Dimethomorph hapten, dimethomorph artificial antigen as well as preparation method and application of dimethomorph hapten

Abstract

The invention discloses a dimethomorph hapten, an artificial antigen and a preparation method and application thereof, and relates to a dimethomorph hapten, an artificial antigen, an antibody, preparation of the dimethomorph hapten, the artificial antigen and the antibody and application of the dimethomorph hapten, the artificial antigen and the antibody to detection of dimethomorph residues in agricultural products. The dimethomorph hapten prepared by the invention completely retains the characteristic structures of o-dimethyl ether phenyl and acyl morpholine, an introduced active group is positioned at a chlorobenzene group end with a weak recognition area, and the electron cloud density of the hapten is almost consistent with that of an original drug; the dimethomorph artificial antigen and the monoclonal antibody have the advantages that the dimethomorph artificial antigen and the monoclonal antibody are high in specificity when being used for ELISA (enzyme-linked immuno sorbent assay) detection, the IC50 value is 0.097 microgram / L, and the immunogenicity of the dimethomorph artificial antigen and the monoclonal antibody are high. The sensitivity of the colloidal gold immunochromatography test strip to dimethomorph in a standard solution is 0.5 mu g / kg, and the detection sensitivity of the colloidal gold immunochromatography test strip to dimethomorph in a sample can reach 5 mu g / kg.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product safety detection, and more specifically, to a dimethomorph hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Dimethomorph is a fungicide specifically targeting oomycetes. Its characteristic action is to disrupt cell wall membrane formation, affecting all stages of the oomycete life cycle. It is a specific agent for controlling oomycete fungal diseases and is highly effective against lower-grade fungal diseases such as downy mildew, downy mildew, late blight, blight (mildew), blight rot, pythium, and blackleg. Due to its increasing use, dimethomorph residues in the environment are becoming increasingly serious, posing a significant health hazard. "GB 2763—2021 Maximum Residue Limits of Pesticides in Food" stipulates a maximum residue limit (MRL) of 0.01 mg / kg for dimethomorph in pineapples and 0.05 mg / kg for potatoes and strawberries. Among relevant detection technologies, dimethomorph is primarily instrumental. However, due to the expensive equipment and instrumentation, lengthy testing times, and the need for specialized personnel, it is not feasible for on-site testing or rapid clinical inspections, significantly inconvenient for routine testing.

[0003] The key to immunoassay detection technology lies in the performance of the antigen and antibody, 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 directly prepared from dimethomorph itself in related technologies suffer from poor sensitivity and low cross-reactivity, failing to meet the practical needs of the current market. Therefore, the development of highly specific dimethomorph haptens or artificial antigens is crucial for rapid, sensitive, and low-cost detection of dimethomorph. Summary of the Invention

[0004] The purpose of the present invention is to provide a dimethomorph hapten, an artificial antigen, an antibody and their preparation and application, for detecting dimethomorph residues in agricultural products.

[0005] According to one aspect of the present invention, there is provided an oxadiazine hapten, the structure of which is shown in formula (I):

[0006]

[0007] According to another aspect of the present invention, there is provided a method for preparing a dimethomorph hapten, comprising the following steps:

[0008] S1. Dimethomorph is reacted with carbon dioxide under alkaline conditions to prepare a dimethomorph hapten as shown in formula (I). The reaction formula is shown in formula (II):

[0009]

[0010] According to another aspect of the present invention, a dimethomorph antigen is provided. The dimethomorph antigen is a conjugate of a dimethomorph hapten and a carrier protein, and its structural formula is shown in Formula (III):

[0011] Among them, protein is a carrier protein.

[0012] In some embodiments, the carrier protein is any one of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin, or hemocyanin.

[0013] According to a fourth aspect of the present invention, a dimethomorph antibody is provided. The dimethomorph antibody is prepared by immunizing an animal with a dimethomorph artificial antigen. The dimethomorph antibody is a dimethomorph monoclonal antibody.

[0014] According to a fifth aspect of the present invention, there is provided the use of dimethomorph haptens and dimethomorph artificial antigens in immunological detection of dimethomorph for non-disease diagnosis purposes.

[0015] According to a sixth aspect of the present invention, there is provided use of a dimethomorph antibody in immunological detection of dimethomorph for non-disease diagnosis purposes.

[0016] According to a seventh aspect of the present invention, a device for detecting dimethomorph is provided. The device is prepared using a dimethomorph artificial antigen and a dimethomorph antibody.

[0017] According to an eighth aspect of the present invention, a method for detecting dimethomorph for non-disease diagnosis purposes is provided. The method comprises detecting dimethomorph residues in agricultural products using a dimethomorph detection device.

[0018] In some embodiments, the produce is a fruit or a vegetable.

[0019] The beneficial effects of the present invention are as follows: (1) the oxadiazine hapten prepared by the present invention completely retains the characteristic structure of o-dimethyl ether phenyl and acylmorpholine, and the introduced active group is located at the end of the chlorophenyl group with weak recognition effect in the region, and the electron cloud density between the hapten and the original drug is almost the same, so that the prepared artificial antigen can well present the characteristic structure of o-dimethyl ether phenyl and acylmorpholine in terms of spatial structure, thereby improving the immunogenicity of the antigen;

[0020] (2) The artificial dimethomorph antigen and monoclonal antibody of the present invention are used for ELISA detection with high specificity, IC 50 The value is 0.097μg / L.

[0021] (3) The artificial dimethomorph antigen and monoclonal antibody of the present invention are used in colloidal gold immunochromatographic technology to quickly and conveniently detect the qualitative detection of dimethomorph. The colloidal gold immunochromatographic test strip prepared in the present invention has a sensitivity of 0.5 μg / kg for dimethomorph in a standard solution and a detection sensitivity of up to 5 μg / kg for dimethomorph in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a mass spectrum of the dimethomorph hapten according to one embodiment of the present invention.

[0023] Figure 2 The synthetic route of the dimethomorph artificial antigen according to one embodiment of the present invention is shown in FIG.

[0024] Figure 3 The figure is a standard curve of an indirect competitive ELISA established based on dimethomorph monoclonal antibody according to one embodiment of the present invention.

[0025] Figure 4 This is a result judgment standard diagram of a dimethomorph detection device according to one embodiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail through specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art are all within the scope of the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents available on the conventional market.

[0027] Example 1 Preparation of dimethomorph hapten

[0028] The method for preparing dimethomorph hapten comprises the following steps:

[0029] 0.92 g of enoylmorpholine (2.37 mmol, CAS: 110488-70-5) was dissolved in 15 mL of anhydrous tetrahydrofuran. 1.42 mL of a 2 M n-butyllithium solution (2.84 mmol, CAS: 109-72-8) was slowly added dropwise at -78°C. The mixture was stirred at this temperature for 1 to 2 hours. Carbon dioxide gas was introduced at a rate of approximately one bubble per second for 30 minutes. After completion of the reaction, the mixture was filtered and dried to obtain 0.42 g of the enoylmorpholine hapten with a yield of 44.68%.

[0030] The prepared dimethomorph hapten was identified by mass spectrometry, and the mass spectrum obtained was shown in Figure 1 .from Figure 1 It can be seen that the molecular ion peak of dimethomorph hapten is ESI-[MH]- : 396, which is the highest peak, consistent with the molecular weight of 397 of the dimethomorph hapten, indicating that the dimethomorph hapten shown in formula (I) was successfully synthesized.

[0031] Example 2 Preparation of dimethomorph artificial antigen

[0032] 2.1 Preparation of dimethomorph artificial antigens with bovine serum albumin as carrier protein

[0033] (1) 10 mg of the dimethomorph hapten prepared in Example 1 was dissolved in 0.2 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 activated hapten ester.

[0034] (2) Weigh 40 mg of bovine serum albumin (BSA) and dissolve it in 4 mL of 0.01 mol / L PBS solution to form a BSA carrier solution. Slowly add the hapten-activated ester dropwise to the BSA carrier solution under stirring and stir at room temperature for 16 to 24 hours.

[0035] (3) The solution obtained in step (2) was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules, thereby obtaining the dimethomorph hapten-BSA conjugate, i.e., the dimethomorph coating antigen, which was aliquoted and stored at 4°C for later use.

[0036] 2.2 Preparation of dimethomorph artificial antigen with lactoferrin as carrier protein

[0037] (1) 10 mg of the dimethomorph 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.

[0038] (2) Weigh 40 mg of lactoferrin (LF) and fully dissolve it in 4 mL of 0.01 mol / L PBS solution to form a lactoferrin carrier solution. Slowly add the above-mentioned hapten-activated ester dropwise to the lactoferrin carrier solution under stirring and stir at room temperature for 16 to 24 hours.

[0039] (3) The solution obtained in step (2) was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules, thereby obtaining the dimethomorph hapten-LF conjugate, i.e., the dimethomorph antigen for immunization. The conjugate was aliquoted and stored at 4°C for later use.

[0040] The synthetic routes of dimethomorph artificial antigens whose carrier protein is bovine serum albumin and dimethomorph artificial antigens whose carrier protein is lactoferrin are shown in Figure 2 .

[0041] Example 3 Preparation of dimethomorph monoclonal antibodies

[0042] The carrier protein prepared in Example 2 was an artificial antigen containing lactoferrin and an oxime-based antigen as the immunogen. After emulsification with an equal volume of Freund's adjuvant, BALB / C mice were immunized. Each mouse was immunized with a dose of 50 to 100 μg. The immunization interval was 2 weeks. After three immunizations, serum titers were detected by collecting blood from the mouse tail vein. If the antibody titer did not meet the requirements, a booster immunization was required. After the antibody titer no longer increased, 100 μg of the complete antigen was used for subcutaneous booster immunization. After 5 days, mouse spleen cells were taken and fused with SP20 cells. The fused cells were screened in HAT culture medium and replaced with complete culture medium for cultivation after 5 days. The cell supernatant was tested with ELISA, and cells in wells with strong positive test results were subjected to limiting dilution cloning culture. After three cloning culture tests, cells in wells that were all positive were hybridoma cells that secreted monoclonal antibodies. After the hybridoma cells were amplified and cultured, they were inoculated into the mouse peritoneal cavity to produce ascites containing the antibody. By purifying ascites using the caprylic acid-ammonium sulfate precipitation method, high-purity and high-specificity dimethomorph monoclonal antibodies can be obtained.

[0043] Example 4 ELISA Performance Evaluation of Dimethomorph Monoclonal Antibodies

[0044] The dimethomorph artificial antigen prepared in Example 2, whose carrier protein was bovine serum albumin, was diluted to 0.1 μg / mL using carbonate buffer at pH 9.6 as the coating diluent. 100 μL / well was added to a polystyrene microplate and coated overnight at 4°C. The plate was then dried. 280 μL / well of 1% BSA was added and the plate was blocked in phosphate buffer at 37°C for 1 hour. The plate was then dried and vacuum-packed for storage.

[0045] The dimethomorph monoclonal antibody prepared in Example 3 was diluted to 0.1 μg / mL using phosphate buffer containing 0.05% sodium azide and pH=7.4, and stored at 4° C. for later use.

[0046] To a microwell ELISA plate coated with an ethylenediamine artificial antigen whose carrier protein is bovine serum albumin, 100 μL / well of the ethylenediamine standard solution was added, and then 20 μL / well of the ethylenediamine monoclonal antibody solution was added, and the reaction was carried out at 37°C for 0.5 h; after drying, 280 μL / well of the washing solution was added, and the plate was washed three times and patted dry; then 100 μL / well of the enzyme-labeled secondary antibody was added, and the plate was reacted at 37°C for 0.5 h; after washing three times again and patting dry, 50 μL / well of the color developing solution A and the color developing solution B were added, respectively, and the plate was reacted at 37°C for 15 min; 1 M sulfuric acid solution was added at 50 μL / well 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.

[0047] Table 1 ELISA test OD values ​​of dimethomorph standard solutions at different concentrations

[0048]

[0049] Based on the data in Table 1, ELISA Calc software was used to perform four-parameter logistic curve fitting to draw the standard curve. Figure 3 The linear equation of the standard curve is: y = (AD) / [1 + (x / C) ^ B] + D, r 2 =0.99929, A=1.27103, B=1.06975, C=0.08397, D=0.09129, x represents the concentration of the test substance, y represents the OD value, and the IC of the dimethomorph antibody is obtained by calculation. 50 The value was 0.097 μg / L, and it was linear in the range of 0.05 to 0.8 μg / L.

[0050] Example 5 Preparation of a dimethomorph detection device

[0051] 5.1 Preparation of a reaction membrane coated with an artificial ethylenediamine antigen and mouse IgG using bovine serum albumin as a carrier protein:

[0052] A nitrocellulose membrane (NC membrane) was used as the reaction membrane. The dimethomorph artificial antigen prepared in Example 2, whose carrier protein was bovine serum albumin, was adjusted to a concentration of 0.05-0.2 mg / mL using coating buffer. The concentration of mouse IgG was also adjusted to 0.05-0.2 mg / mL using coating buffer. The dimethomorph artificial antigen, whose carrier protein was bovine serum albumin, 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. The detection area and the control area were separated by 2.5 mm. The membrane was then placed in a 45°C oven for 12-16 hours and then stored in a constant temperature and humidity chamber until ready for use. The coating buffer used was 0.01 M PBS buffer containing 1% sucrose, 0.05% sodium azide, and a pH of 7.6.

[0053] 5.2 Preparation of microwells containing gold nanoparticle-labeled dimethomorph monoclonal antibodies:

[0054] 5.2.1 Preparation of colloidal gold:

[0055] Dissolve 1g of chloroauric acid in pure water using ultrasonic waves, 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 2mL 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. All glassware should be soaked overnight in a mixture of potassium permanganate and sulfuric acid, then rinsed and dried before use.

[0056] 5.2.2 Labeling of dimethomorph monoclonal antibodies:

[0057] Aliquot 1 mL of nanogold solution into each vial and adjust the pH of the colloidal gold solution with 0.1 mol / L KCO solution. Add 4 μg of dimethomorph monoclonal antibody to the colloidal gold solution 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 value that maintains a red color. Add 10 μL of 10% bovine serum albumin solution for blocking. Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant.

[0058] 5.2.3 Preparation of micropores:

[0059] Add 1 mL of gold diluent containing 2% Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose to reconstitute the gold, dispense 8 μL / well into microwells, dry at 37°C for 16 hours, and store for later use.

[0060] 5.3 Preparation of sample pad:

[0061] 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 0.05M PBST buffer containing 1% sucrose, 0.1% Triton X-ray diffraction, and 0.05% sodium azide.

[0062] 5.4 Assembly of colloidal gold immunochromatographic test card:

[0063] The reaction membrane prepared in step 5.1 is stacked in the middle of the PVC board backing, and the sample pad and absorbent pad prepared in steps 5.2 and 5.3 are stacked at 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 a shell to obtain a dimethomorph colloidal gold immunochromatographic test card.

[0064] Example 6 Sensitivity Determination of Dimethomorph Colloidal Gold Immunochromatographic Detection Card

[0065] Prepare a series of dimethomorph standard solutions of varying concentrations in 0.01M PBS buffer. Then, add 100 μL of each dimethomorph standard solution to the gold-labeled microwells and repeatedly pipette to uniformly redissolve the solution. After 5 minutes of simmering, transfer the solution from the gold-labeled microwells to the sample wells of the dimethomorph colloidal gold immunochromatographic test card prepared in Example 5. Start the timer after sample addition and observe the results after 5 to 8 minutes. After 8 minutes, the result is considered invalid.

[0066] The naked eye judgment standard is shown in Figure 4 Specifically, the T-line color is stronger than the C-line or no significant difference, indicating a negative test result (-); the T-line color is significantly weaker than the C-line or no color is present, indicating a positive test result (+); invalid: the absence of the C-line indicates incorrect operation or an expired test strip. The test was repeated three times. This allows for rapid qualitative detection of dimethomorph using the colloidal gold immunochromatographic test card. Specific results are shown in Table 2 below.

[0067] Table 2 Determination results of dimethomorph standard solutions at different concentrations

[0068]

[0069] As shown in Table 2, the dimethomorph colloidal gold immunochromatographic test card prepared by the present invention has a high sensitivity for dimethomorph detection, which can reach 0.5 μg / L.

[0070] Example 7 Stability Test of Dimethomorph Colloidal Gold Immunochromatographic Test Card

[0071] The colloidal gold qualitative immunochromatographic test card is stored at room temperature. In order to ensure the stability of the test card, the dimethomorph colloidal gold immunochromatographic test card prepared in Example 5 was subjected to an accelerated destructive test. It was placed continuously at room temperature and 45°C for 60 days, and the negative results and the color change of the dimethomorph standard solution were detected on the 0th day, the 5th day, the 10th day, the 20th day, the 30th day, the 40th day, the 50th day and the 60th day, respectively. The experiment was repeated for 3 groups. The results are shown in Table 3 below, where "+" represents positive and "-" represents negative.

[0072] Table 3 Stability results of dimethomorph colloidal gold immunochromatographic test card

[0073]

[0074] As shown in Table 3, after 60 days of sealed storage at room temperature and 45°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 for at least 60 days at 45°C in an accelerated test. Therefore, the dimethomorph colloidal gold qualitative 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.

[0075] Example 8 Detection of Samples Using Dimethomorph Colloidal Gold Immunochromatographic Detection Card

[0076] Take 1g of fruit and vegetable samples (4g of epidermis or cross-section samples for tubers), cut the leafy vegetables into pieces of about 1cm square, put them into a 50mL centrifuge tube, add 7mL of 0.01M PB buffer, shake vigorously and mix for 2min, let the centrifuge tube stand for 2min, and the supernatant is the test solution.

[0077] Take 100 μL of the test solution and add it to the sample well of the dimethomorph colloidal gold immunochromatographic card prepared in Example 5. Start timing after adding the sample. After 5 to 8 minutes, the time is set according to the Figure 4 The judgment criteria for observation results.

[0078] Visual interpretation method: T line color is stronger than C line color or there is no obvious difference with C line color, indicating that the sample is negative (-); positive (+): T line color is significantly weaker than C line color or T line does not show color, indicating that the sample is positive (+); invalid: no C line appears, indicating incorrect operation process or the test strip is invalid.

[0079] Minimum detection limit: Ten blank samples were spiked with dimethomorph standard solution at gradient addition levels of 0, 1.5, 5, 10, and 20 μg / kg. The results are shown in Table 4.

[0080] Table 4 Detection limit results for spiked fruit and vegetable samples

[0081]

[0082] As shown in Table 4, the dimethomorph colloidal gold immunochromatographic test card prepared by the present invention demonstrated good reproducibility in the test results for the 10 samples. When the dimethomorph content in the sample was less than 5 μg / kg, all results were negative; when it was greater than 5 μg / kg, all results were positive. Therefore, the detection limit of the dimethomorph colloidal gold immunochromatographic test card prepared by the present invention for dimethomorph in the sample was 5 μg / kg.

[0083] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A dimethomorph hapten, characterized in that Its structure is shown in formula (I):

2. The method for preparing the dimethomorph hapten according to claim 1, characterized in that: The following steps are involved: S1. Dimethomorph is reacted with carbon dioxide under alkaline conditions to prepare a dimethomorph hapten as shown in formula (I). The reaction formula is shown in formula (II):

3. The dimethomorph artificial antigen is characterized in that The dimethomorph artificial antigen is a conjugate of the dimethomorph hapten according to claim 1 and a carrier protein, and its structural formula is shown in formula (III): Among them, protein is a carrier protein.

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

5. Dimethomorph antibody, characterized in that The dimethomorph antibody is prepared by immunizing an animal with the dimethomorph artificial antigen according to claim 3, and the dimethomorph antibody is a dimethomorph monoclonal antibody.

6. Use of the dimethomorph hapten according to claim 1 and the dimethomorph artificial antigen according to claim 3 in immunological detection of dimethomorph for non-disease diagnosis purposes.

7. Use of the dimethomorph antibody according to claim 5 in immunological detection of dimethomorph for non-disease diagnosis purposes.

8. A detection device for dimethomorph, characterized in that The detection device is prepared using the dimethomorph artificial antigen according to any one of claims 3 or 4 and the dimethomorph antibody according to claim 5.

9. A method for detecting dimethomorph for non-disease diagnosis purposes, characterized in that: The detection method is to detect dimethomorph residues in agricultural products using the dimethomorph detection device according to claim 8.

10. The detection method according to claim 9, characterized in that: The agricultural products are fruits or vegetables.

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