Diflubenzuron hapten, diflubenzuron artificial antigen and preparation method and application thereof
By preparing diflubenzuron hapten and artificial antigen, and combining them with immunochromatography, the problems of expensive equipment and complex operation in existing detection methods have been solved, achieving rapid detection with high sensitivity, which is suitable for on-site detection of diflubenzuron.
Patent Information
- Application Number
- CN202511719075.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-02-24
AI Technical Summary
Existing methods for detecting diflubenzuron rely on expensive instruments and equipment and require professional personnel to operate, making rapid on-site testing impossible and causing inconvenience in daily testing.
Diflubenzuron hapten and artificial antigen were prepared. By designing a synthetic route to retain the characteristic groups of diflubenzuron and conjugating them with a carrier protein, antibodies with high specificity and high sensitivity were prepared and applied to immunochromatographic test strips and kits to achieve rapid detection.
Highly sensitive detection of diflubenzuron has been achieved. The colloidal gold immunochromatographic test strip has a sensitivity of 0.5 µg/kg for diflubenzuron. The kit and test strip can be used for rapid, low-cost on-site detection.
Smart Images
Figure CN121554400A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biochemical technology, and more specifically, to a diflubenzuron hapten, an artificial antigen, and their preparation methods and applications. Background Technology
[0002] Diflubenzuron is a benzoic acid phenyl urea insecticide widely used in fruit trees, vegetables, and grain, cotton, and oil crops such as corn, wheat, rice, cotton, and peanuts. Current detection techniques primarily rely on instrumental methods for diflubenzuron detection. However, due to the high cost of the required equipment, the long testing time, and the need for professional personnel, true on-site and rapid clinical testing is not possible, causing significant inconvenience to daily testing work.
[0003] The key to immunoassay detection technology lies in the performance of antigens and antibodies, and the key to antigens and antibodies is the hapten. Therefore, the structural design of haptens is particularly important to obtain high-performance antigens and antibodies. Thus, developing highly specific and high-crosslinking diflubenzuron haptens or artificial antigens is of paramount importance for rapid, highly sensitive, and low-cost detection methods for diflubenzuron drugs. Summary of the Invention
[0004] To address the problems existing in current detection methods, this invention prepares dicofurine hapten and artificial antigen based on the structure of dicofurine; and thereby prepares dicofurine antibodies that can specifically recognize and have suitable sensitivity, providing a foundation for the development of rapid immunoassay technology and products for dicofurine.
[0005] According to one aspect of the present invention, a diflubenzuron hapten is provided, the structure of which is shown in formula (I): Equation (Ⅰ).
[0006] According to another aspect of the present invention, a method for preparing diflubenzuron hapten is provided, characterized by comprising the following steps: S1. Compound a undergoes chlorination and amination reactions to yield compound b. The structural formula of compound a is shown in formula (II), and the structural formula of compound b is shown in formula (III). Formula (II) Formula (Ⅲ); S2. Compound b is hydrolyzed under alkaline conditions to give compound c, the structural formula of which is shown in formula (Ⅳ): Formula (Ⅳ); S3. The reaction of compound c and compound d yields a diflubenzuron hapten with the structure shown in formula (I), wherein the structural formula of compound d is shown in formula (V): , formula (V).
[0007] According to another aspect of the present invention, a diflubenzuron artificial antigen is provided, which is a conjugate of diflubenzuron hapten and carrier protein, and the structural formula of the diflubenzuron artificial antigen is shown in formula (VI): Formula (VI).
[0008] In some embodiments, the carrier protein is any one of bovine serum albumin, ovalbumin, hemocyanin, or lactoferrin.
[0009] According to a fourth aspect of the invention, the application of diflubenzuron hapten or diflubenzuron artificial antigen in non-disease diagnosis of diflubenzuron in immunological detection is provided.
[0010] In some embodiments, the diflubenzuron artificial antigen includes a diflubenzuron immunogen and a diflubenzuron coating antigen, wherein the diflubenzuron immunogen is obtained by conjugating a diflubenzuron hapten with lactoferrin, and the diflubenzuron coating antigen is obtained by conjugating a diflubenzuron hapten with bovine serum albumin.
[0011] According to a fifth aspect of the present invention, a diflubenzuron antibody is provided, which is prepared by animal immunization with a diflubenzuron artificial antigen, and the diflubenzuron antibody is a diflubenzuron monoclonal antibody.
[0012] According to a sixth aspect of the invention, the application of diflubenzuron antibodies in the non-disease diagnosis of diflubenzuron in immunological detection is provided.
[0013] According to a seventh aspect of the present invention, an immunochromatographic test strip for detecting diflubenzuron is provided, the immunochromatographic test strip having a T line and a C line, the T line being coated with diflubenzuron antigen and the C line being coated with IgG antibody.
[0014] According to an eighth aspect of the present invention, a kit for detecting diflubenzuron is provided, the kit comprising the immunochromatographic test strip card described above for diflubenzuron detection.
[0015] The beneficial effects of the present invention are as follows: (1) The diflubenzuron hapten prepared by the present invention completely retains the three characteristic groups of diflubenzuron: m-difluorophenyl, chlorophenyl and acylurea. The designed coupling arm is directly connected to the benzene ring, so that the small molecule structure on the prepared diflubenzuron artificial antigen not only retains the original characteristics, but also has excellent spatial structure, which is more conducive to presenting the structural characteristics of diflubenzuron drug and improving the immunogenicity of the antigen. (2) The diflubenzuron artificial antigen and monoclonal antibody in this invention have high specificity for ELISA detection, and IC50... 50 The value was 0.14 µg / L; (3) The colloidal gold immunochromatographic test strip prepared in this invention has a sensitivity of 0.5 µg / kg for diflubenzuron in standard solution and a detection sensitivity of up to 5 µg / kg in sample. Attached Figure Description
[0016] Figure 1 This is a mass spectrum of the diflubenzuron hapten according to one embodiment of the present invention.
[0017] Figure 2 This is a synthetic route diagram of the diflubenzuron hapten in Example 1 of the present invention.
[0018] Figure 3 This is an ELISA standard curve based on diflubenzuron monoclonal antibody, representing one embodiment of the present invention. Detailed Implementation
[0019] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.
[0020] Example 1: Synthesis and Identification of Diflubenzuron Hapten
[0021] A method for preparing diflubenzuron hapten includes the following steps:
[0022] S1. Dissolve 500 mg of compound a (CAS No.: 1415124-74-1, 2.31 mmol) in 10 mL of a 1:1 mixture of toluene and thionyl chloride. Add 10 µL of LDMF and react at 60 °C for 1–2 h. After the reaction is complete, evaporate the residue under reduced pressure and dissolve it in 10 mL of dichloromethane. Slowly add the solution dropwise to a mixture containing 10 mL of 25–28% amine water and 10 mL of dichloromethane under ice bath conditions. After the addition is complete, bring the temperature to room temperature and stir for 1–2 h. After the reaction is complete, add 10 mL of 1% sodium carbonate aqueous solution and mix thoroughly. Separate the organic phase and extract it once with 20 mL of 1% sodium carbonate aqueous solution. Collect the organic phase and remove the solvent from the organic phase under reduced pressure. Purify the residue by column chromatography to obtain 368 mg of compound b with the structure shown in formula (Ⅲ).
[0023] S2. 368 mg of compound b (1.71 mmol) was dissolved in 3 mL of methanol by stirring. The mixture was then reacted with 3 mL of 6 mol / L lithium hydroxide aqueous solution at RT for 15-20 h. After that, 20 mL of sodium chloride aqueous solution was added, and the mixture was extracted twice with 20 mL of dichloromethane. The pH of the aqueous phase was then adjusted to 4-5 with 4 M hydrochloric acid to precipitate the solid. The solid was filtered and dried to obtain 287 mg of compound c with the structure shown in formula (Ⅳ).
[0024] S3. Dissolve 287 mg (1.43 mmol) of compound c in 1 mL toluene-4 mL N-methylpyrrolidone, stir at 115-120 °C until completely dissolved, add 329 mg of compound d (CAS No.: 104-12-1, 2.14 mmol), and react at this temperature for 15-18 h. After the reaction is complete, cool to room temperature, add 30 mL of purified water, and adjust the pH to 3-4 with HCl. A gray solid precipitates, is filtered, and the solid is washed with ethyl acetate and dried to obtain 206 mg of diflubenzuron hapten with the structure shown in formula (I). The synthetic route of this diflubenzuron hapten is shown in [reference needed]. Figure 2 .
[0025] The prepared diflubenzuron hapten was identified by mass spectrometry, as shown in the attached figure in the instruction manual. Figure 1 .from Figure 1 It can be seen that the molecular ion peak of the diflubenzuron hapten is ESI-MS: 353 (M-1), which is consistent with the molecular weight of the compound 354, indicating that the diflubenzuron hapten with the structure shown in formula (Ⅰ) was successfully prepared.
[0026] Example 2: Synthesis of diflubenzuron immunogen and diflubenzuron coating antigen
[0027] 2.1 Preparation of diflubenzuron-coated precursor
[0028] The diflubenzuron-coated antigen was prepared using the diflubenzuron hapten obtained in Example 1, and the specific method is as follows:
[0029] (1) Take 10 mg of the diflubenzuron hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester;
[0030] (2) Weigh 35 mg of bovine serum albumin (BSA) and dissolve it completely in 3.5 mL of 0.05 mol / L CB buffer solution to form a bovine serum albumin solution. Add the hapten activated ester from step (1) dropwise slowly to the above bovine serum albumin solution while stirring, and stir at room temperature for 16-24 h.
[0031] (3) The solution obtained in step (2) was dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules, and the diflubenzuron coated antigen was obtained, aliquoted and stored at 4℃ for later use.
[0032] 2.2 Preparation of diflubenzuron immunogen
[0033] The diflubenzuron immunogen was prepared using the diflubenzuron hapten obtained in Example 1, and the specific method is as follows:
[0034] (1) Take 10 mg of the diflubenzuron hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester;
[0035] (2) Weigh 40 mg of lactoferrin (LF) and dissolve it completely in 4 mL of 0.05 mol / L CB buffer solution to form a lactoferrin solution. Add the hapten activated ester dropwise slowly to the above lactoferrin solution while stirring, and stir at room temperature for 16-24 h.
[0036] (3) The solution obtained in step (2) was dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules and obtain diflubenzuron immunogen. It was then aliquoted and stored at 4°C for later use.
[0037] Example 3: Preparation and Identification of Diflubenzuron Monoclonal Antibody
[0038] 3.1 Animal Immunization
[0039] The diflubenzuron immunogen prepared in Example 2 was emulsified with an equal volume of Freund's adjuvant and used to immunize BALB / c mice. The immunization dose per mouse was 50 μg to 100 μg, with an interval of 2 weeks between immunizations. After 3 immunizations, serum titers were measured by collecting tail vein blood from the mice. If the antibody titer did not meet the requirements, a booster immunization was required. Once the antibody titer no longer increased, a subcutaneous booster immunization was performed with 100 μg of the complete antigen.
[0040] 3.2 Hybridoma cell preparation
[0041] Five days after the final booster immunization, mouse spleen cells were fused with human osteosarcoma SP20 cells. The fused cells were screened in HAT medium, and after 5 days, the complete medium was replaced with HAT medium for further culture.
[0042] Cell supernatant was analyzed using ELISA. Cells in wells showing strong positive results were then subjected to limiting dilution clonal culture. Cells in wells showing positive results after three clonal culture assays were identified as hybridoma cells secreting monoclonal antibodies.
[0043] 3.3 Monoclonal Antibody Preparation
[0044] After hybridoma cells were cultured to a large scale, they were inoculated into the peritoneal cavity of mice to produce ascites containing antibodies. The ascites was purified by the octanoic acid-ammonium sulfate precipitation method to obtain high-purity and highly specific diflubenzuron monoclonal antibodies.
[0045] Example 4: ELISA performance evaluation of diflubenzuron monoclonal antibody
[0046] The antibody titer and inhibition rate were determined using an indirect competitive ELISA method. The specific method is as follows:
[0047] (1) Antigen coating Using a carbonate buffer solution at pH 9.6 as the coating diluent, the diflubenzuron coating agent prepared in Example 2 was diluted to 0.2 μg / mL and added to a polystyrene microplate at a rate of 100 μL / well. The plate was coated overnight at 4°C, dried, and washed three times with PBST.
[0048] (2) Closed Add 280 μL / well to phosphate buffer containing 1% wt BSA, block at 37°C for 1 h, spin dry, wash three times with PBST, dry and vacuum package for storage.
[0049] (3) Primary antibody dilution The diflubenzuron monoclonal antibody prepared in Example 3 was diluted to 0.2 μg / mL with phosphate buffer containing 0.05% wt sodium azide (pH 7.4) and stored at 4°C for later use.
[0050] (4) Preparation of standard solutions Dissolve diflubenzuron standard in 0.01M PBS to obtain diflubenzuron standard solutions with concentrations of 0, 0.05, 0.15, 0.45, 1.35 and 4.05 μg / L.
[0051] (5) Sample addition and primary antibody incubation Mix 50 μL of diflubenzuron monoclonal antibody with 100 μL of the corresponding diflubenzuron standard solution, react at room temperature for 10 min, then add 100 μL of the mixed solution to each well of an ELISA plate coated with diflubenzuron artificial antigen, react at 37 °C for 0.5 h, and then dry.
[0052] (6) Washing primary antibody Add 280 μL of PBST per well, wash 3 times, and then pat dry.
[0053] (7) Secondary antibody incubation Add 100 μL / well of HRP-labeled goat anti-mouse IgG enzyme-labeled secondary antibody and react at 37℃ for 0.5 h.
[0054] (8) Washing secondary antibodies Add 280 μL of PBST per well, wash three times, and then pat dry.
[0055] (9) Color development Add 100 μL / well of TMB colorimetric solution and react at 37°C for 15 min; then add 50 μL / well of 1 M sulfuric acid to stop the colorimetric reaction.
[0056] (10) Absorbance determination Place the microplate obtained in the previous step into the microplate reader, set the microplate reader to a wavelength of 450 nm and measure the OD value of each well. The specific results are shown in Table 1.
[0057] Table 1. OD values of different concentrations of diflubenzuron standard solutions tested by ELISA
[0058]
[0059] Using the data shown in Table 1, a four-parameter logistic curve was fitted using ELISA Calc software, and the standard curve shown in the figure below was obtained. Its linear equation is as follows: y = (AD) / [1 + (x / C)^B] + D, r² = 0.99813; where A = 1.23357, B = 0.96294, C = 0.13250, D = 0.12949, x represents the concentration of the analyte, and y represents the OD value. The IC50 of the diflubenzuron monoclonal antibody against diflubenzuron was calculated. 50 The value was 0.14 μg / L, and it showed a linear relationship within the range of diflubenzuron concentration from 0.05 μg / L to 4.05 μg / L.
[0060] Example 5: Preparation of diflubenzuron colloidal gold immunochromatographic reagent card
[0061] 5.1 Preparation of reaction membranes coated with artificial antigen and mouse IgG
[0062] Using nitrocellulose membrane (NC membrane) as the reaction membrane, the concentration of the coating buffer for diflubenzuron prepared in Example 2 was adjusted to 0.05~0.2 mg / mL, and the concentration of the coating buffer for mouse IgG was also adjusted to 0.1~0.5 mg / mL. The diflubenzuron coating agent and mouse IgG were sprayed onto the corresponding T line and C line of the reaction membrane at a membrane solution volume of 0.8~1.2 μL / cm, with a spacing of 2.5 mm between the T line and C line. The membrane was placed in a 45℃ oven for 12~16 h and then stored in a constant temperature and humidity incubator for later use. The coating buffer used was a pH=7.4 0.1M PB buffer containing 1% sucrose and 0.05% sodium azide.
[0063] 5.2 Preparation of microporous gold-labeled diflubenzuron monoclonal antibody
[0064] 5.2.1 Preparation of nano-gold solution
[0065] Dissolve 1g of chloroauric acid in pure water and sonicate, then bring the volume to 100mL. Store at 4°C protected from light. Add 2mL of the above solution to 100mL of pure water, heat to boiling, then add 0.5mL of 0.06% sodium citrate solution. Continue heating for 10 minutes, cool to room temperature, and then reconstitute the original volume with pure water. Store at room temperature protected from light. All glassware used must be soaked overnight in a mixture of potassium permanganate and sulfuric acid, then rinsed and dried before use.
[0066] 5.2.2 Labeling of diflubenzuron monoclonal antibodies
[0067] Aliquot 1 mL of gold nanoparticle solution into a vial and adjust the pH of the colloidal gold solution with 0.1 mol / L K₂CO₃. Add 8 μg of diflubenzuron monoclonal antibody to the gold solutions at different pH values and react at room temperature for 5 minutes. Observe the color change of the solution and record the pH value at which the solution remains red. Add 10 μL of 10% bovine serum albumin solution for blocking, centrifuge at 12,000 rpm for 10 minutes, and discard all supernatant.
[0068] 5.2.3 Preparation of micropores
[0069] Add 1 mL of a gold diluent containing 2% Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose to reconstitute the gold. Dispense 8 μL / well into microwells to obtain gold-labeled microwells. Dry at 37 °C for 16 hours and store for later use.
[0070] 5.3 Preparation of Sample Pads
[0071] The cut 30*30cm blank sample pads were immersed in the sample preparation solution for 5 minutes. After immersion, they were removed and dried at 37℃ for 16 hours. They were then placed in a constant temperature and humidity storage box for later use. The sample pad preparation solution used was 0.1M PB buffer containing 0.05% Tween 20, 1% sucrose, 0.5% Triton 405, and 0.05% sodium azide.
[0072] 5.4 Assembly of Colloidal Gold Qualitative Immunochromatographic Test Strips
[0073] The reaction membrane prepared in step 5.1 is stacked in the middle of the PVC board backing, and the sample pad and absorbent pad prepared in step 5.3 are stacked at both ends respectively. The reaction membrane is connected to the absorbent pad and the sample pad respectively. The detection area is close to the sample pad and the control area is close to the absorbent pad to obtain the test paper. The test paper is cut into 3mm test strips and the test strips are loaded into the test paper card to obtain the diflubenzuron colloidal gold immunochromatographic test paper card.
[0074] Example 6 Performance evaluation of diflubenzuron colloidal gold immunochromatographic test strips
[0075] Prepare a series of standard solutions of diflubenzuron at different concentrations of 0, 0.1, 0.5, 1, and 2 μg / L using 0.1 M PB buffer. Then, add 100 μL of the standard solution to the wells of the gold-labeled microparticles prepared in Example 5, and repeatedly pipette to reconstitute the solution. After standing for 3 minutes, transfer the solution from the gold-labeled microparticles to the sample wells of the diflubenzuron colloidal gold immunochromatographic test strip of this invention. Start timing after sample addition, and observe the results after 5-8 minutes. Results are invalid after 8 minutes.
[0076] Visual interpretation method: A stronger T-line than the C-line or no significant difference in color development indicates a negative result (-). A significantly weaker T-line than the C-line or no T-line indicates a positive result (+). Invalid: No C-line appears, indicating incorrect operation or a faulty test strip. This allows for rapid qualitative detection using colloidal gold immunochromatographic test strips. The results for different concentrations of diflubenzuron standard solutions are shown in Table 2.
[0077] Table 2. Determination results of diflubenzuron standard solutions at different concentrations
[0078]
[0079] As shown in Table 2, the diflubenzuron colloidal gold immunochromatographic test strip prepared in this invention has high sensitivity for the detection of diflubenzuron drugs, and the detection limit for diflubenzuron standard solution is 0.5 μg / L.
[0080] Example 7: Stability Test of Diflubenzuron Colloidal Gold Immunochromatographic Test Strips
[0081] The colloidal gold immunochromatographic test strips were stored at room temperature. To ensure the stability of the test strips, accelerated destructive experiments were conducted, placing them continuously at room temperature and 54°C for 60 days. Negative results and color changes of diflubenzuron standard solution were detected on days 0, 5, 10, 20, 30, 40, 50, and 60, respectively. The experiment was conducted in triplicate, and the results are shown in Table 3 below. "+" represents positive and "-" represents negative.
[0082] Table 3. Stability Results of Diflubenzuron Colloidal Gold Immunochromatographic Test Strips
[0083]
[0084] As shown in Table 3, after 60 days of sealed storage at room temperature and 45°C, the T / C colorimetric depth of the colloidal gold qualitative immunochromatographic test strips showed no significant change, indicating that the colloidal gold qualitative immunochromatographic test strips can be stably stored for at least 60 days at 45°C in accelerated experiments. Therefore, the diflubenzuron colloidal gold qualitative immunochromatographic test strips prepared in this invention can be stably stored at room temperature for more than one year, fully meeting the requirements of the market in terms of storage and transportation.
[0085] Example 8: Sample Performance Test of Diflubenzuron Colloidal Gold Immunochromatographic Test Strips
[0086] 8.1 Sample Pretreatment
[0087] Take 2 g of fruit and vegetable samples. Cut leafy vegetables into square pieces about 1 cm in size. For tubers, take cross-sectional samples or their epidermis. Place them in a 50 mL centrifuge tube, add 6 mL of 0.1 M PBS buffer, and vortex vigorously for 2 min (manual vortexing frequency: 120 times / min). After the centrifuge tube has stood for 2 min, the supernatant is the sample solution.
[0088] 8.2 Spiking of Sample Solution
[0089] Eight different varieties of blank samples (Chinese kale, Chinese cabbage, spinach, radish, lychee, plum, apple, and pear) were sequentially spiked with diflubenzuron standard solution to obtain a series of spiked sample solutions with concentrations of 0, 1, 5, 10, and 20 μg / kg.
[0090] 8.2 Measurement and Result Interpretation
[0091] Take 100 μL of the above-mentioned spiked sample solution and add it to the sample well of the diflubenzuron colloidal gold immunochromatographic test strip of the present invention. Start timing after adding the sample and observe the results according to the schematic diagram after 5-8 minutes.
[0092] Visual interpretation: A stronger T line than the C line, or no significant difference between the two, indicates a negative (-) sample. Positive (+): A visible C line, with the T line significantly weaker or absent, indicates a positive (+) sample. Invalid: No C line appears, indicating incorrect procedure or an expired test strip.
[0093] 8.3 Test Results
[0094] The test results of spiked samples from diflubenzuron colloidal gold immunochromatographic test strips are shown in Table 4.
[0095] Table 4. Results of spiked tests on fruit and vegetable samples
[0096]
[0097] As shown in the table above, the colloidal gold immunochromatographic test strip prepared in this invention exhibits good repeatability in the detection results of eight samples. When the diflubenzuron content in the samples is below 5 μg / kg, the detection results are all negative; when the diflubenzuron content is above 5 μg / kg, the detection results are all positive. Therefore, the detection limit for diflubenzuron in samples prepared in this invention is 5 μg / kg.
[0098] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.
Claims
1. Diflubenzuron hapten, characterized in that, Its structure is shown in equation (Ⅰ): Equation (Ⅰ).
2. The method for preparing the diflubenzuron hapten according to claim 1, characterized in that, Includes the following steps: S1. Compound a undergoes chlorination and amination reactions to yield compound b. The structural formula of compound a is shown in formula (II), and the structural formula of compound b is shown in formula (III). Formula (II) Formula (Ⅲ); S2. Compound b is hydrolyzed under alkaline conditions to give compound c, the structural formula of which is shown in formula (Ⅳ): Formula (Ⅳ); S3. The reaction of compound c and compound d yields a diflubenzuron hapten with the structure shown in formula (I), wherein the structural formula of compound d is shown in formula (V): , formula (V).
3. Diflubenzuron artificial antigen, characterized in that, The diflubenzuron artificial antigen is a conjugate of the diflubenzuron hapten and carrier protein as described in claim 1, and the structural formula of the diflubenzuron artificial antigen is shown in formula (VI): Formula (VI).
4. The artificial antigen for diflubenzuron according to claim 3, characterized in that, The carrier protein is any one of bovine serum albumin, ovalbumin, hemocyanin, or lactoferrin.
5. The application of the diflubenzuron hapten of claim 1 or the diflubenzuron artificial antigen of claim 3 in the non-disease diagnosis of the immunological detection of diflubenzuron.
6. The artificial antigen for diflubenzuron according to claim 3, characterized in that, The diflubenzuron artificial antigen includes a diflubenzuron immunogen and a diflubenzuron coating antigen. The diflubenzuron immunogen is obtained by conjugating the diflubenzuron hapten of claim 1 with lactoferrin, and the diflubenzuron coating antigen is obtained by conjugating the diflubenzuron hapten of claim 1 with bovine serum albumin.
7. A diflubenzuron antibody, characterized in that, The diflubenzuron antibody is prepared by animal immunization using the diflubenzuron artificial antigen described in claim 3, and the diflubenzuron antibody is a diflubenzuron monoclonal antibody.
8. The application of the diflubenzuron antibody as described in claim 7 in non-disease diagnosis of diflubenzuron in immunological detection.
9. An immunochromatographic test strip for detecting diflubenzuron, characterized in that, The immunochromatographic test strip has a T line and a C line, the T line is coated with the diflubenzuron-coated antigen as described in claim 6, and the C line is coated with IgG antibody.
10. A kit for detecting diflubenzuron, characterized in that, The kit comprises an immunochromatographic test strip for diflubenzuron detection as described in claim 9.