A diflubenzuron hapten, antigen, antibody, preparation method and application

Abstract

The application discloses a kind of diflufenican hapten, antigen, antibody and its preparation method and application.The application prepares diflufenican hapten as shown in formula (I) and corresponding diflufenican antigen, and specific antibody for detecting diflufenican is prepared by diflufenican antigen, and further constructs the immune analysis method for directly detecting diflufenican pesticide residue, which is particularly suitable for on-site rapid detection, and can meet the needs of regulatory authorities and detection agencies formula (I).

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology for food safety testing, and more specifically, to a diflubenzuron hapten, antigen, antibody, and their preparation methods and applications in immunological detection. Background Technology

[0002] Florasulam is a triazole pyrimidine sulfonamide selective herbicide. Due to its high activity and low dosage, it is widely used for weed control in crops such as wheat and corn, with its application scope and dosage increasing year by year, making it an ideal herbicide for wheat fields. However, its residues can enter the ecosystem and food chain through soil, water sources, or agricultural products, and their long-term accumulation may pose a potential threat to non-target organisms and human health.

[0003] Currently, the detection of diflubenzuron mainly relies on instrumental analysis methods such as liquid chromatography and ultra-high performance liquid chromatography-tandem mass spectrometry. While these methods offer high accuracy, they suffer from technical bottlenecks such as high equipment costs, complex sample pretreatment, and lengthy detection cycles, making them unsuitable for rapid on-site screening of pesticide residues. Immunological detection methods, however, demonstrate unique application potential in pesticide residue monitoring due to their high sensitivity, ease of operation, and lower cost. In particular, immunoassay methods based on antigen-antibody specific recognition maintain detection performance while enabling real-time on-site detection, significantly reducing the skill requirements for specialized operators. Establishing immunological detection techniques and applying them to the detection of diflubenzuron in agricultural products such as wheat and corn hinges on designing and synthesizing suitable diflubenzuron haptens and obtaining antibodies with high specificity and sensitivity. However, there are currently no reports on the synthesis of diflubenzuron haptens.

[0004] Therefore, there is an urgent need in this field to design and develop a suitable diflubenzuron hapten, and thereby establish a corresponding rapid detection method for diflubenzuron, so as to realize the rapid detection of diflubenzuron residues in agricultural products such as wheat and corn through immunological methods. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention designs a diflusulfonamide hapten and a diflusulfonamide antigen based on the structure of diflusulfonamide; and prepares a diflusulfonamide antibody that can specifically recognize and has suitable sensitivity, providing basic raw materials for the development of rapid immunoassay technology and products against diflusulfonamide.

[0006] In a first aspect, the present invention provides a diflubenzuron hapten, wherein the structure of the diflubenzuron hapten is shown in formula (I):

[0007] Formula (I)

[0008] Where n is 3, 4, 5 or 6.

[0009] In a second aspect, the present invention provides a method for preparing the diflubenzuron hapten of the first aspect of the present invention, the method comprising the following steps:

[0010] S1. Reaction of diflubenzuron with a demethoxylating agent generates intermediate 1.

[0011] ;

[0012] S2. Mix the intermediate 1 with (CH3)3C-OC(=O)-(CH2) n -Br reacts to produce intermediate 2, where n is 3, 4, 5, or 6.

[0013] ;

[0014] S3. Remove the tert-butyloxycarbonyl group from intermediate 2.

[0015] .

[0016] In a third aspect, the present invention provides a diflubenzuron antigen comprising the diflubenzuron hapten of the first aspect of the present invention and a carrier protein coupled to the diflubenzuron hapten.

[0017] In a fourth aspect, the present invention provides a diflubenzuron antibody, wherein the diflubenzuron antibody is an antibody that specifically recognizes the diflubenzuron antigen of the third aspect of the present invention.

[0018] In a fifth aspect, the present invention provides an immunoassay device for diflubenzuron, the device comprising an antibody of the fourth aspect of the present invention and an antigen of the third aspect of the present invention.

[0019] In a sixth aspect, the present invention provides the use of the diflubenzuron hapten of the first aspect of the present invention, the diflubenzuron antigen of the third aspect of the present invention, and / or the diflubenzuron antibody of the fourth aspect of the present invention for detecting diflubenzuron residues in agricultural products.

[0020] The beneficial effects of the present invention include at least one or more of the following:

[0021] The chemical reagents used in the preparation of the diflubenzuron hapten provided by this invention are readily available, the operation process is simple, the synthesis steps are concise and effective, the reaction yield is high, and the detection cost is low.

[0022] The antibody prepared using the hapten of this invention exhibits good specificity for diflubenzuron; specifically, the limit of detection for diflubenzuron by the antibody of this invention is 0.01 ng / mL, IC50... 50 The concentration was 0.67 ng / mL, which meets the limits for diflubenzuron in the latest national standard GB2763-2021 "National Food Safety Standard - Maximum Residue Limits for Pesticides in Food" (0.01 mg / kg for wheat, 0.02 mg / kg for corn, and 0.02 mg / kg for fresh corn). Therefore, the diflubenzuron detection device obtained in this way can achieve rapid detection without the need for large instruments such as liquid chromatography or mass spectrometry.

[0023] Compared with existing technologies, the detection device provided by this invention has advantages such as high sensitivity, strong specificity, low cost, simple operation, and short detection time, making it very suitable for the rapid detection of diflubenzuron residues in agricultural products such as wheat and corn. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other implementation schemes can be obtained based on these drawings without creative effort.

[0025] Figure 1 The mass spectrum of the diflubenzuron hapten of the present invention is shown.

[0026] Figure 2 This is an ultraviolet scan image of the diflubenzuron antigen (SF-LF) and its hapten (SF) and carrier protein (LF) in the embodiments of the present invention.

[0027] Figure 3 This is an ultraviolet scan image of the diflubenzuron antigen (SF-BSA) and its hapten (SF) and carrier protein (BSA) in the embodiments of the present invention.

[0028] Figure 4 A standard curve of the diflubenzuron monoclonal antibody of the present invention obtained by indirect competitive ELISA is shown. Detailed Implementation

[0029] The present invention will now be clearly and completely described in conjunction with its embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0031] The expressions "comprising," "including," or "basically / mainly composed of" as used herein are generally understood as open-ended expressions, meaning that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in some cases, the expressions "comprising," "including," or "basically / mainly composed of" may also be understood as closed-ended expressions, meaning that they include only the elements, components, parts, or method steps specifically listed after the expression, and exclude any other elements, components, parts, or method steps. Furthermore, in the context of this invention, many embodiments use the expression "composed of," which should be understood as a closed-ended expression, meaning that it includes only the elements, components, parts, or method steps specifically listed after the expression, and excludes any other elements, components, parts, or method steps.

[0032] As mentioned above, there is an urgent need in the field for a simple and rapid method for detecting diflubenzuron residues in agricultural products.

[0033] Therefore, in a first aspect, the present invention provides a diflubenzuron hapten, wherein the structure of the diflubenzuron hapten is as shown in formula (I):

[0034] Formula (I)

[0035] Where n is 3, 4, 5 or 6.

[0036] In one embodiment, the structure of the diflubenzuron hapten is shown in formula (II):

[0037] Equation (II).

[0038] The inventors unexpectedly discovered that the diflubenzuron hapten of this invention can be coupled with a carrier protein to form a complete diflubenzuron antigen, wherein the key pharmacodynamic groups in diflubenzuron are well exposed and can be coupled with the carrier protein by introducing an active group (-COOH). The formed diflubenzuron antigen has good immunogenicity, and antibodies with good specificity can be obtained after animal immunization.

[0039] In a second aspect, the present invention provides a method for preparing the diflubenzuron hapten of the first aspect of the present invention, the method comprising the following steps:

[0040] S1. Reaction of diflubenzuron with a demethoxylating agent generates intermediate 1.

[0041] ;

[0042] S2. Mix the intermediate 1 with (CH3)3C-OC(=O)-(CH2) n -Br reacts to produce intermediate 2, where n is 3, 4, 5, or 6.

[0043] ;

[0044] S3. Remove the tert-butyloxycarbonyl group from intermediate 2.

[0045] .

[0046] It should be understood that the synthesis of the above compounds can be achieved using chemical synthesis methods known in the art.

[0047] In one embodiment, the structure of the diflubenzuron hapten is shown in formula (II), and the method includes the following steps:

[0048] S1. Diflubenzuron technical grade was dissolved in a mixture of anhydrous dichloromethane and anhydrous tetrahydrofuran. After stirring and dissolving, the solution was cooled to below -20°C, and a dichloromethane solution of boron tribromide (BBr3) was added dropwise. After the reaction was completed, the dichloromethane was evaporated under reduced pressure, and the solution was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried, and purified by column chromatography to obtain intermediate 1.

[0049] ;

[0050] S2. Dissolve intermediate 1, tert-butyl 4-bromobutyrate, and potassium carbonate in DMF. After the reaction is complete, cool to room temperature and evaporate the DMF under reduced pressure. Add pure water / ethyl acetate and stir to dissolve. After standing, separate the organic layer. Extract the aqueous layer with ethyl acetate and combine the organic layers. Add anhydrous sodium sulfate and dry. Finally, filter and concentrate to obtain intermediate 2-1.

[0051] ;

[0052] S3. Take the intermediate 2-1, dissolve it in dichloromethane, add trifluoroacetic acid while stirring at room temperature, and after the reaction is complete, directly evaporate the solvent under reduced pressure and purify by column chromatography to obtain the diflubenzuron hapten shown in formula (II).

[0053] .

[0054] In a third aspect, the present invention provides a diflubenzuron antigen comprising the diflubenzuron hapten of the first aspect of the present invention and a carrier protein coupled to the diflubenzuron hapten.

[0055] In one embodiment, the carrier protein is bovine serum albumin, lactoferrin, human serum albumin, chicken oocyte albumin, bovine lactoferrin, or hemocyanin.

[0056] In a preferred embodiment, the carrier protein is bovine serum albumin or bovine lactoferrin.

[0057] In a more preferred embodiment, the diflubenzuron antigen is a conjugate of the diflubenzuron hapten of formula (II) and bovine serum albumin (BSA), which may also be referred to herein as diflubenzuron antigen-BSA or SF-BSA.

[0058] In a more preferred embodiment, the diflubenzuron antigen is a conjugate of the diflubenzuron hapten of formula (II) and bovine lactoferrin, which may also be referred to herein as diflubenzuron antigen-LF or SF-LF.

[0059] It is understandable that both diflubenzuron antigen-BSA and diflubenzuron antigen-LF can be used as immunization antigens to generate antibodies in animals, or as coating antigens for the detection of diflubenzuron.

[0060] In one specific implementation, diflubenzuron antigen-BSA is used as the coating antigen, and diflubenzuron antigen-LF is used as the immunizing antigen.

[0061] In a fourth aspect, the present invention provides a diflubenzuron antibody, wherein the diflubenzuron antibody is an antibody that specifically recognizes the diflubenzuron antigen of the third aspect of the present invention.

[0062] It should be noted that, in addition to specifically recognizing the diflusulfonamide antigen of the third aspect of the present invention, the diflusulfonamide antibody can also specifically recognize diflusulfonamide itself and the diflusulfonamide hapten of the first aspect of the present invention.

[0063] It should also be noted that, in the embodiments given in this application, the diflubenzuron antibody is obtained by immunizing animals such as mice with the diflubenzuron antigen of the third aspect of the present invention (polyclonal antibody), or by fusing B cells of immunized animals with myeloma cells to form hybridoma cells and then screening them through cloning (monoclonal antibody). However, those skilled in the art will know that the diflubenzuron antibody of the present invention can also be obtained by other methods, such as by recombinant methods.

[0064] In one embodiment, the diflubenzuron antibody is a monoclonal antibody or a polyclonal antibody.

[0065] In a preferred embodiment, the diflubenzuron antibody is a monoclonal antibody.

[0066] In a more preferred embodiment, the diflubenzuron antibody is a monoclonal antibody that specifically recognizes the diflubenzuron antigen containing the diflubenzuron hapten shown in formula (II).

[0067] In this invention, a monoclonal antibody was prepared by using diflubenzuron antigen-LF as the antigen for immunization, with a limit of detection of 0.01 ng / mL and a half-maximum inhibitory concentration (IC50) of 1 / 2. 50 The value was 0.67 ng / mL, and the linear range was 0.05-8.49 ng / mL. The cross-reactivity rate to common structural analogs such as pyrimethanil, chlorpyrimethanil, methoxysulfuron, diflubenzuron, pyrimisulfuron, and penoxsulam was less than 0.01%, indicating that the antibody generated by the hapten of the present invention has good specificity to diflubenzuron and can effectively eliminate interference from other structural analogs.

[0068] In a fifth aspect, the present invention provides an immunoassay device for diflubenzuron, the device comprising a diflubenzuron antibody according to a fourth aspect of the present invention and a diflubenzuron antigen according to a third aspect of the present invention.

[0069] In one embodiment, the device is a colloidal gold immunochromatographic assay device, comprising a sample pad, a colloidal gold conjugating pad, a nitrocellulose membrane, and an absorbent pad, wherein a detection line and a control line are sequentially arranged on the nitrocellulose membrane, and the detection line is made of diflubenzuron antigen according to the third aspect of the present invention.

[0070] In one specific embodiment, the detection line is made from the diflubenzuron antigen-BSA of the present invention.

[0071] In one embodiment, the device is an enzyme-linked immunosorbent assay (ELISA) device, which includes a diflubenzuron antibody according to the fourth aspect of the present invention, a diflubenzuron antigen according to the third aspect of the present invention, a second antibody corresponding to the diflubenzuron antibody according to the fourth aspect of the present invention, an ELISA plate, a diflubenzuron standard solution, and a TMB colorimetric solution.

[0072] In one embodiment, the diflubenzuron antibody is obtained by immunizing animals with the diflubenzuron antigen of the diflubenzuron hapten shown in formula (II) of the present invention, fusing the B cells of the immunized animals with myeloma cells to form hybridoma cells, and then screening by cloning. However, it is understood that the preparation method of the diflubenzuron antibody of the present invention is not limited to this. For example, after obtaining the heavy chain and light chain sequences of the diflubenzuron antibody, the diflubenzuron antibody of the present invention can be produced by recombinant methods.

[0073] In one specific implementation, the diflubenzuron antibody is a monoclonal antibody obtained by immunizing animals with the diflubenzuron antigen-LF of the present invention, fusing the B cells of the immunized animals with myeloma cells to form hybridoma cells, and then screening through cloning.

[0074] As described above, the diflubenzuron antibody of the present invention can not only specifically recognize the diflubenzuron antigen of the third aspect of the present invention, but also specifically recognize diflubenzuron itself and the diflubenzuron hapten of the first aspect of the present invention, which provides a basis for using the antibody in the detection of diflubenzuron.

[0075] In a sixth aspect, the present invention provides the use of the diflubenzuron hapten of the first aspect of the present invention, the diflubenzuron antigen of the third aspect of the present invention, and / or the diflubenzuron antibody of the fourth aspect of the present invention for detecting diflubenzuron residues in agricultural products.

[0076] In one implementation, the agricultural products include wheat, corn, oats, rice, soybeans, and peanuts.

[0077] As described above, the diflubenzuron hapten, antigen, and antibody of this invention enable rapid detection of diflubenzuron residues in various agricultural products, which can greatly meet the actual detection requirements in the field and provide core reagents for the subsequent development of immunoassay methods for diflubenzuron.

[0078] Example

[0079] The present invention will be further described in detail through specific embodiments. 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.

[0080] Example 1: Synthesis and Identification of Diflubenzuron Hapten

[0081] The preparation method of diflubenzuron hapten includes the following steps:

[0082] S1. Add 1.5 g of diflubenzuron technical grade to a 250 mL reaction flask, dissolve it with 10 mL of anhydrous dichloromethane and 10 mL of anhydrous tetrahydrofuran, and then cool to below -20°C. Slowly add 3.2 g of BBr3 dichloromethane solution, and after the addition is complete, slowly raise the temperature to room temperature to continue the reaction. After the reaction is complete, evaporate most of the dichloromethane under reduced pressure, then add ethyl acetate to dissolve it, and then quench the solution in ice water. Extract the aqueous layer multiple times with ethyl acetate and wash it multiple times with saturated brine. Finally, concentrate and purify by column chromatography to obtain 0.8 g of white powder intermediate 1 (5-hydroxydiflubenzuron).

[0083] .

[0084] S2. In a 500 mL reaction flask, add 0.8 g of intermediate 1, 0.5 g of tert-butyl 4-bromobutyrate, and 0.14 g of potassium carbonate. Dissolve the mixture in 20 mL of DMF by stirring, and react overnight at 30 °C. After the reaction is complete, cool to room temperature and evaporate the DMF under reduced pressure. Add pure water / ethyl acetate to the obtained concentrate and stir to dissolve. After standing, separate the organic layer. Extract the aqueous layer multiple times with ethyl acetate, then combine the organic layers and dry with anhydrous sodium sulfate. Finally, filter and concentrate to obtain 0.5 g of intermediate 2-1 (tert-butyl 5-hydroxydiflubenzuron-4-butyrate).

[0085] .

[0086] S3. Add 0.5 g of the above intermediate 2-1 to a 50 mL reaction flask, dissolve in 4 mL of dichloromethane, add 1 mL of trifluoroacetic acid while stirring at room temperature, and react at room temperature for 3 h. After the starting material has reacted completely, evaporate the solvent under reduced pressure, and directly purify by column chromatography to obtain 0.35 g of the final product, which is the diflubenzuron hapten shown in formula (II).

[0087] .

[0088] The obtained diflubenzuron hapten was identified by mass spectrometry, and the mass spectrum obtained is shown below. Figure 1 As shown in the mass spectrum, the molecular ion peak of the diflubenzuron hapten is at 430.26 [M+H]. + The result is consistent with the molecular weight of the diflubenzuron hapten, which is 431.35, indicating that the diflubenzuron hapten shown in formula (II) was successfully synthesized.

[0089] Example 2: Preparation and identification of diflubenzuron immunogen and coating antigen

[0090] Preparation of diflubenzuron immunogen: Weigh 20 mg of diflubenzuron hapten and dissolve it in 1 mL of dimethyl sulfoxide (DMSO). Add 40 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mg of N-hydroxysuccinimide (NHS), and activate at room temperature for 1 h to prepare the activated solution. Add the activated solution dropwise to 40 mg of bovine lactoferrin (LF) (dissolved in 3.0 mL of PBS, pH 7.4), and stir overnight at 4 °C. Dialyze with PBS for 3 days (changing the medium 3 times a day) to remove excess reagents, and obtain the diflubenzuron immunogen. Aliquot the immunogen and store it at -20 °C.

[0091] Preparation of the diflubenzuron-coated antigen: Weigh 15 mg of diflubenzuron hapten and dissolve it in 1 mL of dimethyl sulfoxide (DMSO). Add 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 15 mg of N-hydroxysuccinimide (NHS) and activate for 1 h. Add the activated solution dropwise to 30 mL of bovine serum albumin (BSA) (dissolved in 3.0 mL of carbonate buffer, pH 9.6) and stir at 4 °C for 4 h. Dialyze with PBS for 3 days. After dialysis, centrifuge at 4000 rpm for 5 min, collect the supernatant to obtain the diflubenzuron-coated antigen, aliquot it, and store it at -20 °C.

[0092] Identification of the diflubenzuron immunogen and coating antigen: The carrier protein, diflubenzuron hapten, diflubenzuron hapten-LF conjugate, and diflubenzuron hapten-BSA conjugate were prepared into 1.0 mg / mL solutions using 0.01 mol / L PBS buffer (pH 7.4 and 0.01 mol / L, respectively). The solution was zeroed with PBS buffer at pH 7.4. The wavelengths were scanned using a UV spectrophotometer in the range of 200–400 nm. The absorption spectra of the carrier protein, diflubenzuron hapten, diflubenzuron hapten-LF conjugate, and diflubenzuron hapten-BSA conjugate are shown below. Figure 2-3 As shown in the figure, the diflubenzuron hapten successfully coupled with the carrier proteins LF and BSA.

[0093] Example 3: Preparation and Identification of Diflubenzuron Monoclonal Antibody

[0094] Animal Immunization: Healthy 6-8 week old BALB / c mice were selected for immunization. The diflubenzuron immunogen prepared in Example 2 was mixed and emulsified with an equal amount of Freund's adjuvant, and then injected into BALB / c mice via multiple sites including the neck, back, and subcutaneous injections (the first immunization used Freund's complete adjuvant, and booster immunizations used Freund's incomplete adjuvant). A second immunization was administered 2 weeks later, followed by booster immunizations every 2 weeks. One week after the fourth booster immunization, blood was collected from the tail of the mice, and serum titers were determined using an indirect competitive ELISA. When the titer no longer increased, a booster immunization was administered via intraperitoneal injection. Blood was collected from the heart 3 days later, incubated in water for 0.5-1 h, centrifuged at 4°C and 10,000 rpm for 15 min, and the supernatant was used as antiserum, which is the polyclonal antibody.

[0095] Cell fusion and cloning: Selecting polyclonal antibodies with high titers and IC50 values. 50 Hybridization was performed in mice with low viral load. Polyethylene glycol (PEG) was used to mix spleen cells from immunized BALB / c mice with SP2 / 0 myeloma cells for cell fusion. After subcloning and four limiting dilutions, a portion of the positive hybridoma cells was cryopreserved, and the other portion was injected into the peritoneal cavity of mice to generate ascites. Ascites was collected seven days later and purified using a protein G immunoaffinity column to obtain monoclonal antibodies.

[0096] Identification of monoclonal antibodies: Carbonate buffer (CBS, pH=9.6) was used as the dilution buffer for the diflubenzuron coating antigen, phosphate buffer (PBS, 0.01 M, pH=7.4) was used as the dilution buffer for both the monoclonal antibodies and standards, and Tween phosphate buffer (PBST, 0.01 M) was used as the dilution buffer for the horseradish peroxidase-labeled goat anti-mouse solution. The diflubenzuron coating antigen was diluted to 1 μg / mL and added to 100 μL per well of a 96-well microplate, incubated overnight at 4°C. After washing twice with PBST, 120 μL of 5% bovine serum albumin was added to each well, and the plate was incubated at 37°C for 3 h. The plate was then centrifuged and dried at 37°C for 1 h. The resulting monoclonal antibodies were diluted 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively, and 1 μg / mL diflubenzuron standard was added. The above solution was added to a 96-well plate coated with diflubenzuron antigen using a standard indirect competitive ELISA method. The plate was incubated at 37°C for 40 min, washed 5 times with PBST, and then 100 μL of horseradish peroxidase-labeled goat anti-mouse solution (5000-fold diluted) was added. The plate was incubated at 37°C for 30 min, washed 5 times with PBST, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate chromogenic solution was added. The plate was incubated again at 37°C for 10 min, and the reaction was terminated with 10% concentrated sulfuric acid. The absorbance data were read on a 450 nm microplate reader, and a standard curve was plotted. The standard inhibition curve of diflubenzuron is shown below. Figure 4 As shown.

[0097] Example 4: Specificity evaluation of diflubenzuron monoclonal antibody

[0098] Using an indirect competitive ELISA method, the limit of detection (LOD) of the obtained diflubenzuron monoclonal antibody against diflubenzuron was determined to be 0.01 ng / mL, and the IC50 was [missing value]. 50 The effective concentration was 0.67 ng / mL, with a linear range of 0.05–8.49 ng / mL. Furthermore, its IC50 values ​​against pyrimethanil, chlorpyrifos, methoxysulfuron, diflubenzuron, pyrazosulfuron, and penoxsulam were also validated. 50 and cross-reactivity rate.

[0099] Cross-reactivity rate = (IC50 of diflubenzuron) 50 ICs of other similar types 50 The result is shown in Table 1, calculated as 100% × 100%.

[0100] Table 1

[0101]

[0102] Example 5: Colloidal gold test strip for the detection of diflubenzuron

[0103] This embodiment provides a colloidal gold test strip, comprising a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a detection line (T line) and a control line (C line) sequentially arranged on it. The colloidal gold conjugate pad is coated with a diflubenzuron monoclonal antibody prepared according to Example 3. The detection line is obtained by streaking with diflubenzuron-coated antigen. The control line is obtained by streaking with goat anti-mouse IgG secondary antibody. The assembly method of the colloidal gold test strip can be any method commonly used in the art.

[0104] The principle of the colloidal gold test strip for detecting diflubenzuron in this embodiment is as follows: It utilizes the indirect competition method to detect the presence of diflubenzuron in the sample. If the sample does not contain diflubenzuron, the test line remains unchanged, while the control line develops color. If the sample contains diflubenzuron, both the test line and the control line develop color. The detection of diflubenzuron can be achieved using methods commonly used in the art.

[0105] Example 6: Diflubenzuron Enzyme-Linked Immunosorbent Assay Kit

[0106] This embodiment provides a diflubenzuron enzyme-linked immunosorbent assay kit, which includes diflubenzuron monoclonal antibody prepared according to Example 3, an ELISA plate, diflubenzuron coated antigen, diflubenzuron standard solution, goat anti-mouse IgG secondary antibody and TMB colorimetric solution.

[0107] The principle of the diflubenzuron ELISA kit for detecting diflubenzuron is as follows: The indirect competitive ELISA method is used to detect the diflubenzuron content in the sample. The microwells of the ELISA plate are pre-coated with diflubenzuron-coated antigen. Diflubenzuron standard solution or the sample to be tested, diflubenzuron monoclonal antibody, goat anti-mouse IgG secondary antibody, and TMB chromogenic solution are added to construct a diflubenzuron standard inhibition curve. Based on the diflubenzuron standard inhibition curve and the absorbance value of the sample to be tested, the diflubenzuron content in the sample is determined. The detection of diflubenzuron can be achieved using methods commonly used in this field.

[0108] 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. A bispyribac-sodium hapten, wherein the structure of the bispyribac-sodium hapten is shown as formula (I): Formula (I); wherein n is 3, 4, 5 or 6.

2. The bispyribac-sodium hapten according to claim 1, wherein the structure of the bispyribac-sodium hapten is shown as formula (II): Formula (II).

3. A method for preparing the bispyribac-sodium hapten of claim 1, the method comprising the following steps: S1. reacting bispyribac-sodium with a demethoxy reagent to form intermediate 1, ； S2. reacting said intermediate 1 with (CH3)3C-O-C(=O)-(CH2) n -Br to form intermediate 2, wherein n is 3, 4, 5 or 6, ； S3. removing the tert-butyloxycarbonyl group in the intermediate 2, 。 4. A bispyribac-sodium antigen comprising the bispyribac-sodium hapten of claim 1 or 2 and a carrier protein coupled to the bispyribac-sodium hapten.

5. The bispyribac-sodium antigen according to claim 4, wherein the carrier protein is bovine serum albumin, lactoferrin, human serum albumin, chicken egg white albumin, bovine lactoferrin or hemocyanin.

6. Use of the bispyribac-sodium hapten according to claim 1 or 2, or the bispyribac-sodium antigen according to claim 4 or 5 for detecting bispyribac-sodium residues in agricultural products.

7. The use according to claim 6, wherein the agricultural products comprise wheat, corn, oat, rice, soybean, peanut.

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