Specific artificial antigen of herbicide alachlor as well as preparation method and application of specific artificial antigen

Abstract

The invention provides a herbicide alachlor specific artificial antigen and a preparation method and application thereof, and belongs to the technical field of pesticide residue detection. The preparation method comprises the following steps: (1) dissolving alachlor and an alkali reagent in an organic solvent, adding 3-mercaptopropionic acid as a derivative agent, and reacting to obtain alachlor hapten; and (2) dissolving the alachlor hapten in a polar solvent, adding an activator for activation, and then dropwise adding the activated hapten solution into a carrier protein solution for coupling to obtain the alachlor specific artificial antigen. The hapten is synthesized by adopting a one-step method, the steps are simplified, the post-treatment is simple, the yield can reach more than 80%, and the hapten retains characteristic functional groups of alachlor and has strong immunogenicity after being coupled with carrier protein. The alachlor monoclonal antibody prepared from the artificial antigen is good in specificity and low in cross reaction rate, the IC50 value is 5 ng / mL, and alachlor residues in food and environmental water samples can be rapidly detected.

Description

Technical Field

[0001] This invention belongs to the field of pesticide residue detection technology, and in particular relates to a specific artificial antigen for the herbicide metolachlor, its preparation method and application. Background Technology

[0002] Alachlor, an amide-based selective pre-emergence herbicide for dryland applications, is of low toxicity to humans and animals. Previously, it was believed that alachlor had no teratogenic or carcinogenic effects; however, since the 1990s, more research has suggested that alachlor is a carcinogen. Herbicide residues in agricultural products and the environment have attracted widespread attention, making residue detection technology a focus of research. Developing reliable, sensitive, rapid, and practical pesticide residue detection methods is a crucial prerequisite for herbicide residue detection.

[0003] Currently, the detection methods for metolachlor at home and abroad mainly focus on GC-MS, HPLC-MS and other methods. Although these methods have high accuracy and sensitivity, the instruments and equipment are relatively expensive, the analysis time is long and the cost is high, the sample pretreatment steps are complicated, and the professional skills required of the experimental personnel are also high. Therefore, it is difficult to meet the rapid detection of large batches of samples. In contrast, antibody-based immunoassay methods are simple and fast, and can achieve high-throughput screening of a large number of samples.

[0004] However, herbicides, as small-molecule haptens, lack immunogenicity. Suitable haptens need to be chemically derivatized or synthesized de novo, and then chemically conjugated to prepare artificial antigens before animals can be immunized. The preparation process for herbicide antibodies is more complex than that for antibodies against large-molecule whole antigens. The design, synthesis, and preparation of artificial whole antigens are crucial factors affecting the preparation of herbicide antibodies and the establishment of immunoassay methods. Some studies have obtained the metolachlor hapten through several chemical reactions, and although antibodies were prepared, the hapten synthesis steps are too complex, posing a significant challenge for bioanalysts and making implementation difficult. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a specific artificial antigen for the herbicide metolachlor, its preparation method and application. The metolachlor hapten can be obtained in one step, the post-processing is simple, the yield is high, and the specificity is good with a low cross-reactivity rate.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for preparing a specific artificial antigen of the herbicide metolachlor, comprising the following steps: (1) Synthesis of metolachlor hapten: Metolachlor and alkaline reagent are dissolved in an organic solvent, 3-mercaptopropionic acid is added as a derivatizing agent, and the mixture is refluxed and stirred at 70~80℃ for 1.5~2.5h. After the reaction is complete, the organic solvent is removed, the pH is adjusted to 3~5, and the metolachlor hapten is obtained by separation. (2) Synthesis of artificial antigen: The metolachlor hapten obtained in step (1) is dissolved in a polar solvent, activated by adding an activator, and then the activated hapten solution is added dropwise to the carrier protein solution. The reaction is stirred at room temperature for 18-22 hours. After dialysis, the metolachlor-specific artificial antigen is obtained.

[0007] Preferably, in step (1), the alkaline reagent is potassium hydroxide, the organic solvent is anhydrous ethanol, and the molar ratio of metolachlor, potassium hydroxide and 3-mercaptopropionic acid is 1:(2.5~3.5):(1.0~1.5).

[0008] Preferably, in step (2), the activator is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; the carrier protein is bovine serum albumin or ovalbumin; the polar solvent is 70% acetonitrile solution; dialysis is performed using 0.01M phosphate buffer, the dialysis temperature is 4°C, and the dialysis time is 2-4 days.

[0009] The present invention also provides a metolachlor-specific artificial antigen prepared by the above preparation method, wherein the artificial antigen is a coupling complex of metolachlor hapten and carrier protein.

[0010] Preferably, the metolachlor hapten contains a carboxyl functional group; when the carrier protein is bovine serum albumin, it forms a metolachlor immunogen; when the carrier protein is ovalbumin, it forms a metolachlor coating antigen.

[0011] This invention also provides the application of the above-mentioned metolachlor-specific artificial antigen in the preparation of metolachlor monoclonal antibodies.

[0012] Preferably, the metolachlor-specific artificial antigen is used as an immunogen to immunize mammals, thereby stimulating the animal's immune system to produce specific antibodies against metolachlor.

[0013] This invention also provides a method for preparing metolachlor monoclonal antibody, comprising the following steps: (1) Immunization of animals: The above-mentioned metolachlor-specific artificial antigen is used to immunize mammals to obtain immunized animals; (2) Cell fusion: Spleen cells from immunized animals are fused with myeloma cells to obtain hybridoma cells; (3) Screening and cloning: Immunoassay was used to screen positive hybridoma cells that could secrete metolachlor-specific antibodies, and hybridoma cell lines that stably secreted antibodies were obtained through cloning culture. (4) Antibody preparation: The hybridoma cell line was cultured and the metolachlor monoclonal antibody was isolated and purified.

[0014] Preferably, in step (1), the mammal is a Balb / c mouse and the immunization dose is 100~200μg / mouse; in step (2), the fusion ratio of spleen cells to myeloma cells is 5:1~10:1 and the myeloma cells are SP2 / 0 myeloma cells; in step (3), the immunoassay method is indirect competitive ELISA and the clonal culture adopts limiting dilution method.

[0015] Preferably, in step (4), hybridoma cells are cultured using an incremental culture method, and antibodies are purified using the octanoic acid-saturated ammonium sulfate method.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention enables the one-step synthesis of metolachlor hapten, which significantly reduces the operational difficulty and shortens the reaction cycle compared to the complex multi-step synthesis process in the prior art. Furthermore, the post-processing is simple and the yield is as high as 80% or more.

[0017] This invention directly designs and synthesizes a hapten using metolachlor as the core, fully preserving the characteristic functional groups of metolachlor and exhibiting higher structural homology with it. The artificial antigen formed after conjugation with a carrier protein can precisely stimulate the animal immune system to produce specific antibodies against metolachlor, effectively reducing the cross-reactivity rate. The metolachlor monoclonal antibody prepared based on this invention, as measured by ic-ELISA, has an IC50 value... 50 With a value of 5 ng / mL, the sensitivity meets the requirements for residue detection and can be used to establish an indirect competitive enzyme-linked immunosorbent assay (ELISA). Attached Figure Description

[0018] Figure 1 This is a mass spectrometry analysis report of the metolachlor hapten of this invention. Detailed Implementation

[0019] This invention provides a method for preparing a specific artificial antigen of the herbicide metolachlor, comprising the following steps: (1) Synthesis of metolachlor hapten: Metolachlor and alkaline reagent are dissolved in an organic solvent, 3-mercaptopropionic acid is added as a derivatizing agent, and the mixture is refluxed and stirred at 70~80℃ for 1.5~2.5h. After the reaction is complete, the organic solvent is removed, the pH is adjusted to 3~5, and the metolachlor hapten is obtained.

[0020] In this invention, potassium hydroxide is preferred as the alkaline reagent, and the molar ratio of metolachlor, potassium hydroxide and 3-mercaptopropionic acid is more preferably 1:(2.5~3.5):(1.0~1.5), and even more preferably 1:3:1.2.

[0021] In this invention, it is preferred to reflux and stir the reaction at 75°C for 2 hours; the preferred organic solvent is anhydrous ethanol, and it is even more preferred to monitor the reaction by TLC (UV, V petroleum ether: V ethyl acetate = 10:1) and then evaporate the ethanol to dryness after the reaction is complete.

[0022] In this invention, it is preferred to use HCl to adjust the pH, more preferably the pH is 3 to 4, and even more preferably the metolachlor hapten is obtained by filtration and drying.

[0023] (2) Synthesis of artificial antigen: The metolachlor hapten obtained in step (1) is dissolved in a polar solvent, activated by adding an activator, and then the activated hapten solution is added dropwise to the carrier protein solution. The reaction is stirred at room temperature for 18-22 hours. After dialysis, the metolachlor-specific artificial antigen is obtained.

[0024] In this invention, the preferred polar solvent is a 70% acetonitrile solution.

[0025] In this invention, the activator is preferably a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and more preferably the mass ratio of metolachlor hapten, EDC and NHS is 1:(1.3~1.4):(1.0~1.02), more preferably 1:1.38:1.02; preferably, the activator is stirred and activated at room temperature for 2 hours.

[0026] In this invention, the preferred carrier protein is bovine serum albumin or ovalbumin; more preferably, the concentration of the carrier protein solution is 10 mg / mL, the pH of the phosphate buffer is 7.4, and the volume ratio of the hapten solution to the carrier protein solution is 1:10.

[0027] In this invention, it is preferred that dialysis is performed using 0.01M phosphate buffer, the dialysis temperature is 4°C, and the dialysis time is 2 to 4 days; it is further preferred that after dialysis, the solution is centrifuged at 10,000 rpm for 10 min, and the supernatant is taken to obtain metolachlor artificial antigen.

[0028] The present invention also provides a metolachlor-specific artificial antigen prepared by the above preparation method, which is a coupling complex of metolachlor hapten and carrier protein.

[0029] In this invention, bovine serum albumin is preferred as the carrier protein to form a metolachlor immunogen; ovalbumin is preferred as the carrier protein to form a metolachlor coating antigen.

[0030] The present invention also provides the application of the above-mentioned metolachlor-specific artificial antigen in the preparation of metolachlor monoclonal antibodies.

[0031] In this invention, it is preferable to use a metolachlor-specific artificial antigen as an immunogen to immunize mammals, thereby stimulating the animal's immune system to produce specific antibodies against metolachlor.

[0032] This invention also provides a method for preparing metolachlor monoclonal antibody, comprising the following steps: (1) Immunizing animals: Immunize mammals with the above-mentioned metolachlor-specific artificial antigen to obtain immunized animals.

[0033] In this invention, the preferred mammal is Balb / c mouse, and the immunization dose is 100-200 μg / mouse, more preferably 150 μg / mouse.

[0034] (2) Cell fusion: Spleen cells from immunized animals are fused with myeloma cells to obtain hybridoma cells.

[0035] In this invention, SP2 / 0 myeloma cells are preferred, and the fusion ratio of spleen cells to myeloma cells is more preferably 5:1 to 10:1, and more preferably 8:1.

[0036] (3) Screening and cloning: positive hybridoma cells that can secrete metolachlor-specific antibodies are screened using immunoassay methods, and hybridoma cell lines that stably secrete antibodies are obtained through cloning culture.

[0037] In this invention, the preferred immunoassay method is the indirect competitive ELISA method, and the clonal culture adopts the limiting dilution method.

[0038] (4) Antibody preparation: The hybridoma cell line was cultured and the metolachlor monoclonal antibody was isolated and purified.

[0039] In this invention, hybridoma cells are preferably cultured using an incremental culture method. More preferably, the hybridoma cells are placed in a cell culture medium and cultured at 37°C. The cell culture medium consists of RPMI 1640 medium supplemented with fetal bovine serum and sodium bicarbonate, such that the final concentration of fetal bovine serum in the cell culture medium is 20% (mass fraction), the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction), and the pH is 7.4. The antibody is preferably purified by the octanoic acid-saturated ammonium sulfate method.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1 Metolachlor hapten synthesis Accurately weigh 269 mg of metolachlor and 168 mg of KOH and dissolve them in 20 mL of anhydrous ethanol. The solution is then sonicated to aid dissolution. Next, add 100 μL of 3-mercaptopropionic acid (3-MPA) and reflux the mixture in a constant-temperature oil bath (75 °C) with stirring for 2 h. After complete reaction by TLC monitoring (UV, V petroleum ether:V ethyl acetate = 10:1), the ethanol is evaporated to dryness. The pH is adjusted to 3-4 with 1 N HCl under ice bath conditions, resulting in a white precipitate. This precipitate is filtered, and the filter cake is dried to obtain a white oily substance.

[0042] The structure of the hapten was identified by mass spectrometry, and the hapten was successfully synthesized. Figure 1 As shown.

[0043] Example 2 Synthesis of Metolachlor Artificial Antigen Accurately weigh 6.1 mg of the metolachlor hapten from Example 1, dissolve it in 200 μL of 70% acetonitrile, add 8.4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6.2 mg of N-hydroxysuccinimide (NHS), and activate at room temperature for 2 h with stirring. Dissolve 20 mg of bovine serum albumin (BSA) in 2 mL of phosphate buffer (0.01 M PBS, pH 7.4). Then, slowly add the hapten solution dropwise to the carrier protein solution with stirring. After reacting at room temperature for 20 h, dialyze with 0.01 M PBS at 4 °C for 3 days. After dialysis, centrifuge at 10,000 rpm for 10 min and collect the supernatant to obtain the metolachlor artificial antigen.

[0044] The metolachlor hapten, BSA, and corresponding artificial antigens were identified by ultraviolet (200-400 nm) scanning. BSA showed a characteristic peak at 280 nm, metolachlor hapten showed a characteristic peak at 250-260 nm, and metolachlor artificial antigen showed a characteristic peak at 250 nm. A significant shift was observed in the curves of the three, indicating that the reaction product was a complex of the carrier protein BSA and the metolachlor hapten, proving that the coupling was successful.

[0045] The synthesis method of the metolachlor coating agent is the same as that of the immunogen, except that the coupling protein BSA is replaced with OVA.

[0046] Example 3 Preparation of metolachlor monoclonal antibody 1. Animal immunization The immunogen obtained in Example 2 was injected into Balb / c mice at a dose of 150 μg per mouse to induce the production of antiserum.

[0047] 2. Cell fusion and cloning Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 8:1 (number-matching). The cell supernatant was analyzed using an indirect competitive ELISA method to screen for positive wells. The positive wells were then cloned using a limiting dilution method until a hybridoma cell line stably secreting monoclonal antibodies was obtained.

[0048] 3. Cell cryopreservation and thawing Hybridoma cells were prepared into 1×10⁻⁶ cells using cryopreservation solution. 6 Cell suspensions of cells / mL were stored long-term in liquid nitrogen. Upon thawing, the cryovials were removed and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture flasks for incubation.

[0049] 4. Preparation and purification of monoclonal antibodies Incremental culture method: Hybridoma cells were placed in cell culture medium (RPMI 1640 medium with 20% fetal bovine serum and 0.2% sodium bicarbonate, pH 7.4) and cultured at 37°C. The culture medium was purified by the caprylic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which were then stored at -20°C.

[0050] Example 4 Identification of metolachlor monoclonal antibodies The coating agent was serially diluted from 1 μg / mL with 0.05 M pH 9.6 carbonate buffer, 100 μL / well, and incubated at 37°C for 2 h. The solution in the plate was then discarded, and the plate was washed three times with washing buffer for 3 min each time. After patting dry, 200 μL / well of blocking buffer was added, and the plate was incubated at 37°C for 2 h. After washing, the plate was dried for later use. The antiserum was serially diluted from 1:1000 and added to each well at 100 μL / well, and incubated at 37°C for 30 min. After thorough washing, 100 μL / well of 1:3000 HRP goat anti-mouse IgG was added, and the plate was incubated at 37°C for 30 min. The plate was removed, thoroughly washed, and 100 μL of TMB chromogenic solution was added to each well. The plate was incubated at 37°C in the dark for 15 min. 50 μL of stop solution was added to each well to terminate the reaction, and the OD of each well was measured using a microplate reader. 450 value.

[0051] The IC50 of the monoclonal antibody metolachlor was determined by ic-ELISA. 50 A value of 5 ng / mL indicates good sensitivity to metolachlor and can be used for metolachlor immunoassay detection.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a specific artificial antigen of the herbicide metolachlor, characterized in that, Includes the following steps: (1) Synthesis of metolachlor hapten: Metolachlor and alkaline reagent are dissolved in an organic solvent, 3-mercaptopropionic acid is added as a derivatizing agent, and the mixture is refluxed and stirred at 70~80℃ for 1.5~2.5h. After the reaction is complete, the organic solvent is removed, the pH is adjusted to 3~5, and the metolachlor hapten is obtained by separation. (2) Synthesis of artificial antigen: The metolachlor hapten obtained in step (1) is dissolved in a polar solvent, activated by adding an activator, and then the activated hapten solution is added dropwise to the carrier protein solution. The reaction is stirred at room temperature for 18-22 hours. After dialysis, the metolachlor-specific artificial antigen is obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the alkaline reagent is potassium hydroxide, and the organic solvent is anhydrous ethanol; the molar ratio of metolachlor, potassium hydroxide and 3-mercaptopropionic acid is 1:(2.5~3.5):(1.0~1.5).

3. The preparation method according to claim 1, characterized in that, In step (2), the activator is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; the carrier protein is bovine serum albumin or ovalbumin; the polar solvent is 70% acetonitrile solution; dialysis is performed using 0.01M phosphate buffer, the dialysis temperature is 4°C, and the dialysis time is 2-4 days.

4. A metolachlor-specific artificial antigen prepared by the preparation method according to any one of claims 1 to 3, wherein the artificial antigen is a coupling complex of metolachlor hapten and carrier protein.

5. The metolachlor-specific artificial antigen according to claim 4, characterized in that, The metolachlor hapten contains a carboxyl functional group; when the carrier protein is bovine serum albumin, it forms a metolachlor immunogen; when the carrier protein is ovalbumin, it forms a metolachlor coating antigen.

6. The use of the metolachlor-specific artificial antigen as described in claim 4 or 5 in the preparation of metolachlor monoclonal antibodies.

7. The application according to claim 6, characterized in that, The metolachlor-specific artificial antigen was used as an immunogen to immunize mammals, stimulating the animal's immune system to produce specific antibodies against metolachlor.

8. A method for preparing a metolachlor monoclonal antibody, characterized in that, Includes the following steps: (1) Immunized animals: Immunize mammals with the metolachlor-specific artificial antigen as described in claim 4 or 5 to obtain immunized animals; (2) Cell fusion: Spleen cells from immunized animals are fused with myeloma cells to obtain hybridoma cells; (3) Screening and cloning: Immunoassay was used to screen positive hybridoma cells that could secrete metolachlor-specific antibodies, and hybridoma cell lines that stably secreted antibodies were obtained through cloning culture. (4) Antibody preparation: The hybridoma cell line was cultured and the metolachlor monoclonal antibody was isolated and purified.

9. The preparation method according to claim 8, characterized in that, In step (1), the mammal is a Balb / c mouse, and the immunization dose is 100~200μg / mouse; in step (2), the fusion ratio of spleen cells to myeloma cells is 5:1~10:1, and the myeloma cells are SP2 / 0 myeloma cells; in step (3), the immunoassay method is indirect competitive ELISA, and the clonal culture adopts the limiting dilution method.

10. The preparation method according to claim 8, characterized in that, In step (4), hybridoma cells are cultured using the incremental culture method, and antibodies are purified by the octanoic acid-saturated ammonium sulfate method.

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