succinic anhydride and an organic solvent for a Friedel-Crafts acylation reaction.

[0011] Preferably, the molar ratio of the cyproconazole, aluminum trichloride, and succinic anhydride is 1:(2-3):(1-1.2);

[0012] The Friedel-Crafts acylation reaction is carried out at a temperature of 80–90 °C for 4–5 h.

[0013] The present invention also provides a cyazofamid antigen, which is obtained by coupling a carrier protein with the cyazofamid hapten prepared by the method described above or the preparation method described above.

[0014] Preferably, the carrier protein is ovalbumin; when the carrier protein is ovalbumin, the structural formula of the cyazofamid antigen is shown in Formula II:

[0015]

[0016] Preferably, the carrier protein is bovine serum albumin; when the carrier protein is bovine serum albumin, the structural formula of the cyazofamid antigen is shown in Formula III:

[0017]

[0018] The present invention also provides a method for preparing the cyproconazole antigen described in the above scheme, comprising the following steps:

[0019] The hapten, coupling agent, and polar solvent described in the above scheme are mixed and activated to obtain an activated hapten solution.

[0020] The activated hapten solution and the carrier protein buffer solution were mixed and coupled to obtain the cyazofamid antigen.

[0021] The present invention also provides a cyazofamid antibody, which is obtained by immunizing a host animal with the cyazofamid antigen described in the above scheme.

[0022] The present invention also provides a test strip or kit for detecting cyazofamid, containing the cyazofamid antibody described in the above-described scheme.

[0023] The present invention also provides the application of the cyproconazole antibody or the test strip or kit described above in the detection of cyproconazole.

[0024] This invention provides a cyazofamid hapten with the structural formula shown in Formula I. This invention introduces an active group at the meta position of the chlorine atom on the benzene ring of cyazofamid. Compared to existing haptens, the hapten provided by this invention completely mimics the structure of cyazofamid, thus enhancing the immunogenicity of the antigen.

[0025] This invention also provides a method for preparing the cyazofamid hapten described in the above scheme. This invention obtains the cyazofamid hapten through Friedel-Crafts acylation reaction, which has a short synthetic route, readily available raw materials, mild conditions, simple operation, and high product purity.

[0026] This invention also provides a cyazofamid antigen, obtained by conjugating the cyazofamid hapten described in the above-described scheme with a carrier protein. The cyazofamid hapten provided by this invention, after conjugation with the carrier protein, yields an antigen with strong immunogenicity; after immunization in mice, the antibody titer is 5.0 × 10⁻⁶. 4 Half-inhibitory concentration (IC50) 50 The limit of detection for cyazofamid in dry tobacco samples was approximately 2.5 μg / g, with a concentration of around 50 ppb. Attached Figure Description

[0027] Figure 1 The image shows the 1H NMR spectrum of the cyazofamid hapten prepared in Example 1 of this invention. Detailed Implementation

[0028] This invention provides a cyazofamid hapten having the structure shown in Formula I:

[0029]

[0030] The present invention also provides a method for preparing the cyazofamid hapten described in the above-mentioned scheme, comprising the following steps:

[0031] A Friedel-Crafts acylation reaction was carried out by mixing cyproconazole, aluminum trichloride, succinic anhydride, and an organic solvent to obtain cyproconazole hapten.

[0032] In this invention, the molar ratio of cyproconazole, aluminum trichloride, and succinic anhydride is preferably 1:(2-3):(1-1.2), more preferably 1:(2.2-2.5):(1-1.1); the aluminum trichloride is preferably anhydrous aluminum trichloride; the Friedel-Crafts acylation reaction is preferably carried out at a temperature of 80-90°C and a time of 4-5 h; the organic solvent is preferably chloromethane, more preferably anhydrous dichloromethane; in a specific embodiment of this invention, the Friedel-Crafts acylation reaction is preferably carried out under reflux conditions, and the reaction progress is monitored by TLC.

[0033] In a specific embodiment of the present invention, it is preferable to first dissolve aluminum trichloride in an organic solvent, add succinic anhydride, stir for 25-30 minutes, then add cyproconazole, and then carry out the reaction under reflux conditions.

[0034] In this invention, the reaction formula for the Friedel-Crafts acylation reaction is shown in Formula A:

[0035]

[0036] After the Friedel-Crafts acylation reaction is completed, the present invention preferably cools the resulting reaction solution to room temperature and then pours it into ice and stirs for 10-15 minutes. The solution is then separated into two layers: an organic layer and an aqueous layer. The aqueous layer is extracted with dichloromethane. The extracted organic layer and the separated organic layers are combined and evaporated to dryness. The residue is then subjected to column chromatography to obtain the cyazofamid hapten. The column chromatography is preferably performed using a dry loading method. The column chromatography preferably includes a first elution and a second elution. The eluent for the first elution is preferably an organic solvent obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 15:1. The eluent for the second elution is preferably an organic solvent obtained by mixing ethyl acetate and petroleum ether in a volume ratio of 1:2. The present invention preferably uses the first elution to remove impurities, followed by the second elution to remove the cyazofamid hapten. The eluent obtained from the second elution is then evaporated to dryness to obtain the cyazofamid hapten.

[0037] The present invention also provides a cyazofamid antigen, which is obtained by coupling a carrier protein with the cyazofamid hapten prepared by the method described above or the preparation method described above.

[0038] In this invention, the carrier protein is ovalbumin; when the carrier protein is ovalbumin, the structural formula of the cyazofamid antigen is shown in Formula II:

[0039]

[0040] In a specific embodiment of the present invention, the cyproconazole antigen with the structure shown in Formula II is a cyproconazole coated antigen.

[0041] In this invention, the carrier protein is bovine serum albumin; when the carrier protein is bovine serum albumin, the structural formula of the cyazofamid antigen is shown in Formula III:

[0042]

[0043] In a specific embodiment of the present invention, the cyproconazole antigen with the structure shown in Formula III is a cyproconazole immunogen.

[0044] The present invention also provides a method for preparing the cyproconazole antigen described in the above scheme, comprising the following steps:

[0045] The hapten, coupling agent, and polar solvent described in the above scheme are mixed and activated to obtain an activated hapten solution.

[0046] The activated hapten solution and the carrier protein buffer solution were mixed and coupled to obtain the cyazofamid antigen.

[0047] This invention involves activating a mixture of the cyproconazole hapten, coupling agent, and polar solvent described in the above-mentioned scheme to obtain an activated hapten solution. In this invention, the polar solvent is preferably dioxane or dimethyl sulfoxide, and the preferred ratio of the cyproconazole hapten to the polar solution is 10 mg: 100 μL. The coupling agent is preferably dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), or isobutyl chloroformate. When the coupling agent is isobutyl chloroformate, tripropylamine is preferably added during the activation process, whereby tripropylamine acts as an acid-binding agent. The activation temperature is preferably 25–27°C, more preferably 25°C, and the activation time is preferably 12–24 h.

[0048] In a specific embodiment of the present invention, when preparing the cyazofamid coated antigen (Formula II), the coupling agent preferably used isobutyl chloroformate (with tripropylamine added simultaneously), and the organic solvent preferably is dioxane; the preferred ratio of tripropylamine to cyazofamid hapten is 40 μL:10 mg; the preferred molar ratio of isobutyl chloroformate to cyazofamid hapten is (1-1.2):1. In a specific embodiment of the present invention, it is preferred to dissolve the cyazofamid hapten in dioxane, then add tripropylamine, and then add the dioxane solution of isobutyl chloroformate under ice bath conditions. After reacting for 0.5-1 h, the mixture is transferred to room temperature for activation. After activation, no further treatment is required to obtain the activated hapten solution.

[0049] In this invention, when preparing the cyazofamid immunogen (Formula III), the activator preferably used is DCC and NHS, and the organic solvent preferably used is dimethyl sulfoxide; the molar ratio of DCC to cyazofamid hapten is preferably (2-2.5):1, and the molar ratio of NHS to cyazofamid hapten is preferably (1-1.2):1; in a specific embodiment of this invention, it is preferable to first dissolve the cyazofamid hapten in dimethyl sulfoxide, and then add NHS and DCC for activation. After activation, it is preferable to filter the obtained activated product to remove dicyclohexylurea, and the resulting clear and transparent filtrate is the activated hapten solution.

[0050] After obtaining the activated hapten solution, the present invention mixes the activated hapten solution with a carrier protein buffer solution for a coupling reaction to obtain the cyazofamid antigen. In the present invention, when preparing the cyazofamid coated antigen, the preferred carrier protein is OVA, and when preparing the cyazofamid immunogen, the preferred carrier protein is BSA; the molar ratio of the carrier protein to the cyazofamid hapten is preferably (45-50):1; the carrier protein buffer solution is preferably obtained by dissolving the carrier protein in BB buffer solution, and the concentration of the BB buffer solution is preferably 0.02 mol / L; the present invention preferably adds the activated hapten solution dropwise to the carrier protein buffer solution, and after the addition is complete, the reaction is carried out at room temperature.

[0051] In this invention, the temperature of the coupling reaction is preferably 25-27°C, more preferably 25°C, and the time of the coupling reaction is preferably 2-3 hours.

[0052] In this invention, the synthesis process of the cyproconazole-coated antigen (Formula II) is as shown in Formula B:

[0053]

[0054] In this invention, the synthesis process of the cyazofamid immunogen (Formula III) is as shown in Formula C:

[0055]

[0056] After the coupling reaction is completed, the present invention preferably centrifuges the obtained reaction solution, aspirates the supernatant, and dialyzes the supernatant to obtain the cyazofamid antigen. In the present invention, the molecular weight cutoff of the dialysis bag is preferably 8000-14000, and the dialysis bag is preferably treated with EDTA solution and washed with distilled water before use; the concentration of the EDTA solution is preferably 0.2 mol / L; when preparing the cyazofamid coated antigen, the dialysis solution is preferably PBS buffer, and the concentration of the PBS buffer is preferably 0.02 mol / L; when preparing the cyazofamid immunogen, the dialysis solution is preferably PB buffer, and the concentration of the PB buffer is preferably 0.02 mol / L; the dialysis time is preferably 3 days, with the dialysis solution changed twice a day. After dialysis, the solution in the dialysis bag is preferably precipitated, aliquoted, and frozen for later use.

[0057] This invention also provides a cyazofamid antibody, obtained by immunizing a host animal with the cyazofamid antigen described in the above scheme; the host animal is preferably a mouse; this invention does not have special requirements for the immunization method, and any method well known to those skilled in the art can be used; in a specific embodiment of this invention, it is preferred to use an incomplete Freund's adjuvant to emulsify the cyazofamid immunogen dilution before immunizing the host animal; the diluent used for the cyazofamid immunogen dilution is preferably a sterilized 1% sodium chloride solution.

[0058] In a specific embodiment of the present invention, the preferred process for preparing cyazofamid antibody is as follows:

[0059] The host animals were immunized with the emulsified cyazofamid immunogen to obtain immunized animals. The serum of the immunized animals was tested using the ELISA method. Once the serum parameters met the requirements, monoclonal antibodies were prepared according to the monoclonal antibody preparation method.

[0060] Spleen cells were extracted from the immunized animal and fused with myeloma cells of the same lineage to obtain fused cells;

[0061] The fused cells were screened for hybridomas.

[0062] The limiting dilution method was used to culture hybridoma cells for clonalization. Immunological methods were used to screen for positive hybridoma cells that could produce the required monoclonal antibodies, and then the cells were cloned and expanded.

[0063] The present invention also provides a test strip or kit for detecting cyazofamid, containing the cyazofamid antibody described in the above-described scheme.

[0064] This invention also provides the application of the cyproconazole antibody or the test strip or kit described above in the detection of cyproconazole; the preferred method for detecting cyproconazole is the ELLSA kit method or the colloidal gold method. Immunizing mice with the cyproconazole antigen of this invention yields antibodies with high titers and high sensitivity; applying these antibodies to the detection of cyproconazole can improve the accuracy and sensitivity of the detection method.

[0065] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0066] Example 1

[0067] Synthesis of cyazofamid hapten:

[0068] 6.37 g of anhydrous aluminum trichloride was dissolved in 100 mL of dichloromethane, and 2.28 g of succinic anhydride was added. The reaction was allowed to proceed for 30 min, followed by the addition of 5.52 g of cyproconazole. The mixture was then heated under reflux in an oil bath for 4 h. After the reaction was complete, the reaction solution was cooled to room temperature and poured into ice. The mixture was stirred thoroughly for 10 min, and the lower layer was separated using a separatory funnel. The aqueous layer was extracted once with dichloromethane. The organic phases were combined, evaporated to dryness, and the mixture was loaded onto a column using a dry mixing method. The product was eluented with ethyl acetate:petroleum ether (1:2) and the solvent was evaporated to dryness. This yielded the cyproconazole hapten, with a purity of 96.1% as determined by high-performance liquid chromatography (HPLC). Figure 1 The obtained 1H NMR spectrum of the cyazofamid hapten is shown below. Figure 1 It can be determined that the product has the target structure.

[0069] Synthesis of the cyproconazole coating agent:

[0070] Dissolve 20 mg of cyazofamid hapten in 200 μL of 1,4-dioxane, add 80 μL of tripropylamine, and add 200 μL of 1,4-dioxane solution containing isobutyl chloroformate (containing 8.15 mg of isobutyl chloroformate) under ice bath conditions. React for 1 h, then allow to return to room temperature and react overnight to obtain an activated hapten solution. Weigh 30 mg of OVA and dissolve it in 1.5 mL of 0.02 mol / L BB buffer. After clarification, add the prepared activated hapten solution dropwise. After the addition is complete, react at room temperature for 2 h. Then centrifuge the reaction solution, collect the supernatant, and dialyze it in a dialysis bag for 3 days using 0.02 mol / L PBS buffer. Change the dialysate twice a day. After dialysis, aspirate the solution from the dialysis bag, aliquot it, and freeze it for later use.

[0071] Synthesis of cyazofamid immunogen:

[0072] Dissolve 20 mg of hapten in 200 μL of DMSO, add 15.9 mg of NHS and 17 mg of DCC, and react overnight at 25°C. The next day, filter the reaction solution to remove dicyclohexylurea, obtaining a clear and transparent liquid, which is the activated hapten solution. Weigh 40 mg of BSA and dissolve it in 3 mL of 0.02 mol / L PB buffer. After clarification, add the prepared activated hapten solution dropwise. After the addition is complete, react at room temperature for 2 hours. Then centrifuge the reaction solution, collect the supernatant, and add it to a dialysis bag for dialysis for 3 days. The dialysis solution is 0.02 mol / L PB buffer. Change the dialysis solution twice a day. After dialysis, aspirate the solution from the dialysis bag, aliquot it, and freeze it for later use.

[0073] Example 2

[0074] Hapten synthesis:

[0075] Dissolve 7g of anhydrous aluminum trichloride in 100mL of dichloromethane, add 2.52g of succinic anhydride, react for 30min, add 6.07g of cyazofamid, and then heat in an oil bath under reflux for 4h. After the reaction is complete, cool the reaction solution to room temperature, then pour the reaction solution into ice, stir thoroughly for 10min, separate the lower layer solution using a separatory funnel, extract the aqueous layer once with dichloromethane, combine the organic phases, evaporate to dryness, dry-mix the sample and load it onto a column, and wash out the product with ethyl acetate:petroleum ether = 1:2 solvent. Evaporate the solvent to dryness to obtain cyazofamid hapten, and determine its purity to be 97.5% by high performance liquid chromatography.

[0076] Synthesis of the cyproconazole coating agent:

[0077] Dissolve 30 mg of cyazofamid hapten in 300 μL of 1,4-dioxane, add 120 μL of tripropylamine, and add 200 μL of 1,4-dioxane solution containing isobutyl chloroformate (12.2 mg chloroformate) under ice bath conditions. React for 1 h, then allow to react overnight at room temperature to obtain an activated hapten solution. Weigh 70.6 mg of OVA and dissolve it in 1.5 mL of 0.02 mol / L BB buffer. After clarification, add the prepared activated hapten solution dropwise. After the addition is complete, react at room temperature for 2 h. Then centrifuge the reaction solution, collect the supernatant, and dialyze it in a dialysis bag for 3 days using PBS as the dialysate. Change the dialysate twice a day. After dialysis, aspirate the solution from the dialysis bag, aliquot it, and freeze it for later use.

[0078] Synthesis of cyazofamid immunogen:

[0079] Dissolve 30 mg of cyazofamid hapten in 300 mL of DMSO, add 10.3 mg of NHS and 30.8 mg of DCC, and react overnight at 25 °C. The next day, filter the reaction solution to remove dicyclohexylurea, and obtain a clear and transparent liquid, which is the activated hapten solution. Weigh 110 mg of BSA and dissolve it in 3 mL of 0.02 mol / L PB buffer. After clarification, add the prepared activated hapten solution dropwise. After the addition is complete, react at room temperature for 2 hours. Then centrifuge the reaction solution, collect the supernatant, and add it to a dialysis bag for dialysis for 3 days. The dialysis solution is 0.02 mol / L PB buffer. Change the dialysis solution twice a day. After dialysis, aspirate the solution from the dialysis bag, aliquot it, and freeze it for later use.

[0080] Example 3

[0081] Synthesis of cyazofamid hapten:

[0082] Dissolve 6.87 g of anhydrous aluminum trichloride in 100 mL of dichloromethane, add 2.47 g of succinic anhydride, react for 30 min, add 5.96 g of cyazofamid, and then heat in an oil bath under reflux for 4 h. After the reaction is complete, cool the reaction solution to room temperature, then pour the reaction solution into ice, stir thoroughly for 10 min, separate the lower layer solution using a separatory funnel, extract the aqueous layer once with dichloromethane, combine the organic phases, evaporate to dryness, dry-mix the sample and load it onto a column, and wash out the product with ethyl acetate:petroleum ether = 1:2 solvent. Evaporate the solvent to dryness to obtain cyazofamid hapten, and determine its purity to be 95% by high performance liquid chromatography.

[0083] Synthesis of the cyproconazole coating agent:

[0084] Dissolve 15 mg of hapten in 150 μL of 1,4-dioxane, add 90 μL of tripropylamine, and add 200 μL of 1,4-dioxane solution containing 6.1 mg of isobutyl chloroformate under ice bath conditions. React for 1 h, then allow to react overnight at room temperature to obtain an activated hapten solution. Weigh 36 mg of OVA and dissolve it in 1.5 mL of 0.02 mol / L BB buffer. After clarification, add the prepared activated hapten solution dropwise. After the addition is complete, react at room temperature for 2 h. Then centrifuge the reaction solution, collect the supernatant, and dialyze it in a dialysis bag for 3 days using 0.02 mol / L PBS buffer. Change the dialysate twice a day. After dialysis, aspirate the solution from the dialysis bag, aliquot it, and freeze it for later use.

[0085] Synthesis of cyazofamid immunogen:

[0086] Dissolve 15 mg of hapten in 150 μL of DMSO, add 5.15 mg of NHS and 15.3 mg of DCC, and react overnight at 25 °C. Filter the reaction solution to remove dicyclohexylurea, obtaining a clear and transparent liquid, which is the activated hapten solution. Weigh 51.6 mg of BSA and dissolve it in 3 mL of 0.02 mol / L PB buffer. After clarification, add the prepared activated hapten solution dropwise. After the addition is complete, react at room temperature for 2 hours. Then centrifuge the reaction solution, collect the supernatant, and add it to a dialysis bag for dialysis for 3 days. The dialysis solution is 0.02 mol / L PB buffer. Change the dialysis solution twice a day. After dialysis, aspirate the solution from the dialysis bag, aliquot it, and freeze it for later use.

[0087] Test Example 1: ELISA Test

[0088] Immunize mice:

[0089] Fifteen purebred BALB / c mice, aged 4-6 weeks, were selected and labeled 1-5 with different colors. Six 2mL syringes were used; three syringes were used to draw 0.5mL of incomplete Freund's adjuvant, and the other three were used to draw 0.5mL of cyproconazole immunogen solution (diluted with sterile 1% sodium chloride solution) containing 50μg, 100μg, and 200μg of cyproconazole immunogen, respectively. The syringes were labeled, and the injection tips of one incomplete Freund's adjuvant syringe and one immunogen syringe were connected together with an infusion tubing (the length should be sufficient to connect the two syringe tips). The syringes were then pushed back and forth with both hands until complete emulsification (emulsification is indicated by a white, milky liquid inside; slight resistance can be felt during pushing; and it forms small droplets that do not spread when dropped into water). Emulsification should not be stopped until complete. After emulsification, the mice were injected subcutaneously (on the back and abdomen) at multiple sites, with approximately 0.2mL injected into each mouse. This method was repeated every two weeks, for a total of three cycles. Blood samples were then collected for testing.

[0090] Blood collection method: Grasp the mouse's scalp by the thumb, index, and middle fingers of the left hand, and fix the tail with the little and ring fingers; gently press the skin around the eye to be removed to make the eyeball congested and protrude; insert a glass capillary tube vertically into the protrusion of the mouse's eyeball, and gently rotate it left and right until blood flows into the capillary (about 2 / 3 of the way through the capillary); remove the capillary tube and blow the blood into a 0.5 mL centrifuge tube (store at low temperature); after blood collection, bake in a 37℃ oven for 30 minutes, then centrifuge in a low-temperature high-speed centrifuge (10,000 rpm) for 10 minutes to separate the serum for ELISA testing.

[0091] Monoclonal antibody preparation process:

[0092] Cell fusion

[0093] Mice were euthanized using carbon dioxide gas, and their spleens were aseptically removed and prepared as a suspension by pressing and grinding in a petri dish. The prepared syngeneic myeloma cells were then mixed with mouse spleen cells in a specific ratio, and polyethylene glycol (PEG), a fusion promoter, was added. Under the influence of PEG, various lymphocytes fused with myeloma cells to form hybridoma cells.

[0094] Selective culture

[0095] The purpose of selective culture is to screen for fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells, lacking hypoxanthine-guanine-phosphoribotransferase, cannot synthesize DNA via the salvage pathway and die. Unfused lymphocytes, although possessing hypoxanthine-guanine-phosphoribotransferase, cannot survive long-term in vitro and gradually die. Only fused hybridoma cells, having acquired hypoxanthine-guanine-phosphoribotransferase from spleen cells and possessing the unlimited proliferation characteristic of myeloma cells, can survive and proliferate in HAT medium.

[0096] Screening and cloning of hybridoma-positive clones

[0097] Only a small fraction of hybridoma cells grown in HAT medium secrete monoclonal antibodies specific to a given target; therefore, screening and cloning are essential. A limiting dilution method was used for the clonal culture of hybridoma cells. Sensitive, rapid, and specific immunological methods were employed to screen for positive hybridoma cells capable of producing the desired monoclonal antibodies, followed by clonal amplification. After comprehensive identification of the immunoglobulin type, subclass, specificity, affinity, epitope recognizing the antigen, and molecular weight of the secreted monoclonal antibodies, the cells were promptly cryopreserved.

[0098] Large-scale preparation of monoclonal antibodies

[0099] Balb / c mice were pretreated by intraperitoneal injection of 0.5 mL of liquid paraffin or norphyrane. One to two weeks later, hybridoma cells were inoculated into the peritoneum. The hybridoma cells proliferated within the mouse peritoneum, producing and secreting monoclonal antibodies. After approximately one to two weeks, abdominal distension was observed. Abdominal fluid was then aspirated using a syringe to obtain a large amount of monoclonal antibodies.

[0100] The reagent formulation used in the ELISA test is as follows:

[0101] Blocking solution (1L volume): 2.5g skim milk powder, 8g sodium chloride, 0.6g potassium dihydrogen phosphate, 5.8g disodium hydrogen phosphate, 50g sucrose, 0.9g potassium chloride, 0.5mL preservative, 50mL bovine serum, and the remainder is water.

[0102] Enzyme dilute (16L): Sodium chloride 128g, potassium dihydrogen phosphate 9.48g, disodium hydrogen phosphate 92.8g, glycerol 800mL, bovine serum 3200mL, preservative 12mL, and the remainder is water.

[0103] Diluted sample (4L): Sodium chloride 23.38g, potassium chloride 0.118g, potassium dihydrogen phosphate 3.48g, sodium dihydrogen phosphate 2.19g, disodium hydrogen phosphate 20.64g, Triton 40mL, preservative 1.5mL, balance water.

[0104] Substrate A solution (100 mL): 0.48 g citric acid, 0.05 g urea peroxide, 90 mL glacial acetic acid, and the remainder is water.

[0105] Substrate B solution (400 mL): 0.164 g citric acid, 0.2 g TMB hydrochloride, 4 mL NN, 14 mL methanol, 160 μ L 1 mol / L hydrochloric acid, and the remainder is water.

[0106] 20× Concentrated Washing Solution (40L): Sodium chloride 2560g, disodium hydrogen phosphate 928g, potassium dihydrogen phosphate 80g, potassium chloride 1.44g, preservative 12mL, Tween 20 800mL, balance is water.

[0107] The specific testing method is as follows:

[0108] (1) Antigen coating: Dilute the cyproconazole coating agent to 1K ratio with CB buffer, add 100 μL / well to the microplate, cover with the membrane and coat at 37℃ for 2 h.

[0109] (2) Blocking: After coating, remove the coating solution and wash it with 250 μL / well of washing working solution (20× concentrated washing solution diluted to 1×) or wash it once with a washing pot. Soak for 30 seconds, then remove the solution and pat dry with absorbent paper or a towel. Add 150 μL / well of blocking solution and place in a 37℃ constant temperature incubator for 2 hours. Remove the solution, remove the blocking solution, and pat dry for later use.

[0110] (3) Dilution of different standard concentrations: The 100 μg / mL of cyproconazole purchased directly was diluted with 0.02 mol / L PBS buffer to 0 ppb (directly PBS buffer), 10 ppb, 50 ppb and 500 ppb.

[0111] (4) Dilution of enzyme-labeled secondary antibody working solution: Dilute the purchased enzyme-labeled secondary antibody solution to a concentration of 1:1K with enzyme dilution buffer.

[0112] (5) Dilution of serum or monoclonal antibody working solution: Dilute serum or prepared monoclonal antibody with sample dilution buffer at concentrations of 1:1K, 1:5K, 1:10K, and 1:20K.

[0113] (6) Testing steps: The test shall be conducted according to the chessboard method. The specific operation steps are as follows:

[0114] 1) Add standard / sample: Add 50 μL of standard / sample to the corresponding microwell.

[0115] 2) Add serum or monoclonal antibody working solution: Add 50 μL of serum or monoclonal antibody working solution per well, gently shake to mix, cover with a cover plate and incubate at 37°C in the dark for 30 min.

[0116] 3) Washing and adding enzyme-labeled secondary antibody working solution: Carefully peel off the cover film, shake off the liquid in the wells, add 250 μL of washing working solution per well, wash thoroughly 4-5 times, with an interval of 10 seconds each time, pour off the washing solution in the wells, pat dry with absorbent paper (air bubbles that are not removed after patting can be punctured with an unused pipette tip), add 100 μL of enzyme-labeled secondary antibody working solution per well, cover with the cover film, and incubate at 37°C in the dark for 30 min.

[0117] 4) Washing the plate: Carefully peel off the cover film, shake off the liquid in the well, add 250μL of washing working solution per well, wash thoroughly 4-5 times, with an interval of 10 seconds each time, pour off the washing solution in the well, and pat dry with absorbent paper (air bubbles that are not removed after patting can be punctured with an unused pipette tip).

[0118] 5) Color development: Add 50 μL each of substrate solution A and solution B per well, gently shake to mix, cover with a cover plate and incubate at 37°C in the dark for 15 min.

[0119] 6) Measurement: Add 50 μL of stop solution per well, gently shake to mix, and set the microplate reader to 450 nm to measure the OD value of each well.

[0120] When the relevant technical parameters are met, the maximum dilution factor of the antibody is approximately 2.0, which corresponds to the antibody titer. A standard curve is plotted with the standard concentration on the x-axis and the corresponding absorbance value on the y-axis. The half-maximal inhibitory concentration (IC50) of cyproconazole is then calculated based on the standard curve. 50 value).

[0121] The test results are shown in Tables 1 to 5.

[0122] Table 1.1 Serum test data after immunization

[0123]

[0124] Table 2. Serum test data after immunization

[0125]

[0126] Table 3. Serum test data after immunization

[0127]

[0128] Table 4 Whole serum test data

[0129]

[0130] Table 5. Test data for preparing monoclonal antibodies

[0131]

[0132] As shown in Tables 1-3, the concentration of cyazofamid antibody in serum gradually increases with the number of immunizations. Table 4 shows that the serum concentration of cyazofamid antibody has essentially reached 5W, IC50. 50 The titer is less than 50 ppb; calculations based on the results in Table 5 show that the titer of the prepared monoclonal antibody is 5 × 10⁻⁶. 4 Half-inhibitory concentration (IC50) 50 The value is within 50 ppb.

[0133] Test Example 2 Colloidal Gold Test

[0134] The testing method is as follows:

[0135] (1) Plating: The coating antigen was diluted with 0.02 mol / L PB buffer (pH 7.2) at ratios of 1:10, 1:20, 1:50, and 1:100 to prepare the T-line coating solution. The secondary antibody was diluted with 0.02 mol / L PB + 10% BSA buffer (pH 7.2) at a ratio of 1:60 to prepare the C-line coating solution. Plating was performed at a rate of 1 μL / cm. After plating, the plates were baked at 37°C for 12 hours (using Shanghai Jieyi 30×6cm backing plates). 2PVC board and absorbent paper (Shanghai Jiening Spec30*20cm); NC film (Shanghai Jiening JN-140).

[0136] (2) Gold Standard Pad Processing Standard: Shanghai Jieyi ordinary glass fiber cut into 30×1cm pieces 2 For size, each strip was treated with 1.5 mL of 0.02 mol / L PB + 10% BSA buffer at pH 7.2 and dried at 37°C overnight (or more than 12 hours).

[0137] (3) Sample pad treatment standard: Shanghai Jiening SF-06 non-woven fabric is cut into 30*1.7cm pieces. 2 For size, each strip was treated with 2 mL of 0.02 mol / L PB + 0.5% Tween-20 + 1% sucrose buffer at pH 7.2 and dried at 37°C overnight (or more than 12 hours).

[0138] (4) Gold spraying: Dilute the serum or monoclonal antibody 1:10 with purified water and mix well. Take 1 mL of colloidal gold solution with 40 nm particles in 4 tubes respectively, add 30 μL of 0.02 mol / L K2CO3 to adjust the pH, mix well, and then add 10 μL, 20 μL, 30 μL and 40 μL of diluted serum or monoclonal antibody solution respectively. Mix well and let it react at room temperature for 30 min. Then add 25 μL of blocking solution (20% BSA aqueous solution) to each tube and let it react at room temperature for 20 min. Centrifuge at 10000 rpm at low temperature (2-8℃) for 10 min, remove the supernatant, and add 100 μL of reconstitution solution to the residue for reconstitution (reconstitution solution is 1 mL of supernatant after centrifugation + 50 μL of 40% sucrose aqueous solution mixed well). Spray gold at a rate of 3 μL / cm and bake at 37℃ for 2 h.

[0139] (5) Pretreatment method for dried tobacco leaf samples

[0140] 1) Before testing, dry tobacco leaf samples are dried and then ground into fine powder.

[0141] 2) Weigh 1 ± 0.05 g of sample into a 10 mL polystyrene centrifuge tube, add 5 mL of methanol, cover the tube, shake manually for 30 s, and let stand to separate the layers.

[0142] 3) Take 100 μL of sample supernatant and add it to a 2 mL polystyrene centrifuge tube. Add 1800 μL of diluent (pH 7.2, 0.02 mol / L PB), shake to mix, and the sample solution to be tested is obtained.

[0143] (6) Methods for testing dried tobacco leaf samples

[0144] 1) Take 70 μL of the sample solution to be tested or use a dropper to vertically drop 3 drops into the sample well.

[0145] 2) Timing begins when the liquid begins to flow. The reaction time is 10 minutes. The results are interpreted using analytical software, a colloidal gold reader, or visual inspection. This invention uses five dried tobacco leaf samples, designated 1#, 2#, 3#, 4#, and 5#. Azoxystrobin standard is added to each dried tobacco leaf sample, controlling the concentration of azoxystrobin in 1g of dried tobacco leaf to be 0ppb, 2500ppb, 5000ppb, and 10000ppb, respectively. The test results are shown in Table 6.

[0146] Table 6. Detection Limit Test Data for Monoclonal Antibody Preparation and Colloidal Gold Preparation from Dried Tobacco Leaves

[0147]

[0148] Take dried tobacco leaf sample #4 and conduct 5 parallel tests. The test results are shown in Table 7.

[0149] Table 7. Parallelism test data for the preparation of monoclonal antibodies and colloidal gold from dried tobacco leaves.

[0150]

[0151] The results in Table 6 show that the colloidal gold prepared using the monoclonal antibody of the present invention has a detection limit of about 2500 ng / g for cyazofamid in dried tobacco samples, and has high sensitivity. The data in Table 7 show that the detection method provided by the present invention has good parallelism.

[0152] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cyazofamid hapten, characterized in that, It has the structure shown in Equation I:

2. The method for preparing the cyazofamid hapten according to claim 1, characterized in that, Includes the following steps: The cyproconazole hapten was obtained by mixing cyproconazole, aluminum trichloride, succinic anhydride and an organic solvent and carrying out a Friedel-Crafts acylation reaction.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the cyproconazole, aluminum trichloride, and succinic anhydride is 1:(2-3):(1-1.2). The Friedel-Crafts acylation reaction is carried out at a temperature of 80–90 °C for 4–5 h.

4. A cyazofamid antigen, characterized in that, The hapten prepared by the method described in claim 1 or the method described in claim 2 or 3 is coupled with a carrier protein; the carrier protein is ovalbumin or bovine serum albumin.

5. The cyazofamid antigen according to claim 4, characterized in that, When the carrier protein is ovalbumin, the structural formula of the cyazofamid antigen is shown in Formula II:

6. The cyazofamid antigen according to claim 4, characterized in that, When the carrier protein is bovine serum albumin, the structural formula of the cyazofamid antigen is shown in Formula III:

7. The method for preparing the cyazofamid antigen according to any one of claims 4 to 6, characterized in that, Includes the following steps: The hapten, coupling agent, and polar solvent described in claim 1 are mixed and activated to obtain an activated hapten solution. The activated hapten solution and the carrier protein buffer solution were mixed and coupled to obtain the cyazofamid antigen.

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