A pendimethalin hapten, antigen, antibody, preparation method and application

Abstract

The present invention relates to the technical field of immunoassay, and provides a pendimethalin hapten, antigen, antibody, preparation method and application thereof. The present invention introduces an active group to the 2-methyl group of pendimethalin. After the obtained pendimethalin hapten is conjugated with a carrier protein, it has high immunogenicity. After immunizing a host animal, antibodies with high titer and high sensitivity can be produced. The pendimethalin hapten of the present invention is obtained by three steps of nitration reaction, substitution reaction and condensation reaction using 2-methyl-4-chlorobenzylamine as a raw material. The preparation method provided by the present invention is simple to operate, has mild conditions, easily available raw materials, and the product has high yield and high purity. The results of the examples show that after the pendimethalin antigen of the present invention is injected and immunized in mice, the antibody titer is 1.0×10 4 , and the half inhibitory concentration (IC 50 ) is about 0.5 ppb. The minimum detection limit of the colloidal gold reagent for pendimethalin in dry tobacco leaf samples is 10 ng / g.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to a pendimethalin hapten, antigen, antibody, preparation method and application thereof. Background Art

[0002] Pendimethalin is an aniline herbicide developed by American Cyanamid Company, which is used to control annual gramineous weeds and some broad-leaved weeds, and is applicable to rice, cotton, corn, tobacco, peanuts, vegetables and orchard crops. Due to its good herbicidal effect and high safety, it has been widely promoted and applied. However, pendimethalin is a low-toxic compound. Skin contact may cause ulceration, and after being absorbed by the human body, it may cause symptoms such as cyanosis, nausea, dizziness and headache. Therefore, the harm of pendimethalin residues in agricultural products to the body cannot be ignored, and it is very necessary to develop a rapid detection method for pendimethalin.

[0003] Currently, the common methods for detecting pendimethalin in foods and agricultural products include liquid chromatography, liquid chromatography-mass spectrometry, and immunoassay. Although the detection results of liquid chromatography and liquid chromatography-mass spectrometry are accurate, they require expensive instrument equipment, and the operation is complex, with high requirements for operators, and are mostly limited to use in professional laboratories. Immunoassay mainly includes competitive enzyme-linked immunosorbent assay (ELISA) and colloidal gold method. Compared with the above two methods, immunoassay is simple, rapid in operation, does not require professional personnel to operate, only basic instrument equipment is needed, and at the same time, because it is suitable for screening a large number of samples, it can be widely used in the safety control of raw materials in food processing enterprises and on-site supervision by industrial and commercial law enforcement departments, and is a very valuable rapid screening method for food safety.

[0004] However, currently, the immunoassay for pendimethalin generally has the problems of insufficient antibody detection sensitivity and low titer. Summary of the Invention

[0005] In view of this, the present invention provides a pendimethalin hapten, antigen, antibody, preparation method and application thereof. After the antigen obtained by conjugating the pendimethalin hapten provided by the present invention with a carrier protein is immunized in a host animal, it can produce antibodies with high titer and high sensitivity, which is beneficial to improving the accuracy of detecting pendimethalin by immunoassay.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] A pendimethalin hapten has the structure shown in Formula I:

[0008]

[0009] The present invention also provides a preparation method for the pendimethalin hapten described in the above solution, including the following steps:

[0010] 2-Methyl-4-chlorobenzylamine, sulfuric acid and nitric acid are mixed for nitration reaction to obtain dinitro-2-methyl-4-chlorobenzylamine; the structure of the dinitro-2-methyl-4-chlorobenzylamine is shown as formula B;

[0011] The dinitro-2-methyl-4-chlorobenzylamine, 3-aminopentane, a basic reagent and an organic solvent are mixed for substitution reaction to obtain a hapten precursor; the structure of the hapten precursor is shown as formula C;

[0012] The hapten precursor, pyridine and succinic anhydride are mixed for condensation reaction to obtain the pendimethalin hapten with the structure shown as formula I;

[0013]

[0014] Preferably, the nitration reaction includes a first stage and a second stage carried out in sequence. The temperature in the first stage is 45-48 °C and the time is 2-2.5 h. The temperature in the second stage is 60-65 °C and the time is 4-4.5 h;

[0015] The temperature of the substitution reaction is 80-85 °C and the time is 4-4.5 h;

[0016] The temperature of the condensation reaction is 75-80 °C and the time is 12-24 h.

[0017] Preferably, the molar ratio of nitric acid to 2-methyl-4-chlorobenzylamine is (2-2.2):1;

[0018] The molar ratio of 3-aminopentane to dinitro-2-methyl-4-chlorobenzylamine is (1-1.2):1;

[0019] The molar ratio of succinic anhydride to the hapten precursor is (1-1.2):1.

[0020] The present invention also provides a pendimethalin antigen, which is obtained by conjugating the pendimethalin hapten described in the above scheme or the pendimethalin hapten prepared by the preparation method described in the above scheme with a carrier protein.

[0021] Preferably, the carrier protein is ovalbumin or bovine serum albumin; when the carrier protein is ovalbumin, the structural formula of the pendimethalin antigen is shown as formula II:

[0022]

[0023] When the carrier protein is bovine serum albumin, the structural formula of the pendimethalin antigen is shown as formula III:

[0024]

[0025] The present invention also provides a method for preparing the pendimethalin antigen described in the above solution, which includes the following steps:

[0026] Mix the pendimethalin hapten, coupling agent and polar solvent described in the above solution for activation to obtain a hapten activation solution;

[0027] Mix the hapten activation solution and the buffer solution of the carrier protein for a coupling reaction to obtain the pendimethalin antigen.

[0028] The present invention also provides a pendimethalin antibody, which is obtained by immunizing a host animal with the pendimethalin antigen described in the above solution.

[0029] The present invention also provides a test strip or kit for detecting pendimethalin, which contains the pendimethalin antibody described in the above solution.

[0030] The present invention also provides the application of the pendimethalin antibody described in the above solution or the test strip or kit described in the above solution in detecting pendimethalin.

[0031] The present invention provides a pendimethalin hapten, and its structural formula is shown in Formula I. The present invention introduces an active group at the 2-position methyl of pendimethalin. After the obtained pendimethalin hapten is coupled with a carrier protein, the resulting antigen has high immunogenicity. After immunizing a host animal, it can produce antibodies with high titer and high sensitivity, thereby improving the accuracy of detecting pendimethalin by immunoassay.

[0032] The present invention also provides a method for preparing the pendimethalin hapten described in the above solution. The present invention uses 5-chloro-2-methylbenzylamine as a raw material and obtains the pendimethalin hapten through three steps of nitration reaction, substitution reaction and condensation reaction. The preparation method provided by the present invention is simple in operation, mild in conditions, easy to obtain raw materials, and has high product yield and high purity. The results of the examples show that when using the method of the present invention to prepare the pendimethalin hapten, the product yield reaches 95%, and the purity reaches 95%.

[0033] The present invention also provides a pendimethalin antigen, which is obtained by coupling the pendimethalin hapten described in the above solution with a carrier protein. After the pendimethalin hapten provided by the present invention is coupled with a carrier protein, the resulting antigen has strong immunogenicity. The results of the examples show that after immunizing mice with the pendimethalin antigen of the present invention, the antibody titer produced is 1.0×10 4 , and the half-inhibitory concentration (IC 50 ) is about 0.5 ppb, and the lowest detection limit of colloidal gold for pendimethalin in dry tobacco leaf samples is 10 ng / g. Description of the Drawings

[0034] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the pendimethalin hapten prepared in Example 1 of the present invention. Detailed implementation mode

[0035] The present invention provides a pendimethalin hapten with a structure shown in Formula I:

[0036]

[0037] The present invention also provides a preparation method of the pendimethalin hapten described in the above solution, including the following steps:

[0038] Mix 2-methyl-4-chlorobenzylamine, sulfuric acid and nitric acid for nitration reaction to obtain dinitro-2-methyl-4-chlorobenzylamine; the structure of the dinitro-2-methyl-4-chlorobenzylamine is shown in Formula B;

[0039] Mix the dinitro-2-methyl-4-chlorobenzylamine, 3-aminopentane, basic reagent and organic solvent for substitution reaction to obtain a hapten precursor; the structure of the hapten precursor is shown in Formula C;

[0040] Mix the hapten precursor, pyridine and succinic anhydride for condensation reaction to obtain a pendimethalin hapten with a structure shown in Formula I;

[0041]

[0042] In the present invention, the synthesis route of the pendimethalin hapten is shown in Chem1:

[0043]

[0044] The preparation method of the pendimethalin hapten will be described in detail below with reference to Chem1.

[0045] In the present invention, 2-methyl-4-chlorobenzylamine (the structural formula is shown in Formula A in Chem1), sulfuric acid and nitric acid are mixed for nitration reaction to obtain dinitro-2-methyl-4-chlorobenzylamine; the structure of the dinitro-2-methyl-4-chlorobenzylamine is shown in Formula B; the structure of the 2-methyl-4-chlorobenzylamine is shown in Formula A in Chem1; in the present invention, the nitration reaction preferably includes a first stage and a second stage in sequence, the temperature of the first stage is preferably 45-48°C, the time is preferably 2-2.5 h, the temperature of the second stage is preferably 60-65°C, and the time is preferably 4-4.5 h.

[0046] In the present invention, the molar ratio of the nitric acid to 2-methyl-4-chlorobenzylamine is preferably (2 to 2.2):1; the nitric acid is preferably fuming nitric acid, and the sulfuric acid is preferably concentrated sulfuric acid; the present invention has no special requirements for the concentrated sulfuric acid and fuming nitric acid, and the concentrated sulfuric acid and fuming nitric acid well-known to those skilled in the art can be used, specifically 98wt% concentrated sulfuric acid and 90wt% to 97.5wt% fuming nitric acid.

[0047] In the present invention, it is preferred to first dissolve 2-methyl-4-chlorobenzylamine in concentrated sulfuric acid, stir at room temperature for 30 min, and then dropwise add a mixed solution of concentrated sulfuric acid and fuming nitric acid to the obtained solution. After the dropping is completed, carry out the nitration reaction in two stages under oil bath conditions in sequence. During the reaction process, it is preferred to monitor the reaction progress by TLC method; the dosage ratio of the 2-methyl-4-chlorobenzylamine to the concentrated sulfuric acid for dissolution is preferably 1 g:30 to 50 mL; the volume ratio of the concentrated sulfuric acid to the fuming nitric acid in the mixed solution of concentrated sulfuric acid and fuming nitric acid is preferably 1:1.

[0048] After the nitration reaction is completed, in the present invention, it is preferred to pour the obtained reaction solution into ice cubes, filter after the solid product precipitates, and wash the filter cake with sodium hydroxide solution until the washing solution is neutral, and dry the washed filter cake to obtain dinitro-2-methyl-4-chlorobenzylamine.

[0049] After obtaining dinitro-2-methyl-4-chlorobenzylamine, in the present invention, the dinitro-2-methyl-4-chlorobenzylamine, 3-aminopentane, basic reagent and organic solvent are mixed to carry out a substitution reaction to obtain a hapten precursor; the structure of the hapten precursor is shown in formula C. In the present invention, the molar ratio of the 3-aminopentane to the dinitro-2-methyl-4-chlorobenzylamine is preferably (1 to 1.2):1; the basic reagent is preferably sodium hydroxide, and the sodium hydroxide is preferably used in the form of an aqueous sodium hydroxide solution, and the concentration of the aqueous sodium hydroxide solution is preferably 2 mol / L; the molar ratio of the dinitro-2-methyl-4-chlorobenzylamine to the sodium hydroxide is preferably (2 to 2.5):1; the organic solvent is preferably tetrahydrofuran, and the present invention has no special requirements for the dosage of the organic solvent, as long as the substitution reaction can proceed smoothly.

[0050] In the present invention, the temperature of the substitution reaction is preferably 80 to 85 °C, and the time is preferably 4 to 4.5 h; in the specific embodiments of the present invention, the substitution reaction is preferably carried out under reflux conditions.

[0051] In a specific embodiment of the present invention, it is preferred to first dissolve dinitro-2-methyl-4-chlorobenzylamine in an organic solvent to obtain a solution of dinitro-2-methyl-4-chlorobenzylamine, dissolve 3-aminopentane in an aqueous sodium hydroxide solution to obtain a 3-aminopentane-sodium hydroxide mixed solution, and then drop the solution of dinitro-2-methyl-4-chlorobenzylamine into the 3-aminopentane-sodium hydroxide mixed solution, and carry out a substitution reaction under the condition of oil bath reflux. During the reaction process, TLC is used to monitor the reaction progress.

[0052] After the substitution reaction is completed, in the present invention, it is preferred to distill the obtained reaction solution under reduced pressure to remove the organic solvent, dissolve the obtained residue in water, extract the obtained solution with petroleum ether to obtain an aqueous phase; after adjusting the pH value of the aqueous phase to 6.5 - 7, extract it with ethyl acetate to obtain an ethyl acetate phase; concentrate the ethyl acetate phase, and subject the obtained concentrate to column chromatography purification to obtain the hapten precursor; the number of times of extraction with petroleum ether is preferably 4 times, and the aqueous phases obtained from the 4 extractions are combined; the number of times of extraction with ethyl acetate is preferably 4 times, and the ethyl acetate phases obtained from the 4 extractions are combined; the eluent used for the column chromatography purification is preferably a mixed solvent of ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is preferably 1:1.

[0053] After obtaining the hapten precursor, in the present invention, the hapten precursor, pyridine and succinic anhydride are mixed to carry out a condensation reaction to obtain the pendimethalin hapten having the structure shown in Formula I. In the present invention, the molar ratio of succinic anhydride to the hapten precursor is preferably (1 - 1.2):1; there is no special requirement for the dosage of pyridine in the present invention, as long as it can dissolve the hapten precursor and enable the condensation reaction to proceed smoothly.

[0054] In the present invention, the temperature of the condensation reaction is preferably 75 - 80 °C, the time is preferably 12 - 24 h, and TLC method is preferably used to monitor the reaction progress during the reaction process.

[0055] After the condensation reaction is completed, in the present invention, it is preferred to distill the obtained reaction solution to dryness under reduced pressure, mix the obtained first residue with toluene and then distill to dryness again under reduced pressure, dissolve the obtained second residue in ethyl acetate and then carry out column chromatography purification to obtain the pendimethalin hapten; the eluent used for the column chromatography purification is preferably a mixed solvent of ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is preferably 2:1.

[0056] The present invention also provides a pendimethalin antigen, which is obtained by conjugating the pendimethalin hapten described in the above scheme or the pendimethalin hapten prepared by the preparation method described in the above scheme with a carrier protein.

[0057] In the present invention, the carrier protein is preferably ovalbumin or bovine serum albumin; when the carrier protein is ovalbumin, the structural formula of the pendimethalin antigen is shown in Formula II:

[0058]

[0059] When the carrier protein is bovine serum albumin, the structural formula of the pendimethalin antigen is shown in Formula III:

[0060]

[0061] In a specific embodiment of the present invention, the pendimethalin antigen with the structure shown in Formula II is the pendimethalin coating antigen, and the pendimethalin antigen with the structure shown in Formula III is the pendimethalin immunogen.

[0062] The present invention also provides a method for preparing the pendimethalin antigen described in the above solution, comprising the following steps:

[0063] Mix the pendimethalin hapten, coupling agent and polar solvent described in the above solution for activation to obtain a hapten activation solution;

[0064] Mix the hapten activation solution and the buffer solution of the carrier protein for a coupling reaction to obtain the pendimethalin antigen.

[0065] In the present invention, the pendimethalin hapten, coupling agent and polar solvent described in the above solution are mixed for activation to obtain a hapten activation solution. In the present invention, the polar solvent is preferably DMF, and the dosage ratio of the pendimethalin hapten to the polar solution is preferably 10 mg: 200 μL; the coupling agent is preferably dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), or (1-ethyl-(3-dimethylaminopropyl) carbodiimide) (EDC) and N-hydroxysuccinimide (NHS); the activation temperature is preferably 25-30 °C, and the time is preferably 12-24 h.

[0066] In the present invention, when preparing the pendimethalin coating antigen (Formula II), the coupling agent used is preferably DCC and NHS; the molar ratio of DCC to the pendimethalin hapten is preferably (2-2.5): 1, and the molar ratio of NHS to the pendimethalin hapten is preferably (1-1.2): 1; in a specific embodiment of the present invention, it is preferred to first dissolve the pendimethalin hapten in DMF, then add NHS and DCC for activation, and after the activation is completed, it is preferred to centrifuge the obtained activation product, and the supernatant is the hapten activation solution.

[0067] In the present invention, when preparing the pendimethalin immunogen (Formula III), the coupling agent used is preferably EDC and NHS; the molar ratio of EDC to the pendimethalin hapten is preferably (2 - 2.5):1; the molar ratio of NHS to the pendimethalin hapten is preferably (1 - 1.2):1; in the specific embodiments of the present invention, it is preferred to first dissolve the pendimethalin hapten in DMF, then add NHS and EDC for activation, and after the activation is completed, it is preferred to centrifuge the obtained activated product, and the supernatant is the hapten activation solution.

[0068] After obtaining the hapten activation solution, the present invention mixes the hapten activation solution and the buffer solution of the carrier protein for a coupling reaction to obtain the pendimethalin antigen. In the present invention, when preparing the pendimethalin coating antigen, the carrier protein used is preferably OVA, and when preparing the pendimethalin immunogen, the carrier protein used is preferably BSA; the molar ratio of the carrier protein to the pendimethalin hapten is preferably (40 - 50):1; the buffer solution of the carrier protein is preferably obtained by dissolving the carrier protein in the buffer solution. When the carrier protein is OVA, the buffer solution is preferably PB buffer solution, and the concentration of the PB buffer solution is preferably 0.02 mol / L. When the carrier protein is BSA, the buffer solution is preferably PB buffer solution, and the concentration of the PB buffer solution is preferably 0.02 mol / L. The present invention preferably adds the hapten activation solution dropwise to the buffer solution of the carrier protein, and after the addition is completed, the reaction is carried out at room temperature.

[0069] In the present invention, the temperature of the coupling reaction is preferably 25 - 30 °C, and the time is preferably 4 - 6 h.

[0070] In the present invention, the synthesis process of the pendimethalin coating antigen (Formula II) is shown as Chem2:

[0071]

[0072] In the present invention, the synthesis process of the pendimethalin immunogen (Formula III) is shown as Chem3:

[0073]

[0074] After the coupling reaction is completed, the present invention preferably centrifuges the obtained reaction solution, aspirates the supernatant, and dialyzes the supernatant to obtain the pendimethalin antigen. In the present invention, the molecular weight cut-off of the dialysis bag used for dialysis is preferably 8,000 to 14,000, and the dialysis bag is preferably treated with an EDTA solution and washed with distilled water before use; the concentration of the EDTA solution is preferably 0.2 mol / L; when preparing the pendimethalin coating antigen, the dialysis solution for dialysis is preferably a PBS buffer solution, and the concentration of the PBS buffer solution is preferably 0.02 mol / L; when preparing the pendimethalin immunogen, the dialysis solution for dialysis is preferably a PB buffer solution, and the concentration of the PB buffer solution is preferably 0.02 mol / L; the dialysis time is preferably 3 days, and the dialysis solution is changed every 8 h. After dialysis, it is preferred to precipitate the solution in the dialysis bag and store it frozen at a low temperature for later use.

[0075] The present invention also provides a pendimethalin antibody obtained by immunizing a host animal with the pendimethalin antigen described in the above solution; the host animal is preferably a mouse; the present invention has no special requirements for the immunization method, and a method well-known to those skilled in the art can be used; in a specific embodiment of the present invention, it is preferred to emulsify the pendimethalin immunogen dilution with incomplete Freund's adjuvant and then immunize the host animal; the diluent used in the pendimethalin immunogen dilution is preferably a sterilized 1% sodium chloride solution.

[0076] In a specific embodiment of the present invention, the process for preparing the pendimethalin antibody is preferably as follows:

[0077] Immunize the host animal with the emulsified pendimethalin antibody to obtain an immunized animal, and use the ELISA method to test the serum of the immunized animal. When the serum parameters meet the requirements, perform monoclonal antibody preparation according to the monoclonal antibody preparation method;

[0078] Extract the spleen cells of the immunized animal and fuse them with syngeneic myeloma cells to obtain fused cells;

[0079] Perform hybridoma screening on the fused cells;

[0080] Use the limiting dilution method for clonal culture of hybridoma cells, and use immunological methods to screen out positive hybridoma cells that can produce the required monoclonal antibody and perform clonal amplification.

[0081] The present invention also provides a test strip or kit for detecting pendimethalin, which contains the pendimethalin antibody described in the above solution.

[0082] The present invention also provides the application of the pendimethalin antibody described in the above solution or the test strip or kit described in the above solution in the detection of pendimethalin; the method for detecting pendimethalin is preferably the ELLSA kit method or the colloidal gold method. By immunizing mice with the pendimethalin antigen of the present invention, the obtained antibody has a high titer and high sensitivity. When it is applied to the detection of pendimethalin, the accuracy and sensitivity of the detection method can be improved.

[0083] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0084] Example 1

[0085] Synthesis of pendimethalin hapten:

[0086] 100 mL of concentrated sulfuric acid was added to 5.29 g of 2-methyl-4-chlorobenzylamine in batches and slowly. After the addition, the mixture was stirred at room temperature for 30 min, and then a mixture of 30 mL of concentrated sulfuric acid and fuming nitric acid (the volume ratio of concentrated sulfuric acid to fuming nitric acid in the mixture was 1:1) was slowly added dropwise. After the addition, the reaction was carried out in an oil bath at 45 °C for 2 h, and then the temperature was raised to 60 °C and reacted for 4 h. After detecting that the reaction was complete by TLC, the reaction solution was poured into ice cubes, and a large amount of solid was precipitated. The solid was filtered by suction, and the filter cake was washed with 1 wt% NaOH solution until the pH value of the washing solution was neutral. The filter cake was placed in the air to dry, and 5.33 g of white solid, namely dinitro-2-methyl-4-chlorobenzylamine (Formula B), was obtained.

[0087] 5.33 g of dinitro-2-methyl-4-chlorobenzylamine was dissolved in 200 mL of dry tetrahydrofuran. Separately, 2.54 g of 3-aminopentane was dissolved in 2 mol / L NaOH solution, and then the tetrahydrofuran solution of 2-methyl-4-chlorobenzylamine was slowly added dropwise to the sodium hydroxide solution of 3-aminopentane. After the addition, the reaction was refluxed in an oil bath at 85 °C for 4 h. After detecting the reaction was complete by TLC, the reaction solution was distilled under reduced pressure to remove tetrahydrofuran. 100 mL of water was added to the residue, and it was extracted with petroleum ether 4 times (200 mL of petroleum ether was used each time). The aqueous phase was taken, and the pH value of the aqueous phase was adjusted to 6.5 with 2 mol / L hydrochloric acid, and then it was extracted with ethyl acetate 4 times (200 mL of ethyl acetate was used each time). The organic phases were combined and concentrated to a solution volume of about 200 mL. Silica gel was added, the sample was mixed, and the sample was loaded onto the column by the dry method. The product was eluted with an ethyl acetate-petroleum ether mixed solvent (ethyl acetate: petroleum ether = 1:1), and the solvent was removed to obtain 4.2 g of a yellowish-brown oily liquid, namely the hapten precursor (Formula C).

[0088] Dissolve 4.2 g of the hapten precursor in pyridine, add 2.82 g of succinic anhydride, and react at 75 °C overnight. Detect by TLC method. After the reaction is complete, perform vacuum distillation. After evaporation to dryness, add toluene to the residue and evaporate to dryness again to fully remove pyridine. Dissolve the residue in ethyl acetate, load the sample wet onto a column, and elute with a mixed solvent of ethyl acetate - petroleum ether (ethyl acetate: petroleum ether = 2:1) to obtain the fraction of the main UV spot, yielding 3.1 g of a pale yellow oily liquid, which is the pendimethalin hapten. Determined by high performance liquid chromatography, the purity of the hapten is 92.1%. Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the obtained pendimethalin hapten. According to Figure 1 it can be determined that the product has the target structure.

[0089] Synthesis of pendimethalin coating antigen:

[0090] Dissolve 12 mg of the pendimethalin hapten in 200 μL of DMF, add 5 mg of NHS and 15 mg of DCC, and react at 25 °C overnight. The next day, centrifuge the reaction solution and take the supernatant (i.e., the hapten activation solution) for standby. Separately, dissolve 30 mg of OVA in 2 mL of 0.02 mol / L PBS buffer. After clarification, slowly add the hapten activation solution dropwise. After addition, react at 25 °C for 4 h, then centrifuge the reaction solution, take the supernatant, add it to a dialysis bag and dialyze for 3 days. The dialysis solution is 0.02 mol / L PBS buffer, and the dialysis solution is changed every 8 h. After dialysis, collect the product and store it frozen at low temperature for standby.

[0091] Synthesis of pendimethalin immunogen:

[0092] Dissolve 12 mg of the hapten in 200 μL of DMF, add 5 mg of NHS and 15 mg of EDC, and react at 25 °C overnight. The next day, centrifuge the reaction solution and take the supernatant (i.e., the hapten activation solution) for standby. Separately, dissolve 44 mg of BSA in 2 mL of 0.02 mol / L PB buffer. After clarification, slowly add the hapten activation solution dropwise. After addition, react at 25 °C for 4 h, then centrifuge the reaction solution, take the supernatant, add it to a dialysis bag and dialyze for 3 days. The dialysis solution is 0.02 mol / L PB buffer, and the dialysis solution is changed every 8 h. After dialysis, collect the product and store it frozen at low temperature for standby.

[0093] Example 2

[0094] Synthesis of pendimethalin hapten:

[0095] 100 mL of concentrated sulfuric acid was slowly added in batches to 6.21 g of 2-methyl-4-chlorobenzylamine. After the addition was complete, the mixture was stirred at room temperature for 30 min. Then, a mixture of 36 mL of concentrated sulfuric acid and fuming nitric acid with a volume ratio of 1:1 was slowly added dropwise. After the addition was complete, the reaction was carried out in an oil bath at 45 °C for 2 h, and then the temperature was raised to 60 °C for 4 h. After the reaction was detected to be complete by TLC, the reaction solution was poured into ice cubes, and a large amount of solid was precipitated. The solid was filtered by suction, and the filter cake was washed with 1 wt% NaOH solution until the pH of the washing solution was neutral. The filter cake was placed in the air to dry, and 6.19 g of white solid was obtained, which was dinitro-2-methyl-4-chlorobenzylamine (Formula B).

[0096] 6.19 g of dinitro-2-methyl-4-chlorobenzylamine was dissolved in 200 mL of dry tetrahydrofuran. Separately, 3.62 g of 3-aminopentane was dissolved in 2 mol / L NaOH solution. Then, the tetrahydrofuran solution of dinitro-2-methyl-4-chlorobenzylamine was slowly added dropwise to the sodium hydroxide solution of 3-aminopentane. After the addition was complete, the reaction was refluxed in an oil bath at 85 °C for 4 h. After the reaction was detected to be complete by TLC, the reaction solution was distilled under reduced pressure to remove tetrahydrofuran. 100 mL of water was added to the residue, and the mixture was extracted with petroleum ether 4 times (200 mL of petroleum ether was used each time). The aqueous phase was taken, and the pH of the aqueous phase was adjusted to 6.5 with 2 mol / L hydrochloric acid. Then, the mixture was extracted with ethyl acetate 4 times (200 mL of ethyl acetate was used each time). The organic phases were combined and concentrated to about 200 mL of solution. Silica gel was added, and the sample was mixed and loaded onto the column by dry method. The product was eluted with a mixed solvent of ethyl acetate - petroleum ether (ethyl acetate: petroleum ether = 1:1), and 5.42 g of yellowish-brown oily liquid was obtained after desolvation, which was the hapten precursor (Formula C).

[0097] 4.2 g of the hapten precursor was dissolved in pyridine, 2.82 g of succinic anhydride was added, and the reaction was carried out at 75 °C overnight. After the reaction was detected to be complete by TLC, the reaction was distilled under reduced pressure. After evaporation to dryness, toluene was added to the residue, and it was evaporated to dryness again to completely remove pyridine. The residue was dissolved in ethyl acetate and loaded onto the column by wet method. The component with the main UV point was eluted with a mixed solvent of ethyl acetate - petroleum ether (ethyl acetate: petroleum ether = 2:1), and 4.36 g of light yellow oily liquid was obtained, which was the pendimethalin hapten. The purity of the pendimethalin hapten was determined by high performance liquid chromatography to be 94.8%.

[0098] Synthesis of pendimethalin coating antigen:

[0099] Dissolve 15 mg of pendimethalin hapten in 220 μL of DMF, add 5 mg of NHS and 15 mg of DCC, and react overnight at 25 °C. The next day, centrifuge the reaction solution and take the supernatant (i.e., the hapten activation solution) for standby. Separately, dissolve 37.5 mg of OVA in 2 mL of 0.02 mol / L PBS buffer. After clarification, slowly add dropwise the hapten activation solution. After addition, react at 25 °C for 4 h. Then centrifuge the reaction solution, take the supernatant, add it to a dialysis bag and dialyze for 3 days. The dialysis solution is 0.02 mol / L PBS solution, and the dialysis solution is changed every 8 h. After dialysis, collect the product and store it frozen at low temperature for standby.

[0100] 5: Synthesis of immunogen

[0101] Dissolve 15 mg of pendimethalin hapten in 220 μL of DMF, add 5 mg of NHS and 14.5 mg of EDC, and react overnight at 25 °C. The next day, centrifuge the reaction solution and take the supernatant (i.e., the hapten activation solution) for standby. Separately, dissolve 56 mg of BSA in 2 mL of 0.02 mol / L PB buffer. After clarification, slowly add dropwise the hapten activation solution. After addition, react at 25 °C for 4 h. Then centrifuge the reaction solution, take the supernatant, add it to a dialysis bag and dialyze for 3 days. The dialysis solution is 0.02 mol / L PB buffer, and the dialysis solution is changed every 8 h. After dialysis, collect the antigen and store it frozen at low temperature for standby.

[0102] Example 3

[0103] Synthesis of pendimethalin hapten:

[0104] Slowly add 100 mL of concentrated sulfuric acid to 5.09 g of 2-methyl-4-chlorobenzylamine in batches. After addition, stir at room temperature for 30 min. Slowly add dropwise a mixture of 36 mL of concentrated sulfuric acid and fuming nitric acid, and the volume ratio of concentrated sulfuric acid to fuming nitric acid in the mixture is 1:1. After addition, react in an oil bath at 45 °C for 2 h, then raise the temperature to 60 °C and react for 4 h. After detecting the completion of the reaction by TLC, pour the reaction solution into ice cubes, a large amount of solid precipitates, filter by suction, wash the filter cake with 1 wt% NaOH solution until the pH of the washing solution is neutral, and place the filter cake in the air to dry to obtain 5.02 g of white solid, which is dinitro-2-methyl-4-chlorobenzylamine (Formula B).

[0105] 5.02 g of 2-methyl-4-chloro-2,4-dinitrobenzylamine was dissolved in 200 mL of dry tetrahydrofuran. Separately, 2.73 g of 3-aminopentane was dissolved in 2 mol / L NaOH solution. Then, the tetrahydrofuran solution of 2-methyl-4-chloro-2,4-dinitrobenzylamine was slowly added dropwise to the sodium hydroxide solution of 3-aminopentane. After the addition, the reaction was refluxed at 85 °C in an oil bath for 4 h. Detection by TLC showed that after the reaction was complete, the reaction solution was distilled under reduced pressure to remove tetrahydrofuran. 100 mL of water was added to the residue, and extraction was carried out 4 times with petroleum ether (200 mL of petroleum ether was used each time). The aqueous phase was taken, and the pH of the aqueous phase was adjusted to 6.5 with 2 mol / L hydrochloric acid. Then, extraction was carried out 4 times with ethyl acetate (200 mL of ethyl acetate was used each time). The organic phases were combined and concentrated until the solution remained about 200 mL. Silica gel was added, the sample was mixed, and the sample was loaded onto the column by the dry method. The product was eluted with a mixed solvent of ethyl acetate - petroleum ether (ethyl acetate: petroleum ether = 1:1), and after desolvation, 2.24 g of a yellowish-brown oily liquid was obtained, which was the hapten precursor (Formula C).

[0106] 2.24 g of the hapten precursor was dissolved in pyridine, 1.62 g of succinic anhydride was added, and the reaction was carried out at 75 °C overnight. Detection by TLC showed that after the reaction was complete, the reaction was distilled under reduced pressure. After evaporation to dryness, toluene was added to the residue, and evaporation was carried out again to completely remove pyridine. The residue was dissolved in ethyl acetate, and the sample was loaded onto the column by the wet method. The component of the main UV spot was eluted with a mixed solvent of ethyl acetate - petroleum ether (ethyl acetate: petroleum ether = 2:1), and 1.89 g of a pale yellow oily liquid was obtained, which was the pendimethalin hapten. The purity of the pendimethalin hapten was determined by high performance liquid chromatography to be 96.3%.

[0107] Synthesis of pendimethalin coating antigen:

[0108] 11 mg of the pendimethalin hapten was dissolved in 200 μL of DMF, 3.83 mg of NHS and 11.5 mg of DCC were added, and the reaction was carried out at 25 °C overnight. The next day, the reaction solution was centrifuged, and the supernatant (i.e., the hapten activation solution) was reserved for use. Separately, 27.7 mg of OVA was dissolved in 2 mL of 0.02 mol / L PBS buffer solution. After clarification, the hapten activation solution was slowly added dropwise. After the addition, the reaction was carried out at 25 °C for 4 h. Then, the reaction solution was centrifuged, and the supernatant was taken and added to a dialysis bag for dialysis for 3 days. The dialysis solution was 0.02 mol / L PBS solution, and the dialysis solution was changed every 8 h. After dialysis was completed, the product was collected and stored frozen at low temperature for standby.

[0109] Synthesis of pendimethalin immunogen:

[0110] Dissolve 11 mg of pendimethalin hapten in 200 μL of DMF, add 3.83 mg of NHS and 10.7 mg of EDC, and react overnight at 25°C. The next day, centrifuge the reaction solution and take the supernatant (i.e., the hapten activation solution) for standby. Separately, dissolve 30 mg of BSA in 2 mL of 0.02 mol / L PB buffer. After clarification, slowly add dropwise the hapten activation solution. After adding, react at 25°C for 4 h, then centrifuge the reaction solution, take the supernatant, add it to a dialysis bag and dialyze for 3 days. The dialysis solution is 0.02 mol / L PB, and the dialysis solution is changed every 8 h. After dialysis, collect the product and store it frozen at low temperature for standby.

[0111] Test Example 1 ELISA Test

[0112] Immunize the mice:

[0113] Select 15 purebred BALB / c mice, 4 - 6 weeks old, and label them 1 - 5 with different colors. Take 6 2-mL syringes, 3 of which suck 0.5 mL of incomplete Freund's adjuvant, and the other 3 respectively suck 0.5 mL of pendimethalin immunogen solution containing 50 μg, 100 μg, and 200 μg of pendimethalin immunogen (diluted with 1% sterilized sodium chloride solution). After labeling, connect the injection heads of one incomplete Freund's adjuvant syringe and one immunogen syringe together with an infusion tube (the length should be appropriate to connect the two syringe heads together), and then push the syringes back and forth with both hands until complete emulsification (the emulsification is marked as the internal liquid being white and milky, and there is a slight resistance during the pushing process, and it forms small drops in water without spreading). During the emulsification process, do not stop until emulsification is complete, otherwise it cannot be emulsified well. After emulsification is completed, perform multiple subcutaneous injections (on the back and abdomen) on the mice, and each mouse is injected with about 0.2 mL. Repeat the same method every 2 weeks, and start blood collection and testing after 3 times;

[0114] Blood collection method: Grasp the scalp of the mouse's neck with the left thumb, index finger, and middle finger, and fix the tail with the little finger and ring finger; gently press the skin of the eye to be removed to make the eyeball congest and protrude; insert a glass capillary vertically into the protruding part of the mouse's eyeball, and gently turn it left and right until blood flows into the capillary (about 2 / 3 of the capillary), then take it out and use an ear syringe to blow the blood in the capillary into a 0.5-mL centrifuge tube (stored at low temperature); after blood collection, place it in an oven at 37°C for 30 min, and then place it in a low-temperature high-speed centrifuge (10,000 revolutions / min) and centrifuge for 10 min to separate the serum for ELISA method testing.

[0115] Process for preparing monoclonal antibody:

[0116] Cell fusion

[0117] Mice were killed with carbon dioxide gas, and the spleen was removed aseptically, squeezed and ground in a plate to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in proportion, and the fusion promoter polyethylene glycol was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.

[0118] Selective training

[0119] The purpose of selective culture is to screen fused hybridoma cells, using HAT selective medium. In HAT medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using the salvage pathway. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the characteristics of myeloma cells that can proliferate indefinitely.

[0120] Screening and cloning of hybridoma positive clones

[0121] Among the hybridoma cells grown in HAT medium, only a few are cells that secrete the predetermined specific monoclonal antibodies, so they must be screened and cloned. The limited dilution method is used for cloning and culture of hybridoma cells. A sensitive, rapid, and specific immunological method is used to screen out positive hybridoma cells that can produce the required monoclonal antibodies, and clone and expand them. After a comprehensive identification of the immunoglobulin type, subclass, specificity, affinity, epitope of the antigen recognized, and molecular weight of the monoclonal antibodies secreted, they are frozen in time.

[0122] Large-scale production of monoclonal antibodies

[0123] Take Balb / c mice and first inject 0.5ml of liquid paraffin or pristane intraperitoneally for pretreatment. After 1-2 weeks, inoculate hybridoma cells intraperitoneally. Hybridoma cells proliferate in the mouse peritoneal cavity and produce and secrete monoclonal antibodies. After about 1-2 weeks, the mouse abdomen can be seen to swell. Draw ascites with a syringe to obtain a large amount of monoclonal antibodies.

[0124] The reagent formula used in the ELISA test is as follows:

[0125] Blocking solution (1L volume): 2.5g skim milk powder, 8g sodium chloride, 0.6g potassium dihydrogen phosphate, 5.8g disodium hydrogen phosphate, 50g sucrose, 0.9g potassium chloride, 0.5mL preservative, 50mL bovine serum, and the balance is water.

[0126] Enzyme Diluent (16L): 128 g of sodium chloride, 9.48 g of potassium dihydrogen phosphate, 92.8 g of disodium hydrogen phosphate, 800 mL of glycerol, 3200 mL of bovine serum, 12 mL of preservative, with the balance being water.

[0127] Sample Diluent (4L): 23.38 g of sodium chloride, 0.118 g of potassium chloride, 3.48 g of potassium dihydrogen phosphate, 2.19 g of sodium dihydrogen phosphate, 20.64 g of disodium hydrogen phosphate, 40 mL of Triton X-100, 1.5 mL of preservative, with the balance being water.

[0128] Substrate Solution A (100mL): 0.48 g of citric acid, 0.05 g of urea peroxide, 90 mL of glacial acetic acid, with the balance being water.

[0129] Substrate Solution B (400mL): 0.164 g of citric acid, 0.2 g of TMB hydrochloride, 4 mL of N-N, 14 mL of methanol, 160 μL of 1mol / L hydrochloric acid, with the balance being water.

[0130] 20× Concentrated Wash Solution (40L): 2560 g of sodium chloride, 928 g of disodium hydrogen phosphate, 80 g of potassium dihydrogen phosphate, 1.44 g of potassium chloride, 12 mL of preservative, 800 mL of Tween 20, with the balance being water.

[0131] The specific test method is as follows:

[0132] (1) Antigen Coating: Dilute the pendimethalin coating antigen with CB buffer at a ratio of 1K, and then add 100 μL / well to the microplate wells of the enzyme-linked immunosorbent assay (ELISA) plate. Cover with a cover membrane and incubate at 37 °C for 2 h.

[0133] (2) Blocking: After coating, remove and discard the coating solution, then wash with the washing working solution (diluted 20× concentrated wash solution to 1×), 250 μL / well or wash once with a wash bottle, soak for 30 s, discard, and pat dry with absorbent paper or a towel. Add 150 μL / well of the blocking solution, place in a 37 °C incubator for 2 h, remove, discard the blocking solution, and pat dry for later use.

[0134] (3) Dilution of Different Standard Concentrations: Dilute the directly purchased 100 μg / mL pendimethalin high standard with 0.02mol / L PBS buffer to 0 ppb (directly PBS buffer), 1 ppb, 10 ppb, and 20 ppb.

[0135] (4) Dilution of the Enzyme-Labeled Secondary Antibody Working Solution: Dilute the directly purchased enzyme-labeled secondary antigen solution with enzyme diluent buffer to a concentration of 1:1K.

[0136] (5) Dilution of Serum or Monoclonal Antibody Working Solution: Dilute the serum or prepared monoclonal antibody with sample diluent buffer at concentrations of 1:1K, 1:5K, 1:1W, and 1:2W.

[0137] (6) Detection step: The test is carried out according to the checkerboard method, and the specific operation steps are as follows:

[0138] 1) Add standard product / sample: Add 50 μL of standard product / sample to the corresponding microplate wells.

[0139] 2) Add serum or monoclonal antibody working solution: Add 50 μL / well of serum or monoclonal antibody working solution, gently mix by oscillation, cover with a cover plate membrane, and react in a dark environment at 37 °C for 30 min.

[0140] 3) Wash the plate and add enzyme-labeled secondary antibody working solution: Carefully remove the cover plate membrane, shake off the liquid in the wells, add 250 μL / well of washing working solution, wash thoroughly 4 - 5 times, with a 10 s interval each time, pour out the washing liquid in the plate wells, pat dry with absorbent paper (bubbles not removed after patting dry can be pricked with an unused pipette tip), add 100 μL / well of enzyme-labeled secondary antibody working solution, cover with a cover plate membrane, and react in a dark environment at 37 °C for 30 min.

[0141] 4) Wash the plate: Carefully remove the cover plate membrane, shake off the liquid in the wells, add 250 μL / well of washing working solution, wash thoroughly 4 - 5 times, with a 10 s interval each time, pour out the washing liquid in the plate wells, pat dry with absorbent paper (bubbles not removed after patting dry can be pricked with an unused pipette tip).

[0142] 5) Color development: Add 50 μL / well of each of substrate solution A and B, gently mix by oscillation, cover with a cover plate membrane, and react in a dark environment at 37 °C for 15 min.

[0143] 6) Measurement: Add 50 μL / well of stop solution, gently mix by oscillation, set the microplate reader at 450 nm, and measure the OD value of each well.

[0144] When the relevant technical parameters are met and the OD value is about 2.0, the maximum dilution multiple of the antibody is the antibody titer; with the standard product concentration as the abscissa and the corresponding absorbance value as the ordinate, plot a standard curve, and calculate the half inhibitory concentration (IC 50 value) of pendimethalin according to the standard curve.

[0145] The test results are shown in Tables 1 - 6.

[0146] Table 1 Test data of serum collected after immunization 1

[0147]

[0148] Table 2 Test data of serum collected after immunization 2

[0149]

[0150] Table 3 Test data of serum collected after immunization 3

[0151]

[0152] Table 4 Serum test data after immunization

[0153]

[0154] Table 5 Whole serum test data

[0155]

[0156] Table 6 Monoclonal antibody preparation test data

[0157]

[0158] It can be seen from the data in Tables 1 to 4 that as the number of immunizations increases, the concentration of pendimethalin antibody in the serum gradually increases; it can be seen from the data in Table 5 that the concentration of pendimethalin in the serum has basically reached 1W, and the IC 50 is about 1 ppb; according to the results in Table 6, the calculation shows that the titer of the prepared monoclonal antibody is 1.0×10 4 , and the half-inhibitory concentration (IC 50 ) is about 0.5 ppb.

[0159] Test Example 2 Colloidal gold test

[0160] The test method is as follows:

[0161] (1) Plate scribing: Dilute the coating antigen with PB buffer solution with a pH of 7.2 and a concentration of 0.1 mol / L at ratios of 1:10, 1:20, 1:50, and 1:100 respectively as the T-line coating solution, and dilute the secondary antibody with PB + 10% BSA buffer solution with a pH of 7.2 and a concentration of 0.02 mol / L at a ratio of 1:60 as the C-line coating solution. Scribe the plates at a rate of 1 μL / cm, and after scribing, bake at 37°C for 12 h (the backboard uses a 30*6 cm 2 PVC plate from Shanghai Jieyi, the absorbent paper uses Spec30*20 cm from Shanghai Jiening, and the NC membrane uses JN-140 from Shanghai Jiening).

[0162] (2) Gold label pad treatment standard: Cut ordinary glass fiber from Shanghai Jieyi into 30*1 cm 2 size, treat each strip with 1.5 mL of PB + 10% BSA buffer solution with a pH of 7.2 and a concentration of 0.02 mol / L, and dry at 37°C overnight (more than 12 h).

[0163] (3) Sample pad treatment standard: Cut non-woven fabric SF-06 from Shanghai Jiening into 30*1.7 cm 2Dimensions: Treat each one with 2 mL of PB buffer (pH 7.2, 0.02 mol / L) + 0.5% Tween-20 + 1% sucrose buffer, and dry overnight at 37°C (for more than 12 h).

[0164] (4) Gold-labeling: First, dilute the serum or monoclonal antibody 1:10 with pure water and mix well. Take 4 tubes of 1 mL colloidal gold solution with 40 nm-sized particles respectively, add 30 μL of 0.02 mol / L K2CO3 to adjust the pH, mix well, then add 10 μL, 20 μL, 30 μL, and 40 μL of the diluted serum or monoclonal antibody dilution respectively, mix well, let it react at room temperature for 30 min, then add 25 μL of blocking solution (20% BSA aqueous solution) respectively, let it react at room temperature for 20 min, centrifuge at 10,000 rpm at low temperature (2 - 8°C) for 10 min, remove the supernatant, dissolve the residue with 300 μL of reconstitution solution (the reconstitution solution is 1 mL of the supernatant after centrifugation + 50 μL of 40% sucrose aqueous solution mixed well), add 50 μL / well to the microplate and lyophilize according to the lyophilization process;

[0165] (5) Pretreatment method for dry tobacco leaf samples

[0166] 1) Before detection, dry the dry tobacco leaf samples and grind them into fine powder.

[0167] 2) Weigh 1 ± 0.05 g of the sample and put it into a 10 mL polystyrene centrifuge tube, add 5 mL of methanol, cover the lid, manually shake for 30 s, and let it stand for layering.

[0168] 3) Take 100 μL of the sample supernatant and add it to a 2 mL polystyrene centrifuge tube, add 400 μL of dilution solution (PB buffer, pH 7.2, 0.02 mol / L), shake and mix well to obtain the sample solution to be detected.

[0169] (6) Detection method for dry tobacco leaf samples

[0170] 1) Vertically drop all the sample solutions to be detected into the sample addition holes;

[0171] 2) Start timing when the liquid flows, react for 10 min, and use the analysis software, colloidal gold reader or visual inspection to judge the results; Five dry tobacco leaf samples are used in the present invention, denoted as 1#, 2#, 3#, 4# and 5# respectively; Pendimethalin standard is added to the dry tobacco leaf samples, and the added concentrations of pendimethalin in 1 g of dry tobacco leaf are controlled to be 0 ppb, 10 ppb, 20 ppb and 40 ppb respectively.

[0172] The test results are shown in Table 7.

[0173] Table 7 Test data of the detection limit of monoclonal antibody preparation, colloidal gold preparation and dry tobacco leaf

[0174]

[0175] Take the dry tobacco leaf sample No. 2 and conduct 5 parallel tests. The test results are shown in Table 8.

[0176] Table 8 Parallel test data of the colloidal gold for monoclonal antibody preparation from dry tobacco leaves

[0177]

[0178]

[0179] The results in Table 7 show that when using the colloidal gold for monoclonal antibody preparation of the present invention, the lowest detection limit for pendimethalin in the dry tobacco leaf sample is about 10 ng / g, with high sensitivity; it can be seen from the data in Table 8 that the detection method provided by the present invention has good parallelism.

[0180] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A pendimethalin hapten, characterized in that, It has the structure shown in Formula I:

2. The preparation method of the pendimethalin hapten according to claim 1, characterized in that, It includes the following steps: Mix 2-methyl-4-chlorobenzylamine, sulfuric acid and nitric acid for nitration reaction to obtain dinitro-2-methyl-4-chlorobenzylamine; the structure of the dinitro-2-methyl-4-chlorobenzylamine is shown in Formula B; Mix the dinitro-2-methyl-4-chlorobenzylamine, 3-aminopentane, basic reagent and organic solvent for substitution reaction to obtain a hapten precursor; the structure of the hapten precursor is shown in Formula C; Mix the hapten precursor, pyridine and succinic anhydride for condensation reaction to obtain the pendimethalin hapten with the structure shown in Formula I; 3. The preparation method according to claim 2, characterized in that, The nitration reaction includes a first stage and a second stage carried out in sequence. The temperature of the first stage is 45-48 °C and the time is 2-2.5 h. The temperature of the second stage is 60-65 °C and the time is 4-4.5 h; The temperature of the substitution reaction is 80-85 °C and the time is 4-4.5 h; The temperature of the condensation reaction is 75-80 °C and the time is 12-24 h.

4. The preparation method according to claim 2, wherein The molar ratio of nitric acid to 2-methyl-4-chlorobenzylamine is (2-2.2):1; The molar ratio of 3-aminopentane to dinitro-2-methyl-4-chlorobenzylamine is (1-1.2):1; The molar ratio of succinic anhydride to hapten precursor is (1-1.2):

1.

5. A pendimethalin antigen, characterized in that, It is obtained by coupling the pendimethalin hapten described in Claim 1 or the pendimethalin hapten prepared by the preparation method described in any one of Claims 2-4 with a carrier protein; the carrier protein is ovalbumin or bovine serum albumin.

6. The pendimethalin antigen according to claim 5, wherein When the carrier protein is ovalbumin, the structural formula of the pendimethalin antigen is shown in Formula II: When the carrier protein is bovine serum albumin, the structural formula of the pendimethalin antigen is shown in Formula III:

7. The preparation method of the pendimethalin antigen according to claim 5 or 6, characterized in that, It includes the following steps: Mix the pendimethalin hapten described in Claim 1, a coupling agent and a polar solvent for activation to obtain a hapten activation solution; Mix the hapten activation solution and a buffer solution of the carrier protein for coupling reaction to obtain the pendimethalin antigen.

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