Trifluralin hapten, complete antigen, artificial antigen and antibody as well as preparation method and application of trifluralin hapten, complete antigen, artificial antigen and antibody

Abstract

The invention discloses a trifluralin hapten, a complete antigen, an artificial antigen and an antibody as well as a preparation method and application thereof, and relates to the technical field of food safety detection. The needle trifluralin hapten provided by the invention almost retains the complete structure of trifluralin, also has an antibody which can react with bovine serum albumin, human serum albumin, hemocyanin and ovalbumin, and can be further coupled with different carrier proteins to be respectively used as an artificial immunogen and a coating antigen so as to prepare a trifluralin detection antibody; the trifluralin antibody is more beneficial for stimulating animal immune response to generate an antibody with higher specificity and sensitivity, and the trifluralin antibody has high sensitivity and good specificity and can be used for rapidly detecting residual trifluralin in animal food.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, in particular to trifluralin hapten, complete antigen, artificial antigen and antibody, and preparation methods and applications thereof. Background Art

[0002] Trifluralin is an important selective pre-emergence dinitroaniline herbicide, widely used in crops such as soybeans, barley, vegetables and fruits to control annual grass weeds and some broadleaf weeds. Although trifluralin is a low-toxic pesticide, long-term exposure experiments have shown that it can cause damage to the liver and kidneys. High-dose exposure can cause low fetal weight and height and increase the risk of miscarriage. Due to the widespread use of trifluralin pesticides, its residues are widely present in soil and water, especially in aquatic products (fish, shrimp, crab), which poses a huge hidden danger to the safety of aquatic products.

[0003] In order to avoid the potential harm of trifluralin herbicides, many countries have restricted its use. In 2015, the European Union established new limit standards for trifluralin, and the maximum residue limit (MRLs) of trifluralin in fruits, vegetables, grains and animal-derived foods was 0.01 mg / kg. Japan's maximum residue limit for trifluralin in imported aquatic products is 0.001 mg / kg. At present, the detection of trifluralin in agricultural products usually uses instrumental analysis methods, such as ultraviolet spectroscopy, high performance liquid chromatography and liquid-mass spectrometry, gas chromatography, capillary electrophoresis, etc. The above analysis methods are accurate and sensitive, but they are difficult to promote comprehensively due to expensive instruments and high operating costs. Practice has proved that using rapid detection methods to screen a large number of samples and then confirming the positive samples with instruments is an effective way to improve detection efficiency and reduce costs, which is in line with my country's national conditions. Among the existing rapid detection technologies, immunoassay technology based on antigen-antibody specific recognition is considered to be the most competitive and challenging ultra-trace detection technology in the 21st century because of its advantages such as sensitivity, rapidity, accuracy, low cost and on-site detection.

[0004] There are only a few reports on fluryl immunoassay in foreign countries. Bruce Riggle first attempted to construct an enzyme-linked immunosorbent assay for fluryl in 1991, with a linear range of 100 to 1000 ppb (ng / mL). At the same time, it can detect some fluryl metabolites, but the sensitivity is low. At present, there are no relevant reports on fluryl immunoassay in China. There is a lack of fluryl pesticide residue immunoassay kits in the domestic testing market. Most drugs, toxins, and environmental pollutants now have relatively small molecular weights and are haptens. Most haptens need to be coupled with proteins to produce immunogenicity. At present, the conventional method for preparing hapten antibodies is to select the original drug with toxicological significance as the analyte, design and synthesize a hapten that retains the molecular structure characteristics or similar structural characteristics of the analyte and carries an active group, and couple the hapten with a macromolecular protein carrier to prepare an artificial immunogen, and prepare specific antibodies against the hapten by animal immunization. The provision of haptens is the most critical step in immunochemical analysis. Reasonable hapten design and preparation will be conducive to improving the quality of antibodies. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a fluralin hapten, a complete antigen, an artificial antigen and an antibody, and a preparation method and application thereof. A hapten which almost retains the complete structure of fluralin and has a carboxyl group is synthesized from scratch, and is further coupled with different carrier proteins as an artificial immunogen and a coating source, thereby preparing a fluralin detection antibody. The fluralin detection antibody has high sensitivity and good specificity, and can be used for rapid detection of fluralin residues in animal food.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: Trifluralin hapten, characterized in that its structural formula is as follows:

[0007]

[0008] Further, the synthesis path is as follows:

[0009]

[0010] Trifluralin complete antigen, wherein the trifluralin complete antigen is a conjugate formed by chemical reaction between the carboxyl group in the trifluralin hapten and the amino group in the carrier protein to form an amide bond connection, and the structural formula of the trifluralin complete 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 method for preparing the trifluralin artificial antigen comprises the following steps:

[0014] S1, synthetic trifluralin hapten;

[0015] S2, synthesis of trifluralin hapten activated ester;

[0016] S3. Use the fluralin hapten activated ester to dialyze to obtain the fluralin artificial antigen.

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

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

[0019] S12, take 2.5 g of di-n-propylamine, add it into the reaction bottle and dilute it with 20 mL of dichloromethane;

[0020] S13, under temperature control, slowly add liquid A into the reaction bottle, after adding, raise the temperature to 40-45°C, and react for 6 hours. After the reaction, spin dry the dichloromethane;

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

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

[0023] S21. In a 5 mL glass bottle, take 24 mg of trifluralin hapten and dissolve it in 1 mL 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 respectively, mix the two together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution C;

[0025] S23, add solution C dropwise to solution B under ice bath condition, and then stir at room temperature overnight. After terminating the reaction, the hapten activated ester is obtained.

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

[0027] S31, take 4 mg of hapten activated ester, add it to 8 mg of hemocyanin dissolved in PBS (0.01 mol / L, pH=7.4), and react overnight at 4 degrees;

[0028] S32, placing the reaction solution into a dialysis bag, and fully dialyzing it with a 0.01 mol / L PBS solution to obtain an immunogen;

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

[0030] S34. The reaction solution was placed in a dialysis bag and dialyzed with 0.01 mol / L PBS solution to obtain the coating source.

[0031] The method for preparing trifluralin antibody is characterized by comprising the following steps:

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

[0033] The second step is to fuse the spleen cells of the boosted immunized mice with myeloma cells, screen out hybridoma cells that can secrete specific antibodies, and expand the culture;

[0034] The third step is to inject the expanded cultured hybridoma cells into mice that have been injected with paraffin in advance, collect the ascites, and obtain monoclonal antibodies.

[0035] The present invention provides application of the trifluralin antibody in detecting trifluralin residues.

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

[0037] For flufluralin, a hapten that almost retains the complete structure of flufluralin and has a carboxyl group was synthesized from scratch. It was further coupled with different carrier proteins as artificial immunogens and coating agents, and then flufluralin detection antibodies were prepared. The antibodies have high sensitivity and good specificity, and can be used to quickly detect flufluralin residues in animal food.

[0038] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the synthesis pathway of the trifluralin hapten of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In the description of the present invention, it should be understood that the terms "opening", "upper", "lower", "thickness", "top", "middle", "length", "inside", "all around" and the like indicating orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the components or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0042] See also Figure 1 The purpose of the present invention is to obtain a highly sensitive fluryl antibody. For fluryl, a hapten that almost retains the complete structure of fluryl and has a carboxyl group is synthesized from scratch, and further coupled with different carrier proteins as artificial immunogens and coatings, thereby preparing a fluryl detection antibody, which has high sensitivity and good specificity and can be used to quickly detect fluryl residual in animal food.

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

[0044] In this embodiment, take 2.5g of 4-chloro-3,5-dinitrobenzoic acid, dissolve it in 10mL of dichloromethane to obtain liquid A; take 2.5g of di-n-propylamine, add it into the reaction bottle and dilute it with 20mL of dichloromethane. Under temperature control, slowly add liquid A into the reaction bottle; after the addition is completed, raise the temperature to 40-45°C and react for 6h. After the reaction is completed, spin-dry the dichloromethane. Add 20mL of dilute sodium hydroxide solution to dissolve the product, extract it twice with ethyl acetate, adjust the pH of the aqueous layer to about 4 with 1mol / L hydrochloric acid, and a yellow precipitate will precipitate. Filter and dry the precipitate to obtain the target hapten, i.e., fluralin hapten;

[0045] In a 5mL glass bottle, take 31mg of hapten and dissolve it in 1mL of N,N-dimethylformamide to prepare liquid B; weigh 20mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12mg of N-hydroxysuccinimide respectively, mix them together and dissolve them in 0.5mL of N,N-dimethylformamide to prepare liquid C; add liquid C dropwise to liquid B under ice bath conditions, and then stir at room temperature overnight. After terminating the reaction, the hapten activated ester is obtained;

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

[0047] Take 25 mg of hapten activated ester and add it to 100 mg BSA dissolved in PBS (0.01 mol / L, pH = 7.4) solution, and react overnight at 4 degrees; then put the reaction solution into a dialysis bag and dialyze it with 0.01 mol / L PBS solution to obtain an artificial antigen (coating source).

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

[0049] Balb / C female mice, about 8 weeks old, were selected as immunized animals and were numbered 1, 2, 3, and 4. Immunization was performed by subcutaneous multiple injections at the back of the neck.

[0050] Immunization procedure: Initial immunization: To improve the immunogenicity of the antigen, the immunogen emulsified with Freund's complete adjuvant is used. The same volume of immunogen and Freund's complete adjuvant are mixed and emulsified, and the dose is 0.1 mg. Reinforcement immunization: Reinforcement immunization is carried out on the 21st and 42nd days after the initial immunization, with a dose of 0.05 mg and the same emulsification method. After that, the booster immunization is carried out every 21 days, for a total of 5 immunizations. Starting from the second booster immunization, blood samples are collected from the animals 10 days after each immunization for serum titer determination and specificity determination.

[0051] Antibody preparation: Select mice with high titer and strong specificity for cell fusion. After 7 days, screen the cultured cells to find the cell wells that meet the requirements, subclone them, and finally obtain a single cell line. After the cells are expanded and cultured, inject them into the mouse peritoneal cavity. After about 7 days, extract the mouse peritoneal effusion and purify the protein to obtain the desired target antibody, that is, trifluralin antibody.

[0052] Steps of serum titer test method:

[0053] 1) The prepared trifluralin coating source was dissolved in a pH 9.6 Na2CO3-NaHCO3 buffer, and the trifluralin coating source was diluted to 1.0 μg / mL as the coating solution. 100 μL was added to each well of a 96-well microplate, and the plate was placed at 4°C overnight or incubated at 37°C for 2 to 3 hours, and then washed once with PBST (phosphate buffer 0.05% (V / V)) and Tween20 washing solution;

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

[0055] 3) Add antiserum and standard: dilute the antiserum in a gradient; dissolve the trifluralin standard in an appropriate amount of DMF to prepare a 1.0 mg / mL standard solution, and store at 4°C for later use. Titer column: add 50uL of blank dilution, 50uL of gradient diluted antiserum and 50uL of goat anti-mouse enzyme-labeled secondary antibody to each well; Inhibition column: add 50uL of standard solution, 50uL of gradient diluted antiserum and 50uL of goat anti-mouse enzyme-labeled secondary antibody to each well, and incubate at 37°C for 30 minutes;

[0056] 4) Wash the microplate three times with washing solution, discard the liquid in the wells, spin dry with a microplate dehydrator, add 50uL substrate A and 50uL substrate B, and react at room temperature for 15 minutes;

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

[0058] The invention uses 4-chloro-3,5-dinitrobenzoic acid as a starting material and prepares a hapten having most of the structure of trifluralin through one-step chemical synthesis, and the molecular weight of the hapten is 311.

[0059] Preparation of monoclonal antibodies

[0060] 1. Animal immunization

[0061] Balb / C female mice, about 8 weeks old, were selected as immunized animals and were numbered 1, 2, 3, and 4. Immunization was performed by subcutaneous multiple injections at the back of the neck.

[0062] Immunization procedure: Initial immunization: To improve the immunogenicity of the antigen, the immunogen emulsified with Freund's complete adjuvant is used. The same volume of immunogen and Freund's complete adjuvant are mixed and emulsified, and the dose is 0.1 mg. Reinforcement immunization: Reinforcement immunization is carried out on the 21st and 42nd days after the initial immunization, with a dose of 0.05 mg and the same emulsification method. After that, the booster immunization is carried out every 21 days, for a total of 5 immunizations. Starting from the second booster immunization, blood samples are collected from the animals 10 days after each immunization for serum titer determination and specificity determination.

[0063] 2. Analysis of antiserum effect

[0064] (1) Starting from the third booster immunization, on the 8th day after each immunization, 30uL of blood was collected from the mouse tail, 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, the steps of which are as follows:

[0066] S1. Coating: Dilute 5000 times the 5mg / mL coating antigen with coating buffer and make a blank control group. Add 100uL / well to the ELISA plate and incubate it in a 4℃ refrigerator overnight. Then wash once with PBST (phosphate buffer 0.05% (V / V) and Tween20 washing solution.

[0067] S2. Blocking: Pour out the liquid in the wells, wash the plate once, add 200 μL of washing solution to each well, spin dry, add 200 μL of blocking solution to each well, block at 37°C for 2h, spin dry, and place in a 4°C refrigerator for later use.

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

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

[0070] S5. Determination: Use an enzyme-linked immunosorbent assay to determine the absorbance of each well at A450nm. Select the antiserum dilution multiple with an absorbance value in the range of 1.5 to 2.0 as the antiserum titer. The effect of the antiserum is determined by its inhibition rate. Under the same drug concentration, the higher the inhibition rate, the higher the sensitivity of the antibody to the drug.

[0071] 3. Cell fusion and screening of positive hybridomas

[0072] (1) Recovery of myeloma cells: Remove myeloma cells from liquid nitrogen and quickly thaw them in a 37°C water bath. Centrifuge at 1000 rpm for 5 min after thawing. Pour off the supernatant in an ultra-clean workbench and add about 1 mL of complete culture medium to the cell pellet to disperse the cells. Use a pipette to remove the cells and mix them with complete culture medium. Place them in a 25 cm2 culture flask and expand to 4 to 6 flasks. During this period, the medium needs to be changed several times. When the cells in each culture flask cover the bottom, they can be used for cell fusion.

[0073] (2) Preparation of feeder cells: Balb / C mice were killed by cervical dislocation 1 day before cell fusion, and after being soaked in 75% alcohol for 5 min, they were moved 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 moved to a 9 cm culture dish. DMEM basal culture medium was added, and the rinsing liquid was repeatedly aspirated and rinsed with a syringe. The rinsing liquid was transferred to a 50 mL centrifuge tube and centrifuged at 1200 r / min for 5 min. The supernatant was discarded, and the cell pellet at the bottom was resuspended with complete culture medium and added to a 96-well cell culture plate, 100 uL per well.

[0074] (3) Preparation of spleen cells: Balb / c mice that have been immunized multiple times and have passed blood tests are bled from their orbits to collect serum, which is then disinfected by soaking in 75% alcohol for 5 minutes and moved to a clean bench for dissection. The spleen is removed aseptically, rinsed with DMEM basal culture medium, and placed in a culture dish for later use. Use a disposable syringe to absorb the culture medium, hold the removed spleen with tweezers in the left hand, insert the syringe into the spleen with the right hand, and slowly inject the culture medium to wash out the cells in the spleen. Repeat until the spleen turns from dark red to colorless and transparent, and then discard the spleen. Collect the mixed culture medium into a 50mL centrifuge tube, seal it, and centrifuge it at 1200r / min for a total of 6 minutes. After centrifugation, discard the supernatant and set aside.

[0075] (4) Cell fusion: Mix the myeloma cells and immune spleen cells after centrifugation in a ratio of about 1:6, add 25 mL of basal culture medium, seal the tube and centrifuge at 1500 r / min for 8 minutes. Discard the supernatant after centrifugation and set aside. Discard the supernatant of the myeloma cells and immune spleen cells mixed by centrifugation, and use a gun to suck off the excess culture medium with the mouth of the centrifuge tube facing downward. Loosen the precipitated cells with your fingers, place the centrifuge tube in 37°C warm water, use the gun tip to suck up 1 mL of PEG preheated to 37°C, and slowly add PEG to the precipitated cells within 1 minute. After adding each drop of PEG, gently stir it with the gun tip to mix it evenly. Let it stand for 1 minute, preheat the complete culture medium, and add 10 mL within 2 minutes, accompanied by gentle stirring, and add along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 1000r / min for 10 minutes, discard the supernatant, add HAT complete medium, use an elbow pipette to gently aspirate the liquid, stir gently, and evenly add it to the 10 96-well culture plates containing feeder cells prepared the day before. Keep the volume of HAT medium containing feeder cells and HAT medium containing fusion cells in each well the same.

[0076] (5) Screening of positive hybridomas: Change the medium with HAT culture medium once within 3 to 4 days after fusion. After 7 days, extract the supernatant in the multi-well culture plate and detect the specific antibodies in the culture medium by indirect ELISA. Select the positive hybridoma cells with high titer and strong drug inhibition. Screen the positive wells with the best fusion effect and mark them. Under sterile conditions, pick the cells growing in clusters in the positive wells with a microscope and transfer them to a 96-well culture plate pre-plated with feeder cells. Each original well is cloned into 96 wells. After the cells adhere to the wall and cover 1 / 4 of the bottom of the well, take the supernatant and test it with icELISA. The titer and inhibition rate are also used as the measurement indicators. The strong positive ones are subcloned by limiting dilution method. Repeat this process for 3 to 4 rounds (note that the positive well cells picked out in each round need to be expanded and cultured, and then frozen for future use) until each well on each plate is positive and the titer and inhibition are similar after detection. At this point, the hybridoma cell line is successfully established, and a hybridoma cell line that can stably secrete uniform antibodies is obtained. Single cell clones were picked and those that tested positive were transferred to 24-well cell culture plates or cell culture dishes for expansion and cryopreservation in time.

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

[0078] After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are usually prepared in large quantities by in vitro culture and in vivo monoclonal antibody induction in animals. The conventional practice is to inject liquid lysate into the peritoneal cavity of more than ten Balb / c mice over 8 weeks old in advance, with a dose of 0.5 mL / mouse, and then inject hybridoma cells into the peritoneal cavity of the mice 1 to 2 weeks later. After inoculation of cells, the state of the mice is observed every day, especially from the 7th day, the abdominal cavity of the mice will swell, and ascites is collected aseptically with a disposable syringe before the death of the mice. The collected ascites is centrifuged at 12000r / min for 10 minutes, the upper fat and the lower fibrin are removed, and the middle layer is collected. The titer and inhibition rate are determined by icELISA method, and after purification, it is stored at -20℃ for later use.

[0079] Establishment of an indirect competitive enzyme-linked immunosorbent assay for trifluralin

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

[0081] S1. Coating: Dilute 5 mg / mL of coating antigen 10,000 times with coating buffer, and make a blank control group. Add 100uL / well to the ELISA plate and incubate it in a 4°C refrigerator overnight. Then wash once with PBST (phosphate buffered saline 0.05% (V / V) and Tween20 washing solution;

[0082] S2. Blocking: Pour out the liquid in the wells, wash the plate once, add 200 μL of washing solution to each well, spin dry, add 200 μL of blocking solution to each well, block at 37°C for 2h, spin dry, and place in a 4°C refrigerator for later use.

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

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

[0085] S5. Determination: Use an enzyme-linked immunosorbent assay (ELISA) to determine the absorbance of each well at A450nm.

[0086] S6. Calculation: The IC50 value of the inhibition curve was calculated using the GraphPad Prism7.0 fitting module.

[0087] 2. Prepare 2.43ng / mL, 0.81ng / mL, 0.27ng / mL, 0.09ng / mL, and 0.03ng / mL trifluralin standard solutions to establish a standard curve for the indirect enzyme-linked immunosorbent assay. After the conditions were optimized, the method had an IC50 of 0.36ng / mL for trifluralin, and a linear detection range of 0.03 to 2.43ng / mL.

[0088] Determination of antibody cross-reactivity rate

[0089] The optimal coating antigen concentration and the optimal antiserum dilution multiple obtained in the above scheme were used to perform an indirect competitive ELISA experiment with trifluralin, diuron, linuron, and meturon as competitive standards to detect the specificity of trifluralin monoclonal antibodies. The half-maximal inhibitory concentration (IC50) and cross-reaction rate (CR) values ​​are listed in the following table:

[0090]

[0091] The experimental results show that the trifluralin monoclonal antibody only recognizes trifluralin and does not recognize 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 article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0093] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A trifluralin hapten, characterized in that Its structural formula is as follows:

2. The trifluralin hapten according to claim 1, characterized in that The synthesis path is as follows:

3. Fluralin complete antigen, characterized in that: The trifluralin complete antigen is a conjugate formed by chemically reacting the carboxyl group in the trifluralin hapten of claim 1 with the amino group in the carrier protein to form an amide bond connection. The structural formula of the trifluralin complete antigen is as follows: The carrier protein is selected from at least one of bovine serum albumin, human serum albumin, hemocyanin and ovalbumin.

4. A method for preparing a trifluralin artificial antigen, characterized in that: The following steps are involved: S1, synthetic trifluralin hapten; S2, synthesis of trifluralin hapten activated ester; S3. Use the fluralin hapten activated ester to dialyze to obtain the fluralin artificial antigen.

5. The method for preparing the trifluralin artificial antigen according to claim 4, characterized in that: The S1 specifically includes the following steps: S11. Prepare solution A: dissolve 2.5 g of 4-chloro-3,5-dinitrobenzoic acid in 10 mL of dichloromethane; S12, take 2.5 g of di-n-propylamine, add it into the reaction bottle and dilute it with 20 mL of dichloromethane; S13, under temperature control, slowly add liquid A into the reaction bottle, after adding, raise the temperature to 40-45°C and react for 6 hours. After the reaction, spin dry the dichloromethane; S14. Add 20 mL of dilute sodium hydroxide solution to dissolve the product, extract twice with ethyl acetate, adjust the pH of the aqueous layer to about 4 with 1 mol / L hydrochloric acid, filter the precipitated yellow precipitate and dry the precipitate to obtain the trifluralin hapten.

6. The method for preparing the trifluralin artificial antigen according to claim 5, characterized in that: The S2 specifically includes the following steps: S21. In a 5 mL glass bottle, take 24 mg of trifluralin hapten and dissolve it in 1 mL 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 respectively, mix the two together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution C; S23, add solution C dropwise to solution B under ice bath condition, and then stir at room temperature overnight. After terminating the reaction, the hapten activated ester is obtained.

7. The method for preparing the trifluralin artificial antigen according to claim 6, characterized in that: The S3 specifically includes the following steps: S31, take 4 mg of hapten activated ester, add it to 8 mg of hemocyanin dissolved in PBS (0.01 mol / L, pH=7.4), and react overnight at 4 degrees; S32, placing the reaction solution into a dialysis bag, and fully dialyzing it with a 0.01 mol / L PBS solution to obtain an immunogen; S33, take 12 mg of the hapten activated ester, add it to a PBS (0.01 mol / L, pH=7.4) solution containing 100 mg of BSA, and react overnight at 4 degrees; S34. The reaction solution was placed in a dialysis bag and dialyzed with 0.01 mol / L PBS solution to obtain the coating source.

8. A method for preparing a trifluralin antibody, characterized in that: The following steps are involved: Step 1: Select female Balb / C mice of appropriate age for immunization, measure the titer and inhibition rate of antiserum after the 4th and 5th immunizations, and boost immunization 3 days before cell fusion; The second step is to fuse the spleen cells of the boosted immunized mice with myeloma cells, screen out hybridoma cells that can secrete specific antibodies, and expand the culture; The third step is to inject the expanded cultured hybridoma cells into mice that have been injected with paraffin in advance, collect the ascites, and obtain monoclonal antibodies.

9. Use of the trifluralin antibody according to claim 8 in detecting trifluralin residues.

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