Bifenthrin hapten, antigen, antibody, preparation method and application thereof

Abstract

The present invention relates to the field of immunoassay technology and provides a bifenthrin hapten, antigen, antibody, preparation method, and application thereof. The bifenthrin hapten provided by the present invention completely retains the molecular structure of bifenthrin. After coupling with a carrier protein, the immunogenicity of the resulting antigen is significantly improved, and the prepared antibody has stronger specificity and higher sensitivity. The results of the examples show that the bifenthrin hapten provided by the present invention is coupled with a carrier protein, and the resulting antigen, after injection into mice for immunization, produces an antibody titer of 1×10 4 , half inhibitory concentration (IC 50 ) is about 20ppb, and the minimum detection limit of colloidal gold for bifenthrin in dry tobacco leaf samples is about 1.25μg / g.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a bifenthrin hapten, antigen, antibody, and a preparation method and application thereof. Background Art

[0002] Bifenthrin is a pyrethroid insecticide and acaricide used to control crop pests, termites, wool moths, and pests in tea gardens. Bifenthrin can cause certain damage to the skin and mucous membranes, nervous system, digestive system, etc. through direct contact, inhalation through the respiratory tract, or ingestion through the digestive tract. At present, the commonly used detection methods for bifenthrin residues are mainly instrumental analysis methods, such as high performance liquid chromatography (HPLC), gas chromatography (GC), and chromatography-mass spectrometry (HPLC-MS-MS and GC-MS). The use of instruments to detect bifenthrin has the advantages of accuracy and sensitivity. However, instrument detection has the following disadvantages: (1) Sample processing requires derivatization, and the pretreatment procedure is cumbersome; (2) The instruments required for detection are expensive and the detection cost is high. The detection conditions are very harsh in some small and medium-sized cities and even rural areas; (3) Personnel engaged in detection need to undergo special training and have relevant professional level; (4) The maintenance cost of the instrument is high; (5) It is not suitable for rapid detection such as on-site sampling.

[0003] Enzyme-linked reaction (ELRA) adsorption assay (ELISA) involves adsorbing a known antigen or antibody onto the surface of a solid support, allowing the enzyme-labeled antigen-antibody reaction to occur on the solid surface. This method is a biochemical analytical procedure based on the highly selective reaction between antibodies and antigens or haptens. ELISA offers the advantages of simplicity, rapidity, and low cost, making it particularly suitable for testing large quantities of samples and has seen rapid growth in recent years.

[0004] The enzyme-linked reaction adsorption assay for cypermethrin has been reported, but the immunogenicity of the antigens used in the current enzyme-linked reaction adsorption assay is generally not strong, resulting in poor antibody specificity and sensitivity, which in turn affects the accuracy of the test results. Summary of the Invention

[0005] In view of this, the present invention provides a bifenthrin hapten, antigen, antibody, preparation method, and application thereof. The antigen obtained by coupling the bifenthrin hapten provided by the present invention with a carrier protein has strong immunogenicity. After immunizing a host animal with the hapten, the antibody obtained has strong specificity and high sensitivity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a bifenthrin hapten having a structure shown in Formula I:

[0008]

[0009] The present invention provides a method for preparing the bifenthrin hapten described in the above scheme, comprising the following steps:

[0010] p-Aminophenylboronic acid, 3-bromo-2-methylbenzyl alcohol, an alkaline reagent, a palladium catalyst and an organic solvent are mixed to perform a Suzuki reaction to obtain a first intermediate; the structure of the first intermediate is shown in Formula A;

[0011]

[0012] The first intermediate, pyridine, trifluorochlorochrysanthemoyl chloride and an organic solvent are mixed to perform a first coupling reaction to obtain a second intermediate, the structure of which is shown in Formula B;

[0013]

[0014] The second intermediate, pyridine, succinic anhydride and 4-dimethylaminopyridine are mixed to perform a second coupling reaction to obtain the bifenthrin hapten having the structure shown in Formula I.

[0015] Preferably, the molar ratio of p-aminophenylboronic acid to 3-bromo-2-methylbenzyl alcohol is 1:(1-1.2); the molar ratio of p-aminophenylboronic acid to alkaline reagent is 1:(0.5-1); the molar ratio of p-aminophenylboronic acid to palladium catalyst is 1:(0.5-1);

[0016] The molar ratio of the first intermediate to trifluorochlorochrysanthemoyl chloride is 1:(1-1.2);

[0017] The molar ratio of the second intermediate to succinic anhydride is 1:(1-1.2).

[0018] Preferably, the temperature of the Suzuki reaction is 78-80°C and the time is 16-18 hours;

[0019] The first coupling comprises a first stage and a second stage, wherein the reaction temperature of the first stage is 0-5°C and the reaction time is 2-2.5h; the reaction temperature of the second stage is room temperature and the reaction time is preferably 4-5h;

[0020] The temperature of the second coupling reaction is 120-125° C., and the time is 12-24 hours.

[0021] The present invention also provides a bifenthrin antigen, which is obtained by coupling the bifenthrin hapten described in the above scheme or the bifenthrin hapten prepared by the preparation method described in the above scheme with a carrier protein.

[0022] Preferably, the carrier protein is ovalbumin or bovine serum albumin; when the carrier protein is ovalbumin, the structural formula of the bifenthrin antigen is as shown in Formula II:

[0023]

[0024] When the carrier protein is bovine serum albumin, the structural formula of the bifenthrin antigen is shown in Formula III:

[0025]

[0026] The present invention also provides a method for preparing the bifenthrin antigen described in the above scheme, comprising the following steps:

[0027] The bifenthrin hapten, coupling agent and polar solvent described in the above scheme are mixed and activated to obtain an activated hapten solution;

[0028] The activated hapten solution and the buffer solution of the carrier protein are mixed to carry out a coupling reaction to obtain the bifenthrin antigen.

[0029] The present invention also provides a bifenthrin antibody obtained by immunizing a host animal with the bifenthrin antigen described in the above scheme.

[0030] The present invention also provides a test paper or a kit for detecting bifenthrin, which contains the bifenthrin antibody described in the above scheme.

[0031] The present invention also provides use of the bifenthrin antibody described in the above scheme or the test paper or kit described in the above scheme in detecting bifenthrin.

[0032] The present invention provides a bifenthrin hapten, the structural formula of which is shown in Formula I. Reported molecular modification schemes for bifenthrin typically involve modification at the chrysanthemic acid site or derivatization with 2,4-diphenylmethanol, which fail to fully retain the entire molecular structure of bifenthrin, resulting in weak immunogenicity of the antigen. The present invention, however, introduces a carboxyl group from the terminal benzene ring of bifenthrin for coupling with a carrier protein to prepare an antigen. The bifenthrin hapten provided by the present invention fully retains the molecular structure of bifenthrin. After coupling with the carrier protein, the immunogenicity of the resulting antigen is significantly improved, and the prepared antibody has stronger specificity and higher sensitivity.

[0033] The present invention also provides a method for preparing the bifenthrin hapten described in the above scheme. The method uses p-aminophenylboronic acid and 3-bromo-2-methylbenzyl alcohol as raw materials, synthesizes a first intermediate (an amino derivative of 2-biphenylmethanol) via a Suzuki reaction, then couples it with trifluorochloric acid to obtain a second intermediate, and finally couples it with succinic anhydride to obtain the bifenthrin hapten. The preparation method provided by the present invention has simple steps, is easy to operate, and produces a high-purity product.

[0034] The present invention also provides a bifenthrin antigen, which is obtained by coupling the bifenthrin hapten described in the above scheme with a carrier protein. After the bifenthrin hapten provided by the present invention is coupled with the carrier protein, the resulting antigen has strong immunogenicity. After injection into mice for immunization, the antibody titer produced is 1×10 4 , half inhibitory concentration (IC 50 ) is about 50ppb, and the minimum detection limit of colloidal gold for bifenthrin in dry tobacco leaf samples is about 1.25μg / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the bifenthrin hapten prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] The present invention provides a bifenthrin hapten having a structure shown in Formula I:

[0037]

[0038] The present invention also provides a method for preparing the bifenthrin hapten described in the above scheme, comprising the following steps:

[0039] p-Aminophenylboronic acid, 3-bromo-2-methylbenzyl alcohol, an alkaline reagent, a palladium catalyst and an organic solvent are mixed to perform a Suzuki reaction to obtain a first intermediate; the structure of the first intermediate is shown in Formula A;

[0040]

[0041] The first intermediate, pyridine, trifluorochlorochrysanthemoyl chloride and an organic solvent are mixed to perform a first coupling reaction to obtain a second intermediate, the structure of which is shown in Formula B;

[0042]

[0043] The second intermediate, pyridine, succinic anhydride and 4-dimethylaminopyridine are mixed to perform a second coupling reaction to obtain the bifenthrin hapten having the structure shown in Formula I.

[0044] In the present invention, the synthesis route of the bifenthrin hapten is shown in Chem1:

[0045]

[0046] The synthesis method of bifenthrin hapten is described in detail below with reference to Chem1.

[0047] The present invention mixes p-aminophenylboronic acid, 3-bromo-2-methylbenzyl alcohol, an alkaline reagent, a palladium catalyst and an organic solvent to carry out a Suzuki reaction to obtain a first intermediate; the structure of the first intermediate is shown in formula A. In the present invention, the molar ratio of p-aminophenylboronic acid to 3-bromo-2-methylbenzyl alcohol is preferably 1:(1-1.2), more preferably 1:(1-1.1); the molar ratio of p-aminophenylboronic acid to the alkaline agent is preferably 1:(0.5-1), more preferably 1:(0.6-0.8); the molar ratio of p-aminophenylboronic acid to the palladium catalyst is preferably 1:(0.5-1), more preferably 1:(0.6-0.8); the alkaline agent is preferably potassium carbonate, and the potassium carbonate is preferably used in the form of an aqueous potassium carbonate solution, and the concentration of the potassium carbonate aqueous solution is preferably 2 mol / L; the palladium catalyst is preferably tetrakistriphenylphosphine palladium; the organic solvent for the Suzuki reaction is preferably anhydrous ethanol, methanol, acetonitrile, DMF, DMSO, acetone or dichloromethane, more preferably anhydrous ethanol.

[0048] In the present invention, the temperature of the Suzuki reaction is preferably 0-5°C, and the time is preferably 16-18 hours; the Suzuki reaction is preferably carried out under nitrogen protection; in a specific embodiment of the present invention, the Suzuki reaction is preferably carried out under reflux conditions, and TLC is preferably used for monitoring during the reaction.

[0049] In a specific embodiment of the present invention, it is preferred to first dissolve p-aminophenylboronic acid in an organic solvent, then add 3-bromo-2-methylbenzyl alcohol, an alkaline reagent and a palladium catalyst, seal the reaction system, replace the air in the system with nitrogen, and then heat to carry out the Suzuki reaction.

[0050] After the Suzuki reaction is completed, the present invention preferably cools the resulting reaction solution to room temperature, removes the organic solvent by distillation under reduced pressure, mixes the residue with water, adjusts the pH value of the resulting mixed solution to 6.6-7, and then extracts with ethyl acetate to obtain an organic phase; the organic phase is dried over anhydrous sodium sulfate and then distilled under reduced pressure to remove the solvent to obtain a first intermediate; the reagent used to adjust the pH value of the mixed solution is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 2 mol / L.

[0051] After obtaining the first intermediate, the present invention mixes the first intermediate, pyridine, trifluorochlorochrysanthemoyl chloride, and an organic solvent to perform a first coupling reaction to obtain a second intermediate, the structure of which is shown in Formula B. In the present invention, the molar ratio of the first intermediate to pyridine is preferably 1:1, and the pyridine is an acid-binding agent; the molar ratio of the first intermediate to trifluorochlorochrysanthemoyl chloride is preferably 1:(1-1.2); and the organic solvent for the first coupling reaction is preferably methyl chloride, more preferably dichloromethane.

[0052] In the present invention, the first coupling reaction preferably includes a first stage and a second stage carried out sequentially. The reaction temperature of the first stage is preferably 0-5°C, and the reaction time is preferably 2-2.5 hours. In a specific embodiment of the present invention, the first stage is preferably carried out under ice bath conditions; the second stage is preferably carried out at room temperature, and the reaction time is preferably 4-5 hours. The present invention preferably uses TLC to monitor the reaction progress.

[0053] In a specific embodiment of the present invention, the first intermediate is preferably first dissolved in an organic solvent, the resulting solution is cooled to 0-5°C, the air in the reaction apparatus is then replaced with nitrogen, and then the organic solution of trifluorochlorochrysanthemoyl chloride is added dropwise under nitrogen protection. After the addition is complete, the first and second stage reactions are carried out in sequence.

[0054] After the first coupling reaction is completed, the present invention preferably performs vacuum distillation on the obtained reaction solution to remove the organic solvent and pyridine in the system, and the residue is dissolved in ethyl acetate and then subjected to column chromatography to obtain a second intermediate; the target component in the purified product obtained by column chromatography separation is the third ultraviolet absorption point from top to bottom. The present invention preferably uses a first developing agent to flush out the first two components, and then uses a second developing agent to flush out the target component, and the eluate of the obtained target component is vacuum distilled to obtain the second intermediate; the first developing agent is preferably a solvent obtained by mixing ethyl acetate and petroleum ether in a volume ratio of 1:15, and the second developing agent is a solvent obtained by mixing ethyl acetate and petroleum ether in a volume ratio of 1:5.

[0055] After obtaining the second intermediate, the present invention mixes the second intermediate, pyridine, succinic anhydride, and 4-dimethylaminopyridine (DMAP) to perform a second coupling reaction to obtain a bifenthrin hapten having the structure represented by Formula I. In the present invention, the molar ratio of the second intermediate to succinic anhydride is 1:1-1.2; the molar ratio of the second intermediate to 4-dimethylaminopyridine is preferably 1:0.1-0.12; the 4-dimethylaminopyridine serves as a catalyst; the pyridine serves as a solvent to dissolve the second intermediate and also as an acid-binding agent; the molar concentration of the second intermediate in the pyridine is 3-3.5 mol / l; and the pyridine is preferably anhydrous pyridine.

[0056] In the present invention, the temperature of the second coupling reaction is preferably 120-125° C., and the time is preferably 12-24 h. In a specific embodiment of the present invention, the second coupling reaction is preferably carried out under reflux conditions.

[0057] After the second coupling reaction, the present invention preferably distills the obtained reaction solution under reduced pressure, dissolves the obtained residue with water, adjusts the pH value of the obtained solution to 6.5-7.5, extracts with ethyl acetate, combines the organic phases, and dries with anhydrous sodium sulfate. The ethyl acetate is removed by distillation under reduced pressure, and the residue is dissolved with ethyl acetate. Silica gel of the same mass as the residue is added, and the ethyl acetate is removed by distillation under reduced pressure. The product is adsorbed on the silica gel in the form of powder, and the powder is loaded into a column. The length of the blank silica gel in the column is about two-thirds of the effective length of the column. The product is then purified by silica gel column chromatography to obtain the bifenthrin hapten. The purified product obtained by the silica gel column chromatography is preferably firstly treated with a first developing solvent to remove impurities above the product point, and then the target product is collected with a second developing solvent. The first developing solvent is preferably a solvent obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 10:1, and the second developing solvent is preferably a solvent obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0058] The present invention also provides a bifenthrin antigen, which is obtained by coupling the bifenthrin hapten described in the above scheme or the bifenthrin hapten prepared by the preparation method described in the above scheme with a carrier protein.

[0059] Preferably, the carrier protein is ovalbumin or bovine serum albumin; when the carrier protein is ovalbumin, the structural formula of the bifenthrin antigen is as shown in Formula II:

[0060]

[0061] When the carrier protein is bovine serum albumin, the structural formula of the bifenthrin antigen is shown in Formula III:

[0062]

[0063] In a specific embodiment of the present invention, the bifenthrin antigen represented by formula II is a bifenthrin coating antigen, and the bifenthrin antigen represented by formula III is a biphenyl polyester immunogen.

[0064] The present invention also provides a method for preparing the bifenthrin antigen described in the above scheme, comprising the following steps:

[0065] The bifenthrin hapten, coupling agent and polar solvent described in the above scheme are mixed and activated to obtain an activated hapten solution;

[0066] The activated hapten solution and the buffer solution of the carrier protein are mixed to carry out a coupling reaction to obtain the bifenthrin antigen.

[0067] The present invention activates the bifenthrin hapten described in the above scheme by mixing a coupling agent and a polar solvent to obtain an activated hapten solution. In the present invention, the polar solvent is preferably DMF, and the amount ratio of the bifenthrin hapten to the polar solution is preferably 10 mg:30-50 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, and the tripropylamine acts as an acid-binding agent. The activation temperature is preferably room temperature, and the activation time is preferably 12-24 hours.

[0068] In the present invention, when preparing the bifenthrin coating (Formula II), the coupling agents used are preferably DCC and NHS; the molar ratio of DCC to bifenthrin hapten is preferably (1.5-2):1, and the molar ratio of NHS to bifenthrin hapten is preferably (1-1.2):1; in a specific embodiment of the present invention, the bifenthrin hapten is preferably first dissolved in DMF, and then NHS and DCC are added for activation. After the activation is completed, the obtained activated product is preferably filtered to remove dicyclohexylurea, and the obtained clear and transparent filtrate is the activated hapten solution.

[0069] In the present invention, when preparing the bifenthrin immunogen (Formula III), the coupling agent used is preferably isobutyl chloroformate (tripropylamine is added simultaneously); the dosage ratio of tripropylamine to bifenthrin hapten is preferably 150-200 μL:1 mmol; and the molar ratio of isobutyl chloroformate to bifenthrin hapten is preferably (1-1.2):1. In a specific embodiment of the present invention, the bifenthrin hapten is preferably dissolved in DMF, followed by the addition of tripropylamine, and then the isobutyl chloroformate solution in DMF is added in an ice bath. After reacting for 1 hour, the mixture is transferred to room temperature for activation.

[0070] After obtaining the activated hapten solution, the present invention mixes the activated hapten solution with a buffer solution of a carrier protein to perform a coupling reaction to obtain the bifenthrin antigen. In the present invention, when preparing the bifenthrin coating source, the carrier protein used is preferably OVA, and when preparing the bifenthrin immunogen, the carrier protein used is preferably BSA; the molar ratio of the carrier protein to the bifenthrin hapten is preferably (40-50):1; the buffer solution of the carrier protein is preferably obtained by dissolving the carrier protein in a CB buffer solution, and the concentration of the CB buffer solution is preferably 0.02 mol / L; the present invention preferably adds the activated hapten solution dropwise to the buffer solution of the carrier protein, and the reaction is carried out at room temperature after the addition is complete.

[0071] In the present invention, the coupling reaction temperature is preferably room temperature, and the time is preferably 2 to 2.5 hours.

[0072] In the present invention, the synthesis process of the bifenthrin coating (Formula II) is shown in Chem2:

[0073]

[0074] In the present invention, the synthesis process of the bifenthrin immunogen (Formula III) is shown in Chem3:

[0075]

[0076] After the coupling reaction is completed, the present invention preferably centrifuges the resulting reaction solution, removes the supernatant, and dialyzes the supernatant to obtain the bifenthrin antigen. In the present invention, the molecular weight cutoff of the dialysis bag is preferably 8000 to 14000, and the dialysis bag is preferably treated with an EDTA solution and rinsed with distilled water before use; the concentration of the EDTA solution is preferably 0.2 mol / L; the dialysate for dialysis is preferably PBS buffer, and the concentration of the PBS buffer is preferably 0.02 mol / L; the dialysis time is preferably 3 days, with the dialysate replaced twice a day. After the dialysis is completed, the solution in the dialysis bag is preferably precipitated and aliquoted and frozen for later use.

[0077] The present invention also provides a bifenthrin antibody, which is obtained by immunizing a host animal with the bifenthrin antigen described in the above scheme; the host animal is preferably a mouse; the present invention has no special requirements for the immunization method, and methods well known to those skilled in the art can be used; in a specific embodiment of the present invention, it is preferred to use incomplete Freund's adjuvant to emulsify the bifenthrin immunogen dilution before immunizing the host animal; the diluent used for the bifenthrin immunogen dilution is preferably a sterilized 1% sodium chloride solution.

[0078] In a specific embodiment of the present invention, the process for preparing the bifenthrin antibody is preferably as follows:

[0079] Immunizing host animals with the emulsified bifenthrin immunogen to obtain immune animals, performing serum tests on the immune animals using the ELISA method, and preparing monoclonal antibodies according to the monoclonal antibody preparation method when the serum parameters meet the requirements;

[0080] extracting spleen cells from the immunized animal and fusing them with syngeneic myeloma cells to obtain fused cells;

[0081] performing hybridoma screening on the fusion cells;

[0082] The limiting dilution method is used to clone and culture hybridoma cells, and immunological methods are used to screen positive hybridoma cells that can produce the required monoclonal antibodies and then clone and expand them.

[0083] The present invention also provides a test paper or a kit for detecting bifenthrin, which contains the bifenthrin antibody described in the above scheme.

[0084] The present invention also provides the use of the bifenthrin antibody, test paper, or kit described in the above embodiment for detecting bifenthrin; the method for detecting bifenthrin is preferably the ELLSA kit method or the colloidal gold method. Immunizing mice with the bifenthrin antigen of the present invention produces antibodies with high titer and sensitivity. Application of these antibodies in the detection of bifenthrin can improve the accuracy and sensitivity of the detection method.

[0085] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0086] Example 1

[0087] Synthesis of bifenthrin hapten: 4.75 g of p-aminophenylboronic acid was dissolved in anhydrous ethanol, and 7.67 g of 3-bromo-2-methylbenzyl alcohol, 34.7 mL of 2 mol / L potassium carbonate solution, and 481 mg of tetrakistriphenylphosphine palladium were added. The reaction system was sealed and the air in the system was replaced with nitrogen. This was repeated three times. The reaction was heated under reflux in an oil bath for 16 h. During this period, the reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to room temperature and the ethanol was removed by vacuum distillation. Water was added to the residue and the pH was adjusted to 7. The residue was then fully extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain 5.52 g of a light yellow oily liquid, which was the first intermediate (Formula A).

[0088] 5.52 g of the first intermediate was dissolved in dichloromethane, first cooled to 0°C in an ice bath, and then the air in the bottle was replaced with nitrogen. Under nitrogen protection, a dichloromethane solution of trifluorochlorochrysanthemoyl chloride (7.43 g) and 2.04 g of anhydrous pyridine were slowly added dropwise. After the addition, the ice bath was kept for 2 hours, and then the ice bath was removed and the reaction was continued for 4 hours. The reaction was detected by TLC. After the reaction was complete, the dichloromethane and pyridine were removed by vacuum distillation, and the residue was dissolved in ethyl acetate and dry-loaded on the column. The target component was the third ultraviolet absorption point from top to bottom. EA:PE = 1:15 was first used to flush out the first two components. After the flushed developing agent had no ultraviolet absorption, it was changed to EA:PE = 1:5 to flush out the third component. 8.83 g of light yellow solid was obtained by vacuum distillation, which was the second intermediate (Formula B).

[0089] 4.26 g of the second intermediate was dissolved in anhydrous pyridine, 1.07 g of succinic anhydride and 0.769 g of DMAP were added, and the reaction was refluxed at 120° C. overnight. The next day, the reaction was detected and the raw materials were basically reacted completely. The reaction solution was distilled under reduced pressure to fully remove pyridine, the residue was dissolved with water, the pH was adjusted to about 7, and extracted with ethyl acetate until no major product point was found. The product was purified by silica gel column chromatography with a developing solvent of PE:EA=10:1 to remove trace impurities above the product point. The developing solvent was then changed to PE:EA=3:1, and the main ultraviolet point was collected to obtain the bifenthrin hapten. The purity was determined to be 92.3% by high performance liquid chromatography. Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the obtained bifenthrin hapten, according to Figure 1 It can be confirmed that the product has the target structure.

[0090] Synthesis of Bifenthrin Coating Source:

[0091] 12 mg of bifenthrin hapten was dissolved in 40 μL DMF, 4.13 mg of NHS and 13.6 mg of DCC were added, and the mixture was reacted at 25°C overnight. The next day, the reaction solution was filtered to remove dicyclohexylurea to obtain a clear and transparent liquid for use. 29 mg of OVA was weighed and dissolved in 0.02 mol / L CB buffer. After clarification, the prepared DMF activation solution of the hapten was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 2 h. The reaction solution was then centrifuged, the supernatant was aspirated, and dialyzed in a dialysis bag treated with 0.2 mol / L EDTA solution and washed with distilled water for 3 days. The dialysate was PBS, and the dialysate was changed twice a day. After the dialysis was completed, the solution in the dialysis bag was aspirated to obtain the bifenthrin coating material, which was aliquoted and frozen for later use.

[0092] Synthesis of Bifenthrin Immunogen:

[0093] 10 mg of bifenthrin hapten was dissolved in 30 μL DMF, 10 μL tripropylamine was added, and a solution of 34.3 mg of isobutyl chloroformate dissolved in 30 μL DMF was added under ice bath, and the reaction was carried out for 1 hour, and then the reaction was carried out at room temperature overnight. The next day, 29 mg of BSA was weighed and dissolved in 0.02 mol / L CB buffer. After clarification, the prepared DMF activation solution of the hapten was added dropwise. After the addition was completed, the reaction was carried out at room temperature for 2 hours, and then the reaction solution was centrifuged, the supernatant was aspirated, and dialyzed for 3 days in a dialysis bag treated with 0.2 mol / L EDTA solution and washed with distilled water. The dialyzate was 0.02 mol / L PBS, and the dialyzate was changed twice a day. After the dialysis was completed, the solution in the dialysis bag was aspirated to obtain the bifenthrin immunogen, which was aliquoted and frozen for later use.

[0094] Example 2

[0095] Synthesis of bifenthrin hapten: 2.73 g of p-aminophenylboronic acid was dissolved in anhydrous ethanol, and 4.42 g of 3-bromo-2-methylbenzyl alcohol, 19.9 mL of 2 mol / L potassium carbonate solution, and 275 mg of tetrakistriphenylphosphine palladium were added. The reaction system was sealed and the air in the system was replaced with nitrogen. This was repeated three times. The reaction was heated in an oil bath and refluxed for 16 h. The reaction was monitored by TLC during this period. After the reaction was complete, the reaction solution was cooled to room temperature and the ethanol was removed by vacuum distillation. Water was added to the residue and the pH was adjusted to 7. The residue was then fully extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain 3.48 g of a light yellow oily liquid, which was the first intermediate (Formula A).

[0096] 3.48 g of the first intermediate was dissolved in dichloromethane, first cooled to 0°C in an ice bath, and then the air in the bottle was replaced with nitrogen. Under nitrogen protection, a dichloromethane solution of trifluorochlorochrysanthemoyl chloride (4.68 g) and 1.29 g of anhydrous pyridine were slowly added dropwise. After the addition was completed, the ice bath was kept for 2 hours, and then the ice bath was removed and the reaction was continued for 4 hours. The reaction was detected by TLC. After the reaction was complete, the dichloromethane and pyridine were removed by vacuum distillation, and the residue was dissolved in ethyl acetate and dry-loaded on the column. The target component was the third ultraviolet absorption point from top to bottom. EA:PE = 1:15 was first used to flush out the first two components. After the flushed developing agent had no ultraviolet absorption, it was changed to EA:PE = 1:5 to flush out the third component. 5.35 g of light yellow solid was obtained by vacuum distillation, which was the second intermediate (Formula B).

[0097] 5.35 g of the second intermediate was dissolved in anhydrous pyridine, 1.34 g of succinic anhydride and 0.18 g of DMAP were added, and the reaction was refluxed at 120° C. overnight. The next day, the reaction was detected and the raw materials had basically reacted completely. The reaction solution was distilled under reduced pressure to fully remove pyridine. The residue was dissolved with water, the pH was adjusted to about 7, and extraction was performed with ethyl acetate until no major product spots were found. The product was purified by silica gel column chromatography with a developing solvent of PE:EA=10:1 to remove trace impurities above the product spot. The developing solvent was then changed to PE:EA=3:1, and the main ultraviolet spot was collected to obtain 4.49 g of cypermethrin hapten, which was determined to have a purity of 98.4% by high performance liquid chromatography.

[0098] Synthesis of Bifenthrin Coating Source:

[0099] 15 mg of bifenthrin hapten was dissolved in 40 μL DMF, 3.8 mg of NHS and 11.5 mg of DCC were added, and the mixture was reacted at 25°C overnight. The next day, the reaction solution was filtered to remove dicyclohexylurea to obtain a clear and transparent liquid for use. 31 mg of OVA was weighed and dissolved in 0.02 mol / L CB buffer. After clarification, the prepared DMF activation solution of the hapten was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 2 h. The reaction solution was then centrifuged, the supernatant was aspirated, and dialyzed in a dialysis bag treated with 0.2 mol / L EDTA solution and washed with distilled water for 3 days. The dialysate was PBS, and the dialysate was changed twice a day. After the dialysis was completed, the solution in the dialysis bag was aspirated to obtain the bifenthrin coating material, which was aliquoted and frozen for later use.

[0100] Synthesis of Bifenthrin Immunogen:

[0101] 15 mg of bifenthrin hapten was dissolved in 30 μL DMF, 10 μL tripropylamine was added, and a solution of 4.5 mg of isobutyl chloroformate dissolved in 30 μL DMF was added under ice bath, and the reaction was carried out for 1 hour, and then the reaction was carried out at room temperature overnight. The next day, 46 mg of BSA was weighed and dissolved in 0.02 mol / L CB buffer. After clarification, the prepared DMF activation solution of the hapten was added dropwise. After the addition was completed, the reaction was carried out at room temperature for 2 hours, and then the reaction solution was centrifuged, the supernatant was aspirated, and dialyzed for 3 days in a dialysis bag treated with 0.2 mol / L EDTA solution and washed with distilled water. The dialyzate was 0.02 mol / L PBS, and the dialyzate was changed twice a day. After the dialysis was completed, the solution in the dialysis bag was aspirated to obtain the bifenthrin immunogen, which was packaged and frozen for later use.

[0102] Example 3

[0103] Synthesis of bifenthrin hapten: 5.36 g of p-aminophenylboronic acid was dissolved in anhydrous ethanol, and 8.65 g of 3-bromo-2-methylbenzyl alcohol, 39 mL of 2 mol / L potassium carbonate solution, and 540 mg of tetrakistriphenylphosphine palladium were added. The reaction system was sealed and the air in the system was replaced with nitrogen. The reaction was repeated three times and heated under reflux in an oil bath for 16 h. The reaction was monitored by TLC during the reaction. After the reaction was complete, the reaction solution was cooled to room temperature and the ethanol was removed by vacuum distillation. Water was added to the residue and the pH was adjusted to 7. The residue was then fully extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain 6.83 g of a light yellow oily liquid, which was the first intermediate (Formula A).

[0104] 6.83 g of the first intermediate was dissolved in dichloromethane, first cooled to 0°C in an ice bath, and then the air in the bottle was replaced with nitrogen. Under nitrogen protection, a dichloromethane solution of trifluorochlorochrysanthemoyl chloride (9.19 g) and 2.53 g of anhydrous pyridine were slowly added dropwise. After the addition, the ice bath was kept for 2 hours, and then the ice bath was removed and the reaction was continued for 4 hours. The reaction was detected by TLC. After the reaction was complete, the dichloromethane and pyridine were removed by vacuum distillation, and the residue was dissolved in ethyl acetate and dry-loaded on the column. The target component was the third ultraviolet absorption point from top to bottom. EA:PE = 1:15 was first used to flush out the first two components. After the flushed developing agent had no ultraviolet absorption, it was changed to EA:PE = 1:5 to flush out the third component. 2.78 g of light yellow solid was obtained by vacuum distillation, which was the second intermediate (Formula B).

[0105] 2.78 g of the second intermediate was dissolved in anhydrous pyridine, 0.699 g of succinic anhydride and 0.093 g of DMAP were added, and the reaction was refluxed at 120° C. overnight. The next day, the reaction was detected, and the raw materials had basically reacted completely. The reaction solution was distilled under reduced pressure to fully remove pyridine. The residue was dissolved with water, the pH was adjusted to about 7, and extraction was performed with ethyl acetate until no major product spots were found. The product was purified by silica gel column chromatography with a developing solvent of PE:EA=10:1 to remove trace impurities above the product spot. The developing solvent was then changed to PE:EA=3:1, and the main ultraviolet spots were collected to obtain 1.65 g of cypermethrin hapten, which was determined to have a purity of 95.2% by high-performance liquid chromatography.

[0106] Synthesis of Bifenthrin Coating Source:

[0107] 18 mg of bifenthrin hapten was dissolved in 40 μL DMF, 4.6 mg of NHS and 13.6 mg of DCC were added, and the mixture was reacted at 25°C overnight. The next day, the reaction solution was filtered to remove dicyclohexylurea to obtain a clear and transparent liquid for later use. 36.9 mg of OVA was weighed and dissolved in 0.02 mol / L CB buffer. After clarification, the prepared DMF activation solution of the hapten was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 2 h. The reaction solution was then centrifuged, the supernatant was aspirated, and dialyzed in a dialysis bag treated with 0.2 mol / L EDTA solution and washed with distilled water for 3 days. The dialysate was 0.02 mol / l PBS, and the dialysate was changed twice a day. After the dialysis was completed, the solution in the dialysis bag was aspirated to obtain the bifenthrin coating material, which was aliquoted and frozen for later use.

[0108] Synthesis of Bifenthrin Immunogen:

[0109] 18 mg of bifenthrin hapten was dissolved in 30 μL DMF, 10 μL tripropylamine was added, and a solution of 5.46 mg of isobutyl chloroformate dissolved in 30 μL DMF was added under ice bath, and the reaction was carried out for 1 hour, and then the reaction was carried out at room temperature overnight. The next day, 55 mg of BSA was weighed and dissolved in 0.02 mol / L CB buffer. After clarification, the prepared DMF activation solution of the hapten was added dropwise. After the addition was completed, the reaction was carried out at room temperature for 2 hours, and then the reaction solution was centrifuged, the supernatant was aspirated, and dialyzed for 3 days in a dialysis bag treated with 0.2 mol / L EDTA solution and washed with distilled water. The dialyzate was 0.02 mol / L PBS, and the dialyzate was changed twice a day. After the dialysis was completed, the solution in the dialysis bag was aspirated to obtain the bifenthrin immunogen, which was aliquoted and frozen for later use.

[0110] Test Example 1 ELISA test

[0111] Immunize mice:

[0112] Fifteen purebred BALB / c mice, aged 4-6 weeks, were selected and color-coded 1-5. Six 2 mL syringes were used. Three were filled with 0.5 mL of incomplete Freund's adjuvant and the other three were filled with 0.5 mL of a bifenthrin immunogen solution containing 50 μg, 100 μg, or 200 μg of bifenthrin immunogen (diluted with sterile 1% sodium chloride solution). After labeling, the syringes of one incomplete Freund's adjuvant syringe and one immunogen syringe were connected with infusion tubing (the length was appropriate to allow the two syringes to be aligned). The syringes were then manually pushed back and forth until complete emulsification (emulsification is indicated by a white, milky liquid, slight resistance during pushing, and small, non-spreading droplets). The emulsification process was continued until complete. Multiple subcutaneous injections (on the back and abdomen) were administered to the mice, with approximately 0.2 mL injected per mouse. The same procedure was repeated every two weeks. Blood tests were performed after three injections.

[0113] Blood collection method: Grab the mouse's neck scalp with the thumb, index finger, and middle finger of the left hand, and fix the tail with the little finger and ring finger; gently press the eye skin to be removed to make the eyeball congested and protruding; use a glass capillary to vertically insert it into the protruding part of the mouse eyeball, and gently rotate it left and right until blood flows into the capillary (approximately 2 / 3 of the capillary), take out the blood in the capillary and blow it into a 0.5mL centrifuge tube (stored at low temperature) with an ear bulb; after blood collection is completed, bake in a 37°C oven for 30 minutes, then place it in a low-temperature high-speed centrifuge (10,000 rpm) and centrifuge for 10 minutes to separate the serum for ELISA testing.

[0114] Monoclonal antibody preparation process:

[0115] Cell fusion

[0116] Mice are killed using carbon dioxide gas, and the spleens are aseptically removed and crushed in a dish to prepare a splenocyte suspension. Prepared syngeneic myeloma cells are mixed with mouse splenocytes in appropriate proportions, and the fusogenic agent polyethylene glycol is added. Under the influence of polyethylene glycol, various lymphocytes fuse with myeloma cells to form hybridomas.

[0117] Selective cultivation

[0118] The purpose of selective culture is to screen for fused hybridoma cells, using HAT selective medium. In HAT medium, unfused myeloma cells, lacking the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPT), cannot utilize the salvage pathway to synthesize DNA and die. Although unfused lymphocytes possess HPT, they cannot survive long-term in vitro and gradually die. Only fused hybridoma cells, which inherit HPT from spleen cells and possess the indefinite proliferation characteristics of myeloma cells, can survive and proliferate in HAT medium.

[0119] Screening and cloning of hybridoma positive clones

[0120] Only a minority of hybridoma cells grown in HAT medium secrete the desired specific monoclonal antibody. Therefore, screening and cloning are essential. Limited dilution is used for hybridoma cell cloning. Sensitive, rapid, and specific immunological methods are used to screen for positive hybridoma cells that produce the desired monoclonal antibody and then clone and expand them. After comprehensive characterization of the immunoglobulin type, subclass, specificity, affinity, antigen epitope recognition, and molecular weight of the secreted monoclonal antibody, the cells are promptly cryopreserved.

[0121] Large-scale production of monoclonal antibodies

[0122] Balb / c mice are pretreated with an intraperitoneal injection of 0.5 ml of liquid paraffin or pristane. One to two weeks later, hybridoma cells are inoculated intraperitoneally. The hybridoma cells proliferate within the mouse's peritoneal cavity and produce and secrete monoclonal antibodies. After approximately one to two weeks, the mouse's abdomen will become visibly distended. Ascites fluid is withdrawn with a syringe to obtain large quantities of monoclonal antibodies.

[0123] The reagent formula used in the ELISA test is as follows:

[0124] Blocking solution (1 L volume): 2.5 g skim milk powder, 8 g sodium chloride, 0.6 g potassium dihydrogen phosphate, 5.8 g disodium hydrogen phosphate, 50 g sucrose, 0.9 g potassium chloride, 0.5 mL preservative, 50 mL bovine serum, and the balance is water.

[0125] Enzyme dilute (16L): 128g sodium chloride, 9.48g potassium dihydrogen phosphate, 92.8g disodium hydrogen phosphate, 800mL glycerol, 3200mL bovine serum, 12mL preservative, and the balance is water.

[0126] Sample dilution (4 L): sodium chloride 23.38 g, potassium chloride 0.118 g, potassium dihydrogen phosphate 3.48 g, sodium dihydrogen phosphate 2.19 g, sodium hydrogen phosphate 20.64 g, Triton 40 mL, preservative 1.5 mL, and the balance is water.

[0127] Substrate A solution (100 mL): 0.48 g citric acid, 0.05 g urea peroxide, 90 mL glacial acetic acid, and the balance water.

[0128] Substrate B solution (400 mL): 0.164 g citric acid, 0.2 g TMB hydrochloride, 4 mL NN, 14 mL methanol, 160 μL 1 mol / L hydrochloric acid, and the balance water.

[0129] 20× concentrated washing solution (40 L): 2560 g sodium chloride, 928 g disodium hydrogen phosphate, 80 g potassium dihydrogen phosphate, 1.44 g potassium chloride, 12 mL preservative, 800 mL Tween 20, and the balance water.

[0130] The specific test methods are as follows:

[0131] (1) Antigen coating: Dilute the cypermethrin coating antigen to a 1K ratio with CB buffer, add 100 μL / well to the ELISA microplate, cover with a cover film, and incubate at 37°C for 2 h.

[0132] (2) Blocking: After coating, remove and discard the coating solution and use washing working solution (20× concentrated washing solution diluted to 1×) at 250 μL / well or wash once with a washing pot, soak for 30 seconds, discard the solution and pat dry with absorbent paper or towel, add blocking solution at 150 μL / well, place in a 37°C constant temperature incubator for reaction for 2 hours, remove and discard the blocking solution and pat dry for use.

[0133] (3) Dilution of different standard concentrations: Use 0.02 mol / L PBS buffer to dilute the directly purchased 100 μg / mL high-standard cypermethrin to 0 ppb (directly PBS buffer), 5 ppb, 20 ppb, and 50 ppb.

[0134] (4) Dilution of enzyme-labeled secondary antibody working solution: Use enzyme dilution buffer to dilute the purchased enzyme-labeled secondary antigen solution to a concentration of 1:1K.

[0135] (5) Dilution of serum or monoclonal antibody working solution: dilute the serum or prepared monoclonal antibody to a concentration of 1:1K, 1:5K, 1:1W, or 1:5W using sample dilution buffer.

[0136] (6) Testing steps: Test according to the chessboard method. The specific steps are as follows:

[0137] 1) Add standard / sample: Add 50 μL of standard / sample to the corresponding microwells.

[0138] 2) Add serum or monoclonal antibody working solution: Add 50 μL / well of serum or monoclonal antibody working solution, gently shake to mix, cover the plate with a cover film, and incubate at 37°C in a dark environment for 30 min.

[0139] 3) Wash the plate and add enzyme-labeled secondary antibody working solution: Carefully peel off the cover film, shake off the liquid in the wells, add 250μL / well of washing working solution, wash thoroughly 4-5 times, with 10 seconds between each wash, discard the washing solution in the wells, pat dry with absorbent paper (air bubbles that are not removed after patting dry can be punctured with an unused pipette tip), add 100μL / well of enzyme-labeled secondary antibody working solution, cover the plate with cover film, and incubate at 37℃ in a dark environment for 30 minutes.

[0140] 4) Washing: Carefully peel off the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, and wash thoroughly 4-5 times, with 10 seconds between each wash. Pour off the washing solution in the wells and pat dry with absorbent paper (air bubbles that are not removed after patting dry can be punctured with an unused pipette tip).

[0141] 5) Color development: Add 50 μL / well of substrate solution A and solution B, gently shake to mix, cover the plate with a cover film, and incubate at 37°C in a dark environment for 15 minutes.

[0142] 6) Determination: Add 50 μL / well of stop solution, gently shake to mix, and set the microplate reader to measure the OD value of each well at 450 nm.

[0143] Under the condition that relevant technical parameters are met, when the OD value is about 2.0, the maximum dilution multiple of the antibody is the antibody titer; the standard concentration is used as the horizontal axis and the corresponding absorbance value is used as the vertical axis to draw a standard curve, and the half-maximal inhibitory concentration (IC50) of bifenthrin is calculated based on the standard curve. 50 value).

[0144] The test results are shown in Tables 1 to 5.

[0145] Table 1. Serum test data after immunization

[0146]

[0147] Table 2. Serum test data after immunization

[0148]

[0149] Table 3 3 Serum test data after immunization

[0150]

[0151] Table 4 Whole serum test data

[0152]

[0153] Table 5 Preparation of monoclonal antibody test data

[0154]

[0155] According to the data in Tables 1 to 3, it can be seen that the concentration of bifenthrin antibodies in serum gradually increases with the increase in the number of immunizations; according to the data in Table 4, it can be seen that the concentration of bifenthrin antibodies in serum has basically reached 1W, IC 50 Less than 20 ppb; According to the results in Table 5, the titer of the prepared monoclonal antibody was 1×10 4 , half inhibitory concentration (IC 50 ) is less than 20ppb.

[0156] Test Example 2 Colloidal Gold Test

[0157] The test method is as follows:

[0158] (1) Streaking: Dilute the coated antigen with PB buffer (pH 7.2, 0.02 mol / L) at a ratio of 1:10, 1:20, 1:50, and 1:100 as the T-line coating solution. Dilute the secondary antibody with PB+10% BSA buffer (pH 7.2, 0.02 mol / L) at a ratio of 1:60 as the C-line coating solution. Streak the plate at a volume of 1 μL / cm. After streaking, bake at 37°C for 12 h (the back plate was prepared with Shanghai Jieyi 30*6 cm 2 Line PVC board, absorbent paper is Shanghai Jie Ning Spec30*20cm, NC film is Shanghai Jie Ning JN-140).

[0159] (2) Gold Standard Mat Processing Standard: Shanghai Jieyi ordinary glass fiber cut into 30*1cm 2 The size was determined by treating each strip with 1.5 mL of PB+10% BSA buffer (pH 7.2, 0.02 mol / L) and drying at 37°C overnight (more than 12 h).

[0160] (3) Sample pad processing standard: Shanghai Jie Ning SF-06 non-woven fabric cut into 30*1.7cm 2 Size, each strip was treated with 2 mL of PB + 0.5% Tween-20 + 1% sucrose buffer, pH 7.2, 0.02 mol / L, and dried at 37°C overnight (more than 12 h).

[0161] (4) Gold labeling: Dilute the serum or monoclonal antibody 1:10 with purified water and mix well. Take 4 tubes of 1 mL colloidal gold solution with 40 nm particles, add 30 μL of 0.02 mol / L K2CO3 to adjust the pH, add 10 μL, 20 μL, 30 μL, and 40 μL of diluted serum or monoclonal antibody diluent, mix well, and let it react at room temperature for 30 min. Then add 25 μL of blocking solution (20% BSA aqueous solution) and let it react at room temperature for 20 min. Centrifuge at 10,000 rpm for 10 min at low temperature (2-8°C), 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). Spray gold at a volume of 3 μL / cm and bake at 37°C for 2 h.

[0162] (5) Pretreatment method of dry tobacco leaf samples

[0163] 1) Before testing, dry tobacco leaf samples were dried and ground into fine powder.

[0164] 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 seconds, and allow to stand to separate layers.

[0165] 3) Take 100 μL of the sample supernatant and add it to a 2 mL polystyrene centrifuge tube. Add 500 μL of diluent (pH 7.2, 0.02 mol / L PB) and shake to mix. This is the sample solution to be tested.

[0166] (6) Dry tobacco leaf sample detection method

[0167] 1) Take 70 μL of the sample solution or use a dropper to vertically drop 3 drops into the sample well.

[0168] 2) The timing was started when the liquid began to flow, and the reaction lasted for 10 minutes. The results were interpreted using analytical software, a colloidal gold reader, or visual inspection. Five dry tobacco leaf samples, designated 1#, 2#, 3#, 4#, and 5#, were used. A bifenthrin standard was added to the dry tobacco leaf samples to control the bifenthrin concentrations per gram of dry tobacco leaf to be 0 ppb, 1250 ppb, 2500 ppb, and 5000 ppb, respectively. The test results are shown in Table 6.

[0169] Table 6 Detection limit test data of colloidal gold prepared from dry tobacco leaves using monoclonal antibodies

[0170]

[0171] Dry tobacco leaf sample 3# was taken and tested five times in parallel. The test results are shown in Table 7.

[0172] Table 7 Parallel test data for preparing monoclonal antibodies and colloidal gold from dry tobacco leaves

[0173]

[0174] The results in Table 6 show that the colloidal gold prepared by using the monoclonal antibody prepared by the present invention has a minimum detection limit of about 1250 ng / g for bifenthrin in dry tobacco leaf samples, with high sensitivity. According to the data in Table 7, it can be seen that the detection method provided by the present invention has good parallelism.

[0175] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a bifenthrin hapten, characterized in that: The bifenthrin hapten has a structure shown in Formula I: Formula I; The preparation method comprises the following steps: p-Aminophenylboronic acid, 3-bromo-2-methylbenzyl alcohol, an alkaline reagent, a palladium catalyst and an organic solvent are mixed to perform a Suzuki reaction to obtain a first intermediate; the structure of the first intermediate is shown in Formula A; Formula A; The first intermediate, pyridine, trifluorochlorochrysanthemoyl chloride and an organic solvent are mixed to perform a first coupling reaction to obtain a second intermediate, the structure of which is shown in Formula B; Formula B; The second intermediate, pyridine, succinic anhydride and 4-dimethylaminopyridine are mixed to perform a second coupling reaction to obtain the bifenthrin hapten having the structure shown in Formula I.

2. The preparation method according to claim 1, characterized in that The molar ratio of the p-aminophenylboronic acid to 3-bromo-2-methylbenzyl alcohol is 1:(1-1.2); the molar ratio of the p-aminophenylboronic acid to the alkaline reagent is 1:(0.5-1); and the molar ratio of the p-aminophenylboronic acid to the palladium catalyst is 1:(0.5-1). The molar ratio of the first intermediate to trifluorochlorochrysanthemoyl chloride is 1:(1-1.2); The molar ratio of the second intermediate to succinic anhydride is 1:(1-1.2).

3. The preparation method according to claim 1, characterized in that The temperature of the Suzuki reaction is 78-80°C and the time is 16-18 hours; The first coupling reaction includes a first stage and a second stage, wherein the reaction temperature of the first stage is 0-5°C and the reaction time is 2-2.5 hours; the reaction temperature of the second stage is room temperature and the reaction time is 4-5 hours; The temperature of the second coupling reaction is 120-125° C., and the time is 12-24 hours.

4. A bifenthrin antigen, characterized in that The bifenthrin hapten prepared by the preparation method according to any one of claims 1 to 3 is coupled with a carrier protein.

5. The bifenthrin antigen according to claim 4, characterized in that The carrier protein is ovalbumin or bovine serum albumin; when the carrier protein is ovalbumin, the structural formula of the bifenthrin antigen is as shown in Formula II: Formula II; When the carrier protein is bovine serum albumin, the structural formula of the bifenthrin antigen is shown in Formula III: Formula III.

6. The method for preparing the bifenthrin antigen according to claim 4 or 5, characterized in that: The following steps are involved: Mixing the bifenthrin hapten prepared by the preparation method according to any one of claims 1 to 3, a coupling agent and a polar solvent for activation to obtain an activated hapten solution; The activated hapten solution and the buffer solution of the carrier protein are mixed to carry out a coupling reaction to obtain the bifenthrin antigen.

7. A bifenthrin antibody, characterized in that: The method is obtained by immunizing a host animal with the bifenthrin antigen according to any one of claims 4 to 5.

8. A test paper or kit for detecting bifenthrin, characterized in that: Containing the bifenthrin antibody according to claim 7.

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