Diafenthiuron hapten, artificial antigen, preparation method, antibody and application thereof

Abstract

The present invention discloses a diafenthiuron hapten, an artificial antigen, preparation methods, antibodies, and applications thereof. The structural formula of the diafenthiuron hapten is #imgabs0#. The hapten, artificial antigen, antibodies to immunized animal products, and the test kit prepared in this invention exhibit high sensitivity and specificity, are simple to operate, have a short detection time, are not limited by specialized personnel, are inexpensive, and are suitable for grassroots deployment. This technology fills a gap in domestic rapid detection of diafenthiuron. The application of this technology can significantly accelerate detection and has significant social benefits in controlling diafenthiuron pesticide poisoning in edible agricultural products, reducing pesticide over-limits, improving the qualified rate of commercially available vegetables, and ensuring food safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality detection, and in particular relates to a diafenthiuron hapten, an artificial antigen, a preparation method thereof, an antibody and applications thereof. Background Art

[0002] Difenthiuron is a new, highly effective thiourea insecticide and acaricide with contact, stomach, systemic, and fumigant properties, and also exhibits a limited ovicidal effect. This product is highly effective against a variety of insect and acaricide pests, including red spider mites on citrus and apple trees, diamondback moths and cabbage loopers on cruciferous vegetables, and tea leafhoppers.

[0003] Currently, diafenthiuron is primarily detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, this method is complex, tedious, costly, and requires high technical skills for pretreatment, making it unsuitable for rapid on-site testing. Rapid diagnostic test strips based on colloidal gold immunochromatographic technology (referred to as colloidal gold test strips) have been gradually applied in fields such as food safety supervision, biomedicine, and animal and plant quarantine. Compared with instrumental methods, they are simpler, faster, require no testing equipment, and are low-cost and pollution-free. They are also gradually being used to detect pesticide residues in edible agricultural products. Therefore, immunochromatographic testing provides a new analytical method for the rapid on-site detection of diafenthiuron residues.

[0004] However, diafenthiuron has a relative molecular mass of less than 1000, making it a small molecule and non-immunogenic, unable to directly induce the production of specific antibodies. Only by modifying the hapten structure and coupling it to a macromolecular carrier to create an artificial complete antigen can an immune response and the production of specific antibodies be induced. Furthermore, the diafenthiuron hapten design must avoid the production of byproducts, and the titer, specificity, and detection limit of the antibodies obtained from immunized animals must meet the requirements for preparation into a test kit. Summary of the Invention

[0005] The purpose of the present invention is to overcome at least one deficiency of the prior art and to provide a diafenthiuron hapten, an artificial antigen, a preparation method thereof, an antibody and applications thereof.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a diafenthiuron hapten, the structural formula of the diafenthiuron hapten is as follows:

[0008]

[0009] In a second aspect, the preparation method of the diafenthiuron hapten described in the first aspect of the present invention has the following synthetic route:

[0010]

[0011] The following steps are involved:

[0012] 1) Compound a and compound b are added, and compound c is obtained after distillation and recrystallization;

[0013] 2) Compound c undergoes bromination reaction with bromine and iron in aqueous solution, followed by distillation, separation, drying, and sealed heating to obtain compound d;

[0014] 3) Compound d undergoes a substitution reaction with compound e under alkaline conditions, followed by acidification, purification, and drying to obtain the diafenthiuron hapten.

[0015] In some examples, the temperature of the bromination reaction in step 2) is 20-30° C., and the reaction time is 1-5 h.

[0016] In some examples, the mass ratio of compound d to compound e in step 3) is 1.9 to 2.1.

[0017] In a third aspect, the present invention provides a diafenthiuron artificial antigen, which is obtained by coupling the diafenthiuron hapten described in the first aspect to a carrier protein.

[0018] In some examples, the diafenthiuron artificial antigen is prepared by the carbodiimide method.

[0019] In some examples, the carrier protein is selected from any one of hemocyanin, bovine serum albumin, rabbit serum albumin, or chicken ovalbumin.

[0020] In a fourth aspect, the present invention provides a diafenthiuron antibody, which is obtained by immunizing an animal with the diafenthiuron artificial antigen described in the third aspect.

[0021] In a fifth aspect, the invention provides a use of the diafenthiuron artificial antigen described in the third aspect in the preparation of a diafenthiuron immunoassay kit, a colloidal gold immunochromatographic test strip, or a time-resolved fluorescence test strip.

[0022] The beneficial effects of the present invention are:

[0023] The antibodies prepared by the present invention against haptens and artificial antigens in animal products, and the resulting test kits, have the advantages of high sensitivity and specificity, simple operation, short detection time, no need for specialized personnel, low cost, and suitability for grassroots promotion. These advantages fill the gap in domestic rapid detection of diafenthiuron. The application of this technology can greatly accelerate detection speed and has significant social benefits in controlling the occurrence of diafenthiuron pesticide poisoning in edible agricultural products, reducing excessive pesticide levels, increasing the qualified rate of commercially available vegetables, and ensuring food safety. DETAILED DESCRIPTION

[0024] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0025] Example 1: Synthesis of diafenthiuron hapten

[0026] 3.85 g of compound c was placed in a round-bottom flask, 200 mL of purified water and 2 g of iron powder were added, and 20 mL of 1 mol / L bromine water was added. The temperature was maintained at 20-25° C. and stirring was continued for 1 hour to complete the bromination reaction. The reaction mixture was heated and distilled, separated, and dried to obtain compound d. 500 mg of compound d and 500 mg of compound e were placed in a 250 mL round-bottom flask, 80 mL of distilled water was added, and heated and stirred to dissolve. After heating and reflux for 1-2 hours, 15 mL of 3 mol / L sodium hydroxide solution was dropwise added, and heating and reflux were continued for 6 hours. The mixture was cooled to room temperature. 15 mL of 2 mol / L hydrochloric acid solution was added to stop the reaction, and the mixture was dried to obtain diafenthiuron hapten.

[0027] Example 2: Synthesis of artificial antigens for immunization

[0028] Take 10 mg of diafenthiuron hapten and dissolve it in 500 μL DMF, add 5 mg NHS and 20 mg EDC, react at room temperature in the dark for 12 hours, centrifuge at 10000 rpm for 10 minutes, take the supernatant and dry it, and dissolve the residue in 200 μL DMF. This solution is liquid A; take 5 mg of BSA and fully dissolve it in 5 mL of 0.2 mol / L NaHCO3 solution, then slowly add liquid A drop by drop to the BSA solution, seal it and stir it at room temperature in the dark for 6 hours; the reaction product is placed in a 4°C chromatography cabinet with 0.01 mol / L PBS for 3 days, centrifuge and collect the supernatant, which is the diafenthiuron artificial antigen, and store it at -20°C.

[0029] Example 3: Synthesis of artificial antigens for coating

[0030] Take 10 mg of diafenthiuron hapten and dissolve it in 500 μL DMF, add 5 mg NHS and 20 mg EDC, react at room temperature in the dark for 12 hours, centrifuge at 10000 rpm for 10 minutes, take the supernatant and dry it, and dissolve the residue in 200 μL DMF. This solution is liquid A; take 5 mg of OVA and fully dissolve it in 5 mL of 0.2 mol / L NaHCO3 solution, then slowly add liquid A drop by drop to the BSA solution, seal it and stir it at room temperature in the dark for 6 hours; the reaction product is placed in a 4℃ chromatography cabinet with 0.01 mol / L PBS for 3 days, centrifuge and collect the supernatant, which is the diafenthiuron artificial antigen, and store it at -20℃.

[0031] Example 4: Preparation of monoclonal antibodies

[0032] 1. Animal Immunization

[0033] For the first immunization, a 100 μg mixture of artificial antigen and Freund's complete adjuvant (CFA) was administered subcutaneously at multiple sites on the back of female BALB / c mice. Three weeks later, mice were immunized with a 100 μg mixture of artificial antigen and Freund's incomplete adjuvant (CFA) at a dose of 80 μg. Three weeks later, mice were immunized using the same method at a dose of 60 μg. Seven to ten days after the third immunization, mouse sera were tested using an enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 1.

[0034] Table 1 Mouse immune effect table

[0035]

[0036] As shown in Table 1, the antibody titer and inhibitory effect of immunized mouse 5 were the best, and it was used for subsequent cell fusion screening.

[0037] 2. Establishment of hybridoma cell lines

[0038] Aseptically harvest spleen cells from immunized mice and fuse them with NS0 myeloma cells. The fused cells are cultured in HAT selective medium in 96-well cell culture plates. After 7-10 days, hybridoma culture supernatants are collected and positive hybridoma cells are screened by indirect ELISA using the coated artificial antigen as the detection target. Microplates are coated with an appropriately diluted coated artificial antigen (50 μL / well) overnight at 4°C. Blocking is performed with 5% porcine serum at 37°C for 1 hour. Culture supernatants from the cells to be tested (50 μL / well) are added. HAT medium and mouse immune serum serve as negative and positive controls. A 1:1000 horseradish peroxidase-conjugated goat anti-mouse IgG antibody (50 μg / well) is added and incubated at 37°C for 1 hour. After each reaction, the plates are washed thoroughly with PBS containing 0.05% Tween-20. Color development is performed with the substrate TMB, and the reaction is terminated with 1 mol / L H2SO4 (50 μL / well). The OD value of each well is measured at 450 nm using a microplate reader. Select the clones with strong positive results and vigorous cell growth and perform three consecutive limiting dilution cloning. After expansion culture, freeze the cells.

[0039] 3. Preparation of Monoclonal Antibodies

[0040] Monoclonal antibodies were prepared by inducing ascites in vivo. The peritoneal fluid was collected from multiparous BALB / c female mice sensitized with pristane or liquid paraffin (0.5 mL / mouse). After 7 to 10 days, when the abdomen of the mouse was significantly enlarged, the ascites was collected aseptically with a 16-gauge needle. Usually 5 to 20 mL of ascites could be collected from each mouse. After centrifugation, the supernatant was collected, aliquoted, and stored at -20°C for later use.

[0041] IV. Antibody titer determination

[0042] The antibody titer of mouse ascites was determined by indirect ELISA, following the procedure of step 1. The results showed that the titer of diafenthiuron monoclonal antibody was >15K.

[0043] 5. Detection limit determination

[0044] The half inhibitory concentration (IC50) of diafenthiuron on mouse ascites was determined by indirect competitive ELISA. 50 ), with reference to step 1. The results showed that the minimum detection limit of the diafenthiuron monoclonal antibody against the diafenthiuron standard was 3 μg / L, and the half-maximal inhibition concentration was 8.7 μg / L.

[0045] 6. Cross-reactivity determination

[0046] The detection limits for the target and related antigens in mouse ascites were determined using an indirect competitive ELISA, and the cross-reactivity rates of the monoclonal antibody with related drugs were calculated. The results showed that the cross-reactivity rates of the diafenthiuron monoclonal antibody with diafenthiuron and other insecticides and acaricides were: 100% for diafenthiuron; and less than 1% for bromocyprodinil, abamectin, spirodiclofen, fenpyroxithrin, lambda-cyhalothrin, cyprodinil, thiamethoxam, cyazofamid, triclopyralid, indoxacarb, imidacloprid, and fenoxystrobin. This demonstrates the high specificity of the prepared antibody.

[0047] Comparative Example 1:

[0048] Take 1-tert-butyl-3-(2,6-diisopropyl-4-phenoxyphenyl)thiourea, add 10mmol HNO3 and 10mmol H2SO4 mixed acid solution, react under 60℃ sealed conditions for 3h, stop the reaction, and wash with water to obtain compound f;

[0049]

[0050] Take 10 mmol and dissolve it in 100 mL 1 mol / L HCL solution, add 5 g zinc powder, react under 65 ° C sealed conditions for 1 hour, stop the reaction, add water and wash to obtain the comparative example hapten, that is, hapten formula (3).

[0051]

[0052] The hapten reacts with nitrous acid at low temperature to form a diazonium salt, which then forms an azo-coupled comparative artificial antigen with the tyrosine and histidine residues on the carrier protein molecule, namely, the artificial antigen formula (4).

[0053]

[0054] Animal immunization was performed according to Example 4, and then the mouse serum was detected by enzyme-linked immunosorbent assay and compared with the artificial antigen of Example.

[0055] Table 2 Artificial antigen immune effect table

[0056]

[0057] Table 2 shows that the inhibitory effect of artificial antigen immunized animals on diafenthiuron is the worst and they are not suitable for cell fusion.

[0058] Example 6

[0059] 1. Preparation of diafenthiuron colloidal gold test card

[0060] Preparation of colloidal gold: Take 100mL of ultrapure water and place it in a 500mL clean conical flask, add 1mL of 1% (W / V) citric acid and boil; quickly add 1mL of freshly prepared 1% (W / V) citric acid solution while stirring, boil for about 3min until the color of the solution changes from yellow to purple-red, and continue boiling for 2min; wait for the solution to cool to room temperature, make up to 100mL with ultrapure water, adjust the pH to 9.0 with 0.2mol / LK2CO3, and store at 4℃ in the dark.

[0061] Colloidal gold labeling of monoclonal antibodies: Take 2 mL of the monoclonal antibody IgG to be labeled, add 10 mL of colloidal gold solution, and react at room temperature for 10 minutes; then add 1 mL of 20 mol / L sodium borate solution containing 10% (w / v) BSA and react for 10 minutes; after centrifugation, remove the supernatant and resuspend the colloidal gold in 20 mol / L sodium borate solution containing 1% (w / v) BSA. Store at 4°C in the dark.

[0062] Preparation of test membrane: Cut nitrocellulose membrane into 2.5 cm × 30 cm strips and place them on the platform of the gold spray instrument; dilute the artificial antigen and rabbit anti-mouse IgG to 1 mg / mL with PBS, and spray the artificial antigen and rabbit anti-mouse IgG solutions at 1 μL / cm in the center of the test membrane to form the test line and quality control line; dry in a 42°C drying oven for 30 min; place in an aluminum foil bag, add desiccant, and store sealed at 4°C.

[0063] Preparation of conjugate pad: Cut glass wool into 1.5 cm × 30 cm strips and place them on the gold spraying instrument platform; take 1 mL of colloidal gold label and add 2 mL of 0.02 mol / L sodium borate solution containing 2% (W / V) BSA, 3% (W / V) sucrose, 0.5 mol / L NaCl, 0.1% (V / V) Tween-20 and 0.1% (W / V) preservative; spray the anti-colloidal gold label solution onto the glass wool at 15 μL / cm; place in a 50°C drying oven to dry for 30 min; place in an aluminum foil bag, add desiccant, and store sealed at 4°C.

[0064] Preparation of sample pads: Cut glass wool into 1.5 cm × 30 cm strips and soak the glass wool strips in a PBS solution containing 0.1 mol / L NaCl, 0.1% (v / v) Tween-20, and 0.1% (w / v) preservative. Dry the strips in a 50°C drying oven for 30 min. Place the strips in an aluminum foil bag, add a desiccant, and seal them for storage at room temperature.

[0065] Assembly of the test card: Cut the PVC board into 7.5cm×30cm long boards as the support board, stick the test membrane, conjugate pad, sample pad, absorbent paper, etc. on the PVC board, and then cut it into 0.3cm test strips. Put the test paper into the card shell to make the test card.

[0066] 2. Preparation of Extract

[0067] 0.1 mol / L PBS (pH 7.6) solution containing 0.1% (V / V) Tween-20, 0.1% (W / V) preservative, and 5% (V / V) methanol.

[0068] 3. Sample Pretreatment

[0069] Before testing, the sample needs to be returned to room temperature (20-30°C). Weigh 2g ± 0.1g of the chopped and mixed sample into a 50mL centrifuge tube. (Note: The sample should be chopped into pieces smaller than 1cm square and mixed evenly.)

[0070] Pipette 5 mL of the extract into a centrifuge tube, cover the tube tightly, and shake vigorously (frequency: 120 times / min). Wash the sample thoroughly for 2 minutes, and let it stand for 2 minutes to obtain a clear sample solution, which is the solution to be tested.

[0071] 4. Testing steps

[0072] Before testing, return the test card and the sample solution to room temperature (20°C-30°C); tear open the aluminum foil bag and remove the test card from the aluminum foil bag; use a pipette to slowly add 100uL of the above test solution to the sample well (S) on the test card and start the timer; the result should be read within 5-8 minutes; the result read after other times will be invalid.

[0073] 5. Result determination

[0074] Negative (-): The T line is darker than or the same dark as the C line (control line), indicating that the concentration of diafenthiuron in the sample is lower than the detection limit or does not contain diafenthiuron.

[0075] Positive (+): The T line is lighter than the C line, or the T line is not colored, indicating that the concentration of diafenthiuron in the sample is higher than or equal to the detection limit. The lighter the T line is than the C line, the higher the concentration of diafenthiuron in the sample.

[0076] Invalid: No C line appears, which may be due to improper operation or the test card is invalid. Please read the instructions again and retest with a new test card.

[0077] 6. Minimum detection limit test

[0078] Cabbage and apple samples tested negative for diafenthiuron. 2.0 g of each chopped sample was weighed into a 50 mL centrifuge tube, and different concentrations of diafenthiuron standard solution were added. The samples were tested using a colloidal gold immunochromatographic detection kit for diafenthiuron. The test results are shown in Table 3. The detection limit for diafenthiuron was 0.1 mg / kg, which is lower than the maximum residue limit specified in GB 2763-2021 National Food Safety Standard: Maximum Residue Limits of Pesticides in Food, making it suitable for rapid screening.

[0079] Table 3 Minimum detection limit test results

[0080]

[0081] 7. Repeatability test

[0082] A sample of pakchoi was selected. After testing negative for diafenthiuron, 2.0 g of each chopped sample was weighed and placed in a 50 mL centrifuge tube. Different concentrations of diafenthiuron standard solution were added. The sample was tested using three batches of diafenthiuron-based colloidal gold immunochromatographic test kits. Each sample was tested three times. The results are shown in Table 4. This shows that the test kit has good reproducibility.

[0083] Table 4 Repeatability test results

[0084]

[0085] 8. Stability test

[0086] The diafenthiuron colloidal gold immunochromatographic test kit was stored in a 37°C oven, and its minimum detection limit was regularly tested to observe changes in the test kit's performance. The results are shown in Table 5. This shows that the test kit is relatively stable and can be stored at room temperature for at least one year.

[0087] Table 5 Stability test results

[0088] date 0mg / kg 0.1mg / kg Equivalent to room temperature storage time Day 1 - + 4 days Day 2 - + 8 days Day 3 - + 12 days Day 4 - + 16 days Day 5 - + 20 days Day 10 - + 40 days Day 15 - + 60 days Day 30 - + 120 days Day 40 - + 160 days Day 50 - + 200 days Day 60 - + 240 days Day 80 - + 320 days Day 100 - + 400 days

[0089] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A diafenthiuron hapten, characterized in that: The structural formula of the diafenthiuron hapten is as follows: 。 2. The method for preparing the diafenthiuron hapten according to claim 1, characterized in that: Its synthetic route is as follows: , including the following steps: 1) Compound a and compound b are added, and compound c is obtained after distillation and recrystallization; 2) Compound c undergoes bromination reaction with bromine and iron in aqueous solution, followed by distillation, separation, drying, and sealed heating to obtain compound d; 3) Compound d undergoes a substitution reaction with compound e under alkaline conditions, followed by acidification, purification, and drying to obtain the diafenthiuron hapten.

3. The preparation method according to claim 2, characterized in that The temperature of the bromination reaction in step 2) is 20-30° C., and the reaction time is 1-5 hours.

4. The preparation method according to claim 2, characterized in that In the step 3), the mass ratio of compound d to compound e is 1.9-2.1:

1.

5. A diafenthiuron artificial antigen, characterized in that The diafenthiuron artificial antigen is obtained by coupling the diafenthiuron hapten described in claim 1 with a carrier protein.

6. The diafenthiuron artificial antigen according to claim 5, characterized in that The diafenthiuron artificial antigen is prepared from the diafenthiuron hapten according to claim 1 and a carrier protein by a carbodiimide method.

7. The diafenthiuron artificial antigen according to claim 5, characterized in that The carrier protein is selected from any one of hemocyanin, bovine serum albumin, rabbit serum albumin or chicken ovalbumin.

8. A diafenthiuron antibody, characterized in that The diafenthiuron antibody is obtained by immunizing an animal with the diafenthiuron artificial antigen according to claim 5.

9. Use of the diafenthiuron artificial antigen according to any one of claims 5 to 7 in the preparation of a diafenthiuron immunoassay kit.

10. Use of the diafenthiuron artificial antigen according to any one of claims 5 to 7 in the preparation of colloidal gold immunochromatographic test strips or time-resolved fluorescence test strips.

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