Cyhalofop-butyl hapten as well as preparation method and application thereof

Abstract

The invention discloses a cyhalofop-butyl hapten as well as a preparation method and application thereof, and belongs to the technical field of biochemical engineering. According to the cyhalofop-butyl hapten, butanol of a cyhalofop-butyl chemical structure is changed into 6-aminocaproic acid, almost all the structures of the cyhalofop-butyl are reserved, the cyhalofop-butyl hapten has an active group carboxyl coupled with protein, and after the cyhalofop-butyl hapten is coupled with carrier protein, an organism can generate an antibody capable of recognizing the cyhalofop-butyl with high titer and high specificity. The hapten compound disclosed by the invention is simple and convenient in synthesis method and relatively high in purity, and the prepared anti-cyhalofop-butyl monoclonal antibody is very high in sensitivity and can be simultaneously used for carrying out rapid and accurate immunodetection and immunoassay on cyhalofop-butyl and cyhalofop oxalic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to a cyhalofop-butyl hapten and a preparation method and application thereof. Background Art

[0002] Cyhalofop-butyl is a newly developed oxyphenoxypropionic acid systemic herbicide developed by Dow Chemical Company in the United States. Its trade name is Clincher, code name is XED-537, and its chemical name is (R)-2-[4-(4Chemicalbook-cyano-2-fluorophenoxy)phenoxy]butyl propionate. It is mainly used in rice nursery fields, direct seeding fields, and transplanting fields to control most vicious grass weeds such as barnyard grass, cyhalofop-butyl, and goosegrass. It can also effectively control weeds that are resistant to dichloroquinoline, sulfonylurea and amide herbicides. It has the characteristics of high efficiency, low toxicity and low residue. Its mechanism of action is to quickly conduct in the plant body and quickly degrade into a series of metabolites: cyhalofop-butyl (ACID), cyhalofop-butyl (AMIDE), cyhalofop-butyl diac (DIACID) and cyhalofop-butyl (DP), among which ACID is a compound with herbicidal activity. ACID accumulates in the intercalary meristem of weeds, thereby effectively inhibiting acetyl-CoA carboxylase, an enzyme necessary for fatty acid biosynthesis in the weeds, thereby achieving the purpose of weed control.

[0003] Due to the widespread use of cyhalofop-butyl, its residues in the environment or plants are too large. Excessive intake of food containing cyhalofop-butyl and its metabolites can cause great harm to the human body. On August 5, 2022, the European Commission issued Regulation (EU) 2022 / 1363, which revised the maximum residue limit of cyhalofop-butyl in certain products, and the maximum residue limit in some products was reduced to 0.01 mg / kg. my country also stipulates that its maximum residue limit in cereals, finished grains and brown rice is 0.1 mg / kg. Whether it is consumed domestically or exported abroad, its residue detection in food cannot be avoided. At present, the detection of cyhalofop-butyl is mainly instrumental methods, but these methods have the disadvantages of expensive instruments, time-consuming detection, and the need for professional technicians to operate. They cannot meet the requirements of modern detection for rapid and accurate detection, so it is necessary to develop an immunoassay method. The fundamental basis of immunoassay methods is to have excellent antigens and antibodies as the basis. Therefore, the purpose of the present invention is to obtain a highly sensitive cyhalofop-butyl antibody. For cyhalofop-butyl, a hapten that retains almost the entire structure of cyhalofop-butyl and has a carboxyl group is synthesized from scratch, and further coupled with different carrier proteins as artificial immunogens and coating sources, thereby preparing a cyhalofop-butyl detection antibody, which has high sensitivity and good specificity and can be used for the rapid analysis and detection of cyhalofop-butyl and cyanocobalamic acid. Summary of the invention

[0004] The purpose of the present invention is to provide a cyhalofop-butyl hapten and a preparation method and application thereof. A hapten which retains almost the entire structure of cyhalofop-butyl and has a carboxyl group is synthesized from scratch, and different carrier proteins are coupled as artificial immunogens and coating sources respectively to prepare cyhalofop-butyl antibodies. The hapten has high sensitivity and good specificity and can be used for rapid analysis and detection of cyhalofop-butyl.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a cyhalofop-butyl hapten, the molecular structure of which is shown in I: .

[0006] Furthermore, the method for preparing the cyhalofop-butyl hapten comprises the following steps: (1) Take cyhalofop-butyl and dissolve it in methanol to obtain liquid A; take sodium hydroxide and dissolve it in pure water to obtain liquid B; slowly add liquid A to liquid B under stirring at room temperature; after the addition is completed, continue to stir the reaction, and after the reaction is completed, evaporate the methanol by rotary evaporation, extract with ethyl acetate, discard the organic layer, adjust the pH value of the water layer to 2.8-3, precipitate a white precipitate, filter and dry the precipitate to obtain cyhalofop-butyl; (2) Take cyanofluoric acid, add thionyl chloride, and reflux under stirring. After the reaction is completed, evaporate the thionyl chloride by rotary evaporation, and then add tetrahydrofuran to dissolve the product to obtain a hapten intermediate; (3) 6-aminocaproic acid is taken, sodium hydroxide solution is added to dissolve, the temperature is lowered, and the hapten intermediate is added dropwise under magnetic stirring. After the reaction is completed, the temperature is naturally restored to room temperature, and the reaction is continued by stirring. After the reaction is complete, the pH value is adjusted to 2.8-3, a white precipitate is precipitated, and the precipitate is filtered and dried to obtain the cyhalofop-butyl hapten. The reaction formula of the method is as follows: .

[0007] Furthermore, in the preparation method of the cyhalofop-butyl hapten, in step (1), the mass volume concentration of liquid A is 0.18-0.2 g / mL, the mass volume concentration of liquid B is 0.08 g / mL, the volume ratio of liquid A to liquid B is 1:1, stirring and reacting for 6-8 hours, and adjusting the pH value with 2 mol / L hydrochloric acid; in step (2), the mass volume ratio of cyhalofop-butyl to thionyl chloride is 1:5, reflux reaction is performed for 6-8 hours, and the mass volume ratio of cyhalofop-butyl to tetrahydrofuran is 1:5; in step (3), the concentration of the sodium hydroxide solution is 2 mol / L / L, the mass volume ratio of 6-aminocaproic acid to the sodium hydroxide solution is 0.065 g / mL, the temperature is lowered to 0-5°C, stirring and reacting for 2-3 hours, and adjusting the pH value with 2 mol / L hydrochloric acid.

[0008] The present invention provides a cyhalofop-butyl artificial antigen, the molecular structure of which is shown in II: .

[0009] Furthermore, in the cyhalofop-butyl artificial antigen, the proteins in structural formula II are at least two of bovine serum albumin, hemocyanin and ovalbumin.

[0010] Furthermore, the preparation method of the cyhalofop-butyl artificial antigen comprises the following steps: M1. Take cyhalofop-butyl artificial hapten, dissolve it in N,N-dimethylformamide to prepare liquid C; weigh 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide respectively, mix them together and dissolve them in N,N-dimethylformamide to prepare liquid D; add liquid D dropwise into liquid C under ice bath condition, then stir at room temperature overnight, and terminate the reaction to obtain hapten activated ester E; M2. Take hapten activated ester E, add it to PBS containing protein 1, and react overnight at 4°C to obtain reaction solution F; put the reaction solution F into a dialysis bag, and fully dialyze it with PBS to obtain an artificial antigen, i.e., an immunogen; M3. Take the hapten activated ester E, add it to the PBS containing protein 2, and react overnight at 4°C to obtain the reaction solution G; put the reaction solution G into a dialysis bag, and dialyze it thoroughly with PBS to obtain the artificial antigen, i.e., the coating antigen.

[0011] Furthermore, in the preparation method of the cyhalofop-butyl artificial antigen, the mass volume ratio of the cyhalofop-butyl artificial hapten to N,N-dimethylformamide in M1 is 40-42 mg / mL, the mass ratio of the cyhalofop-butyl artificial hapten, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 40-42:20:12, the volume ratio of N,N-dimethylformamide in liquid C to the volume ratio of N,N-dimethylformamide in liquid D is 2:1; the mass ratio of the hapten activated ester to protein 1 or protein 2 in M2 and M3 is 1:3-9; the PBS concentration is 0.01 mol / L, and the pH is 7.4.

[0012] Furthermore, the use of cyhalofop-butyl hapten or cyhalofop-butyl artificial antigen in the preparation of cyhalofop-butyl antibody.

[0013] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. The present invention hydrolyzes cyhalofop-butyl under alkaline conditions to generate cyhalofop-butyl acid, sodium hydroxide provides an alkaline environment to promote the hydrolysis of ester bonds, and cyhalofop-butyl acid is precipitated by adjusting the pH value, which is convenient for separation and purification; thionyl chloride reacts with cyhalofop-butyl acid to convert the carboxylic acid into an acyl chloride, which is convenient for subsequent connection with a carrier protein; 6-aminocaproic acid reacts with a hapten intermediate (acyl chloride) to form an amide bond, and the hapten is connected with a carrier protein mimetic (6-aminocaproic acid) to form a soluble hapten; as a small molecule compound, the cyhalofop-butyl hapten itself does not have immunogenicity, but after chemical modification, it can be combined with a macromolecular carrier to form an artificial antigen, thereby inducing the body to produce specific antibodies. The hapten part in the artificial antigen can retain the molecular characteristic structure of cyhalofop-butyl, so that the body can recognize and produce specific antibodies against cyhalofop-butyl.

[0014] 2. The present invention forms a covalent bond by reacting the activated ester with the amino group of the protein, thereby connecting the hapten to the carrier protein and enhancing the immunogenicity; the cyhalofop-butyl monoclonal antibody prepared by the present invention can simultaneously recognize cyhalofop-butyl and its metabolite cyanoxalic acid, but does not recognize other similar herbicides. The cyhalofop-butyl monoclonal antibody has good specificity and can be applied to the detection and analysis of cyhalofop-butyl. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 is the chemical structural formula of the cyhalofop-butyl artificial hapten of the present invention; Figure 2 is the chemical structural formula of the cyhalofop-butyl artificial antigen of the present invention, wherein the protein is at least two of bovine serum albumin, hemocyanin and ovalbumin; Figure 3 The present invention is a reaction formula for preparing the cyhalofop-butyl artificial hapten. DETAILED DESCRIPTION

[0017] 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.

[0018] Example 1

[0019] This embodiment provides a method for preparing a cyhalofop-butyl artificial antigen, comprising the following steps: Among them, the preparation of cyhalofop-butyl hapten refers to Figure 1 , Figure 3 As shown, cyhalofop-butyl was used as the starting material and a hapten with a complete structure of cyhalofop-butyl was prepared through three steps of chemical synthesis, and its molecular weight was 414.4. The specific preparation steps are as follows: (1) Take 3.6 g of cyhalofop-butyl and dissolve it in 20 mL of methanol to obtain liquid A; take 1.6 g of sodium hydroxide and dissolve it in 20 mL of pure water to obtain liquid B; slowly add liquid A to liquid B under stirring at room temperature; after the addition is completed, continue stirring and reacting for 6 hours. After the addition is completed, evaporate the methanol by rotary evaporation, extract with ethyl acetate, discard the organic layer, adjust the pH value of the water layer to 3 with 2 mol / L hydrochloric acid, precipitate a white precipitate, filter and dry the precipitate to obtain cyhalofop-butyl; (2) Take 2 g of cyanofluoric acid, add 10 mL of thionyl chloride, and reflux under stirring. After the reaction is completed, evaporate the thionyl chloride by rotary evaporation, and then add 10 mL of tetrahydrofuran to dissolve the product to obtain a hapten intermediate; (3) Take 1.3 g of 6-aminocaproic acid, add 20 mL of 2 mol / L sodium hydroxide solution to dissolve, cool to 4 ° C, add the hapten intermediate dropwise under magnetic stirring, and after completion, return to room temperature naturally, continue stirring and reacting for 2 hours. After the reaction is complete, adjust the pH value to 3 with 2 mol / L hydrochloric acid to precipitate a white precipitate, filter and dry the precipitate to obtain the cyhalofop-butyl hapten; Among them, the preparation of cyhalofop-butyl artificial antigen, see Figure 2 As shown, the specific preparation steps are as follows: M1. Take 40 mg of cyhalofop-butyl artificial hapten and dissolve it in 1 mL of N,N-dimethylformamide to prepare liquid C; weigh 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of N-hydroxysuccinimide respectively, mix the two together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare liquid D; add liquid D dropwise into liquid C under ice bath condition, then stir at room temperature overnight, and terminate the reaction to obtain hapten activated ester E; M2. Take 4 mg of hapten activated ester E, add it to PBS (0.01 mol / L, pH=7.4) containing 5 mg of hemocyanin, and react overnight at 4°C to obtain reaction solution F; put the reaction solution F into a dialysis bag, and dialyze it thoroughly with 0.01 mol / L PBS to obtain an artificial antigen, i.e., an immunogen; M3. Take 20 mg of hapten activated ester E and add it to PBS (0.01 mol / L, pH=7.4) containing 33 mg of bovine serum albumin. React overnight at 4°C to obtain reaction solution G. Put the reaction solution G into a dialysis bag and dialyze it thoroughly with 0.01 mol / L PBS to obtain the artificial antigen, i.e., the coating antigen.

[0020] Example 2

[0021] This embodiment provides a method for preparing a cyhalofop-butyl artificial antigen, comprising the following steps: Among them, the preparation of cyhalofop-butyl hapten refers to Figure 1 , Figure 3 As shown, cyhalofop-butyl was used as the starting material and a hapten with a complete structure of cyhalofop-butyl was prepared through three steps of chemical synthesis, and its molecular weight was 414.4. The specific preparation steps are as follows: (1) Take 3.8 g of cyhalofop-butyl and dissolve it in 20 mL of methanol to obtain liquid A; take 1.6 g of sodium hydroxide and dissolve it in 20 mL of pure water to obtain liquid B; slowly add liquid A to liquid B under stirring at room temperature; after the addition is completed, continue stirring and reacting for 7 hours. After the addition is completed, evaporate the methanol by rotary evaporation, extract with ethyl acetate, discard the organic layer, adjust the pH value of the water layer to 2.9 with 2 mol / L hydrochloric acid, precipitate a white precipitate, filter and dry the precipitate to obtain cyhalofop-butyl; (2) Take 2 g of cyanofluoric acid, add 10 mL of thionyl chloride, and reflux under stirring. After the reaction is completed, evaporate the thionyl chloride by rotary evaporation, and then add 10 mL of tetrahydrofuran to dissolve the product to obtain a hapten intermediate; (3) Take 1.3 g of 6-aminocaproic acid, add 20 mL of 2 mol / L sodium hydroxide solution to dissolve, cool to 5 ° C, add the hapten intermediate dropwise under magnetic stirring, and after completion, return to room temperature naturally, continue stirring and reacting for 3 hours. After the reaction is complete, adjust the pH value to 2.9 with 2 mol / L hydrochloric acid to precipitate a white precipitate, filter and dry the precipitate to obtain the cyhalofop-butyl hapten; Among them, the preparation of cyhalofop-butyl artificial antigen, see Figure 2 As shown, the specific preparation steps are as follows: M1. Take 41 mg of cyhalofop-butyl artificial hapten and dissolve it in 1 mL of N,N-dimethylformamide to prepare liquid C; weigh 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of N-hydroxysuccinimide respectively, mix the two together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare liquid D; add liquid D dropwise into liquid C under ice bath condition, then stir at room temperature overnight, and terminate the reaction to obtain hapten activated ester E; M2. Take 4 mg of hapten activated ester E, add it to PBS (0.01 mol / L, pH=7.4) containing 10 mg of bovine serum albumin, and react overnight at 4°C to obtain reaction solution F; put the reaction solution F into a dialysis bag, and dialyze it thoroughly with 0.01 mol / L PBS to obtain an artificial antigen, i.e., an immunogen; M3. Take 20 mg of hapten activated ester E, add it to 45 mg of ovalbumin dissolved in PBS (0.01 mol / L, pH=7.4), and react overnight at 4°C to obtain reaction solution G; put the reaction solution G into a dialysis bag, and dialyze it thoroughly with 0.01 mol / L PBS to obtain the artificial antigen, i.e., the coating antigen.

[0022] Example 3

[0023] This embodiment provides a method for preparing a cyhalofop-butyl artificial antigen, comprising the following steps: Among them, the preparation of cyhalofop-butyl hapten refers to Figure 1 , Figure 3 As shown, cyhalofop-butyl was used as the starting material and a hapten with a complete structure of cyhalofop-butyl was prepared through three steps of chemical synthesis, and its molecular weight was 414.4. The specific preparation steps are as follows: (1) Take 4 g of cyhalofop-butyl and dissolve it in 20 mL of methanol to obtain liquid A; take 1.6 g of sodium hydroxide and dissolve it in 20 mL of pure water to obtain liquid B; slowly add liquid A to liquid B under stirring at room temperature; after the addition is completed, continue stirring and reacting for 8 hours. After the addition is completed, evaporate the methanol by rotary evaporation, extract with ethyl acetate, discard the organic layer, adjust the pH value of the water layer to 2.8 with 2 mol / L hydrochloric acid, precipitate a white precipitate, filter and dry the precipitate to obtain cyhalofop-butyl; (2) Take 2 g of cyanofluoric acid, add 10 mL of thionyl chloride, and reflux under stirring. After the reaction is completed, evaporate the thionyl chloride by rotary evaporation, and then add 10 mL of tetrahydrofuran to dissolve the product to obtain a hapten intermediate; (3) Take 1.3 g of 6-aminocaproic acid, add 20 mL of 2 mol / L sodium hydroxide solution to dissolve, cool to 0 ° C, add the hapten intermediate dropwise under magnetic stirring, and after completion, return to room temperature naturally, continue stirring and reacting for 3 hours. After the reaction is complete, adjust the pH value to 2.8 with 2 mol / L hydrochloric acid to precipitate a white precipitate, filter and dry the precipitate to obtain the cyhalofop-butyl hapten; Among them, the preparation of cyhalofop-butyl artificial antigen, see Figure 2 As shown, the specific preparation steps are as follows: M1. Take 42 mg of cyhalofop-butyl artificial hapten and dissolve it in 1 mL of N,N-dimethylformamide to prepare liquid C; weigh 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of N-hydroxysuccinimide respectively, mix the two together and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare liquid D; add liquid D dropwise into liquid C under ice bath condition, then stir at room temperature overnight, and terminate the reaction to obtain hapten activated ester E; M2. Take 4 mg of hapten activated ester E, add it to PBS (0.01 mol / L, pH=7.4) containing 5 mg of hemocyanin, and react overnight at 4°C to obtain reaction solution F; put the reaction solution F into a dialysis bag, and dialyze it thoroughly with 0.01 mol / L PBS to obtain an artificial antigen, i.e., an immunogen; M3. Take 20 mg of hapten activated ester E and add it to 50 mg of ovalbumin dissolved in PBS (0.01 mol / L, pH=7.4), and react overnight at 4°C to obtain reaction solution G; put the reaction solution G into a dialysis bag, and dialyze it thoroughly with 0.01 mol / L PBS to obtain the artificial antigen, i.e., the coating antigen.

[0024] Example 4

[0025] This embodiment provides a method for preparing a cyhalofop-butyl monoclonal antibody, comprising the following steps: 1. Animal immunization Balb / C female mice, about 7 weeks old, were selected as immunized animals and were numbered 1, 2, 3, and 4. Immunization was performed by subcutaneous multiple-point injection at the back of the neck.

[0026] 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 0.05 mg. Reinforcement immunization: Reinforcement immunization is carried out on the 21st and 42nd days after the initial immunization, with a dose of 0.1 mg and the same emulsification method. After that, the reinforcement immunization is carried out every 21 days, for a total of 5 times. Starting from the second reinforcement immunization, blood samples are collected from animals 8 days after each immunization for serum titer determination and specificity determination.

[0027] 2. Analysis of antiserum effect (1) Starting from the third booster immunization, on the 8th day after each immunization, 30 μL of blood was collected from the mouse tail, centrifuged to obtain antiserum, and stored at -20°C for later use.

[0028] (2) The titer and specificity of the antiserum were determined by indirect enzyme-linked immunosorbent assay. The steps are as follows: S1. Coating: Dilute 5 mg / mL of coating antigen 10,000 times with coating buffer, and add 100 μL / well to the ELISA plate as a blank control group, and incubate in a 4°C refrigerator overnight. Then wash once with PBST (phosphate buffered saline 0.05% (V / V) Tween20 washing solution); S2. Blocking: Pour out the liquid in the wells, wash the plate once, add 200μL of washing solution to each well, spin dry, add 200μL of blocking solution (1% BSA-PBS) to each well, block at 37℃ for 2h, spin dry, and place in a 4℃ refrigerator for later use.

[0029] 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; shake and mix, react at 37°C for 30 minutes, wash the plate 3 times with a plate washer, add 200 μL of washing solution to each well, and spin dry.

[0030] 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.

[0031] 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.

[0032] 3. Cell fusion and screening of positive hybridomas (1) Recovery of myeloma cells: Take the myeloma cells out of liquid nitrogen and quickly thaw them in a 37°C water bath. After thawing, centrifuge at 1000 rpm for 5 min. 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 take out 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.

[0033] (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 minutes, 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 minutes. 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 μL per well.

[0034] (3) Preparation of spleen cells: Bleed the orbits of Balb / c mice that have been immunized multiple times and have passed blood tests, collect the serum, and then soak in 75% alcohol for 5 minutes for disinfection, and move to the clean bench for dissection. Remove the spleen aseptically, rinse with DMEM basal culture medium, and place 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 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.

[0035] (4) Cell fusion: Mix the myeloma cells and immune spleen cells after centrifugation in a ratio of about 1:6 in a centrifuge tube, 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 use it for later use. 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 it 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.

[0036] (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 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.

[0037] 4. Large-scale preparation of monoclonal antibodies 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. The conventional practice is to inject liquid paraffin into the peritoneal cavity of 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. In particular, from the 7th day onwards, the abdominal cavity of the mice will swell. Before the death of the mice, ascites is collected aseptically with a disposable syringe. The collected ascites is centrifuged at 12000r / min for 10 minutes to remove the upper fat and lower fibrin, collect the middle layer, and determine its titer and inhibition rate by icELISA. After purification, it is stored at -20°C for later use.

[0038] Example 5

[0039] This embodiment provides a serum titer detection method, comprising the following steps: (1) Dissolve the prepared cyhalofop-butyl coating agent in a pH 9.6 Na2CO3-NaHCO3 buffer solution, dilute the cyhalofop-butyl coating agent to 0.3 μg / mL as the coating solution, add 100 μL to each well of a 96-well microplate, place at 4°C for more than 16 hours or incubate at 37°C for 2 to 3 hours, and wash once with PBST (phosphate buffered saline 0.05% (V / V) Tween20 washing solution); (2) Add 200 μL of 1% BSA in PBS blocking solution to each well, block for 2 hours, and wash once with washing solution; (3) Add antiserum and standard: dilute the antiserum in a gradient; dissolve the cyfluthrin 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 50 μL of blank dilution, 50 μL of gradient diluted antiserum, and 50 μL of goat anti-mouse enzyme-labeled secondary antibody to each well; Inhibition column: add 50 μL of standard solution, 50 μL of gradient diluted antiserum, and 50 μL of goat anti-mouse enzyme-labeled secondary antibody to each well, and incubate at 37°C for 30 minutes; (4) Wash the microplate three times with washing solution, discard the liquid in the wells, spin dry with a microplate dehydrator, add 50 μL substrate A and 50 μL substrate B, and react at room temperature for 15 minutes; (5) Reading determination: Measure the absorbance at dual wavelengths of 450 nm and 630 nm; 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 determined by its inhibition rate.

[0040] Example 6

[0041] This embodiment provides a method for determining cyhalofop-butyl by indirect competitive enzyme-linked immunosorbent assay (icELISA), which specifically comprises the following steps: S1. Coating: Dilute 50,000 times the 5 mg / mL coating antigen with coating buffer and add 100 μL / well to the ELISA plate as a blank control group, incubate overnight in a 4°C refrigerator. Then wash once with PBST (phosphate buffered saline 0.05% (V / V) and Tween20 washing solution; 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.

[0042] 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; shake and mix, react at 37°C for 30 minutes, wash the plate 3 times with a plate washer, add 200 μL of washing solution to each well, and spin dry.

[0043] 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.

[0044] S5. Determination: Use an enzyme-linked immunosorbent assay (ELISA) to determine the absorbance of each well at A450nm.

[0045] S6. Calculation: The IC50 value of the inhibition curve was calculated using the GraphPad Prism10.0 fitting module.

[0046] 1.6 ng / mL, 0.8 ng / mL, 0.4 ng / mL, 0.2 ng / mL, and 0.1 ng / mL cyhalofop-butyl standard solutions were prepared to establish a standard curve for the indirect enzyme-linked immunosorbent assay. After the conditions were optimized, the method had an IC50 of 0.46 ng / mL for cyhalofop-butyl, and a linear detection range of 0.1 to 1.6 ng / mL.

[0047] Experimental example Determination of antibody cross-reactivity rate 1. According to the optimal coating antigen concentration and the optimal antiserum dilution factor obtained in Example 1, an indirect competitive ELISA experiment was performed with cyhalofop-butyl, cyhalofop-butyl, quizalofop-ethyl and fluazifop-butyl as competitive standards to detect the specificity of the cyhalofop-butyl monoclonal antibody. The half-maximal inhibitory concentration (IC50) and cross-reaction rate (CR) values ​​are listed in Table 1.

[0048] Table 1 drug <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Cyhalofop-butyl 0.46 100 Cyanofluoric acid 0.95 47 Quizalofop-Ph-ethyl >100 <1 Haloxyfop-Phthiophene >100 <1 The experimental results show that the cyhalofop-butyl monoclonal antibody can simultaneously recognize cyhalofop-butyl and its metabolite cyanofluoric acid, but does not recognize other similar herbicides, indicating that the cyhalofop-butyl monoclonal antibody of the present invention has good specificity and can be applied to the immunoassay detection of the total residues of cyhalofop-butyl and cyanofluoric acid.

[0049] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0050] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. 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 cyhalofop-butyl hapten, characterized in that: Its molecular structure is shown in I: 。 2. The method for preparing the cyhalofop-butyl hapten according to claim 1, characterized in that: The following steps are involved: (1) Take cyhalofop-butyl and dissolve it in methanol to obtain liquid A; take sodium hydroxide and dissolve it in pure water to obtain liquid B; slowly add liquid A to liquid B under stirring at room temperature; after the addition is completed, continue to stir the reaction, and after the reaction is completed, evaporate the methanol by rotary evaporation, extract with ethyl acetate, discard the organic layer, adjust the pH value of the water layer to 2.8-3, precipitate a white precipitate, filter and dry the precipitate to obtain cyhalofop-butyl; (2) Take cyanofluoric acid, add thionyl chloride, and reflux under stirring. After the reaction is completed, evaporate the thionyl chloride by rotary evaporation, and then add tetrahydrofuran to dissolve the product to obtain a hapten intermediate; (3) 6-aminocaproic acid is taken, sodium hydroxide solution is added to dissolve, the temperature is lowered, and the hapten intermediate is added dropwise under magnetic stirring. After the reaction is completed, the temperature is naturally restored to room temperature, and the reaction is continued by stirring. After the reaction is complete, the pH value is adjusted to 2.8-3, a white precipitate is precipitated, and the precipitate is filtered and dried to obtain the cyhalofop-butyl hapten. The reaction formula of the method is as follows: 。 3. The method for preparing the cyhalofop-butyl hapten according to claim 2, characterized in that: In step (1), the mass volume concentration of liquid A is 0.18-0.2 g / mL, the mass volume concentration of liquid B is 0.08 g / mL, and the volume ratio of liquid A to liquid B is 1:

1. The reaction is stirred for 6-8 hours and the pH is adjusted with 2 mol / L hydrochloric acid.

4. The method for preparing the cyhalofop-butyl hapten according to claim 2, characterized in that: In step (2), the mass volume ratio of cyanofluoric acid to thionyl chloride is 1:5, the reflux reaction is carried out for 6-8 hours, and the mass volume ratio of cyanofluoric acid to tetrahydrofuran is 1:

5.

5. The method for preparing the cyhalofop-butyl hapten according to claim 2, characterized in that: In step (3), the concentration of the sodium hydroxide solution is 2 mol / L, the mass volume ratio of 6-aminocaproic acid to the sodium hydroxide solution is 0.065 g / mL, the temperature is lowered to 0-5°C, the reaction is stirred for 2-3 hours, and the pH is adjusted with 2 mol / L hydrochloric acid.

6. A cyhalofop-butyl artificial antigen, characterized in that: Its molecular structure is shown in II: 。 7. The cyhalofop-butyl artificial antigen according to claim 6, characterized in that: The proteins in structural formula II are at least two of bovine serum albumin, hemocyanin and ovalbumin.

8. The method for preparing the cyhalofop-butyl artificial antigen according to claim 6, characterized in that: The preparation steps are as follows: M1. Take cyhalofop-butyl artificial hapten, dissolve it in N,N-dimethylformamide to prepare liquid C; weigh 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide respectively, mix them together and dissolve them in N,N-dimethylformamide to prepare liquid D; add liquid D dropwise into liquid C under ice bath condition, then stir at room temperature overnight, and terminate the reaction to obtain hapten activated ester E; M2. Take hapten activated ester E, add it to PBS containing protein 1, and react overnight at 4°C to obtain reaction solution F; put the reaction solution F into a dialysis bag, and fully dialyze it with PBS to obtain an artificial antigen, i.e., an immunogen; M3. Take the hapten activated ester E, add it to the PBS containing protein 2, and react overnight at 4°C to obtain the reaction solution G; put the reaction solution G into a dialysis bag, and dialyze it thoroughly with PBS to obtain the artificial antigen, i.e., the coating antigen.

9. The method for preparing the cyhalofop-butyl artificial antigen according to claim 8, characterized in that: The mass volume ratio of cyhalofop-butyl artificial hapten to N,N-dimethylformamide in M1 is 40-42 mg / mL, the mass ratio of cyhalofop-butyl artificial hapten, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 40-42:20:12, the volume ratio of N,N-dimethylformamide in solution C to the volume ratio of N,N-dimethylformamide in solution D is 2:1; the mass ratio of hapten activated ester to protein 1 or protein 2 in M2 and M3 is 1:3-9; the concentration of PBS is 0.01 mol / L, and the pH is 7.

4.

10. Use of a cyhalofop-butyl hapten according to claim 1 or a cyhalofop-butyl artificial antigen according to claim 6 in the preparation of cyhalofop-butyl antibodies.

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