Ethopyrin artificial hapten, artificial antigen, antibody and their application

Abstract

The invention discloses the design and synthesis of haptens of ethofenprox, the development of artificial antigens and antibodies of ethofenprox, and the application thereof in the field of rapid detection and affinity purification column. An artificial antigen with immunogenicity is prepared by the steps of ethofenprox hapten design, linker introduction, carrier introduction, synthesis, purification, etc., and an amide carboxylic acid-modified ethofenprox hapten product is obtained. The ethofenprox hapten of the structure is coupled with keyhole limpet hemocyanin, bovine serum albumin or chicken ovalbumin to synthesize an immunogen and a coating agent. The artificial hapten has high immunoactivity and can stimulate the animal immune system to produce anti-ethofenprox antibodies, and the antibody titer of immune Blab / c mouse serum is 3200. The invention fills the gap that there is no suitable artificial antigen for ethofenprox for preparing specific and highly sensitive antibodies, and establishes a rapid and sensitive immunoassay analysis method for ethofenprox.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay detection of small molecule compounds, and specifically involves organic synthesis, immunochemistry, biochemistry, physical and chemical determination and other technologies. In particular, it involves the design and synthesis of insecticide ether pyrethrin small molecule compound haptens and artificial antigens with functional carboxylic acid groups, mouse immunization, hybridoma cell screening, preparation of specific monoclonal antibodies and establishment of immunoassay methods, which are specifically manifested in the application of ELISA detection methods, rapid detection methods of immunochromatographic test strips and immunoaffinity purification columns, and are applied in the fields of agricultural products and ecological environment. Background Art

[0002] Etofenprox is called an insecticide similar to pyrethroids. It does not contain chrysanthemic acid in its structure, but its spatial structure is similar to that of pyrethroids. In fact, etofenprox does not belong to pyrethroid pesticides, but to ethers. However, it has the advantages of pyrethroid pesticides, with high insecticidal activity and fast knockdown speed. It is suitable for the prevention and control of rice, vegetables, and cotton. It is particularly effective against the Homoptera planthoppers, and also has good effects on a variety of pests such as Lepidoptera, Hemiptera, Orthoptera, Coleoptera, Diptera, and Isoptera. It is especially effective against rice planthoppers. As a low-toxic and safe insecticide, etofenprox is also a designated product after the country prohibits the use of highly toxic pesticides on rice. It is the only pyrethroid pesticide allowed to be registered on rice. Its rapidity and persistence are better than pymetrozine and nitenpyram. Since 2009, ethofenprox has been listed as a key promotion product by the National Agricultural Technology Extension Center, and the plant protection stations in Anhui, Jiangsu, Hubei, Hunan, Guangxi and other places have listed the pesticide as a key promotion variety of the plant protection stations. Comprehensive evaluation of the toxicity of ethofenprox is a low-toxic insecticide, but it is highly toxic to fish and bees and has certain environmental risks. Therefore, we need to correctly analyze the advantages and disadvantages of ethofenprox, and while promoting its use, we should also strengthen its supervision. The effective methods of supervision are mainly strict control of drug use and on-site testing supervision.

[0003] The present invention provides technical support for on-site detection and supervision of chlorpyrifos, and develops a rapid detection and pre-treatment extraction method for chlorpyrifos. At present, the quantitative detection method of chlorpyrifos residues is mainly instrumental analysis methods such as high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry, which have high sensitivity and good accuracy, but have high equipment conditions and complex pre-treatment, and have high technical requirements for analysts, and are difficult to meet the needs of rapid on-site detection of large quantities of samples. The present invention develops an immunochemical analysis technology with chlorpyrifos antigen antibody as the core, overcomes the above-mentioned shortcomings, and has the advantages of high sensitivity, high specificity, fast and convenient use, and environmental friendliness. So far, the monoclonal antibody hybridoma cell technology of chlorpyrifos has not been publicly reported in China, and the specific antibodies produced by it are applied to the rapid detection and analysis of chlorpyrifos, and chlorpyrifos immunoaffinity purification columns are developed for rapid sample pre-treatment. Summary of the invention

[0004] The invention discloses the design and synthesis of ethofenprox hapten, the preparation of ethofenprox artificial antigen and the preparation of monoclonal antibody thereof. The purpose is to provide a preparation method of ethofenprox hapten and corresponding artificial antigen and antibody, and to apply them to rapid immune detection and analysis of ethofenprox in samples, and to use immunoaffinity purification column for rapid sample pretreatment of ethofenprox.

[0005] The present invention selects ether or similar pyrethroid compounds represented by ethotripsy, performs hapten design and structural modification on them, and obtains ethotripsy hapten molecules containing amide carboxylic acid groups. Since the ethotripsy hapten molecules do not have immunogenicity, they need to be carried out through macromolecular proteins as carriers to obtain immunogenicity. Then, a certain number of hapten molecules are connected to protein macromolecules to obtain coatings and immunogens, respectively.

[0006] The uniqueness of the present invention lies in overcoming the difficulty of chemical synthesis of ethofenprox hapten and forming a set of technical routes for the synthesis of ethofenprox hapten. When used for immunizing animals, it can produce specific antibodies with high affinity, and the ELISA method, i.e., enzyme-linked immunosorbent assay method, is established based on this, which can accurately detect the content of ethofenprox. Based on the specific antibody, an agarose immunoaffinity purification column is constructed, which can be used for the rapid extraction pretreatment of trace ethofenprox in samples.

[0007] Another unique feature of the present invention is that keyhole limpet hemocyanin coupled small molecules are selected as immunogens, which increases the coupling ratio of hapten molecules to protein molecules in the coupling reaction and enhances the immunogenicity.

[0008] The structure of ethofenprox is shown below:

[0009]

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is: the ether pyrethrin hapten is an amide carboxylic acid group substituted at the R position on the benzene ring, and R can be one or more of amide propionic acid, amide butyric acid, amide valeric acid, and amide caproic acid derivative groups, and the molecular structure is:

[0011]

[0012] Furthermore, the ether pyrethrin is prepared by nitration, amination, acylation and hydrolysis reaction. The ether pyrethrin hapten is obtained by introducing amide carboxylic acid into ether pyrethrin under strong base catalysis conditions. The molecular structure is:

[0013]

[0014] The present invention also discloses an ethomethrin immunogen (MJZ-KLH or MJZ-BSA), which is synthesized by coupling a hapten with a modified ethomethrin R site structure with a KLH protein or a BSA protein. The molecular structural formulas of the MJZ-KLH and MJZ-BSA are:

[0015]

[0016] 4. The artificial coating antigen of ethoxyfenpropathrin (MJZ-OVA) is synthesized by coupling the artificial hapten with the modified structure of the ethoxyfenpropathrin R site and the OVA protein. The molecular structure of the MJZ-OVA is:

[0017]

[0018] The invention also discloses an ethofenprox antibody, which is a monoclonal antibody capable of generating a specific immune reaction with an ethofenprox immunogen or an ethofenprox artificially coated antigen.

[0019] Anti-ethofenproxil monoclonal antibody is a monoclonal antibody that can produce specific immune reaction with MJZ-KLH, MJZ-BSA or MJZ-OVA, and ethofenproxil compounds.

[0020] An application of an ethofenproxil antibody, which can be used for rapid and sensitive detection of ethofenproxil content in agricultural products and environmental samples, is specifically manifested in the application of ELISA immunoassay method, immunochromatographic test paper rapid detection method and affinity purification column. The application areas include agricultural products such as vegetables, fruits, and meat, as well as environmental samples such as water, soil, and atmosphere.

[0021] Ethofenprox is prepared through a series of reactions including nitration, amination, acylation, and hydrolysis. The synthetic route is as follows:

[0022]

[0023] The preparation method of ethoprolin hapten comprises the following steps:

[0024] (1) dissolving ethoxyfenpropathrin in a 10-20 times equivalent of MeOH aqueous solution, adding 1-2 times molar equivalent of HNO3, heating to 60-80°C under stirring, and reacting to obtain nitroethoxyfenpropathrin;

[0025] (2) adding appropriate amounts of Fe and HCl to carry out catalytic reaction to obtain aminoether pyrethrin;

[0026] (3) further adding 1-2 times the equivalent of acyl chloride methyl ester, stirring the reaction, and obtaining ethoxythrin-amide methyl ester;

[0027] (4) adding 1-2 times the equivalent of NaOH, reacting for 1-2 hours to obtain ethoxyfen-amide n-carboxylic acid, cooling to 5-10° C., filtering with suction, and recrystallizing the filtered solid sample with 1,4-dioxane and drying to obtain ethoxyfen-hapten crystals.

[0028] The preparation method of MJZ-KLH or MJZ-BSA is a carbodiimide method, comprising the following steps:

[0029] (1) Dissolve 0.004 mmoL of ethoxyfenpropathrin hapten in 0.5 mL of DMF, add 0.008-0.02 mmoL of N-hydroxysuccinimide (NHS), stir and react for 15 min, then add 0.004-0.016 mmoL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stir and react at room temperature overnight. The reaction solution is the ethoxyfenpropathrin hapten activation solution.

[0030] (3) Prepare 5-10 mg / mL KLH or BSA solution in 0.01 M carbonate buffer (pH 9.0), add the above HaptenA activation solution dropwise under room temperature stirring, and then change to slow magnetic stirring to react for 3.5 hours. The reaction solution is placed in a pretreated dialysis bag and dialyzed three times with 100 times the volume of 0.01 M phosphate buffer (PBS), changing the solution every 6 hours.

[0031] (4) After the dialysis is completed, the dialysate is taken out, mixed and dispensed into low-adsorption centrifuge tubes to obtain MJZ-KLH or MJZ-BSA, which are then stored at -20°C.

[0032] The preparation method of MJZ-OVA is the carbonyldiimidazole method, which comprises the following steps:

[0033] (1) 0.01 mmol of ethoxyfenpropathrin hapten was weighed and dissolved in 0.5 mL of DMF. 0.02-0.1 mmol of carbonyldiimidazole was added under stirring and reacted at room temperature for 3 h to obtain an ethoxyfenpropathrin hapten activated solution.

[0034] (2) A 10-20 mg / mL OVA solution was prepared in 0.01 M carbonate buffer (pH 9.0), and the above-mentioned etherpyrimidine hapten activation solution was added dropwise under stirring at room temperature. The reaction was continued with stirring for 3 h. The reaction solution was placed in a pretreated dialysis bag and dialyzed three times with 100 times the volume of 0.01 M PBS, with the solution changed every 6 h.

[0035] (3) After dialysis, the dialysate was taken out, mixed and dispensed into low-adsorption centrifuge tubes to obtain MJZ-OVA solution, which was then stored at -20°C.

[0036] The method for preparing anti-ethoprene monoclonal antibodies comprises animal immunization, hybridoma cell screening, and antibody preparation, and includes the following steps:

[0037] (1) Immunization: 6-week-old BALB / c female mice were selected as immunized animals. The immunization method was subcutaneous multi-point injection of Freund's adjuvant. A booster immunization was performed 21 days after the first immunization, and a booster immunization was performed every 14 days, for a total of 3 booster immunizations and 1 final immunization. The specific experimental method is as follows:

[0038] Primary immunization: Dissolve 0.1-0.2 mg MJZ-KLH or MJZ-BSA in 0.9% saline, take an equal volume of Freund's complete adjuvant, put them into syringes, and use an emulsifying tube to connect the syringe to fully emulsify the immunogen MJZ-KLH. Inject the mice subcutaneously at 7 points, including the neck, back, limbs, and abdomen.

[0039] Enhanced immunization: Dissolve 0.05-0.1 mg MJZ-KLH or MJZ-BSA in 0.9% saline, take an equal volume of Freund's complete adjuvant, put them into syringes, connect the syringe with an emulsifying tube to fully emulsify the immunogen MJZ-KLH or MJZ-BSA, and perform intraperitoneal injection immunization on mice.

[0040] Final immunization: 0.05-0.1 mg MJZ-KLH or MJZ-BSA was dissolved in 0.9% saline and directly injected intraperitoneally for immunization.

[0041] (2) Hybridoma cell screening: Regularly monitor the antibody titer of mice. After two booster immunizations, measure the titer of mouse tail blood every other week. The absorbance value OD 450nm ≥1 is positive. Using MJZ-OVA as the coating source, the final immunization was performed when the titer of the test mouse tail blood reached 1:1000-1:4000. The spleen was removed by neck dissection 3 days after the last immunization, and hybridoma cell fusion was performed to screen monoclonal hybridoma cells. The homologous indirect competitive ELISA method was used to coat 5-10μg / mL MJZ-OVA, and 100-500ng / mL of ethoprolin standard solution was used as the competitive drug to select the strong positive OD450nm The cell lines with p ≥ 0.5 and inhibition rate ≥ 50% were subcloned 2-3 times continuously to obtain stable monoclonal hybridoma cell lines.

[0042] (3) Antibody preparation: 7-week-old F1 mice were selected and intraperitoneally injected with 500 μL of pristane per mouse. Seven days later, the stable monoclonal hybridoma cell line (cell density of about 2×10 6 / mL) 500μL / mouse was inoculated into the peritoneal cavity of mice. After 7-10 days, the abdominal cavity of the mice was swollen and the ascites was collected and purified by caprylic acid-ammonium sulfate precipitation method. The purified ascites was coated with MJZ-OVA, and the titer of the antibody and the inhibition rate of ethoprofen were determined by indirect method.

[0043] Ethofenprox ELISA analysis method steps:

[0044] The ethofenproxetine monoclonal antibody is used for rapid detection of ethofenproxetine content in a sample, and the immunoassay is an enzyme-linked immunosorbent assay (ELISA). The ethofenproxetine antibody is used for immunoassay of ethofenproxetine content, comprising the following steps:

[0045] (1) Coating: MJZ-OVA was dissolved in 0.01 mol / L carbonate buffer (pH 9.0), coated on the ELISA plate at 100 μl / well, and stored at 4°C overnight. The plate was then removed and washed three times with 0.01 M PBST.

[0046] (2) Blocking: After coating, 2% skim milk powder was prepared in 0.01 mol / L PBS (pH 7.4), and the ELISA plate was blocked at 300 μl / well. The plate was blocked in a 37°C incubator for 30 minutes, and then washed twice with 0.01 mol / L PBST.

[0047] (3) Competitive reaction: The sample to be tested was dissolved in 0.01 mol / L PBS solution containing 30% methanol, and 50 μl / well of the ELISA plate was added. Then, 50 μl / well of the ethoprolin antibody solution was added. The plate was reacted in a 37°C incubator for 0.5 h, and the plate was washed three times after removal.

[0048] (4) Enzyme-labeled secondary antibody reaction: Rabbit anti-mouse enzyme-labeled secondary antibody was diluted 20,000 times with 0.01 mol / L PBS (pH 7.4), added at 100 μl / well, and reacted in a 37°C incubator for 0.5 h. After removal, the plate was washed 4 times.

[0049] (5) Color development and termination: The color development substrate was tetramethylbenzidine (TMB) solution, 100 μl / well was added during color development, and 2 mol / L H2SO4 50 μl / well was added after 15 minutes of color development to terminate the reaction.

[0050] (6) After color development is completed, place the plate on an ELISA reader and read the OD 450nm Numerical, computational and analytical data.

[0051] Ethofenprox immunochromatographic test strip rapid detection method steps:

[0052] The ethofenproxen coated antigen and ethofenproxen monoclonal antibody are used for rapid detection of ethofenproxen content in agricultural products and environmental samples, and the immunoassay is a colloidal gold immunochromatographic test strip. The ethofenproxen antibody is used in the immunochromatographic test strip for rapid detection, which comprises the following steps:

[0053] (1) The coated antigen MJZ-OVA was streaked on the chromatographic membrane at a concentration of 0.05-0.5 mg / mL as the detection line (T line), and the goat anti-mouse secondary antibody was streaked on the chromatographic membrane at a concentration of 0.05-0.4 mg / mL as the quality control line (C line), and then placed in a forced air drying oven for drying.

[0054] (2) Take the above-mentioned chromatography membrane, assemble it with the sample pad and the absorbent pad to make chromatography test paper, and cut it into 3-4 mm test strips.

[0055] (3) The monoclonal antibody to ethoprofen is labeled with 30 nm colloidal gold particles at a labeling concentration of 2-10 μg / mL to prepare a gold-labeled ethoprofen antibody. 10-50 μL of the prepared gold-labeled antibody is added to a 96-well microplate and freeze-dried or air-dried.

[0056] (4) Prepare a standard solution of ethoprolin with a concentration range of 0.01-10 mg / L, take the test strip in step 2 and the gold-labeled microwell in step 3 for detection, and the sample volume is 100-200 μL.

[0057] (5) When the ethofenproxetine colloidal gold immunochromatographic test strip is used to detect the ethofenproxetine standard solution, when the concentration is 0.5 mg / L or above, it shows a positive result, the T line does not show color, and the C line shows color; when the concentration is lower than 0.5 mg / L, it shows a negative result, the T line shows color, and the C line shows color.

[0058] Preparation steps of ethotripsy immunoaffinity purification column:

[0059] The ethofenproxetine monoclonal antibody is used to prepare an agarose immunoaffinity purification column, and the ethofenproxetine immunoaffinity purification column is used to quickly extract ethofenproxetine from a sample. The preparation of the affinity purification column includes the following steps:

[0060] (1) To prepare agarose gel, dissolve cyanogen bromide-activated agarose gel 4B in 0.1 M carbonate buffer (pH 9.2) at a concentration of 100-300 mg / mL and vortex to dissolve.

[0061] (2) Agarose gel labeled antibody: add ethoprolin monoclonal antibody to the above agarose gel solution at an amount of 0.2-1.0 mg / mL, vortex mix evenly, and let stand for reaction for 1 hour.

[0062] (3) The above monoclonal antibody-labeled agarose gel was loaded into an empty affinity chromatography column, the column head was sealed, and the unbound sites were blocked with 2% skim milk. The column was filled with 2-4 column volumes of skim milk and blocked for 1 hour.

[0063] (4) After sealing, open the sealing cap and elute with 2% skim milk for 2-4 column volumes.

[0064] (5) Remove unbound or loosely bound proteins by eluting with 0.1 M carbonate buffer (pH 9.2) for 10-20 column volumes.

[0065] (6) Remove non-specific proteins and elute with 0.2 M glycine-HCl buffer (pH 2.8) for 5-10 column volumes.

[0066] (7) For the preservation of agarose immunoaffinity chromatography columns, elute with 4-8 column volumes of 0.01 M phosphate buffer (pH 7.2) containing 0.02-0.1% proclin 300 as a preservative, leaving 1-2 mm of solution above the affinity chromatography column for preservation. Install sealing caps at the head and the tail of the column respectively and store at 4°C.

[0067] Compared with the existing analysis technology, the present invention has the following advantages and effects:

[0068] (1) The designed and synthesized ethoxyfenproxin hapten has a high similarity to the target analyte and retains the characteristic structure of ethoxyfenproxin intact, laying a foundation for the preparation of ethoxyfenproxin antibodies with good specificity;

[0069] (2) Experimental verification shows that the synthesis method of the hapten is simple, efficient, and requires few reaction steps. The etherpyrethrin hapten can be synthesized in only four steps, thereby improving the controllability and timeliness of the reaction.

[0070] (3) The antibody against ethoxyfenproxetine has good detection sensitivity. The coating concentration of MJZ-OVA is 1 μg / mL, the antibody concentration is 1 μg / mL, and the methanol content of the ethoxyfenproxetine standard solution is 30%. The ELISA method standard curve was established, and the linear equation was: y=0.1816ln(x)+0.7153, R 2 =0.9712, estimated IC 50 Reached 306ng / mL.

[0071] (4) The present invention is the first to apply ethotripsy monoclonal antibodies to an agarose immunoaffinity purification column, which is used for sample pretreatment, has a high recovery rate, is fast and simple to operate, and the entire operation process can be completed in 30 minutes, thereby improving the pretreatment efficiency.

[0072] Therefore, compared with other methods, the method for synthesizing haptens of the present invention is easier to promote and popularize. The antibody involved in the present invention has high activity and is successfully applied to the ELISA detection method. It is simple and fast to operate. The detection process only takes 1 hour and 15 minutes, and the detection accuracy can reach more than 95%. The immunoaffinity purification column developed by the present invention can be applied to rapid pretreatment and can be reused, with a recovery rate of more than 85%. Therefore, the present invention not only presents a good detection effect under a laboratory environment, but also lays a foundation for the development of a low-cost, high-efficiency, fast-operation immune rapid detection tool, which is suitable for on-site rapid detection needs, has good application prospects, and has both economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0074] Figure 1 Ethofenprox hapten 1 H NMR spectrum.

[0075] Figure 2 This is the HPLC-MS spectrum of ethomethrin hapten.

[0076] Figure 3 The standard curve diagram established for the ethomethrin ELISA analysis method of the present invention.

[0077] Figure 4 This is a color development diagram for detecting the ethomethrin gold-labeled immunochromatographic test paper of the present invention. DETAILED DESCRIPTION

[0078] The following examples of the present invention are only used as further explanations of the present invention and cannot be used as limiting contents or scopes of the present invention. The present invention is now further explained by examples.

[0079] Example 1

[0080] (1) Synthesis of ethoxyfenpropathrin hapten

[0081] In the animal immune system, to obtain antibodies, the antigen must be immunogenic. As a small molecule compound, ethotripsy does not have natural immunogenicity, that is, it cannot stimulate the animal immune system to produce antibodies. We need to artificially modify its structure and couple it with protein to make it immunogenic. The main active groups in the protein coupling method are carboxyl, amino, hydroxyl, and thiol. In the present invention, a carboxylic acid group is introduced into the ethotripsy structure so that it can be condensed and coupled with protein.

[0082] Synthesis method of ether pyrethrin hapten: Weigh 2g of ether pyrethrin and dissolve it in 15 times the volume of MeOH aqueous solution, add 1.5 times the molar equivalent of HNO3, heat to 80°C under stirring, and react to obtain nitro ether pyrethrin. Add appropriate amounts of Fe and HCl for catalytic reaction to obtain amino ether pyrethrin. Further, add 1.5 times the equivalent of methyl acylchloridovalerate and react with stirring to obtain ether pyrethrin-amide valeric acid methyl ester. Add 1.5 times the equivalent of NaOH and hydrolyze for 2 hours to obtain ether pyrethrin-amide valeric acid, cool to 10°C, filter, and the obtained solid sample is recrystallized with 1,4-dioxane. After drying, 0.532g of ether pyrethrin hapten solid is obtained with a purity of 96.2%. Characterized as 1 H NMR(500MHz, CDCl3)δ8.47(d,J=2.3Hz,1H),7.78(s,1H),7.34(m,2H),7.27(q,J=2.9 ,2.1Hz,1H),7.11(m,1H),7.01(dd,J=8.6,2.7Hz,4H),6.94(s,1H),6.90(dd,J=8.2, 2.6Hz,1H),6.77(d,J=8.6Hz,1H),4.47(s,2H),4.07(dd,J=11.1,6.9Hz,2H),3.46(s ,2H),2.42(t,J=6.6Hz,4H),1.77(m,4H),1.45(d,J=7.2Hz,4H),1.33(s,6H).ESI-MS C 31 H 37 NO6,calc.519.2,found:[MH] - 518.2.

[0083] (2) Preparation of artificial cypermethrin antigens MJZ-KLH and MJZ-BSA

[0084] 0.004mmoL of ether pyrethrin hapten was dissolved in 0.5mL DMF, 0.012mmoL N-hydroxysuccinimide (NHS) was added, and the reaction was stirred for 15min, and then 0.006mmoL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added, and the reaction was stirred at room temperature overnight. The reaction solution is the ether pyrethrin hapten activation solution. 5mg / mL KLH solution was prepared with 0.01M carbonate buffer (pH 9.0), and the above HaptenA activation solution was added dropwise under room temperature stirring, and then the reaction was changed to slow magnetic stirring for 3.5h. The reaction solution was placed in a pretreated dialysis bag and dialyzed 3 times with 100 times the volume of 0.01M phosphate buffer (PBS), and the solution was changed every 6h. After the dialysis, the dialysate was taken out, mixed and dispensed into low-adsorption centrifuge tubes to obtain the ethomethrin immunogen MJZ-KLH, which was stored at -20°C.

[0085] Weigh 0.01mmoL of ether pyrethrin hapten and dissolve it in 0.5mL DMF. Add 0.02mmoL carbonyl diimidazole under stirring, and react at room temperature for 3h to obtain ether pyrethrin hapten activation solution. Prepare 10mg / mL BSA solution with 0.01M carbonate buffer (pH 9.0), add the above hapten activation solution dropwise under room temperature stirring, continue stirring and reacting for 3h, put the reaction solution into the pretreated dialysis bag, dialyze 3 times with 100 times volume of 0.01M PBS, and change the solution every 6h. After the dialysis, take out the dialysate, mix it well and divide it into low adsorption centrifuge tubes to obtain the ether pyrethrin coated original MJZ-OVA solution, and store it at -20℃.

[0086] (2) Preparation of hybridoma cell lines with monoclonal antibodies against ethoprolin

[0087] The immunized animals were 6-week-old BALB / c female mice. The immunization method was subcutaneous multi-point Freund's adjuvant injection. The booster immunization was performed 21 days after the first immunization, and the booster immunization was performed every 14 days, for a total of 3 booster immunizations and 1 final immunization. For the first immunization, 0.2 mg MJZ-KLH was dissolved in 0.9% saline, and an equal volume of Freund's complete adjuvant was taken and loaded into syringes. The immunogen MJZ-KLH was fully emulsified with an emulsifying tube docking the syringe, and the mouse was injected subcutaneously at 7 points, including the neck, back, limbs, and abdomen. For the booster immunization, 0.1 mg MJZ-KLH was dissolved in 0.9% saline, and an equal volume of Freund's complete adjuvant was taken and loaded into syringes. The immunogen MJZ-KLH was fully emulsified with an emulsifying tube docking the syringe, and the mouse was intraperitoneally injected. For the final immunization, 0.1 mg MJZ-KLH was dissolved in 0.9% saline and loaded into syringes. The immunogen MJZ-KLH was fully emulsified with an emulsifying tube docking the syringe, and the mouse was intraperitoneally injected.

[0088] The antibody titer was monitored regularly during the mouse immunization process. After two booster immunizations, the titer of the mouse tail blood was measured every other week, and the absorbance value OD was used. 450nm ≥1 is positive. Using MJZ-OVA as the coating source, the final immunization was performed when the titer of the test mouse tail blood reached 1:10000-1:20000. The spleen was removed by neck dissection 3 days after the last immunization, and hybridoma cell fusion was performed to screen monoclonal hybridoma cells. The homologous indirect competitive ELISA method was used to coat 5μg / mL MJZ-OVA, and 100ng / mL of ethoprolin standard solution was used as the competitive drug to select the strong positive OD 450nm The cell lines with p ≥ 0.5 and inhibition rate ≥ 50% were subcloned 2-3 times continuously to obtain stable monoclonal hybridoma cell lines.

[0089] (3) Preparation of monoclonal antibodies against ethoxyfenpropathrin

[0090] Seven-week-old F1 mice were selected and intraperitoneally injected with 500 μL of pristane per mouse. Seven days later, the stable monoclonal hybridoma cell line of ethoprolin obtained by screening (cell density of about 2×10 6 / mL) was inoculated into the abdominal cavity of mice at a dose of 500 μL / mouse. One week later, the abdominal cavity of the mice began to swell, and the mouse ascites was collected and purified by caprylic acid-ammonium sulfate precipitation method to obtain ethomethrin monoclonal antibodies.

[0091] Example 2

[0092] The ethoprofen monoclonal antibody obtained in Example 1 was used to establish an ELISA rapid detection method.

[0093] MJZ-OVA was dissolved in 0.01mol / L carbonate buffer (pH 9.0), coated on the ELISA plate at 100μl / well, and coated overnight in a refrigerated environment at 4℃. After removal, the plate was washed 3 times with 0.01M PBST. After coating, 2% skim milk powder was prepared with 0.01mol / L PBS (pH7.4), and the ELISA plate was blocked at 300μl / well. The plate was blocked in a 37℃ incubator for 30 minutes, and then washed 2 times with 0.01mol / LPBST. The sample to be tested was dissolved in 0.01mol / L PBS solution containing 30% methanol, and 50μl / well was added to the ELISA plate, and then 50μl / well of the ether pyrethrin antibody solution was added. The plate was reacted in a 37℃ incubator for 0.5 hours, and then the plate was washed 3 times after removal. The rabbit anti-mouse enzyme-labeled secondary antibody was diluted 20,000 times with 0.01 mol / L PBS (pH 7.4), added 100 μl / well, reacted in a 37°C incubator for 0.5 hours, and washed 4 times after removal. The color substrate was tetramethylbenzidine (TMB) solution, 100 μl / well was added during color development, and 2 mol / L H2SO4 50 μl / well was added after 15 minutes of color development to terminate. After color development was terminated, the ELISA plate was placed on an ELISA reader to read the OD450nm Numerical, computational and analytical data.

[0094] The inhibition rate of the target analyte concentration on the antibody I = ((Amax-Amin)-(Ai-Amin) / (Amax-Amin)) × 100, the binding rate of the antibody and the antigen B / B0 = (Ai-Amin) / (Amax-Amin), where: Amax is the average absorbance value of the blank well, Amin is the average absorbance value of the BLAB / c mouse serum control well before immunization, and Ai is the average absorbance value of the sample well. Draw a standard curve with the inhibition rate I or the binding rate B / B0 as the ordinate and the analyte concentration C as the abscissa, as shown in the attached figure. Figure 3 The detection limit can stably reach 306μg / L. The target analyte concentrations corresponding to 5% and 95% inhibition rates are used as the minimum detection concentration and the maximum detection concentration, respectively. The detection range is 26μg / L to 3642μg / L.

[0095] GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" stipulates that the limit standard of ethoxyfenpropathrin in vegetables, fruits and meat crops is 0.5-5 mg / kg. The sensitivity of this method is 0.306 mg / kg, which can meet the maximum residue limit (MRL) standard detection requirements. The actual sample detection used leeks (MRL = 1 mg / kg), celery (MRL = 1 mg / kg), radish (MRL = 1 mg / kg), pears (MRL = 0.6 mg / kg), grapes (MRL = 4 mg / kg), beef (MRL = 0.5 mg / kg), water, and soil. Negative samples and ethoxyfenpropathrin positive added samples were set up respectively, with added concentrations of 0.02 mg / kg, 0.2 mg / kg and 2 mg / kg. The sample was extracted with an extract, and the final sample to be tested was diluted 10 times. The concentration of ethoxyfenpropathrin in the blank sample and the added sample was calculated according to the ELISA standard curve, and the results were consistent with the added concentration. The specific data results are shown in Table 1.

[0096] Table 1 ELISA test results of ethofenprox in actual samples

[0097]

[0098] Example 3

[0099] The ethofenproxetine coated antigen and ethofenproxetine antibody obtained in Example 1 were used to prepare an immunochromatographic rapid test strip. The detection limit of ethofenproxetine by the rapid test strip method was 0.5 mg / L, and this method was applied to actual sample detection.

[0100] The actual sample test used leeks, celery, radish, pear, grape, beef, water, and soil. Blank samples and positive samples for fennel were set up respectively, and the samples were extracted with extracting solution. The blank samples and samples with fennel added concentrations below the detection limit of fennel were negative except for the 0.2 mg / kg fennel added concentration group in leek and beef samples, which showed false positives. The test paper test results of samples with fennel added concentrations above the detection limit of fennel were all positive. The specific results are shown in Table 2.

[0101] Table 2 Test results of ethoxyfenpropathrin test paper in actual samples

[0102]

[0103]

[0104] Example 4

[0105] The ethomethrin antibody obtained in Example 1 is used to prepare an agarose immunoaffinity purification column. The ethomethrin affinity purification column is mainly used for rapid extraction of ethomethrin in sample pretreatment.

[0106] Dissolve the cyanogen bromide activated sepharose 4B in 0.1M carbonate buffer (pH9.2) at a concentration of 200mg / mL and vortex to dissolve. Add cypermethrin monoclonal antibody to the agarose gel solution at an amount of 0.2-1.0mg / mL and vortex to mix evenly. Let it stand for 1h to react. Label the agarose gel with cypermethrin antibody. Load the monoclonal antibody-labeled agarose gel into an empty affinity chromatography column, seal the column head, block the unbound sites with 2% skim milk, fill the column with 2 column volumes of skim milk, and block for 1h. After blocking, open the sealing cap and elute with 2% skim milk for 2 column volumes. Elute with 0.1M carbonate buffer (pH9.2) for 10-20 column volumes to remove unbound and loosely bound proteins. Then elute with 0.2M glycine-hydrochloric acid buffer (pH2.8) for 5-10 column volumes to remove non-specific proteins. The agarose immunoaffinity chromatography column is stored and finally eluted with 0.01M phosphate buffer (pH7.2) containing a preservative of 0.02-0.1% proclin300 for 5 column volumes, leaving 1-2mm of solution above the affinity chromatography column, and sealing caps are respectively added to the column head and the column tail to obtain the ethoprofen immunoaffinity purification column.

[0107] The ethomethrin immunoaffinity purification column in this embodiment has application prospects in rapid sample pretreatment. It is used for sample pretreatment and has the advantages of high recovery rate, environmental protection and safety, and rapid and simple operation.

[0108] Finally, it should be stated that the present invention is not limited to the above four embodiments, and may also have other derivative uses. Other variations that can be directly derived or associated with the content disclosed by those skilled in the art should be considered as the protection scope of the present invention.

Claims

1. An artificial hapten of ethoxyfenpropathrin, characterized in that: The molecular structure is: Where n is 1 or 2 or 3 or 4.

2. The artificial hapten of ethoprolin according to claim 1, characterized in that: It is prepared by nitration, amination, acylation and hydrolysis of ethoprolin, and the ethoprolin hapten is obtained by introducing amide carboxylic acid into ethoprolin under strong base catalysis conditions.

3. The ethofenprox immunogen is characterized by: The etherpyrimidine artificial hapten according to claim 1 or 2 is coupled with keyhole limpet hemocyanin (KLH) to form a composite, and the molecular structure is:

4. The ethofenprox immunogen is characterized by: The artificial hapten of ethoprolin according to claim 1 or 2 is coupled with bovine serum albumin (BSA) to form a synthetic product, and the molecular structure is:

5. Ethopyrothrin artificially coated antigen, characterized in that: The ethoxyfenproxetine artificial hapten according to claim 1 or 2 is coupled with chicken ovalbumin (OVA) to form a synthetic ethoxyfenproxetine artificial coating antigen having a molecular structure of:

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