trifluralin haptens, complete antigens, artificial antigens and antibodies, and methods of making and using the same

Abstract

The application discloses trifluralin hapten, complete antigen, artificial antigen and antibody and a preparation method and application thereof, and relates to the technical field of food safety detection. The trifluralin hapten provided by the application almost retains the complete structure of trifluralin, and further has a carboxyl group which can react with bovine serum albumin, human serum albumin, hemocyanin and egg white albumin, so that the trifluralin hapten can be coupled with different carrier proteins to be used as artificial immunogens and coating origins, and then the trifluralin detection antibody is prepared, which is more conducive to stimulating the animal immune response to generate an antibody with stronger specificity and higher sensitivity. The trifluralin antibody has high sensitivity and good specificity, and can be used for rapidly detecting the residual trifluralin in animal food.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to trifluralin hapten, complete antigen, artificial antigen and antibody, as well as their preparation methods and applications. Background Technology

[0002] Trifluralin is an important selective pre-emergence dinitroaniline herbicide widely used on crops such as soybeans, barley, vegetables, and fruits to control annual grasses and some broadleaf weeds. Although trifluralin is classified as a low-toxicity pesticide, long-term exposure studies have shown that it can cause liver and kidney damage. High-dose exposure can lead to low birth weight and height in fetuses and increase the risk of miscarriage. Due to the widespread use of trifluralin pesticides, its residues are ubiquitous in soil and water bodies, particularly accumulating in aquatic products (fish, shrimp, crabs), posing a significant threat to the safety of aquatic food consumption.

[0003] To avoid the potential hazards of trifluralin herbicides, many countries have restricted its use. In 2015, the European Union established new limits for trifluralin, setting the maximum residue limits (MRLs) at 0.01 mg / kg for fruits, vegetables, grains, and animal-derived foods. Japan sets the MRL for trifluralin in imported aquatic products at 0.001 mg / kg. Currently, the detection of trifluralin in agricultural products typically uses instrumental analysis methods, such as ultraviolet spectroscopy, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), and capillary electrophoresis. While these methods are accurate and sensitive, their high instrument costs and operating expenses hinder widespread adoption. Practice has shown that using rapid detection methods for initial screening of large numbers of samples, followed by instrumental confirmation of positive samples, is an effective way to improve detection efficiency and reduce costs, which aligns with my country's national conditions. Among existing rapid detection technologies, immunoassay based on antigen-antibody specific recognition is considered the most competitive and challenging ultra-micro detection technology of the 21st century due to its advantages of sensitivity, speed, accuracy, low cost, and the ability to perform on-site detection.

[0004] Only a few international publications report on the immunoassay of trifluralin. Bruce Riggle first attempted to construct an enzyme-linked immunosorbent assay (ELISA) for trifluralin in 1991, with a linear range of 100–1000 ppb (ng / mL), and could also detect some trifluralin metabolites, but with low sensitivity. Currently, there are no reports on the immunoassay of trifluralin in China. Furthermore, there is a lack of trifluralin pesticide residue immunoassay kits on the domestic market. Most drugs, toxins, and environmental pollutants have relatively small molecular weights and are haptens. Most haptens require protein conjugation to produce immunogenicity. The current conventional method for preparing hapten antibodies is to select a toxicologically significant parent drug as the analyte, design and synthesize a hapten that retains or has similar structural characteristics to the analyte and contains active groups, conjugate the hapten with a large protein carrier to prepare an artificial immunogen, and simultaneously prepare specific antibodies against the hapten through animal immunization. The provision of the hapten is the most critical step in immunochemical analysis; reasonable hapten design and preparation will contribute to improved antibody quality. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides trifluralin hapten, complete antigen, artificial antigen, and antibody, along with their preparation methods and applications. A hapten that almost completely retains the structure of trifluralin while also containing a carboxyl group is synthesized de novo. Different carrier proteins are then coupled to serve as artificial immunogens and coating antigens, respectively, to prepare trifluralin detection antibodies. These antibodies exhibit high sensitivity and specificity and can be used for the rapid detection of trifluralin residues in animal foods.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a trifluralin hapten, characterized by the following structural formula:

[0007]

[0008] Furthermore, its synthetic route is as follows:

[0009]

[0010] The complete trifluralin antigen is a conjugate formed by the chemical reaction between the carboxyl group of the trifluralin hapten and the amino group of the carrier protein, creating an amide bond. The structural formula of the complete trifluralin antigen is as follows:

[0011]

[0012] The carrier protein is selected from at least one of bovine serum albumin, human serum albumin, hemocyanin, and ovalbumin.

[0013] The preparation method of trifluralin artificial antigen includes the following steps:

[0014] S1, Synthetic fluroxypyr hapten;

[0015] S2, Synthetic fluroxypyr hapten activated ester;

[0016] S3. Fluroxane artificial antigen was obtained by dialysis using fluroxane hapten activated ester.

[0017] Furthermore, S1 specifically includes the following steps:

[0018] S11. Preparation of solution A: Dissolve 2.5g of 4-chloro-3,5-dinitrobenzoic acid in 10mL of dichloromethane;

[0019] S12. Take 2.5g of di-n-propylamine, add it to the reaction flask, and dilute it with 20mL of dichloromethane;

[0020] S13. Under controlled temperature conditions, slowly add solution A to the reaction flask. After the addition is complete, raise the temperature to 40-45℃ and react for 6 hours. After the reaction is complete, evaporate the dichloromethane to dryness.

[0021] S14. Dissolve the product in 20 mL of dilute sodium hydroxide solution, extract twice with ethyl acetate, adjust the pH of the aqueous layer to about 4 with 1 mol / L hydrochloric acid, filter and dry the precipitate to obtain the trifluralin hapten.

[0022] Furthermore, S2 specifically includes the following steps:

[0023] S21. In a 5mL glass bottle, take 24mg of fluroxypyr hapten and dissolve it in 1mL of N,N-dimethylformamide to prepare solution B.

[0024] S22. Weigh 24 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 14 mg of N-hydroxysuccinimide, mix them together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution C.

[0025] S23. Under ice bath conditions, solution C is added dropwise to solution B, and then stirred overnight at room temperature. After terminating the reaction, the activated hapten ester is obtained.

[0026] Furthermore, S3 specifically includes the following steps:

[0027] S31. Take 4 mg of hapten activated ester and add it to a PBS (0.01 mol / L, pH=7.4) solution containing 8 mg of hemocyanin. React overnight at 4 degrees Celsius.

[0028] S32. Place the reaction solution into a dialysis bag and dialyze thoroughly with 0.01 mol / L PBS solution to obtain the immunogen;

[0029] S33. Take 12 mg of hapten activated ester and add it to a PBS (0.01 mol / L, pH=7.4) solution containing 100 mg BSA. React overnight at 4 degrees Celsius.

[0030] S34. Place the reaction solution into a dialysis bag and dialyze thoroughly with 0.01 mol / L PBS solution to obtain the coated antigen.

[0031] A method for preparing fluroxypyr antibody, characterized by comprising the following steps:

[0032] Step 1: Select age-appropriate female Balb / C mice for immunization. After the 4th and 5th immunizations, measure the titer and inhibition rate of the antiserum. Boost the immunization 3 days before cell fusion.

[0033] The second step involves fusing spleen cells and myeloma cells from mice that have undergone booster immunization, screening out hybridoma cells that can secrete specific antibodies, and then expanding their culture.

[0034] The third step involves injecting the expanded hybridoma cells into mice that have been pre-injected with paraffin, collecting the ascites fluid, and obtaining monoclonal antibodies.

[0035] This invention provides the application of the above-mentioned fluroxypyr antibody in the detection of fluroxypyr residues.

[0036] The present invention has the following beneficial effects:

[0037] For trifluralin, a hapten that almost retains the complete structure of trifluralin while also containing a carboxyl group was synthesized de novo. It was further coupled with different carrier proteins as artificial immunogens and coating agents, respectively, to prepare trifluralin detection antibodies. These antibodies have high sensitivity and good specificity and can be used for rapid detection of trifluralin residues in animal food.

[0038] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0039] Figure 1 This invention provides the synthetic pathway for the trifluralin hapten. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] In the description of this invention, it should be understood that the terms "opening", "upper", "lower", "thickness", "top", "middle", "length", "inner", "around", etc., which indicate orientation or positional relationship, are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the components or elements referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as limiting this invention.

[0042] Please see Figure 1 The purpose of this invention is to obtain a highly sensitive fluroxypyr antibody. For fluroxypyr, a hapten that almost retains the complete structure of fluroxypyr and also contains a carboxyl group was synthesized de novo. Different carrier proteins were then coupled to serve as artificial immunogens and coating antigens, respectively, to prepare a fluroxypyr detection antibody. This antibody has high sensitivity and good specificity and can be used for the rapid detection of fluroxypyr residues in animal food.

[0043] Specifically, the technical solution designed to achieve the above objectives is to synthesize a trifluralin hapten with the molecular structure shown below. Then, the hapten is linked to the carrier protein to synthesize an artificial antigen, which is then used to immunize animals to obtain antibodies.

[0044] In this embodiment, 2.5 g of 4-chloro-3,5-dinitrobenzoic acid was dissolved in 10 mL of dichloromethane to obtain solution A; 2.5 g of di-n-propylamine was added to the reaction flask and diluted with 20 mL of dichloromethane. Under temperature control, solution A was slowly added to the reaction flask; after the addition was complete, the temperature was raised to 40–45 °C, and the reaction was carried out for 6 hours. After the reaction was complete, the dichloromethane was evaporated to dryness. The product was dissolved in 20 mL of dilute sodium hydroxide solution, extracted twice with ethyl acetate, and the pH of the aqueous layer was adjusted to about 4 with 1 mol / L hydrochloric acid. At this point, a yellow precipitate was formed. The precipitate was filtered and dried to obtain the target hapten, namely the trifluralin hapten.

[0045] In a 5 mL glass bottle, 31 mg of the hapten was dissolved in 1 mL of N,N-dimethylformamide to prepare solution B. 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of N-hydroxysuccinimide were weighed separately and dissolved together in 0.5 mL of N,N-dimethylformamide to prepare solution C. Solution C was added dropwise to solution B under ice bath conditions, and then the mixture was stirred overnight at room temperature. The activated hapten ester was obtained after the reaction was terminated.

[0046] Take 6 mg of the hapten activated ester and add it to a PBS solution (0.01 mol / L, pH=7.4) containing 10 mg of hemocyanin. React overnight at 4 degrees Celsius. Then, put the reaction solution into a dialysis bag and dialyze it thoroughly with 0.01 mol / L PBS solution to obtain the artificial antigen (immunogen).

[0047] Take 25 mg of the activated hapten ester and add it to a PBS solution (0.01 mol / L, pH = 7.4) containing 100 mg BSA. React overnight at 4 degrees Celsius. Then, put the reaction solution into a dialysis bag and dialyze it thoroughly with 0.01 mol / L PBS solution to obtain the artificial antigen (coated antigen).

[0048] The preparation and purification process of fluroxypyr antibody is as follows:

[0049] Balb / C female mice, approximately 8 weeks old, were selected for immunization and numbered 1, 2, 3, and 4. Immunization was performed via subcutaneous injection at multiple points on the back of the neck.

[0050] Immunization Schedule: Primary Immunization: To enhance the immunogenicity of the antigen, an immunogen emulsified with Freund's complete adjuvant was used. Equal volumes of immunogen and Freund's complete adjuvant were mixed and emulsified, with a dose of 0.1 mg. Booster Immunization: Booster immunizations were administered on days 21 and 42 after the primary immunization, with a dose of 0.05 mg, using the same emulsification method. Subsequent booster immunizations were given every 21 days, for a total of 5 immunizations. Starting with the second booster immunization, serum titers and specificities were determined in animals 10 days after each immunization.

[0051] Antibody preparation: High-titer and highly specific mice were selected for cell fusion. After 7 days, the cultured cells were screened, and cell wells meeting the requirements were identified. Subcloning was then performed to obtain a single cell line. After cell expansion culture, the cells were injected into the peritoneal cavity of mice. After about 7 days, the peritoneal fluid was aspirated, and protein purification was performed to obtain the desired target antibody, namely fluroxypyr antibody.

[0052] The steps of serum titer testing:

[0053] 1) Dissolve the prepared trifluralin coating agent in a pH 9.6 Na2CO3-NaHCO3 buffer solution, dilute the trifluralin coating agent to 1.0 μg / mL as the coating solution, add 100 μL to each well of a 96-well microplate, incubate overnight at 4°C or at 37°C for 2-3 hours, and wash once with 0.05% (V / V) PBST (phosphate buffer) and Tween 20 washing solution.

[0054] 2) Add 200 μL of PBS blocking buffer containing 3% skim milk powder to each well, block for 2 hours, and wash once with washing buffer;

[0055] 3) Add antiserum and standards: Dilute the antiserum serially; dissolve the trifluralin standard in an appropriate amount of DMF to prepare a 1.0 mg / mL standard solution, and store at 4℃ for later use. For the titer series: Add 50 μL of blank diluent, 50 μL of serially diluted antiserum, and 50 μL of goat anti-mouse enzyme-labeled secondary antibody to each well; for the inhibition series: Add 50 μL of standard solution, 50 μL of serially diluted antiserum, and 50 μL of goat anti-mouse enzyme-labeled secondary antibody to each well, and incubate at 37℃ for 30 minutes.

[0056] 4) Wash the microplate three times with washing solution, shake off the liquid in the wells, dry it with a microplate dehydrator, add 50 μL of substrate A and 50 μL of substrate B, and react at room temperature for 15 minutes.

[0057] 5) Reading determination: The absorbance value is measured at dual wavelengths of 450nm and 630nm; the antiserum dilution factor with an absorbance value in the range of 1.5 to 2.0 is selected as the antiserum titer, and the effect of the antiserum is obtained from its inhibition rate.

[0058] This invention uses 4-chloro-3,5-dinitrobenzoic acid as a starting material to prepare a hapten with most of the structure of trifluralin through a one-step chemical synthesis. The hapten has a molecular weight of 311.

[0059] Preparation of monoclonal antibodies

[0060] 1. Animal immunization

[0061] Balb / C female mice, approximately 8 weeks old, were selected for immunization and numbered 1, 2, 3, and 4. Immunization was performed via subcutaneous injection at multiple points on the back of the neck.

[0062] Immunization Schedule: Primary Immunization: To enhance the immunogenicity of the antigen, an immunogen emulsified with Freund's complete adjuvant was used. Equal volumes of immunogen and Freund's complete adjuvant were mixed and emulsified, with a dose of 0.1 mg. Booster Immunization: Booster immunizations were administered on days 21 and 42 after the primary immunization, with a dose of 0.05 mg, using the same emulsification method. Subsequent booster immunizations were given every 21 days, for a total of 5 immunizations. Starting with the second booster immunization, serum titers and specificities were determined in animals 10 days after each immunization.

[0063] 2. Analysis of antiserum efficacy

[0064] (1) Starting from the third booster immunization, on the 8th day after each immunization, 30uL of blood was collected from the tail of the mouse, centrifuged to obtain antiserum, and stored at -20℃ for later use.

[0065] (2) The titer and specificity of the antiserum were determined by indirect enzyme-linked immunosorbent assay (ELISA). The steps are as follows:

[0066] S1. Coating: Dilute 5 mg / mL of the coating antigen 5000 times with coating buffer and prepare a blank control group. Add 100 μL / well to the microplate and incubate overnight at 4°C. Then wash once with 0.05% PBST (phosphate buffer, v / v) and Tween 20 wash buffer.

[0067] S2. Sealing: Pour off the liquid in the wells, wash the plate once, add 200 μL of washing solution to each well, spin dry, add 200 μL of sealing solution to each well, seal at 37°C for 2 hours, spin dry, and store in a refrigerator at 4°C for later use.

[0068] S3. Sample addition: Add 50 μL of standard diluent, diluted enzyme-labeled secondary antibody and diluted serum to each control well; add 50 μL of diluted standard, diluted enzyme-labeled secondary antibody and diluted serum to each sample well; vortex to mix, react at 37℃ for 30 min, wash the plate 3 times with a plate washer, add 200 μL of washing buffer to each well, and spin dry.

[0069] S4. Color development: Add 50 μL of color development solution A and 50 μL of color development solution B to each well, place in an oven at 37 °C for 15 min, and then add 50 μL of stop solution (1 mol / L H2SO4) to each well.

[0070] S5. Measurement: The absorbance at 450 nm in each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The antiserum dilution factor with absorbance values ​​in the range of 1.5 to 2.0 was selected as the antiserum titer. The effectiveness of the antiserum was determined by its inhibition rate. At the same drug concentration, the higher the inhibition rate, the higher the antibody's sensitivity to the drug.

[0071] 3. Screening for cell fusion and positive hybridomas

[0072] (1) Resuscitating myeloma cells: Remove myeloma cells from liquid nitrogen and quickly place them in a 37°C water bath to thaw. After thawing, centrifuge at 1000 r / min for 5 min. Discard the supernatant in a clean bench and add about 1 mL of complete culture medium to the cell pellet. Disperse the cells, remove them with a pipette and mix them with the complete culture medium. Place them in 25 cm2 culture flasks and expand to 4-6 flasks. Change the medium several times during this period. When the cells in each culture flask cover the bottom, they can be used for cell fusion.

[0073] (2) Feeder cell preparation: Balb / C mice were euthanized by cervical dislocation one day before cell fusion. After soaking in 75% alcohol for 5 min, the mice were transferred to a clean bench for dissection. The abdomen was cut open, the abdominal skin was peeled off, the peritoneum was cut open, the spleen was removed, and the cells were transferred to a 9 cm culture dish. DMEM basal medium was added, and the cells were repeatedly aspirated and rinsed with a syringe. The rinsing solution was transferred to a 50 mL centrifuge tube, centrifuged at 1200 r / min for 5 min, and the supernatant was discarded. The cell pellet at the bottom was resuspended in complete culture medium and added to a 96-well cell culture plate, 100 μL per well.

[0074] (3) Spleen cell preparation: Blood was drawn from the orbital region of Balb / c mice that had been repeatedly immunized and passed blood tests. Serum was collected, then disinfected by soaking in 75% alcohol for 5 minutes, and transferred to a clean bench for dissection. The spleen was aseptically removed, rinsed with DMEM basal medium, and placed in a culture dish for later use. Using a disposable syringe, the culture medium was drawn up. The spleen was held with forceps in the left hand, and the syringe was inserted into the spleen in the right hand to slowly inject the culture medium to wash out the cells. This process was repeated until the spleen changed from dark red to colorless and transparent, at which point the spleen was discarded. The mixed culture medium was collected in a 50 mL centrifuge tube, sealed, and centrifuged at 1200 rpm for 6 minutes. The supernatant was discarded after centrifugation and the mixture was ready for use.

[0075] (4) Cell fusion: Myeloma cells and immune spleen cells (after centrifugation and removal of supernatant) were mixed in a centrifuge tube at a ratio of approximately 1:6. 25 mL of basal culture medium was added, the tube was sealed, and centrifuged at 1500 r / min for 8 min. The supernatant was discarded after centrifugation. The supernatant of the centrifuged and mixed myeloma cells and immune spleen cells was discarded, and excess culture medium was removed by pipetting the centrifuge tube with the opening facing down. The precipitated cells were loosened by tapping with fingers. The centrifuge tube was placed in 37°C warm water. 1 mL of PEG preheated to 37°C was pipetted in and slowly added to the precipitated cells over 1 min. After each drop of PEG was added, the mixture was gently stirred with the pipette tip to ensure even mixing. The mixture was allowed to stand for 1 min. The complete culture medium was preheated, and 10 mL of the preheated medium was added over 2 min, stirring gently along the wall to separate the PEG. Seal the centrifuge tubes and centrifuge at 1000 rpm for 10 minutes. Discard the supernatant, add HAT complete medium, gently aspirate the liquid with a bent pipette, stir gently, and evenly add it to 10 96-well culture plates containing feeder cells prepared the day before. Ensure that the volume of HAT medium containing feeder cells and HAT medium containing fusion cells is the same in each well.

[0076] (5) Screening of positive hybridomas: Change the medium with HAT medium once within 3-4 days after fusion. After 7 days, extract the supernatant from the multi-well culture plate and detect the specific antibodies in the culture medium using indirect ELISA. Select positive hybridoma cells with high titers and strong drug inhibition. Screen the positive wells with the best fusion effect and mark them. Under sterile conditions, pick the clustered cells in the positive wells with a microscope and transfer them to a 96-well culture plate pre-coated with feeder cells. Each original well is cloned into 96 wells. After the cells adhere to the wall and grow to cover 1 / 4 of the bottom of the well, take the supernatant and detect it with icELISA. Again, use titer and inhibition rate as the evaluation indicators. Select the strong positive cells and perform subcloning using the limiting dilution method. Repeat this process 3-4 times (note that the positive well cells picked in each round need to be expanded in culture and then frozen for later use) until every well in every plate is positive and the titer and inhibition are similar. At this point, the hybridoma cell line is successfully established and a hybridoma cell line that can stably secrete uniform antibodies is obtained. Select single-cell clones; those that test positive for all cells should be transferred to 24-well cell culture plates or cell culture dishes for further culture and then frozen promptly.

[0077] 4. Large-scale preparation of monoclonal antibodies

[0078] After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are typically prepared in large quantities using in vitro culture and in vivo induction methods. The standard procedure is as follows: Liquid calcite is injected intraperitoneally into more than ten Balb / c mice over 8 weeks old at a dose of 0.5 mL per mouse. One to two weeks later, hybridoma cells are injected into the peritoneum of these mice. The mice are observed daily after cell inoculation, especially from day 7 onwards, when the peritoneum becomes distended. Ascites fluid is aseptically collected using a disposable syringe before the mice die. The collected ascites fluid is centrifuged at 12000 rpm for 10 minutes to remove the upper fat and lower fibrin layers. The intermediate layer is collected, and its titer and inhibition rate are determined using icELISA. After purification, the fluid is stored at -20°C for later use.

[0079] Establishment of an indirect competitive enzyme-linked immunosorbent assay (ELISA) method for fluroxypyr.

[0080] 1. The indirect competitive enzyme-linked immunosorbent assay (icELISA) reaction specifically includes the following steps:

[0081] S1. Coating: Dilute 5 mg / mL of the coating antigen 10,000 times with coating buffer and prepare a blank control group. Add 100 μL / well to the microplate and incubate overnight at 4°C. Then wash once with 0.05% PBST (phosphate buffer, v / v) and Tween 20 wash buffer.

[0082] S2. Sealing: Pour off the liquid in the wells, wash the plate once, add 200 μL of washing solution to each well, spin dry, add 200 μL of sealing solution to each well, seal at 37°C for 2 hours, spin dry, and store in a refrigerator at 4°C for later use.

[0083] S3. Sample addition: Add 50 μL of standard diluent, diluted enzyme-labeled secondary antibody and diluted serum to each control well; add 50 μL of diluted standard, diluted enzyme-labeled secondary antibody and diluted serum to each sample well; vortex to mix, react at 37℃ for 30 min, wash the plate 3 times with a plate washer, add 200 μL of washing buffer to each well, and spin dry.

[0084] S4. Color development: Add 50 μL of color development solution A and 50 μL of color development solution B to each well, place in an oven at 37 °C for 15 min, and then add 50 μL of stop solution (1 mol / L H2SO4) to each well.

[0085] S5. Measurement: The absorbance at 450 nm in each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0086] S6. Calculation: Use the graphpad prism 7.0 fitting module to calculate the IC50 value of the suppression curve.

[0087] 2. Prepare trifluralin standard solutions with concentrations of 2.43 ng / mL, 0.81 ng / mL, 0.27 ng / mL, 0.09 ng / mL, and 0.03 ng / mL to establish a standard curve for the indirect enzyme-linked immunosorbent assay (ELISA). After optimization, the method showed an IC50 of 0.36 ng / mL for trifluralin, with a linear detection range of 0.03–2.43 ng / mL.

[0088] Determination of antibody cross-reactivity

[0089] The optimal coating antigen concentration and optimal antiserum dilution obtained from the above schemes were used in an indirect competitive ELISA experiment with trifluralin, diuron, linuron, and metribuzin as competitive standards to detect the specificity of trifluralin monoclonal antibody. The half-maximal inhibitory concentration (IC50) and cross-reactivity (CR) values ​​are listed in the table below:

[0090]

[0091] Experimental results show that the trifluralin monoclonal antibody only recognizes trifluralin and not other similar herbicides, indicating that the trifluralin monoclonal antibody of the present invention has good specificity and can be used for the preliminary detection of trifluralin residues in food.

[0092] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0093] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A trifluralin artificial antigen, characterized in that, The trifluralin artificial antigen is a conjugate formed by the chemical reaction between the carboxyl group of the trifluralin hapten and the amino group of the carrier protein, creating an amide bond. The structural formula of the trifluralin artificial antigen is as follows: ； The carrier protein is selected from at least one of bovine serum albumin, human serum albumin, hemocyanin, and ovalbumin; The structural formula of the trifluralin hapten is as follows: 。 2. The method for preparing trifluralin artificial antigen according to claim 1, characterized in that, Includes the following steps: S1. Synthesize the fluroxypyr hapten, the synthetic route of which is as follows: ； S2, Synthetic fluroxypyr hapten activated ester; S3. Fluroxane artificial antigen was obtained by dialysis using fluroxane hapten activated ester.

3. The method for preparing trifluralin artificial antigen according to claim 2, characterized in that: S1 specifically includes the following steps: S11. Preparation of solution A: Dissolve 2.5g of 4-chloro-3,5-dinitrobenzoic acid in 10mL of dichloromethane; S12. Take 2.5g of di-n-propylamine, add it to the reaction flask, and dilute it with 20mL of dichloromethane; S13. Under controlled temperature conditions, slowly add solution A to the reaction flask. After the addition is complete, raise the temperature to 40-45℃ and react for 6 hours. After the reaction is complete, evaporate the dichloromethane. S14. Dissolve the product in 20 mL of dilute sodium hydroxide solution, extract twice with ethyl acetate, adjust the pH of the aqueous layer to 4 with 1 mol / L hydrochloric acid, filter and dry the precipitate to obtain the trifluralin hapten.

4. The method for preparing trifluralin artificial antigen according to claim 3, characterized in that: S2 specifically includes the following steps: S21. In a 5mL glass bottle, take 24mg of trifluralin hapten and dissolve it in 1mL of N,N-dimethylformamide to prepare solution B. S22. Weigh 24 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 14 mg of N-hydroxysuccinimide, mix them together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution C. S23. Under ice bath conditions, solution C is added dropwise to solution B, and then stirred overnight at room temperature. After terminating the reaction, the activated ester of the hapten is obtained.

5. The method for preparing trifluralin artificial antigen according to claim 4, characterized in that: S3 specifically includes the following steps: S31. Take 4 mg of hapten activated ester and add it to a PBS solution containing 8 mg of hemocyanin. The solution is 0.01 mol / L and pH=7.

4. React overnight at 4 degrees Celsius. S32. Place the reaction solution into a dialysis bag and dialyze thoroughly with 0.01 mol / L PBS solution to obtain the immunogen; S33. Take 12 mg of hapten activated ester and add it to a PBS solution containing 100 mg BSA. The solution concentration is 0.01 mol / L and pH=7.

4. React overnight at 4 degrees Celsius. S34. Place the reaction solution into a dialysis bag and dialyze thoroughly with 0.01 mol / L PBS solution to obtain the coated antigen.

6. A method for preparing antibodies using the trifluralin artificial antigen of claim 1, characterized in that, Includes the following steps: Step 1: Select age-appropriate female Balb / C mice for immunization. After the 4th and 5th immunizations, measure the titer and inhibition rate of the antiserum. Boost the immunization 3 days before cell fusion. The second step involves fusing spleen cells and myeloma cells from mice that have undergone booster immunization, screening out hybridoma cells that can secrete specific antibodies, and then expanding their culture. The third step involves injecting the expanded hybridoma cells into mice that have been pre-injected with paraffin, collecting the ascites fluid, and obtaining monoclonal antibodies.

7. The application of the fluroxypyr antibody of claim 6 in the detection of fluroxypyr residues.

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