Chlorpyrifos hapten, artificial antigen, monoclonal antibody, and preparation method and application thereof

Abstract

The present invention relates to a chlorpyrifos hapten, an artificial antigen, a monoclonal antibody, and a preparation method and application thereof. The chemical structural formula of the chlorpyrifos hapten is: #imgabs0#. The preparation steps are as follows: 3,5,6-trichloropyridine-2-ol and ethyl thiophosphoryl dichloride react to obtain compound C; compound C reacts with ethyl 3-hydroxy-2-(hydroxymethyl)propionate to obtain compound E; and compound E is hydrolyzed under alkaline conditions to obtain the chlorpyrifos hapten. The chlorpyrifos hapten of the present invention not only contains two complete chlorpyrifos structures in each molecule, but also completely retains the characteristic groups of trichloro-2-pyridine and thiophosphorothioate in the chlorpyrifos molecule, so that the electron cloud density between the prepared hapten and the original drug is the same; at the same time, the prepared artificial antigen also enables the small molecule to have a more prominent spatial structure in the carrier protein, thereby improving the immunogenicity of the antigen.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and in particular to a chlorpyrifos hapten, an artificial antigen, a monoclonal antibody, and a preparation method and application thereof. Background Art

[0002] Chlorpyrifos, chemical name O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate, molecular formula C9H 11 Cl3NO3PS is a white crystal with a slight thiol smell. It is a non-systemic broad-spectrum insecticide and acaricide. It is currently widely used in agricultural production. It is one of the new high-efficiency and low-toxic alternatives to highly toxic pesticides such as methamidophos and methyl parathion. It is also an important variety for monitoring pesticide residues in safe agricultural products. As the situation of chlorpyrifos residues in the environment becomes more and more serious, it poses a huge threat to people's health. "GB 2763-2021 Maximum Residue Limits of Pesticides in Food" stipulates that the maximum residue limit standard (MRL) of chlorpyrifos in edible peas is 0.01 mg / kg, and the minimum maximum residue limit standard (MRL) in other fruits and vegetables is 0.02 mg / kg.

[0003] Among relevant detection technologies, chlorpyrifos detection is primarily based on instrumentation. However, due to the high cost of the equipment and instruments, the lengthy testing process, and the need for specialized personnel, it is impossible to truly implement on-site testing and rapid inspections, causing significant inconvenience in daily testing work.

[0004] The key to immunoassay detection technology lies in the performance of antigens and antibodies, and the key to these two is the hapten. Therefore, to obtain high-performance antigens and antibodies, the structural design of the hapten is particularly important. Artificial antigens prepared directly from chlorpyrifos itself suffer from poor sensitivity and low cross-reactivity, failing to meet the practical needs of the current market. Therefore, the development of highly specific chlorpyrifos haptens or artificial antigens is crucial for rapid, highly sensitive, and low-cost detection of chlorpyrifos. Summary of the Invention

[0005] Technical Problem to be Solved: In response to the shortcomings of the prior art, the present invention provides a chlorpyrifos hapten, an artificial antigen, a monoclonal antibody, and a preparation method and application thereof. The chlorpyrifos hapten not only contains two complete chlorpyrifos structures in each molecule, but also completely retains the characteristic groups of trichloro-2-pyridine and phosphorothioate in the chlorpyrifos molecule, so that the electron cloud density between the prepared hapten and the original drug is consistent; at the same time, the prepared artificial antigen also enables the small molecule to have a more prominent spatial structure in the carrier protein, thereby improving the immunogenicity of the antigen.

[0006] Technical solution: A chlorpyrifos hapten, the chemical structure of the chlorpyrifos hapten is:

[0007]

[0008] The preparation method of the above-mentioned chlorpyrifos hapten comprises the following steps:

[0009] 1) Compound a and compound b react to obtain compound c, wherein compound a is 3,5,6-trichloropyridin-2-ol, compound b is ethylthiophosphoryl dichloride, and the structural formula of compound c is

[0010] 2) Compound c reacts with compound d to obtain compound e, wherein compound d is ethyl 3-hydroxy-2-(hydroxymethyl)propionate, and the structural formula of compound e is

[0011] 3) Compound e is hydrolyzed under alkaline conditions to obtain chlorpyrifos hapten.

[0012] A chlorpyrifos artificial antigen, which is obtained by connecting the chlorpyrifos hapten and a protein carrier, and the chemical structure of the chlorpyrifos artificial antigen is:

[0013]

[0014] Among them, protein is a protein carrier.

[0015] The protein carrier mentioned above is bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

[0016] A chlorpyrifos monoclonal antibody is prepared by immunizing animals with the chlorpyrifos artificial antigen.

[0017] Application of the aforementioned chlorpyrifos artificial antigen and the aforementioned chlorpyrifos monoclonal antibody in the preparation of a chlorpyrifos detection kit.

[0018] The above-mentioned chlorpyrifos artificial antigen and chlorpyrifos monoclonal antibody are used in the preparation of a chlorpyrifos detection kit, which includes a chlorpyrifos colloidal gold immunochromatographic test strip, wherein the T line of the chlorpyrifos colloidal gold immunochromatographic test strip is coated with the chlorpyrifos artificial antigen, and the C line is coated with the chlorpyrifos monoclonal antibody.

[0019] Beneficial effects: The chlorpyrifos hapten, artificial antigen, monoclonal antibody, and preparation methods and applications thereof provided by the present invention have the following beneficial effects:

[0020] 1. The hapten prepared by the present invention not only contains two complete chlorpyrifos structures in each molecule, but also completely retains the characteristic trichloro-2-pyridine and phosphorothioate groups in the chlorpyrifos molecule, resulting in a similar electron cloud density between the prepared hapten and the original drug. At the same time, the prepared artificial antigen also gives the small molecule a more prominent spatial structure in the carrier protein, thereby improving the immunogenicity of the antigen.

[0021] 2. The chlorpyrifos artificial antigen and monoclonal antibody of the present invention have strong specificity for ELISA detection, with an IC50 value of 1.25 μg / L.

[0022] 3. The artificial chlorpyrifos antigen and monoclonal antibody of the present invention are used in colloidal gold immunochromatography technology to quickly and conveniently achieve qualitative detection of chlorpyrifos.

[0023] 4. The colloidal gold immunochromatographic test strip prepared in the present invention has a sensitivity of 5 μg / kg for chlorpyrifos in a standard solution and a detection sensitivity of up to 20 μg / kg in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the synthesis reaction equation of the chlorpyrifos hapten in Example 1.

[0025] Figure 2 This is the mass spectrum of the chlorpyrifos hapten in Example 1.

[0026] Figure 3 This is the synthetic reaction equation for the chlorpyrifos artificial antigen in Example 2.

[0027] Figure 4 The standard curve was drawn by using ELISACalc software to fit the four-parameter logistic curve in Example 3.

[0028] Figure 5 This is a diagram showing the visual judgment standard for the chlorpyrifos colloidal gold immunochromatographic test paper card in Example 4. DETAILED DESCRIPTION

[0029] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0030] Example 1

[0031] This embodiment provides a chlorpyrifos hapten, the chemical structure of the chlorpyrifos hapten is:

[0032]

[0033] The synthesis reaction equation of the chlorpyrifos hapten is as follows: Figure 1 shown.

[0034] The preparation steps of the chlorpyrifos hapten are as follows:

[0035] (1) 11.09 g (55.87 mmol, CAS: 37439-34-2) of compound a was placed in a 100 mL round-bottom flask. 20 mL of tetrahydrofuran and 3.58 g (11.18 mmol) of K2CO3 were added in sequence. After thorough stirring, the mixture was heated to 50-60°C. At the same temperature, a mixture of 1.00 g (5.59 mmol, CAS: 1498-64-2) of compound b dissolved in 20 mL of tetrahydrofuran was slowly added dropwise. After the addition was complete, the reaction mixture was reacted at 50-60°C for more than 3-4 h. After the reaction was complete, the solvent was evaporated under reduced pressure, 100 mL of purified water was added to the residue, and the mixture was extracted twice with ethyl acetate. The organic phase was desolvated under reduced pressure, and the residue was purified by column chromatography (30% EA / PE, V / V) to obtain 1.22 g of compound c.

[0036] (2) 1.22 g (3.58 mmol) of compound c was added to 10 mL of tetrahydrofuran solution, followed by the addition of 0.25 g (1.72 mmol, CAS: 99116-11-7) of compound d and 0.43 g (4.29 mmol) of triethylamine. The reaction mixture was reacted at 40-50°C for 2-3 h. After the reaction was complete, the solvent was evaporated under reduced pressure, 50 mL of purified water was added to the residue, and the mixture was extracted twice with ethyl acetate. The organic phase was desolvated under reduced pressure, and the residue was purified by column chromatography (20% EA / PE, v / v) to obtain 0.64 g of compound e.

[0037] (3) 0.64 g (0.85 mmol) of compound e was dissolved in 10 mL of ethanol, and then 10 mL of 6 mol / L lithium hydroxide aqueous solution was added. The resulting reaction mixture was reacted at room temperature for 10 to 12 h, 30 mL of saturated sodium chloride aqueous solution was added, and the mixture was extracted twice with 30 mL of dichloromethane. The aqueous phase was collected and the pH value was adjusted to 4 to 5 with 4 mol / L hydrochloric acid. The solid precipitated, filtered, and dried to obtain 0.36 g of chlorpyrifos hapten (i.e. Figure 1 Chinese formula 1 hapten).

[0038] The mass spectrum of the chlorpyrifos hapten (ESI-MS: 726.8[M-1]) is as follows Figure 2 shown.

[0039] Example 2

[0040] This embodiment provides a chlorpyrifos artificial antigen, the chemical structure of which is:

[0041]

[0042] Among them, protein is a protein carrier.

[0043] The protein carrier is bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

[0044] The synthetic reaction equation of the chlorpyrifos artificial antigen is as follows: Figure 3 shown.

[0045] The steps for preparing the chlorpyrifos artificial antigen with bovine serum albumin as the carrier protein are as follows:

[0046] (1) 10 mg of the chlorpyrifos hapten prepared in Example 1 was dissolved in 0.2 mL of dimethylformamide (DMF) and stirred thoroughly. 10 mg of EDC and 10 mg of N-hydroxysuccinimide (NHS) were added and stirred at room temperature for 4 h to obtain the hapten activated ester.

[0047] (2) Weigh 40 mg of bovine serum albumin (BSA) and fully dissolve it in 4 mL of 0.01 mol / L PBS solution to form a carrier protein solution. Slowly add the hapten activated ester described in step (1) dropwise to the carrier protein solution under stirring, and stir at room temperature for 16 to 24 hours;

[0048] (3) The solution obtained in step (2) was dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules;

[0049] (4) Aliquot and store at 4°C until use.

[0050] The steps for preparing the artificial chlorpyrifos antigen with lactoferrin as carrier protein are as follows:

[0051] (1) 5 mg of the chlorpyrifos hapten prepared in Example 1 was dissolved in 0.1 mL of dimethylformamide (DMF) and stirred thoroughly. 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS) were added and stirred at room temperature for 4 h to obtain the hapten activated ester.

[0052] (2) Weigh 30 mg of lactoferrin (LF) and fully dissolve it in 5 mL of 0.01 mol / L PBS solution to form a carrier protein solution. Slowly add the hapten-activated ester dropwise to the carrier protein solution under stirring and stir at room temperature for 16 to 24 hours.

[0053] (3) The solution obtained in step (2) was dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules;

[0054] (4) Aliquot and store at 4°C until use.

[0055] Example 3

[0056] This example provides a method for preparing a chlorpyrifos monoclonal antibody, and the preparation steps are as follows:

[0057] BALB / C mice were immunized with the artificial chlorpyrifos antigen, containing lactoferrin as the carrier protein, emulsified with an equal volume of Freund's adjuvant. Each mouse received a dose of 50-100 μg of the antigen, with a two-week interval between immunizations. After three immunizations, serum titers were measured by collecting blood from the tail vein. If the antibody titer did not meet the required level, a booster immunization was performed. After the titer ceased to increase, a subcutaneous booster immunization with 100 μg of the complete antigen was performed. Five days later, spleen cells were harvested and fused with SP20 cells. The fused cells were selected in HAT medium and cultured five days later with complete medium replaced with HAT medium. The cell supernatant was assayed by ELISA. Cells from wells with strongly positive results were cloned using the limiting dilution method. After three clone culture assays, cells from wells that consistently tested positive were identified as hybridoma cells secreting monoclonal antibodies. After amplification, the hybridoma cells were inoculated into the peritoneal cavity of mice to produce antibody-containing ascites. By purifying ascites with the caprylic acid-ammonium sulfate precipitation method, high-purity and high-specificity chlorpyrifos monoclonal antibodies can be obtained.

[0058] The application and effect of the chlorpyrifos artificial antigen prepared in Example 2 and the chlorpyrifos monoclonal antibody prepared in Example 3 in ELISA were evaluated. The specific process and results are shown below.

[0059] Using pH 9.6 carbonate buffer as the coating diluent, dilute the chlorpyrifos artificial antigen with bovine serum albumin as the carrier protein to 0.05 μg / mL, add 100 μL / well to the polystyrene microplate, coat overnight at 4°C, spin dry, add 280 μL / well of 1% BSA / phosphate buffer at 37°C for blocking for 1 hour, spin dry, dry, and vacuum pack for storage.

[0060] Chlorpyrifos monoclonal antibody was diluted to 0.1 μg / mL using phosphate buffer containing 0.05% sodium azide, pH 7.4, and stored at 4°C until use.

[0061] The chlorpyrifos standard was dissolved in 0.01 M PBS buffer to prepare chlorpyrifos standard solutions at concentrations of 0, 0.3, 0.9, 2.7, 8.1 and 27.3 μg / L, respectively.

[0062] To a microwell ELISA plate coated with chlorpyrifos artificial antigen, 100 μL / well of chlorpyrifos standard solution was added, and then 20 μL / well of chlorpyrifos monoclonal antibody solution was added, and the reaction was carried out at 37°C for 0.5 h. After drying, 280 μL / well of PBST washing solution was added, and the plate was washed three times and patted dry. Then, 100 μL / well of enzyme-labeled secondary antibody was added, and the plate was reacted at 37°C for 0.5 h. After washing again three times and patting dry, 50 μL / well of TMB color developing solution A and TMB color developing solution B were added, respectively, and the plate was reacted at 37°C for 15 min. 50 μL / well of 1 mol / L sulfuric acid was added to terminate the reaction, and the OD value of each well was measured using a microplate reader set at a wavelength of 450 nm. The results are shown in Table 1 below.

[0063] Table 1

[0064] Concentration (μg / L) 0 0.3 0.9 2.7 8.1 27.3 OD value (chlorpyrifos) 1.72 1.47 0.99 0.58 0.38 0.29

[0065] From the data in Table 1, we can see that the four-parameter logistic curve fitting was performed using ELISACalc software to draw the standard curve (such as Figure 4 As shown), the linear equation of chlorpyrifos is:

[0066] y=(AD) / [1+(x / C)^B]+D,

[0067] Among them, r 2 =0.99916, A=1.72696, B=1.30385, C=0.90769, D=0.28733, x represents the concentration of the analyte, y represents the OD value, and the IC50 value obtained by calculation is 1.25 μg / L, which is linear in the range of 0.2 to 6.4 μg / L.

[0068] Example 4

[0069] This embodiment provides a method for preparing a chlorpyrifos colloidal gold immunochromatographic test paper card, and the preparation steps are as follows:

[0070] (1) Preparation of reaction membrane coated with artificial antigen and mouse IgG:

[0071] Using a nitrocellulose membrane (NC membrane) as the reaction membrane, an artificial antigen with a carrier protein of bovine serum albumin was adjusted to a concentration of 0.05-0.2 mg / mL using coating buffer, and mouse IgG was also adjusted to a concentration of 0.1-0.5 mg / mL using coating buffer. The antigen and mouse IgG were sprayed onto the corresponding detection area (T line) and control area (C line) of the reaction membrane at a membrane volume of 0.8-1.2 μL / cm, with a spacing of 2.5 mm between the detection area and the control area. The membrane was then placed in a 45°C oven for 12-16 hours and stored in a constant temperature and humidity chamber until use. The coating buffer used was PBS buffer containing 1% sucrose and 0.05% sodium azide, with a pH of 7.6 and a concentration of 0.01 mol / L.

[0072] (2) Preparation of nano-gold labeled chlorpyrifos monoclonal antibody micropores:

[0073] 1. Preparation of nanogold solution:

[0074] Dissolve 1g of chloroauric acid in pure water and ultrasonically, then dilute to 100mL. Store at 4°C in the dark until ready for use. Add 2mL of this solution to 100mL of pure water, heat to boiling, and then add 0.5mL of 0.06% sodium citrate solution. Continue heating for 10 minutes. Cool to room temperature, then bring the volume back to the original volume with pure water. Store at room temperature in the dark until ready for use. This will yield the gold nanoparticle solution. All glassware should be soaked overnight in a mixture of potassium permanganate and sulfuric acid, then rinsed and dried before use.

[0075] 2. Labeling of chlorpyrifos monoclonal antibody:

[0076] Aliquot 1 mL of nanogold solution into each vial and adjust the pH of the nanogold solution with 0.1 mol / L K₂CO₃. Add 3 μg of chlorpyrifos monoclonal antibody to the nanogold solutions at different pH values ​​and allow to react at room temperature for 5 minutes. Observe the color change of the solution and record the pH at which the solution remains red. Add 10 μl of 10% bovine serum albumin for blocking. Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant.

[0077] 3. Preparation of micropores:

[0078] Add 1 mL of gold diluent to re-dissolve, dispense 12 μL / well into microwells, dry at 37°C for 16 h, and store for later use.

[0079] The gold diluent used was 0.01 mol / L PBST buffer containing 2% Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose.

[0080] (3) Preparation of sample pad:

[0081] Soak the cut 30*30cm blank sample pad in the sample treatment solution for 5 minutes, take it out and dry it at 37℃ for 16 hours, and place it in a constant temperature and humidity storage box for use. The sample pad treatment solution used is 0.01mol / L PBST buffer containing 0.3% Tween 20, 1% sucrose, 0.5% Triton 405, and 0.05% sodium azide.

[0082] (4) Assembly of chlorpyrifos colloidal gold immunochromatographic test paper card:

[0083] The reaction membrane prepared in step (1) is stacked on the middle of the PVC board backing, and the sample pad and the absorbent pad prepared in step (3) are stacked on both ends respectively. The reaction membrane is connected to the absorbent pad and the sample pad respectively, with the detection area close to the sample pad and the control area close to the absorbent pad to obtain a test paper board. The test paper board is cut into 3 mm test strips, and the test strips are loaded into the test paper card to obtain a chlorpyrifos colloidal gold immunochromatographic test paper card.

[0084] The performance of the chlorpyrifos colloidal gold immunochromatographic test paper card prepared in Example 4 was evaluated, and the specific process and results are shown below.

[0085] A series of chlorpyrifos standard solutions of varying concentrations were prepared in 0.01 mol / L PBS buffer. 100 μL of the standard solutions were then added to the gold-labeled microwells and repeatedly pipetted to uniformly redissolve the solution. After standing for 5 minutes, the solution in the gold-labeled microwells was transferred to the sample wells of the chlorpyrifos colloidal gold immunochromatographic card of the present invention. After sample addition, a timer was started, and the results were observed after 5 to 8 minutes. After 8 minutes, the result was deemed invalid.

[0086] Visual interpretation method: If the color of T line is stronger than that of C line or there is no significant difference with that of C line, it indicates that the test result is negative (-). If the color of T line is significantly weaker than that of C line or T line does not show any color, it indicates that the test result is positive (+). Invalid: No C line appears, indicating an incorrect operation process or the test strip has expired. The test is repeated for 3 groups to achieve rapid qualitative detection of chlorpyrifos colloidal gold immunochromatographic test strips. The judgment criteria are as follows: Figure 5 shown.

[0087] The determination results of chlorpyrifos standard solutions at different concentrations are shown in Table 2 below.

[0088] Table 2

[0089]

[0090] As can be seen from Table 2, by detecting the concentration of the chlorpyrifos standard solution, the colloidal gold immunochromatographic reagent card prepared in the present invention has a high sensitivity for chlorpyrifos detection, which can reach 5 μg / L.

[0091] The stability of the chlorpyrifos colloidal gold immunochromatographic test paper card prepared in Example 4 was tested, and the specific process and results are shown below.

[0092] The chlorpyrifos colloidal gold immunochromatographic test strips were stored at room temperature. To ensure the stability of the test strips, accelerated destructive tests were conducted on the test strips, which were placed at room temperature and 54°C for 60 consecutive days. Negative results and color changes of the chlorpyrifos standard solution were detected on the 0th, 5th, 10th, 20th, 30th, 40th, 50th and 60th days, respectively. The experiment was repeated for three groups. The results are shown in Table 3 below: ("+" represents positive, "-" represents negative)

[0093] Table 3

[0094]

[0095] As shown in Table 3, after 60 days of sealed storage at room temperature and 37°C, the T / C color depth readings of the colloidal gold qualitative immunochromatographic test strips showed no significant change, indicating that the colloidal gold qualitative immunochromatographic test strips can be stably stored at 37°C for at least 60 days in an accelerated test. Therefore, the chlorpyrifos colloidal gold immunochromatographic test strips prepared by the present invention can be stably stored at room temperature for more than one year, fully meeting market requirements for storage and transportation.

[0096] The detection performance of the chlorpyrifos colloidal gold immunochromatographic test paper card prepared in Example 4 on fruit and vegetable samples was tested. The specific process and results are shown below.

[0097] Take 2g of fruit and vegetable sample (4g for tubers), cut leafy vegetables into pieces approximately 1cm square, and take a cross-section sample or the epidermis of tubers. Place the sample in a 50mL centrifuge tube and add 7mL of 0.01mol / L PB buffer. Mix vigorously by shaking for 2 minutes. Let the centrifuge tube stand for 2 minutes. The supernatant is the test solution. If the sample becomes turbid or has excessive impurities after adding the diluent, filter it (or centrifuge at 4000 rpm for 3 minutes) before retesting. Before testing, dilute the sample solution with the diluent in different proportions using a centrifuge tube to obtain the test solution.

[0098] Take 100 μL of the test solution and add it to the sample well of the chlorpyrifos colloidal gold immunochromatographic card of the present invention. Start timing after adding the sample. Figure 5 Observe the results. Visual interpretation: If the T line appears stronger than the C line or has no significant difference in color, the sample is negative (-). Positive (+): If the T line appears significantly weaker than the C line or has no color, the sample is positive (+). Invalid: If the C line does not appear, it indicates an incorrect procedure or the test strip has expired.

[0099] Minimum detection limit: The chlorpyrifos standard was dissolved in methanol and added to the sample in a certain amount according to the spiking requirements so that the concentrations in the sample were 0, 10, 20, 40, and 80 μg / kg, respectively. The results are shown in Table 4.

[0100] Table 4

[0101]

[0102]

[0103] As shown in Table 4 above, the chlorpyrifos colloidal gold immunochromatographic test strips prepared by the present invention showed good reproducibility in the test results of the 10 samples. When the chlorpyrifos content in the samples was less than 20 μg / kg, the results were all negative; when the chlorpyrifos content was greater than 20 μg / kg, the results were all positive. Therefore, the detection limit of the chlorpyrifos colloidal gold immunochromatographic test strips prepared by the present invention in the samples was 20 μg / kg.

[0104] The above is a detailed introduction to the embodiments of the present invention. For those skilled in the art, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A chlorpyrifos hapten, characterized in that The chemical structural formula of the chlorpyrifos hapten is:

2. The method for preparing a chlorpyrifos hapten according to claim 1, characterized in that The preparation steps are as follows: 1) Compound a and compound b react to obtain compound c, wherein compound a is 3,5,6-trichloropyridin-2-ol, compound b is ethylthiophosphoryl dichloride, and the structural formula of compound c is 2) Compound c reacts with compound d to obtain compound e, wherein compound d is ethyl 3-hydroxy-2-(hydroxymethyl)propionate, and the structural formula of compound e is 3) Compound e is hydrolyzed under alkaline conditions to obtain chlorpyrifos hapten.

3. A chlorpyrifos artificial antigen, characterized by: The chlorpyrifos artificial antigen is obtained by connecting the chlorpyrifos hapten and a protein carrier. The chemical structure of the chlorpyrifos artificial antigen is: Among them, protein is a protein carrier.

4. The artificial chlorpyrifos antigen according to claim 3, characterized in that: The protein carrier is bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

5. A chlorpyrifos monoclonal antibody, characterized in that: The chlorpyrifos monoclonal antibody is prepared by immunizing animals with the chlorpyrifos artificial antigen.

6. Use of the chlorpyrifos artificial antigen according to claim 3 or 4 and the chlorpyrifos monoclonal antibody according to claim 5 in the preparation of a chlorpyrifos detection kit.

7. Use of the chlorpyrifos artificial antigen and chlorpyrifos monoclonal antibody according to claim 6 in the preparation of a chlorpyrifos detection kit, characterized in that: The detection kit comprises a chlorpyrifos colloidal gold immunochromatographic test strip, wherein the T line of the chlorpyrifos colloidal gold immunochromatographic test strip is coated with the chlorpyrifos artificial antigen, and the C line is coated with the chlorpyrifos monoclonal antibody.

Images