A difenoconazole hapten, antigen, antibody, detection device and preparation and application thereof

Abstract

The invention discloses a difenoconazole hapten, antigen, antibody, detection device, preparation and application thereof, and relates to a difenoconazole hapten, antigen, antibody, detection device, preparation and application thereof for detecting difenoconazole drug residues in food. The difenoconazole hapten prepared by the invention completely retains the characteristic structures of 3-chlorophenyl 4-chlorophenyl ether, 1,3-dioxolane and 1,2,4-triazole in difenoconazole, improves the immunogenicity of the difenoconazole antigen, and the difenoconazole antigen and monoclonal antibody prepared by the invention are used for ELISA detection with strong specificity, IC 50 The value is 0.06µg / L. The sensitivity of the colloidal gold detection device prepared by the present invention to difenoconazole in the standard solution is 0.5µg / kg, and the sensitivity to difenoconazole in the sample can reach 5µg / kg.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and more specifically, to a difenoconazole hapten, an antigen, an antibody, a detection device, and preparation and application thereof. Background Art

[0002] Difenoconazole is a broad-spectrum, highly effective triazole systemic fungicide, which is widely used in crops such as grains, oilseeds, fruits, vegetables, tea, and Chinese medicinal materials to control diseases. As the situation of difenoconazole residues in the environment becomes increasingly serious, it poses a huge threat to people's health.

[0003] In the relevant detection technology, the detection method of difenoconazole is mainly based on the instrument method, but because the required equipment and instruments are expensive, the detection time is long, and professional personnel are required to operate, it is impossible to realize on-site detection and rapid clinical inspection in a real sense, which brings great inconvenience to daily detection work. The key to immunoassay detection technology is determined by the performance of antigens and antibodies, and the key to antigens and antibodies is haptens. Therefore, to obtain antigens and antibodies with excellent performance, the structural design of haptens is particularly important. The artificial antigens directly prepared based on difenoconazole itself in the related technology have the defects of poor sensitivity and low cross-reaction rate, which cannot meet the actual use needs of the existing market. Therefore, the development of highly specific difenoconazole haptens or artificial antigens is of vital importance for a rapid, highly sensitive and low-cost detection method for difenoconazole. Summary of the invention

[0004] The purpose of the present invention is to provide a difenoconazole hapten, an antigen, an antibody, a detection device and a preparation and application thereof, for detecting difenoconazole residues in food.

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

[0006] , formula (Ⅰ).

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

[0008] S1. Compound a and compound b undergo a substitution reaction to obtain compound c. The reaction formula of this step is shown in formula (II):

[0009] , formula (II);

[0010] S2. Compound c is hydrolyzed under alkaline conditions to obtain the difenoconazole hapten represented by formula (I). The reaction formula of this step is shown in formula (III):

[0011] , formula (III).

[0012] According to another aspect of the present invention, a difenoconazole antigen is provided. The difenoconazole antigen is a conjugate of difenoconazole hapten and a carrier protein, and its structural formula is shown in formula (IV):

[0013] , formula (IV).

[0014] In some embodiments, the carrier protein is any one of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

[0015] According to a fourth aspect of the present invention, a difenoconazole antibody is provided. The difenoconazole antibody is prepared by immunizing an animal with a difenoconazole antigen. The difenoconazole antibody is a difenoconazole monoclonal antibody.

[0016] According to a fifth aspect of the present invention, there is provided the use of a difenoconazole hapten and a difenoconazole antigen in immunological detection of difenoconazole for non-disease diagnosis purposes.

[0017] According to a sixth aspect of the present invention, there is provided a use of a difenoconazole antibody in an immunological detection of difenoconazole for non-disease diagnosis purposes.

[0018] According to a seventh aspect of the present invention, a detection device for difenoconazole is provided. The detection device is prepared using difenoconazole antigen and difenoconazole antibody.

[0019] According to an eighth aspect of the present invention, there is provided a method for detecting difenoconazole for non-disease diagnosis purposes, the method comprising detecting difenoconazole drug residues in food using a difenoconazole detection device.

[0020] In some embodiments, the food is a fruit or a vegetable.

[0021] The beneficial effects of the present invention are as follows: (1) the difenoconazole hapten prepared by the present invention completely retains the characteristic structures of 3-chlorophenyl 4-chlorophenyl ether, 1,3-dioxolane and 1,2,4-triazole, and not only has a good spatial structure, but also has the same electron cloud density as that of difenoconazole, thereby improving the immunogenicity of the difenoconazole antigen;

[0022] (2) The difenoconazole antigen and monoclonal antibody prepared by the present invention are highly specific for ELISA detection, and IC 50 The value is 0.06µg / L;

[0023] (3) The difenoconazole antigen and monoclonal antibody prepared by the present invention are used in colloidal gold immunochromatography technology to quickly and conveniently realize the qualitative detection of difenoconazole. The colloidal gold detection device prepared by the present invention has a sensitivity of 0.5 µg / kg to difenoconazole in the standard solution and a sensitivity of up to 5 µg / kg to difenoconazole in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a mass spectrum of the difenoconazole hapten according to one embodiment of the present invention.

[0025] Figure 2 The figure is a hydrogen nuclear magnetic resonance image of the difenoconazole hapten according to one embodiment of the present invention.

[0026] Figure 3 The present invention is a synthetic route of the difenoconazole antigen according to one embodiment of the present invention.

[0027] Figure 4 The standard curve of the indirect competitive ELISA established based on the difenoconazole monoclonal antibody according to one embodiment of the present invention is shown in FIG.

[0028] Figure 5 This is a result judgment standard diagram of a difenoconazole detection device according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0030] Example 1 Preparation of difenoconazole hapten

[0031] The method for preparing difenoconazole hapten comprises the following steps:

[0032] S1. Take 0.30g (2.36mmol, CAS: 4928-88-5) of compound b in a 50ml round-bottom flask, then add 10ml of acetone, 1.18g (2.83mmol, CAS: 873012-43-2) of compound a, 0.65g (4.72mmol) of K 2 CO 3and 10 mg of n-tetrabutylammonium bromide, stir and mix thoroughly, raise the temperature to 50-60°C and react for 5-8 hours. After the reaction is complete, evaporate the solvent under reduced pressure, add 100 ml of purified water to the residue, extract twice with ethyl acetate, and remove the solvent from the organic phase under reduced pressure to obtain compound c;

[0033] S2. Dissolve compound c in 20 mL of methanol, add 20 mL of 6 mol / L lithium hydroxide aqueous solution, react at room temperature for 10-12 h, add 70 mL of saturated sodium chloride aqueous solution, extract twice with 40 mL of dichloromethane, adjust the pH value of the aqueous phase to 4-5 with 4M hydrochloric acid, precipitate solid, filter and dry to obtain 0.86 g of difenoconazole hapten with a yield of 80.93%.

[0034] The prepared difenoconazole hapten was identified by mass spectrometry, and the obtained mass spectrum is shown in Figure 1 .from Figure 1 It can be seen that the molecular ion peak of difenoconazole hapten is ESI- [MH] - : 449.1, which is the highest peak, consistent with the molecular weight of the difenoconazole hapten of 449.1, indicating that the difenoconazole hapten shown in formula (I) was successfully synthesized.

[0035] The prepared difenoconazole hapten was subjected to hydrogen nuclear magnetic resonance ( 1 H-NMR) determination, the results are as follows Figure 2 As shown: 1 HNMR (CDCl 3 , 500 MHz): 8.44–8.49 (d, 1H), 7.59–7.63 (dd, 1H), 7.37-7.38 (m, 2H), 7.01-7.06 (m, 3H), 6.85-6.88 (m, 1H), 4.81-4.94 (m, 2H), 3.89-4.15 (m, 2H), 3.19-3.36 (m, 1H) and 1.14-1.28 (m, 3H).

[0036] Example 2 Preparation of difenoconazole immunization antigen and coating antigen

[0037] 2.1 Preparation of antigen for difenoconazole immunization

[0038] (1) Take 5 mg of the difenoconazole hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir it thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), and stir it at room temperature for 4 h to obtain the hapten activated ester;

[0039] (2) Weigh 30 mg of lactoferrin (LF) and fully dissolve it in 5 mL of 0.01 mol / L PBS solution to form a lactoferrin carrier solution. Slowly add the above-mentioned hapten activated ester dropwise into the lactoferrin carrier solution under stirring and stir at room temperature for 16 to 24 h.

[0040] (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 being changed 3 times a day to remove unreacted small molecules, thereby obtaining a difenoconazole hapten-LF conjugate, i.e., a difenoconazole immunization antigen, which was packaged and stored at 4°C for later use.

[0041] 2.2 Preparation of difenoconazole coating antigen

[0042] (1) Take 10 mg of the difenoconazole hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), stir it thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), and stir it at room temperature for 4 h to obtain the hapten activated ester;

[0043] (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 bovine serum albumin carrier solution. Slowly add the hapten activated ester of step (1) dropwise into the bovine serum albumin carrier solution under stirring and stir at room temperature for 16 to 24 h.

[0044] (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 being changed 3 times a day to remove unreacted small molecules, thereby obtaining a difenoconazole hapten-BSA conjugate, i.e., a difenoconazole coating antigen, which was aliquoted and stored at 4°C for later use.

[0045] The synthetic route of difenoconazole immunization antigen and coating antigen is shown in Figure 3 .

[0046] Example 3 Preparation of difenoconazole monoclonal antibody

[0047] After emulsifying the fenpropimorph azole immunization antigen prepared in Example 2 with an equal volume of Freund's adjuvant, BALB / C mice were immunized. The immunization dose for each mouse was 50-100 μg, and the immunization interval was 2 weeks. After immunization 3 times, the serum titer was detected by collecting the tail venous blood of the mouse. If the antibody titer does not meet the requirements, it is necessary to strengthen the immunization. After the antibody titer no longer increases, 100 μg of full antigen is used for subcutaneous strengthening immunization. After 5 days, the mouse spleen cells are taken and fused with SP20 cells. The fused cells are screened in HAT culture medium, and after 5 days, the complete culture medium is replaced with HAT culture medium for culture. The cell supernatant is detected by ELISA, and the cells in the wells with strongly positive test results are cloned and cultured by limiting dilution method. After 3 clone culture tests, the cells in the wells that are positive are hybridoma cells that secrete monoclonal antibodies. After the hybridoma cells are amplified and cultured, they are inoculated into the peritoneal cavity of mice to produce ascites containing antibodies. By purifying ascites with the caprylic acid-ammonium sulfate precipitation method, high-purity and high-specificity difenoconazole monoclonal antibodies can be obtained.

[0048] Example 4 ELISA performance evaluation of difenoconazole monoclonal antibody

[0049] Using carbonate buffer at pH = 9.6 as the coating diluent, the difenoconazole coating antigen prepared in Example 2 was diluted to 0.1 μg / mL, and 100 μL / well was added to the polystyrene microplate, coated overnight at 4°C, dried, and 1% BSA solution was added at 280 μL / well. The plates were blocked in phosphate buffer at 37°C for 1 h, dried, and vacuum packed for storage after drying.

[0050] The difenoconazole monoclonal antibody prepared in Example 3 was diluted to 0.06 μg / mL using a phosphate buffer solution containing 0.05% sodium azide and pH=7.4, and stored at 4°C for later use.

[0051] To the microwell ELISA plate coated with fenpropimorph antigen, 100 μL / well of fenpropimorph standard solution was added, and then 20 μL / well of fenpropimorph monoclonal antibody solution was added, and the reaction was carried out at 37°C for 0.5 h; after drying, 280 μL / well of washing solution was added, and the plate was washed 3 times and patted dry; then 100 μL / well of enzyme-labeled secondary antibody was added, and the reaction was carried out at 37°C for 0.5 h; after washing 3 times again and patting dry, 50 μL / well of color development solution A and color development solution B were added respectively, and the reaction was carried out at 37°C for 15 min; 50 μL / well of 1 M sulfuric acid solution was added to terminate the reaction, and the OD value of each well was measured by setting the ELISA reader at a wavelength of 450 nm. The results are shown in Table 1.

[0052] Table 1 OD values ​​of difenoconazole standard solutions at different concentrations

[0053]

[0054] 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 4 , the linear equation of the standard curve is:

[0055] y = (AD) / [1+(x / C)^B]+D, r 2 =0.99968, A=1.33017, B= 1.21611, C= 0.04735, D=0.13820, x represents the concentration of the substance to be tested, y represents the OD value, and the IC of the difenoconazole antibody is obtained by calculation 50 The value was 0.06 μg / L, and it was linear in the range of 0.02~0.32 μg / L.

[0056] Example 5 Preparation of a detection device for difenoconazole

[0057] 5.1 Preparation of reaction membrane coated with difenoconazole coating antigen and mouse IgG:

[0058] Using nitrocellulose membrane (NC membrane) as the reaction membrane, the concentration of the difenoconazole coating antigen prepared in Example 2 was adjusted to 0.05-0.2 mg / mL with a coating buffer, and the concentration of mouse IGg with a coating buffer was also adjusted to 0.05-0.2 mg / mL, and the difenoconazole coating antigen and mouse IGg were sprayed onto the detection area (T line) and control area (C line) corresponding to the reaction membrane according to a membrane liquid volume of 0.8-1.2 μL / cm, with an interval of 2.5 mm between the detection area and the control area, and placed in a 45°C oven for 12-16 hours, and placed in a constant temperature and humidity storage box for use, and the coating buffer used was a 0.01M PBS buffer containing 1% sucrose, 0.05% sodium azide, and pH=7.6;

[0059] 5.2 Preparation of microwells containing gold nanoparticle-labeled monoclonal antibody against difenoconazole:

[0060] 5.2.1 Preparation of colloidal gold:

[0061] Take 1g of chloroauric acid, dissolve it with pure water by ultrasonic, and then make up to 100mL. Store it at 4℃ away from light for later use. Take 4mL of the above solution to 100ml of pure water, heat it to boiling, then add 1.2mL of 0.06% sodium citrate solution, continue heating for 10 minutes, cool it to room temperature, restore it to the original volume with pure water, and store it at room temperature away from light for later use. All glassware used needs to be soaked in a mixture of potassium permanganate and sulfuric acid overnight, and then washed and dried before use.

[0062] 5.2.2 Labeling of difenoconazole monoclonal antibody:

[0063] Dispense 1mL / bottle of nanogold solution and use 0.1 mol / L K 2 CO 3 Adjust the pH value of the colloidal gold solution. Add 5 μg of difenoconazole monoclonal antibody to colloidal gold solutions of different pH values ​​and react at room temperature for 5 minutes. Observe the color change of the solution and record the pH value that keeps the solution red. Add 10 μL of 10% bovine serum albumin solution for blocking, centrifuge at 12,000 rpm for ten minutes, and discard all the clear liquid.

[0064] 5.2.3 Preparation of micropores:

[0065] Add 1 mL of gold diluent containing 2% Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose to reconstitute the solution, dispense 12 μL / well into microwells, dry at 37 °C for 16 hours, and store for later use.

[0066] 5.3 Preparation of sample pad:

[0067] 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 PBS buffer containing 0.05% Tween 20, 1% sucrose, 0.5% PVP40, and 0.05% sodium azide.

[0068] 5.4 Assembly of colloidal gold immunochromatographic test card:

[0069] 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 the test paper card to obtain a fenpropimorph colloidal gold immunochromatographic test card.

[0070] Example 6 Determination of the sensitivity of the difenoconazole colloidal gold immunochromatographic test card

[0071] A series of different concentrations of difenoconazole standard solutions were prepared with 0.01M PBS buffer, and then 100 μL of different concentrations of difenoconazole standard solutions were added to the gold-labeled microwells, and the solutions were repeatedly blown to make them uniformly redissolved. After standing for 5 minutes, the solution in the gold-labeled microwells was transferred to the sample well of the difenoconazole colloidal gold immunochromatographic detection card of the present invention. After adding the sample, the timing was started, and the results could be observed after 5 to 8 minutes. After 8 minutes, it was judged to be invalid.

[0072] The results are judged by the attached Figure 5Specifically, 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 a negative test result (-); if the color of T line is significantly weaker than that of C line or T line does not show color, it indicates a positive test result (+); Invalid: No C line appears, indicating an incorrect operation process or the test strip has expired. The test was repeated for 3 groups to achieve rapid qualitative detection of the colloidal gold immunochromatographic test card of difenoconazole. The specific results are shown in Table 2 below.

[0073] Table 2 Determination results of difenoconazole standard solutions at different concentrations

[0074]

[0075] As shown in Table 2, the difenoconazole colloidal gold immunochromatographic detection card prepared by the present invention has a high sensitivity for difenoconazole detection, which can reach 0.5 μg / L.

[0076] Example 7 Stability Test of Difenoconazole Colloidal Gold Immunochromatographic Test Card

[0077] The colloidal gold qualitative immunochromatographic test card is stored at room temperature. In order to ensure the stability of the test card, the test card was subjected to an accelerated destructive test. It was placed at 45°C for 60 consecutive days, and the negative and color changes of the difenoconazole standard solution were tested on the 0th, 5th, 10th, 20th, 30th, 40th, 50th and 60th days, respectively. The experiment was repeated for 3 groups, and the results are shown in Table 3 below. ("+" represents positive, "-" represents negative)

[0078] Table 3 Stability results of difenoconazole colloidal gold immunochromatographic test card

[0079]

[0080] As shown in Table 3, after the colloidal gold immunochromatographic test card was sealed and stored at 45° C. for 60 days, the T / C color depth interpretation result of the test card did not change significantly, indicating that the colloidal gold immunochromatographic test card can be stably stored for at least 60 days at 45° C. Therefore, the difenoconazole colloidal gold immunochromatographic test card prepared by the present invention can be stably stored at room temperature for more than one year, which can fully meet the market requirements during storage and transportation.

[0081] Example 8 Detection of samples using difenoconazole colloidal gold immunochromatographic test card

[0082] Take 2 g of fruit and vegetable samples (4 g for tubers), cut leafy vegetables into pieces of about 1 cm square, take cross-section samples or epidermis of tubers, put them into a 50 mL centrifuge tube, add 4 mL of 0.01M PB buffer, shake vigorously for 2 minutes, let the centrifuge tube stand for 2 minutes, take 100 μL of supernatant and add it to 400 μL 0.1MPB buffer, shake and mix to obtain the test solution.

[0083] Take 100 μL of the test solution and add it to the sample well of the difenoconazole colloidal gold immunochromatographic detection card prepared in Example 5. Start timing after adding the sample. After 5 to 8 minutes, the time is set according to the attached Figure 5 The judgment criteria for observation results.

[0084] Visual interpretation: 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 sample is negative (-); if the color of T line is significantly weaker than that of C line or there is no color of T line, it indicates that the sample is positive (+); Invalid: No C line appears, indicating an incorrect operation process or the test strip has expired.

[0085] Minimum detection limit: 10 blank samples were subjected to gradient spike detection using the difenoconazole standard solution, with the spike increments being 0, 1.5, 5, 10, and 20 μg / kg, respectively. The results are shown in Table 4.

[0086] Table 4 Detection limits of spiked samples for fruit and vegetable samples

[0087]

[0088] As shown in Table 4, the difenoconazole colloidal gold immunochromatographic detection card prepared by the present invention has good repeatability in the detection results of 10 kinds of fruit and vegetable samples. When the difenoconazole content in the sample is lower than 5 μg / kg, all are negative; when it is higher than 5 μg / kg, all are positive. Therefore, the detection limit of the difenoconazole colloidal gold immunochromatographic detection card prepared by the present invention for the difenoconazole in the sample is 5 μg / kg.

[0089] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

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

2. The method for preparing the difenoconazole hapten according to claim 1, characterized in that: The following steps are involved: S1. Compound a and compound b undergo a substitution reaction to obtain compound c. The reaction formula of this step is shown in formula (II): , formula (II); S2. Compound c is hydrolyzed under alkaline conditions to obtain the difenoconazole hapten represented by formula (I). The reaction formula of this step is shown in formula (III): , formula (III).

3. A difenoconazole antigen, characterized in that The difenoconazole antigen is a conjugate of the difenoconazole hapten according to claim 1 and a carrier protein, and its structural formula is shown in formula (IV): , formula (IV), The carrier protein is any one of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

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

Images