Bixafen hapten and its synthesis method, artificial antigen, antibody, detection device, method and application

Abstract

The present invention provides a bixafen hapten and its synthesis method, artificial antigen, antibody, detection device, method and application, by designing and developing a suitable bixafen hapten, artificial antigen and antibody, and thus establishing a corresponding bixafen rapid detection method, thereby achieving rapid detection of bixafen in food by immunological methods. The chemical reagents used in the bixafen hapten synthesis method are easy to obtain, the operation process is simple, the synthesis steps are concise and effective, the reaction yield is high, and the detection cost is low. The present invention applies an immunological detection method to carry out exclusive detection of bixafen residual in a sample, which has good specificity, high sensitivity, simple operation, and can achieve the advantages of on-site rapid detection, can better meet the needs of on-site supervision and law enforcement of food safety regulatory departments and testing agencies, and provides technical support for relevant law enforcement departments to quickly detect whether bixafen residual in food.

Description

Technical Field

[0001] The present invention relates to the field of immunological detection technology, and in particular to a bixafen hapten and a synthesis method thereof, an artificial antigen, an antibody, a detection device, a method and applications. Background Art

[0002] Bixafen, a succinate dehydrogenase inhibitor (SDHI) fungicide, is widely used in foods such as grains, oils and fats, and mammalian meat.

[0003] Common methods for detecting bixafen include high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). These methods offer high accuracy, but are difficult to implement, require high precision, and are expensive, making them unsuitable for large-scale promotion and application. Furthermore, the equipment used in these methods is complex to operate, expensive, and requires high operator skill. Furthermore, the lack of immediate results makes them unsuitable for rapid testing and monitoring of suspected cases by primary-level food safety regulatory authorities.

[0004] Immunological detection and analysis methods are analytical methods based on the specific recognition and reversible binding reaction of antigens and antibodies. They have the advantages of high sensitivity, high specificity, low instrument requirements, rapidity, simple operation, and low cost. However, the key technology for establishing immunological detection and analysis technology and applying it to the detection of bixafen residues in food is the ability to obtain antibodies with strong specificity and high sensitivity. To achieve this goal, the prerequisite is to design and synthesize a suitable bixafen hapten. However, there are currently no reports on the synthesis of bixafen haptens. Therefore, it is urgent to design and develop a suitable bixafen hapten, and thereby establish a corresponding bixafen rapid detection method, so as to achieve rapid detection of bixafen in food by immunological methods. Summary of the Invention

[0005] The present invention aims to provide a bixafen hapten and a synthesis method thereof, an artificial antigen, an antibody, a detection device, a method and an application thereof. The synthesis method of the present invention can be used to synthesize a bixafen hapten, an artificial antigen and an antibody, etc., which can be applied to an immunological detection method to specifically detect residual bixafen in a sample, with the advantages of good specificity, high sensitivity, simple operation and the ability to achieve rapid on-site detection.

[0006] The present invention solves the technical problem by adopting the following technical solutions.

[0007] The present invention provides a bixafen hapten, the structural formula of the bixafen hapten is:

[0008] (I).

[0009] The present invention provides a method for synthesizing a bixafen hapten, comprising the following steps:

[0010] S1. Under alkaline and heating conditions, ethyl 3-trifluoromethylpyrazole-4-carboxylate and 3-bromopropylene undergo a substitution reaction to obtain a first intermediate, the structural formula of which is:

[0011] ;

[0012] S2. The first intermediate is hydrolyzed to obtain a second intermediate, the structural formula of which is:

[0013] ;

[0014] S3, 2-bromo-4-fluoroaniline and 3,4-dichlorophenylboronic acid undergo a coupling reaction under palladium catalysis to obtain a third intermediate, the structural formula of the third intermediate is:

[0015] ;

[0016] S4, the second intermediate reacts with thionyl chloride to obtain an acyl chloride, and then the acyl chloride reacts with the third intermediate to obtain a fourth intermediate, the structural formula of the fourth intermediate is:

[0017] ;

[0018] S5, the fourth intermediate and tert-butyl acrylate undergo olefin coupling reaction in the presence of a Grubbs catalyst to obtain a fifth intermediate, the structural formula of which is:

[0019] ;

[0020] S6. The fifth intermediate undergoes a hydrolysis reaction to obtain the bixafen hapten.

[0021] The present invention provides an artificial bixafen antigen, comprising a bixafen immune antigen and a bixafen coating antigen, wherein the bixafen immune antigen comprises the bixafen hapten and a first carrier protein coupled to the bixafen hapten, and the bixafen coating antigen comprises the bixafen hapten and a second carrier protein coupled to the bixafen hapten.

[0022] The present invention provides a bixafen antibody, which is prepared by immunizing animals with the bixafen artificial antigen.

[0023] The present invention proposes the use of the bixafen hapten, the bixafen artificial antigen, and the bixafen antibody in immunological detection of bixafen residues.

[0024] The present invention provides a colloidal gold chromatography detection device for bixafen, comprising a test strip and a microporous reaction cup, wherein the test strip comprises a reaction membrane, a sample absorption pad, a water absorbent pad and a bottom plate, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with the bixafen artificial antigen, the quality control line is coated with a goat anti-mouse antibody, and the microporous reaction cup contains the bixafen antibody labeled with colloidal gold.

[0025] The present invention provides a method for detecting bixafen residue in a sample, and uses the bixafen colloidal gold chromatography detection device to detect the bixafen in the sample.

[0026] The beneficial effects of the bixafen hapten and its synthesis method, artificial antigen, antibody, detection device, method and application of the embodiments of the present invention are:

[0027] 1. The present invention designs and develops a suitable bixafen hapten, artificial antigen, and antibody, and establishes a corresponding rapid detection method for bixafen, thereby enabling rapid immunological detection of bixafen in food. The synthesis method of the bixafen hapten uses readily available chemical reagents, a simple operation, concise and efficient synthesis steps, high reaction yield, and low detection costs.

[0028] 2. The present invention utilizes the principle of colloidal gold immunochromatography to qualitatively detect whether bixafen remains in a sample by colorimetry between the test line and the quality control line in a test strip. No large-scale instruments such as liquid chromatography or mass spectrometry are required during application, thereby achieving the purpose of rapid detection. The present invention utilizes an immunological detection method to perform exclusive detection of bixafen residue in a sample, which has the advantages of good specificity, high sensitivity, simple operation, and the ability to achieve on-site rapid detection. This method can better meet the needs of on-site supervision and law enforcement by food safety regulatory authorities and testing agencies, and provides technical support for relevant law enforcement departments to rapidly detect whether bixafen remains in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 is a synthetic flow chart of the bixafen hapten of the present invention;

[0031] Figure 2 Schematic diagram of the cross-sectional structure of the test strip of the bixafen colloidal gold immunochromatographic detection device of the present invention;

[0032] Figure 3 This is a schematic structural diagram of a microporous reaction cup of the bixafen colloidal gold chromatography detection device of the present invention;

[0033] Figure 4 This is the mass spectrum of the bixafen hapten of Example 1 of the present invention;

[0034] Figure 5 This is an absorption curve diagram of the carrier protein, bixafen hapten and bixafen immune antigen of Example 2 of the present invention;

[0035] Figure 6 This is an absorption curve diagram of the carrier protein, bixafen hapten and bixafen coated antigen of Example 2 of the present invention;

[0036] Figure 7 This is a standard curve diagram of the indirect competitive ELISA established for the bixafen monoclonal antibody of Example 3 of the present invention;

[0037] Figure 8 Schematic diagram of the result determination of the bixafen colloidal gold immunochromatographic detection device. DETAILED DESCRIPTION

[0038] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0039] The following describes in detail the bixafen hapten and its synthesis method, artificial antigen, antibody, detection device, method and application of the embodiments of the present invention.

[0040] An embodiment of the present invention provides a bixafen hapten, the structural formula of the bixafen hapten is:

[0041] (I)

[0042] Reference Figure 1 As shown, the present invention provides a method for synthesizing a bixafen hapten, comprising the following steps:

[0043] S1. Under alkaline and heating conditions, ethyl 3-trifluoromethylpyrazole-4-carboxylate and 3-bromopropylene undergo a substitution reaction to obtain a first intermediate, the structural formula of which is:

[0044] .

[0045] Furthermore, in a preferred embodiment of the present invention, the specific steps of the substitution reaction of ethyl 3-trifluoromethylpyrazole-4-carboxylate and 3-bromopropylene under alkaline and heating conditions are as follows: ethyl 3-trifluoromethylpyrazole-4-carboxylate and 3-bromopropylene are added to a reactor, dissolved in DMF, and then potassium carbonate is added. The reaction is stirred continuously at room temperature. After the reaction is completed, water is added, extracted with ethyl acetate, and the organic phase is collected. The organic phase is evaporated to dryness and then purified by column to obtain a first intermediate. The molar ratio of ethyl 3-trifluoromethylpyrazole-4-carboxylate, 3-bromopropylene, and potassium carbonate is 1:1:1.5.

[0046] S2. The first intermediate is hydrolyzed to obtain a second intermediate, the structural formula of which is:

[0047] .

[0048] Furthermore, in a preferred embodiment of the present invention, the hydrolysis reaction of the first intermediate is carried out as follows: the first intermediate is dissolved in ethanol, a sodium hydroxide solution is added, and the reaction is heated under reflux to carry out the hydrolysis reaction. After the reaction is completed, the organic solvent is evaporated to dryness, the mixture is diluted with water, and then extracted with ethyl acetate. The organic phase is evaporated to dryness and purified by column chromatography to obtain the second intermediate.

[0049] S3, 2-bromo-4-fluoroaniline and 3,4-dichlorophenylboronic acid undergo a coupling reaction under palladium catalysis to obtain a third intermediate, the structural formula of the third intermediate is:

[0050] .

[0051] Furthermore, in a preferred embodiment of the present invention, the specific steps for the coupling reaction of 2-bromo-4-fluoroaniline and 3,4-dichlorophenylboronic acid under palladium catalysis are as follows: 2-bromo-4-fluoroaniline, 3,4-dichlorophenylboronic acid, tetrakis(triphenylphosphine)palladium and potassium carbonate are weighed in a flask, THF and water are added, and the mixture is refluxed at 70°C under nitrogen protection. After the reaction is completed, the mixture is extracted with ethyl acetate, and the organic phase is evaporated to dryness and purified by column to obtain a third intermediate. The molar ratio of 2-bromo-4-fluoroaniline, 3,4-dichlorophenylboronic acid, tetrakis(triphenylphosphine)palladium and potassium carbonate is 1:1.1:0.05:2.5.

[0052] S4, the second intermediate reacts with thionyl chloride to obtain an acyl chloride, and then the acyl chloride reacts with the third intermediate to obtain a fourth intermediate, the structural formula of the fourth intermediate is:

[0053] .

[0054] Furthermore, in a preferred embodiment of the present invention, the second intermediate reacts with thionyl chloride to obtain an acid chloride, and then the acid chloride is reacted with the third intermediate in the following specific steps: the second intermediate is refluxed with thionyl chloride to produce an acid chloride. The thionyl chloride is evaporated, dissolved in DCM, and a DCM solution of the third intermediate and triethylamine is added dropwise, and the reaction is stirred at room temperature. After completion of the reaction, water is added for extraction, and the organic phase is evaporated to dryness and column purified to obtain a fourth intermediate. The molar ratio of the second intermediate, the third intermediate, and triethylamine is 1.2:1:0.05:3.5.

[0055] S5, the fourth intermediate and tert-butyl acrylate undergo olefin coupling reaction in the presence of a Grubbs catalyst to obtain a fifth intermediate, the structural formula of which is:

[0056] .

[0057] Furthermore, in a preferred embodiment of the present invention, the specific steps of the olefin coupling reaction between the fourth intermediate and tert-butyl acrylate in the presence of a Grubbs catalyst are as follows: the fourth intermediate, tert-butyl acrylate, Grubbs second-generation catalyst, and cuprous iodide are added to diethyl ether and refluxed under nitrogen. After completion of the reaction, water is added for extraction, and the organic phase is evaporated to dryness and purified by column to obtain a fifth intermediate. The molar ratio of the fifth intermediate, tert-butyl acrylate, Grubbs second-generation catalyst, and cuprous iodide is 1:5:0.1:0.1.

[0058] S6. The fifth intermediate undergoes a hydrolysis reaction to obtain the bixafen hapten.

[0059] Furthermore, in a preferred embodiment of the present invention, the specific steps of the hydrolysis reaction of the fifth intermediate are: dissolving the fifth intermediate with 2 mL of dichloromethane, adding 2 mL of trifluoroacetic acid, reacting at room temperature for 1 hour, evaporating the solvent, adding water, extracting with ethyl acetate, and evaporating the organic phase to dryness to obtain the bixafenthion hapten.

[0060] The present invention designs and develops a suitable bixafen hapten, artificial antigen, and antibody, and establishes a corresponding rapid detection method for bixafen, thereby enabling rapid detection of bixafen in food via immunological methods. The synthesis method of the bixafen hapten uses readily available chemical reagents, a simple operation process, concise and efficient synthesis steps, high reaction yield, and low detection costs.

[0061] The present invention also provides a bixafen artificial antigen, comprising a bixafen immune antigen and a bixafen coating antigen, wherein the bixafen immune antigen comprises the bixafen hapten and a first carrier protein coupled to the bixafen hapten, and the bixafen coating antigen comprises the bixafen hapten and a second carrier protein coupled to the bixafen hapten.

[0062] Furthermore, in a preferred embodiment of the present invention, the first carrier protein and the second carrier protein are both selected from one of bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.

[0063] The present invention also provides a bixafen antibody, which is prepared by immunizing animals with the bixafen artificial antigen.

[0064] Furthermore, in a preferred embodiment of the present invention, the bixafen antibody is a bixafen monoclonal antibody.

[0065] The present invention also provides use of the bixafen hapten, the bixafen artificial antigen, and the bixafen antibody in immunological detection of bixafen residues.

[0066] Reference Figure 2 and Figure 3 As shown, the present invention also provides a colloidal gold chromatography detection device for bixafen, comprising a test strip and a microporous reaction cup, wherein the test strip comprises a reaction membrane, a sample absorption pad, a water absorbent pad and a bottom plate, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with the bixafen artificial antigen, the quality control line is coated with a goat anti-mouse antibody, and the microporous reaction cup contains the bixafen antibody labeled with colloidal gold.

[0067] The present invention also provides a method for detecting bixafen residues in a sample, which uses the bixafen colloidal gold chromatography detection device to detect bixafen in the sample, comprising the following steps:

[0068] (1) Sample pretreatment;

[0069] (2) Detection using a bixafen colloidal gold immunochromatographic detection device;

[0070] (3) Analyze the test results.

[0071] Furthermore, in a preferred embodiment of the present invention, the sample is pork, beef or mutton.

[0072] The present invention utilizes the principle of colloidal gold immunochromatography to qualitatively detect whether bixafen remains in a sample by colorimetry between the test line and the quality control line in a test strip. No large-scale instruments such as liquid chromatography or mass spectrometry are required during application, thereby achieving the purpose of rapid detection. The present invention utilizes an immunological detection method to perform exclusive detection of bixafen remaining in a sample, which has the advantages of good specificity, high sensitivity, simple operation, and the ability to achieve on-site rapid detection. This method can better meet the needs of on-site supervision and law enforcement by food safety regulatory authorities and testing agencies, and provides technical support for relevant law enforcement agencies to rapidly detect whether bixafen remains in food.

[0073] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0074] It should be noted that the reagents used in the present invention can be obtained commercially. For example, the analytical grade bixafen is selected from Sinopharm Chemical Reagent Co., Ltd.; the 98% 3-trifluoromethylpyrazole-4-carboxylic acid ethyl ester is selected from Shanghai Aladdin Biochemical Technology Co., Ltd.; the 97% 3-bromopropene is selected from Sinopharm Chemical Reagent Co., Ltd.; the 98% 2-bromo-4-fluoroaniline is selected from Sinopharm Chemical Reagent Co., Ltd.; the 98% 3,4-dichlorophenylboric acid is selected from Sinopharm Chemical Reagent Co., Ltd.; the 99% thionyl chloride is selected from Sinopharm Chemical Reagent Co., Ltd.; the 99% tert-butyl acrylate is selected from Sinopharm Chemical Reagent Co., Ltd.; the analytical grade trifluoroacetic acid is selected from Sinopharm Chemical Reagent Co., Ltd.; and the 99.8% N,N-dimethylformamide is selected from Sinopharm Chemical Reagent Co., Ltd. N,N-dimethylformamide.

[0075] Example 1

[0076] This embodiment provides a bixafen hapten, which is prepared according to the following steps:

[0077] (1) 4.2 g of ethyl 3-trifluoromethylpyrazole-4-carboxylate and 2.4 g of 3-bromopropylene were added to a reactor and dissolved in 25 mL of DMF. 4.1 g of potassium carbonate was then added. The molar ratio of ethyl 3-trifluoromethylpyrazole-4-carboxylate, 3-bromopropylene, and potassium carbonate was 1:1:1.5. The reaction was stirred at room temperature for 5 h. After the reaction was completed, water was added and the mixture was extracted with ethyl acetate. The organic phase was collected, evaporated to dryness, and purified by column chromatography to obtain 3.5 g of the first intermediate.

[0078] (2) Weigh 2.2 g of the first intermediate, dissolve it in 50 mL of ethanol, add 10 mL of 0.05 mol / L sodium hydroxide solution, heat under reflux, and perform hydrolysis reaction; after the reaction, evaporate the organic solvent, dilute with water, and extract with ethyl acetate. The organic phase is evaporated to dryness and purified by column to obtain 1.3 g of the second intermediate.

[0079] (3) Weigh 1.9 g of 2-bromo-4-fluoroaniline, 2.1 g of 3,4-dichlorophenylboronic acid, 0.6 g of tetrakis(triphenylphosphine)palladium, and 3.5 g of potassium carbonate into a flask. The molar ratio of 2-bromo-4-fluoroaniline, 3,4-dichlorophenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 1:1.1:0.05:2.5. Add THF and water, protect with nitrogen, and reflux at 70°C. After the reaction is complete, extract with ethyl acetate. The organic phase is evaporated to dryness and purified by column chromatography to obtain 2.1 g of the third intermediate.

[0080] (4) 452 mg of the second intermediate was refluxed with thionyl chloride to form an acid chloride. The thionyl chloride was evaporated and dissolved in DCM. A DCM solution of 514 mg of the third intermediate and 708 mg of triethylamine was added dropwise, wherein the molar ratio of the second intermediate, the third intermediate, and triethylamine was 1.2:1:3.5. The mixture was stirred at room temperature. After the reaction was completed, water was added for extraction. The organic phase was evaporated to dryness and purified by column chromatography to obtain 707 mg of the fourth intermediate.

[0081] (5) 442 mg of the fourth intermediate, 640 mg of tert-butyl acrylate, 85 mg of Grubbs' second-generation catalyst, and 19 mg of cuprous iodide were added to 20 mL of ether, protected by nitrogen, and refluxed. The molar ratio of the fourth intermediate, tert-butyl acrylate, Grubbs' second-generation catalyst, and cuprous iodide was 1:5:0.1:0.1. After the reaction was completed, water was added for extraction. The organic phase was evaporated to dryness and purified by column chromatography to obtain 443 mg of the fifth intermediate.

[0082] (6) The fifth intermediate was dissolved in 2 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 1 h. The solvent was evaporated, water was added, and the mixture was extracted with ethyl acetate. The organic phase was evaporated to dryness to obtain 303 mg of bixafenthion hapten.

[0083] The prepared bixafen hapten was identified by mass spectrometry. Figure 4 Shown is the mass spectrum of the bixafen hapten of Example 1. Figure 4 It can be seen that the molecular ion peak of the bixafen hapten is EI-MS (negative) m / z: 500.25 [MH]- and is the highest peak, which is consistent with the molecular weight of the bixafen hapten of 501.03, indicating that the bixafen hapten represented by formula (I) was successfully synthesized.

[0084] Example 2

[0085] This embodiment provides a bixafen immunization antigen and a bixafen coating antigen, which are prepared according to the following steps:

[0086] Preparation of Bixafen Antigen for Immunization: 30 mg of the bixafen hapten prepared in Example 1 was weighed and dissolved in 2 mL of N,N-dimethylformamide (DMF). 18 mg of N-hydroxysuccinimide (NHS) and 26 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) were added and reacted at room temperature for 6 h to prepare an activation solution. 60 mg of bovine serum albumin (BSA) was then dissolved in 2 mL of 0.05 M boric acid buffer (pH 9.0). 1 mL of DMF and 0.5 mL of the activation solution were added. The mixture was reacted at room temperature for 4 h and then dialyzed against PBS (0.02 mol / L, pH 7.4 phosphate buffer). The solution was changed every 4 h for 7 to 8 min. After dialysis, the mixture was centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain the bixafen hapten-BSA conjugate, which was the bixafen immune antigen. The conjugate was aliquoted and stored at -20°C.

[0087] Preparation of bixafen coating antigen: 10 mg of the bixafen hapten prepared in Example 1 was weighed and dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF), followed by the addition of 3 μL of diisopropylethylamine and 5 μL of isobutyl chloroformate, and the reaction was carried out at 0°C for 30 min to prepare solution A. 70 mg of hen egg white albumin (OVA) was dissolved in 7 ml of 0.1 M boric acid buffer to obtain solution B. Solution A was added dropwise to solution B, reacted at room temperature for 4 h, and then dialyzed against PBS (0.02 mol / L, pH = 7.4 phosphate buffer), with the solution changed every 4 h for 7 to 8 times. After dialysis, the solution was centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain the bixafen hapten-OVA conjugate, i.e., the bixafen coating antigen. The solution was aliquoted and stored at -20°C.

[0088] Identification: Carrier protein, bixafen hapten, bixafen immunization antigen, and bixafen coating antigen were prepared into 0.5 mg / mL solutions in PBS buffer (pH 7.4, 0.01 mol / L), adjusted to zero with PBS buffer (pH 7.4, 0.01 mol / L), and scanned with an ultraviolet spectrophotometer at a wavelength of 200-400 nm.

[0089] like Figure 5 Shown is the absorption curve of the carrier protein, bixafen hapten and bixafen immune antigen of Example 2. Figure 6The absorption curves of the carrier protein, bixafen hapten and bixafen coated antigen of Example 2 are shown. Figure 5 and Figure 6 It can be seen that the ultraviolet characteristic absorption peaks of the bixafen immune antigen and the bixafen coated antigen are shifted to varying degrees relative to the bixafen hapten and the carrier protein (BSA or OVA), and it is found that the bixafen immune antigen and the bixafen coated antigen have the characteristic absorption peaks of both the bixafen hapten and the carrier protein, indicating that the bixafen hapten and the carrier protein are successfully coupled, and the bixafen immune antigen and the bixafen coated antigen are successfully prepared.

[0090] Example 3

[0091] This embodiment provides a bixafen monoclonal antibody, which is prepared according to the following steps:

[0092] (1) Animal immunization: Healthy 6- to 8-week-old BALB / c mice were selected for immunization. The bixafen immunization antigen obtained in Example 2 was injected into the BALB / c mice at a dose of 150 μg / mouse to produce antiserum.

[0093] (2) Cell fusion and cloning: spleen cells of immune BALB / c mice were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio). The cell supernatant was determined by indirect competitive ELISA method and positive wells were screened ( Figure 7 The positive wells were cloned using the limiting dilution method to directly obtain hybridoma cell lines that stably secreted bixafen monoclonal antibodies.

[0094] (3) Cell freezing and recovery: The bixafen monoclonal hybridoma cells were prepared into 1×10 6 Cell suspensions of 100 cells / mL can be stored in liquid nitrogen for a long term. When thawing, remove the cryovials and immediately thaw in a 37°C water bath. Centrifuge to remove the cryopreservation solution and transfer to a culture flask for culture.

[0095] (4) Preparation and purification of monoclonal antibodies: BALB / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). Seven days later, 5×10 cells of a stable bixafen-containing monoclonal hybridoma cell line were intraperitoneally injected. 5 After 7 days, the ascites was collected and purified using the caprylic acid-saturated ammonium sulfate method. The resulting bixafen monoclonal antibody was stored at -20°C.

[0096] Example 4

[0097] This embodiment provides a goat anti-mouse antibody, which is prepared according to the following steps:

[0098] Sheep were used as immune animals, and pathogen-free sheep were immunized with the mouse antibody of Example 3 as an immunogen to obtain sheep anti-mouse antibodies.

[0099] Example 5

[0100] This embodiment provides a bixafen colloidal gold chromatography detection device, the preparation method of which mainly includes:

[0101] preparing a microporous reaction cup containing freeze-dried bixafen monoclonal antibody-colloidal gold label;

[0102] preparing a reaction membrane having a detection line coated with a bixafen hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody;

[0103] Assemble the prepared reaction membrane with the sample absorption pad, water absorption pad, and bottom plate to form a test paper;

[0104] The prepared freeze-dried microporous reaction cup containing bixafenthion monoclonal antibody-colloidal gold marker and the test paper are assembled.

[0105] To facilitate the preparation of the detection device, the preparation process of the detection device is described in detail step by step below.

[0106] (1) Preparation of bixafenthion monoclonal antibody-colloidal gold label

[0107] (1.1) Preparation of colloidal gold solution

[0108] Dilute a 1% chloroauric acid solution to 0.01% (mass fraction) with double-distilled deionized water. Place 100 ml of this 0.01% chloroauric acid solution in a conical flask and heat to boiling using a constant-temperature electromagnetic stirrer. Add 1.5 ml of a 1% trisodium citrate solution while continuing to stir at high temperature until the solution turns a translucent red. Cool to room temperature, then return to the original volume with deionized water to obtain a colloidal gold solution. Store at 4°C. The prepared colloidal gold solution should be pure, translucent, free of precipitates and floating matter, and appear wine red when observed in sunlight.

[0109] (1.2) Preparation of bixafenthion monoclonal antibody-colloidal gold label

[0110] Under magnetic stirring, the pH of the colloidal gold solution was adjusted to 7.0 with 0.2 mol / L potassium carbonate solution. The bixafen monoclonal antibody prepared in Example 4 was added to the colloidal gold solution at a standard concentration of 20-50 μg of bixafen monoclonal antibody per milliliter of colloidal gold solution. Stirring and mixing were continued for 30 minutes. After standing for 10 minutes, 10% bovine serum albumin (BSA) solution was added to a concentration of 1% by volume in the colloidal gold solution. The solution was allowed to stand for 10 minutes. Centrifugation was then performed at 12,000 rpm at 4°C for 40 minutes. The supernatant was discarded, and the pellet was resuspended in a reconstitution buffer solution with a volume of 1 / 10 of the initial colloidal gold solution to prepare a bixafen monoclonal antibody-colloidal gold label. The solution was then stored at 4°C until use.

[0111] Reconstitution buffer: 0.02 mol / L phosphate buffer containing 0.1%~0.5% bovine serum albumin (volume percentage), 0.05%~0.2% Tween-20 (mass percentage), 2%~4% trehalose (mass percentage), pH= 7.2.

[0112] (1.3) Preparation of microwell reaction cup

[0113] Add 100 μL of bixafen monoclonal antibody-colloidal gold label to the microporous reaction cup, place it in a freeze dryer, pre-freeze at a cold trap temperature of -50°C for 3 hours, and then vacuum dry it for 6 hours. Then take it out to obtain a microporous reaction cup with freeze-dried bixafen monoclonal antibody-colloidal gold label. Seal it for storage. The freeze-dried amount of bixafen monoclonal antibody-colloidal gold label is 0.20~0.60 μg / mL.

[0114] (1.4) Preparation of reaction membrane

[0115] The bixafen hapten-OVA conjugate was coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody of Example 4 was coated on the reaction membrane to form a quality control line.

[0116] Coating process: Dilute the bixafenthion coating antigen to a concentration of 0.5 mg / mL in 0.01 mol / L, pH 7.2 phosphate buffer and coat the membrane in the test zone (zone T) of the nitrocellulose membrane using a Bio dot stripper at a coating volume of 1.0 μL / cm. Dilute the goat anti-mouse antibody to a concentration of 500 μg / mL in 0.01 mol / L, pH 7.2 phosphate buffer and coat the membrane in the control zone (zone C) of the nitrocellulose membrane using a Bio dot stripper at a coating volume of 1.0 μL / cm. Dry the coated membrane at 50°C for 16 hours and reserve it for production.

[0117] (1.5) Preparation of sample absorption pad

[0118] The sample absorption pad was soaked in 1% bovine serum albumin (volume fraction) and 0.1 mol / L phosphate buffer with a pH of 7.2 for 2 h, and dried at 37°C for 2 h before use.

[0119] (2) Preparation of a colloidal gold chromatography detection device for bixafenthion

[0120] (2.1) Assembly of test strips

[0121] A sample absorption pad, reaction membrane, and water-absorbing pad are sequentially attached to a base plate. The base plate is a PVC base plate, the sample absorption pad is filter paper, the water-absorbing pad is filter paper, and the reaction membrane is a nitrocellulose membrane. The end of the sample absorption pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the water-absorbing pad, the beginning of the sample absorption pad is aligned with the beginning of the base plate, and the end of the water-absorbing pad is aligned with the end of the base plate. The microporous reaction cuvette has a microporous stopper.

[0122] (2.2) Assembly of the Bixafen colloidal gold chromatography test kit

[0123] The test strips prepared in the above steps and the microporous reaction cup are assembled into a test strip box, and stored in an environment of 2-8°C.

[0124] Example 6

[0125] This embodiment provides a method for detecting bixafen residue in a sample, comprising the following steps:

[0126] (1) Preparation of sample extract: Accurately weigh 3.40 g of potassium dihydrogen phosphate and 3.55 g of disodium hydrogen phosphate, dissolve in water and dilute to 1000 mL, then mix thoroughly.

[0127] (2) Sample preparation:

[0128] (2.1) Meat: remove bones, viscera, skin, fat, and fascia.

[0129] (2.2) Take approximately 100 g of muscle, grind and mix thoroughly using a tissue grinder, divide into two equal portions, place them into clean containers as a test sample and a reserve sample, seal them, label them, and store them at -20°C.

[0130] (3) Sample pretreatment: Weigh 2 g (accurate to 0.1 g) of uniform sample into a 10 mL centrifuge tube, add 8 mL of sample extract, vortex or vigorously shake for 5 min, and centrifuge at 4000 r / min for 3 min. The supernatant after centrifugation is the test solution.

[0131] (4) Determination steps: Pipette 200 μL of the test solution into a microporous reaction cup and pipette up and down 5-10 times to mix evenly. Incubate at room temperature for 3 minutes. Insert the test strip into the reaction cup and incubate at room temperature for 3 minutes. Remove the test strip, gently scrape off the sample pad at the bottom of the test strip, and read the result.

[0132] (5) Result determination

[0133] Reference Figure 8 As shown, the result is determined by comparing the color depth of the control line (C line) and the test line (T line).

[0134] Positive: When the control area (C) shows a band and the test area (T) does not show color, it is judged as positive, that is, there is bixafen in the sample, which is indicated by "+";

[0135] Negative: When both the control area and the test area show bands, it is judged as negative, that is, there is no bixafen in the sample, indicated by "-";

[0136] Invalid: When no band is displayed in the control area (C), the test strip is invalid.

[0137] Test Example 1

[0138] Sensitivity refers to the percentage of positive samples detected as positive out of the total number of positive samples at the actual minimum detection level achieved by the method under experimental conditions, i.e., the method's limit of detection (LOD). This test was conducted on blank pork, beef, and mutton matrices to determine the method's limit of detection and sensitivity. The specific steps were as follows:

[0139] The test was conducted on blank pork, beef, and mutton samples. Sample concentrations covered blank, 0.5 times the detection limit, 1 times the detection limit, and 2 times the detection limit, with 50 samples tested at each concentration level. The sensitivity results for each matrix sample are shown in Table 1.

[0140] Table 1 Sample measurement results

[0141]

[0142] As shown in Table 1, when pork, beef, and mutton were spiked at a concentration of 0.5 times the detection limit of 1 mg / kg, the pork and beef test results were all negative, while one positive case was found in mutton. When spiked at concentrations of 1 times the detection limit of 2 mg / kg and 2 times the detection limit of 4 mg / kg, the test results were all positive. This indicates that the detection limit of this test strip is 2 mg / kg, the sensitivity of the pork and beef matrices is 100%, and the sensitivity of the mutton matrix is ​​99%.

[0143] Test Example 2

[0144] The false-negative rate is the maximum probability (expressed as a percentage) of detecting a negative result in a positive sample, at the practical minimum detection level achieved by the method under experimental conditions. The false-positive rate is the maximum probability (expressed as a percentage) of detecting a positive result in a negative sample, at the practical minimum detection level achieved by the method under experimental conditions. This test was conducted on blank pork, beef, and lamb matrices to determine the false-negative and false-positive rates of the method. The specific steps were as follows:

[0145] The test was conducted on blank pork, beef, and mutton samples. Samples were spiked at concentrations ranging from blank to 0.5 times the detection limit, 1 times the detection limit, and 2 times the detection limit. Fifty samples were tested at each concentration level. The false negative and false positive rates for each matrix are shown in Table 2.

[0146] Table 2 Determination results of false negative rate and false positive rate of each matrix sample

[0147]

[0148] As can be seen from Table 2, when testing negative pork, the negative coincidence rate was 100% and the false positive rate was 0%; when testing positive pork, the positive coincidence rate was 100% and the false negative rate was 0%; when testing negative beef, the negative coincidence rate was 100% and the false positive rate was 0%; when testing positive beef, the positive coincidence rate was 100% and the false negative rate was 0%; when testing negative mutton, the negative coincidence rate was 99% and the false positive rate was 1%; when testing positive mutton, the positive coincidence rate was 100% and the false negative rate was 0%.

[0149] In this test example, the false positive rates of the three matrices were all ≤10%, and the false negative rates were all ≤5%, indicating that the test strip of the present invention can be used to quickly detect the amount of bixafenthion residues in livestock meat such as pork, beef, and mutton.

[0150] Test Example 3

[0151] Cross-reactivity refers to the cross-reactivity between a rapid test method and its related products, reflecting the specificity of the product. This test example tested the method's cross-reactivity on three matrix samples: blank pork, beef, and mutton. The specific steps are as follows:

[0152] Using PBS buffer solution (pH 8.0) as the solvent, the detection limit level of the standard substance of bixafen was added, and the standard substances of pesticides with similar functions and structures to bixafen (flupyraclostrobin, benzovindipyruvate, flupyraclostrobin, fluinoxam, and penthiopyrad) at 10-1000 times the concentration were added to prepare test solutions at different concentration levels to verify the cross-reaction of the rapid test product.

[0153] The results showed that the test strip tested positive for 2 mg / kg of bixafen, but negative for 2000 mg / kg of other pesticides with similar functions and structures to bixafen, including flupyrad, benzovintriazole, fluopyram, fluinopyram, and penthiopyrad. This indicates that the test strip has good specificity and no cross-reactivity with these drugs.

[0154] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

Claims

1. A bixafen hapten, characterized in that: The structural formula of the bixafen hapten is: （I）。 2. A method for synthesizing the bixafen hapten according to claim 1, characterized in that: The following steps are involved: S1. Under alkaline and heating conditions, ethyl 3-trifluoromethylpyrazole-4-carboxylate and 3-bromopropylene undergo a substitution reaction to obtain a first intermediate, the structural formula of which is: ； S2. The first intermediate is hydrolyzed to obtain a second intermediate, the structural formula of which is: ； S3, 2-bromo-4-fluoroaniline and 3,4-dichlorophenylboronic acid undergo a coupling reaction under palladium catalysis to obtain a third intermediate, the structural formula of the third intermediate is: ； S4, the second intermediate reacts with thionyl chloride to obtain an acyl chloride, and then the acyl chloride reacts with the third intermediate to obtain a fourth intermediate, the structural formula of the fourth intermediate is: ； S5, the fourth intermediate and tert-butyl acrylate undergo olefin coupling reaction in the presence of a Grubbs catalyst to obtain a fifth intermediate, the structural formula of which is: ； S6. The fifth intermediate undergoes a hydrolysis reaction to obtain the bixafen hapten.

3. A bixafen artificial antigen, characterized in that: It comprises a bixafen immune antigen and a bixafen coating antigen, wherein the bixafen immune antigen comprises the bixafen hapten according to claim 1 and a first carrier protein coupled to the bixafen hapten, and the bixafen coating antigen comprises the bixafen hapten according to claim 1 and a second carrier protein coupled to the bixafen hapten.

4. The bixafen artificial antigen according to claim 3, characterized in that The first carrier protein and the second carrier protein are both selected from one of bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.

5. A bixafen antibody, characterized in that: The bixafen antibody is prepared by immunizing an animal with the bixafen artificial antigen according to any one of claims 3 to 4.

6. The bixafen antibody according to claim 5, characterized in that The bixafen antibody is a bixafen monoclonal antibody.

7. Use of the bixafen hapten according to claim 1, the bixafen artificial antigen according to claim 3 or 4, or the bixafen antibody according to claim 5 or 6 in the preparation of an immunological detection reagent for bixafen residues.

8. A bixafen colloidal gold chromatography detection device, characterized in that: The invention comprises a test strip and a microporous reaction cup, wherein the test strip comprises a reaction membrane, a sample absorption pad, a water absorbent pad and a bottom plate, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with the artificial antigen of bixafen according to any one of claims 3 or 4, the quality control line is coated with a goat anti-mouse antibody, and the microporous reaction cup contains the bixafen antibody according to any one of claims 5 or 6 labeled with colloidal gold.

9. A method for detecting bixafen residue in a sample for non-diagnostic purposes, characterized in that: The bixafen colloidal gold chromatography detection device according to claim 8 is used to detect bixafen in the sample.

10. The detection method according to claim 9, characterized in that: The sample is pork, beef or mutton.

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