Florasulam hapten, florasulam antigen, florasulam antibody and preparation method and application thereof

Abstract

The invention discloses a florasulam hapten, a florasulam antigen, a florasulam antibody as well as a preparation method and application of the florasulam hapten. The florasulam hapten as shown in the formula (I) and the corresponding florasulam antigen are prepared, a specific antibody capable of being used for detecting florasulam is prepared through the florasulam antigen, and an immunoassay method for directly detecting florasulam pesticide residues is further constructed. The method is particularly suitable for on-site rapid detection, and can meet the requirements of supervision departments and detection organizations as shown in the formula (I).

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection of food safety detection, and more specifically, to a florasulam hapten, antigen, antibody, preparation methods thereof, and applications thereof in immunological detection. Background Art

[0002] Florasulam, a selective triazole pyrimidine sulfonamide herbicide, is widely used for weed control in crops such as wheat and corn due to its high activity and low use rate. Its scope and usage are increasing annually, making it an ideal agent for weed control in wheat fields. However, its residues can enter ecosystems and food chains through soil, water, or agricultural products. Long-term accumulation of these residues may pose a potential threat to non-target organisms and human health.

[0003] Currently, the detection of florasulam is primarily based on instrumental analysis methods such as liquid chromatography and ultra-high performance liquid chromatography-tandem mass spectrometry. While these methods offer high accuracy, they face technical bottlenecks such as high equipment costs, complex sample pretreatment, and lengthy testing cycles, making them inadequate for rapid on-site screening of pesticide residues. Immunological assays, however, offer unique potential for pesticide residue monitoring due to their high sensitivity, ease of use, and low cost. Immunoassays based on antigen-antibody specific recognition, in particular, maintain high performance while enabling on-site, immediate testing, significantly reducing the need for specialized operator skills. The key to establishing and applying immunological assays for the detection of florasulam in agricultural products such as wheat and corn lies in the design and synthesis of suitable florasulam haptens and the production of highly specific and sensitive antibodies. However, there are currently no reports on the synthesis of florasulam haptens.

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

[0005] In view of the problems existing in the prior art, the present invention designs the bispyribac hapten and bispyribac antigen based on the structure of bispyribac; and thereby prepares bispyribac antibodies that can specifically recognize and have suitable sensitivity, providing basic raw materials for the development of rapid immune detection technology and products for bispyribac.

[0006] In a first aspect, the present invention provides a florasulam hapten, wherein the structure of the florasulam hapten is shown in formula (I): Formula (I) Wherein, n is 3, 4, 5 or 6.

[0007] In a second aspect, the present invention provides a method for preparing the florasulam hapten of the first aspect of the present invention, the method comprising the following steps: S1. reacting florasulam with a demethoxy reagent to generate intermediate 1, ; S2. The intermediate 1 and (CH3)3C-OC(=O)-(CH2) n -Br reaction generates intermediate 2, where n is 3, 4, 5 or 6, ; S3. removing the tert-butyloxycarbonyl group in the intermediate 2, .

[0008] In a third aspect, the present invention provides a florasulam antigen, which comprises the florasulam hapten of the first aspect of the present invention and a carrier protein coupled to the florasulam hapten.

[0009] In a fourth aspect, the present invention provides a florasulam antibody, wherein the florasulam antibody is an antibody that specifically recognizes the florasulam antigen of the third aspect of the present invention.

[0010] In a fifth aspect, the present invention provides an immunoassay device for florasulam, the device comprising the antibody of the fourth aspect of the present invention and the antigen of the third aspect of the present invention.

[0011] In a sixth aspect, the present invention provides use of the florasulam hapten of the first aspect of the present invention, the florasulam antigen of the third aspect of the present invention, and / or the florasulam antibody of the fourth aspect of the present invention for detecting florasulam residues in agricultural products.

[0012] The beneficial effects of the present invention are at least one or more of the following: The chemical reagents used in the preparation of the florasulam hapten provided by the present invention are easily available, the operation process is simple, the synthesis steps are concise and effective, the reaction yield is high, and the detection cost is low.

[0013] The antibody prepared by the hapten of the present invention has good specificity for florasulam; specifically, the minimum detection limit of the antibody of the present invention for florasulam is 0.01 ng / mL, and the IC 50The concentration was 0.67 ng / mL, meeting the limits for florasulam in the recently promulgated national standard GB2763-2021, "National Food Safety Standard: Maximum Residue Limits of Pesticides in Food" (0.01 mg / kg for wheat, 0.02 mg / kg for corn, and 0.02 mg / kg for fresh corn). The resulting florasulam detection device achieves rapid detection without the need for large instruments such as liquid chromatography or mass spectrometry.

[0014] Compared with the existing technology, the detection device provided by the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, and short detection time. It is very suitable for the rapid detection of florasulam residues in agricultural products such as wheat and corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The mass spectrum of the florasulam hapten of the present invention is shown.

[0017] Figure 2 This is an ultraviolet scanning image of florasulam antigen (SF-LF), its hapten (SF), and carrier protein (LF) in an embodiment of the present invention.

[0018] Figure 3 This is an ultraviolet scanning image of florasulam antigen (SF-BSA), its hapten (SF), and carrier protein (BSA) in an embodiment of the present invention.

[0019] Figure 4 The standard curve of the florasulam monoclonal antibody of the present invention obtained by indirect competitive ELISA is shown. DETAILED DESCRIPTION

[0020] The present invention will be described clearly and completely below in conjunction with the embodiments and accompanying drawings of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments that can be obtained by a person of ordinary skill in the art based on the embodiments of the present invention fall within the scope of protection of the present invention.

[0021] 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 of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0022] The expressions “comprising”, “including” or “essentially / mainly consisting of…” used in this document can generally be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions “comprising”, “including” or “essentially / mainly consisting of…” can also be understood as closed-ended expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In addition, in the context of the present invention, many embodiments use the expression “consisting of…”, which should be understood as a closed-ended expression, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included.

[0023] As described above, there is an urgent need in the art for a method that is simple to operate and can quickly detect florasulam residues in agricultural products.

[0024] Therefore, in a first aspect, the present invention provides a bispyribac hapten, wherein the structure of the bispyribac hapten is shown in formula (I): Formula (I) Wherein, n is 3, 4, 5 or 6.

[0025] In one embodiment, the structure of the florasulam hapten is shown in formula (II): Formula (II).

[0026] The inventors unexpectedly discovered that the florasulam hapten of the present invention can be coupled to a carrier protein to form a complete florasulam antigen. The key pharmacophore of florasulam is well exposed, and the introduction of a reactive group (-COOH) facilitates coupling to the carrier protein. The resulting florasulam antigen exhibits excellent immunogenicity and can produce highly specific antibodies after animal immunization.

[0027] In a second aspect, the present invention provides a method for preparing the florasulam hapten of the first aspect of the present invention, the method comprising the following steps: S1. reacting florasulam with a demethoxy reagent to generate intermediate 1, ; S2. The intermediate 1 and (CH3)3C-OC(=O)-(CH2) n -Br reaction generates intermediate 2, where n is 3, 4, 5 or 6, ; S3. removing the tert-butyloxycarbonyl group in the intermediate 2, .

[0028] It should be understood that the synthesis of the above compounds can be achieved using chemical synthesis methods known in the art.

[0029] In one embodiment, the structure of the florasulam hapten is as shown in formula (II), and the method comprises the following steps: S1. Dissolve the original drug of florasulam in a mixture of anhydrous dichloromethane and anhydrous tetrahydrofuran, stir and dissolve, then cool to below -20°C. Add a dichloromethane solution of boron tribromide (BBr3) dropwise. After the reaction is complete, dichloromethane is evaporated under reduced pressure. Extract with ethyl acetate, wash with saturated sodium chloride solution, dry, and purify by column chromatography to obtain intermediate 1. ; S2. The intermediate 1, tert-butyl 4-bromobutyrate and potassium carbonate were dissolved in DMF. After the reaction, the mixture was cooled to room temperature and evaporated to dryness under reduced pressure. Pure water / ethyl acetate was added and stirred to dissolve. After standing, the organic layer was separated. The aqueous layer was extracted with ethyl acetate and the organic layers were combined, dried over anhydrous sodium sulfate, and finally filtered and concentrated to obtain intermediate 2-1. ; S3. Take the intermediate 2-1, add dichloromethane to dissolve it, add trifluoroacetic acid under stirring at room temperature, and directly evaporate the solvent under reduced pressure after the reaction is completed. Purify it by column chromatography to obtain the bispyribac hapten represented by formula (II). .

[0030] In a third aspect, the present invention provides a florasulam antigen, which comprises the florasulam hapten of the first aspect of the present invention and a carrier protein coupled to the florasulam hapten.

[0031] In one embodiment, the carrier protein is bovine serum albumin, lactoferrin, human serum albumin, chicken ovalbumin, bovine lactoferrin, or hemocyanin.

[0032] In a preferred embodiment, the carrier protein is bovine serum albumin or bovine lactoferrin.

[0033] In a more preferred embodiment, the florasulam antigen is a conjugate of a florasulam hapten of formula (II) and Bovine Serum Albumin (BSA), which can also be referred to herein as florasulam antigen-BSA or SF-BSA.

[0034] In a more preferred embodiment, the florasulam antigen is a conjugate of a florasulam hapten of formula (II) and Bovine Lactoferrin, which can also be referred to herein as florasulam antigen-LF or SF-LF.

[0035] It is understood that both florasulam antigen-BSA and florasulam antigen-LF can be used as immunizing antigen to generate antibodies in animals, or as coating antigen for the detection of florasulam.

[0036] In a particular embodiment, florasulam antigen-BSA is used as coating antigen and florasulam antigen-LF is used as immunizing antigen.

[0037] In a fourth aspect, the present application provides a florasulam antibody, wherein the florasulam antibody is an antibody specifically recognizing a florasulam antigen of the third aspect of the present application.

[0038] It is noted here that in addition to specifically recognizing a florasulam antigen of the third aspect of the present application, the florasulam antibody can also specifically recognize florasulam itself as well as a florasulam hapten of the first aspect of the present application.

[0039] It is further noted that in the examples given in the present application, the florasulam antibody is obtained by immunizing an animal such as a mouse with a florasulam antigen of the third aspect of the present application (polyclonal antibody), by fusing B-cells of the immunized animal with myeloma cells to form hybridoma cells, and by cloning and screening (monoclonal antibody), but the skilled person is aware that the florasulam antibody of the present application can also be obtained by other methods such as by recombinant methods.

[0040] In an embodiment, the florasulam antibody is a monoclonal antibody or a polyclonal antibody.

[0041] In a preferred embodiment, the florasulam antibody is a monoclonal antibody.

[0042] In a more preferred embodiment, the florasulam antibody is a monoclonal antibody specifically recognizing a florasulam antigen comprising a florasulam hapten of formula (II).

[0043] In the present invention, a monoclonal antibody was prepared by using florasulam antigen-LF as an immunization antigen, and the minimum detection limit was 0.01 ng / mL and the half inhibitory concentration IC 50 The value was 0.67 ng / mL, and the linear range was 0.05-8.49 ng / mL. The cross-reactivity rates to common structural analogues such as fluazifop, clomiphenesulfuron, oxazolidinone, bispyribac, formisulfuron, and penoxsulam were all lower than 0.01%, indicating that the antibody generated by the hapten of the present invention has good specificity for bispyribac and can effectively eliminate the interference of other structural analogues.

[0044] In a fifth aspect, the present invention provides an immunoassay device for florasulam, the device comprising the florasulam antibody of the fourth aspect of the present invention and the florasulam antigen of the third aspect of the present invention.

[0045] In one embodiment, the device is a colloidal gold immunochromatographic detection device, which includes a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorbent pad, wherein a detection line and a quality control line are sequentially provided on the nitrocellulose membrane, and the detection line is made of the bispyribac antigen of the third aspect of the present invention.

[0046] In a specific embodiment, the test line is made of the florasulam antigen-BSA of the present invention.

[0047] In one embodiment, the device is an enzyme-linked immunosorbent assay device, which includes the florasulam antibody of the fourth aspect of the present invention, the florasulam antigen of the third aspect of the present invention, a second antibody corresponding to the florasulam antibody of the fourth aspect of the present invention, an ELISA plate, a florasulam standard solution, and a TMB colorimetric solution.

[0048] In one embodiment, the florasulam antibody is obtained by immunizing an animal with a florasulam antigen comprising the florasulam hapten represented by Formula (II) of the present invention, fusing B cells from the immunized animal with myeloma cells to form hybridoma cells, and then screening by cloning. However, it should be understood that the method for preparing the florasulam antibody of the present invention is not limited to this. For example, after obtaining the heavy and light chain sequences of the florasulam antibody, the florasulam antibody of the present invention can be produced by recombinant means.

[0049] In a specific embodiment, the florasulam antibody is a monoclonal antibody obtained by immunizing an animal with the florasulam antigen-LF of the present invention, fusing B cells of the immunized animal with myeloma cells to form hybridoma cells, and then screening them through cloning.

[0050] As described above, the florasulam antibody of the present invention can not only specifically recognize the florasulam antigen of the third aspect of the present invention, but can also specifically recognize florasulam itself and the florasulam hapten of the first aspect of the present invention, which provides a basis for using the antibody in the detection of florasulam.

[0051] In a sixth aspect, the present invention provides use of the florasulam hapten of the first aspect of the present invention, the florasulam antigen of the third aspect of the present invention, and / or the florasulam antibody of the fourth aspect of the present invention for detecting florasulam residues in agricultural products.

[0052] In one embodiment, the agricultural products include wheat, corn, oats, rice, soybeans, and peanuts.

[0053] As described above, the bispyribac hapten, antigen and antibody of the present invention can be used to quickly detect bispyribac residues in various agricultural products, which can greatly meet the actual detection requirements in this field and provide core reagents for the subsequent development of immunoassay methods for bispyribac.

[0054] Example The present invention will be further described in detail by way of specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of protection of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art are all within the meaning of the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available raw materials and reagents.

[0055] Example 1: Synthesis and identification of florasulam hapten The preparation method of florasulam hapten comprises the following steps: S1. Add 1.5 g of florasulam technical to a 250 mL reaction flask, stir and dissolve with 10 mL of anhydrous dichloromethane and 10 mL of anhydrous tetrahydrofuran, then cool to below -20°C. Slowly add 3.2 g of BBr3 dichloromethane solution dropwise. After completion of the addition, slowly raise the temperature to room temperature to allow the reaction to proceed. After the reaction is complete, evaporate most of the dichloromethane under reduced pressure, then add ethyl acetate to dissolve, and then pour the solution into ice water to quench. The aqueous layer is extracted multiple times with ethyl acetate and washed multiple times with saturated brine. Finally, it is concentrated and purified by column chromatography to obtain 0.8 g of white powder intermediate 1 (5-hydroxy florasulam). .

[0056] S2. Add 0.8 g of intermediate 1, 0.5 g of tert-butyl 4-bromobutyrate, and 0.14 g of potassium carbonate to a 500 mL reaction flask, stir and dissolve with 20 mL of DMF, and react at 30°C overnight. After the reaction, cool to room temperature and evaporate the DMF under reduced pressure. Add pure water / ethyl acetate to the obtained concentrate, stir and dissolve. After standing, separate the organic layer, extract the aqueous layer several times with ethyl acetate, then combine the organic layers and add anhydrous sodium sulfate to dry. Finally, filter and concentrate to obtain 0.5 g of intermediate 2-1 (tert-butyl 5-hydroxybispyribac-4-butyrate). .

[0057] S3. Add 0.5 g of the intermediate 2-1 to a 50 mL reaction flask, dissolve it 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 reaction is complete, evaporate the solvent under reduced pressure and directly purify it by column to obtain 0.35 g of the final product, which is the bispyribac hapten represented by formula (II): .

[0058] The obtained florasulam hapten was identified by mass spectrometry, and the obtained mass spectrum was as follows: Figure 1 As shown in the mass spectrum, the molecular ion peak of florasulam hapten is 430.26[M+H] + , which is consistent with the molecular weight of the florasulam hapten, 431.35, indicating that the florasulam hapten represented by formula (II) was successfully synthesized.

[0059] Example 2: Preparation and identification of florasulam immunogen and coating Preparation of the florasulam immunogen: 20 mg of the florasulam hapten was dissolved in 1 mL of dimethyl sulfoxide (DMSO). 40 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mg of N-hydroxysuccinimide (NHS) were added and activated at room temperature for 1 h to prepare an activation solution. This solution was added dropwise to 40 mg of bovine lactoferrin (LF) dissolved in 3.0 mL of PBS, pH 7.4, and stirred overnight at 4°C. The immunogen was dialyzed against PBS for 3 days (with three changes of PBS daily) to remove excess reagents. The resulting solution was aliquoted and stored frozen at -20°C.

[0060] Preparation of diflufenican-coated antigen: 15 mg of diflufenican hapten was dissolved in 1 mL of dimethyl sulfoxide (DMSO), and 30 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 15 mg of N-hydroxysuccinimide (NHS) were added, and activated for 1 h. The activated solution was added dropwise to 30 mL of bovine serum albumin BSA (dissolved in 3.0 mL of carbonate buffer, pH 9.6), and stirred at 4°C for 4 h. After dialysis with PBS for 3 days, centrifugation was performed at 4000 rpm for 5 min, and the supernatant was obtained, and the diflufenican-coated antigen was obtained, which was divided and stored at -20°C.

[0061] Identification of diflufenican immunogen and coated antigen: the carrier protein, diflufenican hapten, diflufenican hapten-LF conjugate, and diflufenican hapten-BSA conjugate were respectively prepared into a 1.0 mg / mL solution with PBS buffer at pH=7.4, 0.01 mol / L, and adjusted to zero with PBS at pH=7.4, 0.01 mol / L. The absorption spectra of the carrier protein, diflufenican hapten, diflufenican hapten-LF conjugate, and diflufenican hapten-BSA conjugate were obtained by scanning with a UV spectrophotometer at a wavelength of 200-400 nm, as shown in Figure 2-3 From the graph, it can be seen that the diflufenican hapten has been successfully conjugated with the carrier proteins LF and BSA.

[0062] Example 3: Preparation and identification of diflufenican monoclonal antibody Animal immunization: healthy 6-8 week old BALB / c mice were selected for immunization, and the diflufenican immunogen prepared in Example 2 was mixed with an equal amount of Freund's adjuvant to emulsify uniformly, and then the BALB / c mice were immunized by subcutaneous injection at multiple points on the neck and back (Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the booster immunization). The second immunization was performed 2 weeks later, and the booster immunization was performed every 2 weeks thereafter. One week after the fourth booster immunization, blood was taken from the tail of the mouse, and the serum titer was determined by indirect competitive ELISA. When the titer no longer increased, intraperitoneal booster immunization was performed. Three days later, the blood was collected from the heart, and water bathed for 0.5-1 h, and then centrifuged at 10000 rpm for 15 min at 4°C, and the supernatant was obtained, which was the antiserum, i.e., the polyclonal antibody.

[0063] Cell fusion and cloning: the polyclonal antibody with high titer and IC 50The researchers used mice with low expression levels to fuse the cells. Immunized BALB / c mouse spleen cells were mixed with SP2 / 0 myeloma cells using polyethylene glycol (PEG) for cell fusion. After subcloning and four limiting dilutions, a portion of the positive hybridoma cells was cryopreserved, and another portion was injected into the mouse peritoneal cavity to generate ascites. Seven days later, the ascites was collected and purified using a protein G immunoaffinity column to obtain monoclonal antibodies.

[0064] Monoclonal antibody identification: Carbonate buffered saline (CBS, pH 9.6) was used as a diluent for the florasulam coating antigen, phosphate buffered saline (PBS, 0.01 M, pH 7.4) was used as a diluent for the monoclonal antibody and standards, and phosphate-buffered saline (PBST, 0.01 M) was used as a diluent for the horseradish peroxidase-conjugated goat anti-mouse solution. The florasulam coating antigen was diluted to 1 μg / mL and added to a 96-well microtiter plate (100 μL per well) for overnight incubation at 4°C. After washing the plate twice with PBST, 120 μL of 5% bovine serum albumin was added to each well and incubated in a 37°C waterbath for 3 h. The plate was then shaken dry and dried at 37°C for 1 h. The monoclonal antibody was diluted 1000-, 2000-, 4000-, 8000-, 16000-, 32000-, and 64000-fold, respectively, and a 1 μg / mL florasulam standard was added. The above solution was added to a 96-well plate coated with a bispyribac-coated antigen according to the general indirect competitive ELISA method, incubated in a 37°C water bath for 40 min, washed 5 times with PBST, and then 100 μL of a 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse solution was added. The plate was incubated in a 37°C water bath for 30 min, washed 5 times with PBST, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate color development solution was added. The plate was incubated in a 37°C water bath for 10 min again. 10% concentrated sulfuric acid was added to terminate the reaction. The absorbance data was read on a microplate reader at a wavelength of 450 nm. A standard curve was drawn. The obtained standard inhibition curve of bispyribac is shown as follows. Figure 4 shown.

[0065] Example 4: Evaluation of the specificity of the monoclonal antibody against florasulam The indirect competitive ELISA method was used to determine the minimum detection limit of the obtained monoclonal antibody against florasulam to be 0.01 ng / mL, and the IC 50 The linear range was 0.05-8.49 ng / mL. In addition, the IC values ​​of fluazifop, chloranil, methoxysulfuron, bisfluazur, pyrasulfuron, and penoxsulam were also verified. 50 and cross-reaction rate.

[0066] Cross-reactivity ratio = (IC 50 / Other analog ICs 50 ) × 100%, the specific results are shown in Table 1: Table 1

[0067] Example 5 Colloidal gold test strip for florasulam detection This example provides a colloidal gold test strip, comprising a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and a water-absorbing pad. The nitrocellulose membrane is provided with a test line (T line) and a quality control line (C line), respectively. The colloidal gold conjugate pad is coated with the florasulam monoclonal antibody prepared according to Example 3. The test line is streaked with the florasulam-coated antigen. The quality control line is streaked with a goat anti-mouse IgG secondary antibody. The colloidal gold test strip can be assembled using methods commonly used in the art.

[0068] The colloidal gold test strip for florasulam in this embodiment detects florasulam using the principle of indirect competition to detect whether the test sample contains florasulam. If the test sample does not contain florasulam, the test line does not develop color, while the quality control line does. If the test sample does contain florasulam, both the test line and the quality control line develop color. Detection of florasulam can be achieved using methods commonly used in the art.

[0069] Example 6: Enzyme-linked immunosorbent assay kit for bispyribac This example provides a florasulam enzyme-linked immunosorbent assay kit, which comprises the florasulam monoclonal antibody prepared according to Example 3, an ELISA plate, a florasulam coating antigen, a florasulam standard solution, a goat anti-mouse IgG secondary antibody, and a TMB colorimetric solution.

[0070] The principle of the florasulam enzyme-linked immunosorbent assay (ELISA) kit for detecting florasulam is to use an indirect competitive ELISA method to detect the florasulam content in the test sample. The florasulam coating antigen is pre-coated in the microwells of the ELISA plate. A florasulam standard solution or the test sample, a florasulam monoclonal antibody, a goat anti-mouse IgG secondary antibody, and a TMB colorimetric solution are added to create a florasulam standard inhibition curve. Based on the florasulam standard inhibition curve and the absorbance value of the test sample, the florasulam content in the test sample is determined. Detection of florasulam can be achieved by operating using methods commonly used in the art.

[0071] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A hapten of florasulam, wherein the structure of the hapten of florasulam is as shown in formula (I): Formula (I); in, n is 3, 4, 5 or 6.

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

3. A method for preparing the florasulam hapten according to claim 1 or 2, comprising the following steps: S1. reacting florasulam with a demethoxy reagent to generate intermediate 1, ； S2. The intermediate 1 and (CH3)3C-OC(=O)-(CH2) n -Br reaction generates intermediate 2, where n is 3, 4, 5 or 6, ； S3. removing the tert-butyloxycarbonyl group in the intermediate 2, 。 A florasulam antigen, comprising the florasulam hapten according to claim 1 or 2 and a carrier protein coupled to the florasulam hapten. 5 . The florasulam antigen according to claim 4 , wherein the carrier protein is bovine serum albumin, lactoferrin, human serum albumin, chicken ovalbumin, bovine lactoferrin or hemocyanin. 6 . A florasulam antibody, wherein the florasulam antibody is an antibody that specifically recognizes the florasulam antigen according to claim 4 or 5 . The florasulam antibody according to claim 6 , wherein the florasulam antibody is a polyclonal antibody or a monoclonal antibody.

8. An immunoassay device for florasulam, comprising the florasulam antibody according to claim 6 or 7 and the florasulam antigen according to claim 4 or 5.

9. The immunoassay device according to claim 8, wherein the device is a colloidal gold immunochromatographic detection device, comprising a sample pad, a colloidal gold binding pad, a nitrocellulose membrane, and a water-absorbing pad, wherein a detection line and a quality control line are sequentially provided on the nitrocellulose membrane, and the detection line is made of the florasulam antigen according to claim 4 or 5.

10. The immunoassay device according to claim 8, wherein the device is an enzyme-linked immunosorbent assay device, comprising the florasulam antibody according to claim 6 or 7, the florasulam antigen according to claim 4 or 5, a second antibody corresponding to the florasulam antibody according to claim 6 or 7, an ELISA plate, a florasulam standard solution, and a 3,3',5,5'-tetramethylbenzidine colorimetric solution.

11. Use of the florasulam hapten according to claim 1 or 2, the florasulam antigen according to claim 4 or 5, and / or the florasulam antibody according to claim 6 or 7 for detecting florasulam residues in agricultural products.

12. The use according to claim 11, wherein the agricultural products include wheat, corn, oats, rice, soybeans, and peanuts.

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