A kit for detecting picoxystrobin, and its preparation method and application

Abstract

The invention discloses a kit for detecting picoxystrobin, a preparation method and application thereof. The picoxystrobin hapten prepared by the invention retains the characteristic structure of picoxystrobin to the greatest extent, significantly enhances the immunogenicity of the picoxystrobin hapten, and the obtained picoxystrobin monoclonal antibody has high sensitivity. Through ELISA detection technology, the minimum detection limit of picoxystrobin is 0.5 μg / L. The preparation method of the picoxystrobin hapten has simple reaction operation, easy control of reaction conditions, and high purity and yield of the prepared picoxystrobin hapten. The picoxystrobin fluorescent quantitative immunochromatographic test strip prepared by the invention has a detection sensitivity of up to 1 μg / L, high precision, can be stably stored at room temperature for more than one year, is conducive to storage and transportation, and can realize rapid quantitative detection of picoxystrobin.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochemistry, and in particular to a kit for detecting picoxystrobin, a preparation method thereof and an application thereof. Background Art

[0002] Picoxystrobin, chemical name is (E)-3-methoxy-2-{2-[6-(trifluoromethyl)-2-pyridyloxymethyl]phenyl} methyl acrylate, molecular formula: C 18 H 16 F3NO4, relative molecular weight: 367.32. Picoxystrobin is a methoxyacrylate fungicide, which was first developed by Syngenta in 2001. After DuPont acquired the product in 2006, it began to promote and apply it on a large scale around the world. Picoxystrobin was officially registered by the Ministry of Agriculture of China in 2012. This is a systemic fungicide, mainly used to prevent and control crop diseases such as leaf blight, leaf rust, glumes blight, brown spot, powdery mildew, web spot, white rot and downy mildew. Therefore, picoxystrobin is widely used and has been reported in agricultural products such as tobacco, wheat, rice, grapes, peppers, bananas, potatoes and cucumbers. At the same time, experts and scholars have also paid attention to the research on the residues of picoxystrobin. Experiments have shown that picoxystrobin has good stability and low mobility in the soil, but long-term and high-frequency application can easily lead to the accumulation of picoxystrobin in the soil. In 2017, the European Commission issued a decision to gradually abolish the registration and use of picoxystrobin, mainly due to concerns about the risks of teratogenicity and cytotoxicity of its metabolite IN-H8612, and its metabolite IN-QDY68 poses a high risk to aquatic organisms and mammals that feed on earthworms. Currently, China, Canada, the United States and the European Union have all established maximum residue limits (MRLs) for picoxystrobin in agricultural products. As a new fungicide, picoxystrobin has broad-spectrum fungicidal activity and the strongest systemic activity among strobilurin fungicides. It also has the advantages of good crop safety and low toxicity, and its application will become more and more extensive.

[0003] At present, the main detection methods for picoxystrobin are chromatographic detection methods such as LC, GC and GC-MS. These methods are expensive and require professional personnel to operate. Although they are highly accurate, the detection is cumbersome, the cost is high, and it is difficult to conduct on-site detection, which cannot meet the current market demand for simplicity and speed; although the enzyme-linked immunosorbent assay method has high sensitivity and high throughput, it also requires professional personnel to operate, and the detection time is long, which also cannot meet the needs of on-site rapid screening; the colloidal gold method has the advantages of high throughput, simple operation, short detection time and no need for professional technicians to operate, but because it is difficult to perform quantitative analysis, it cannot meet the needs of increasingly heavy emergencies and emergency monitoring work. Therefore, there is an urgent need for a detection method that is simple to operate, highly sensitive, high-throughput, rapid and quantitative. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a kit for detecting picoxystrobin and a preparation method and application thereof.

[0005] The first object of the present invention is to provide the use of the compound of formula II as a picoxystrobin hapten, the structural formula of formula II is as follows:

[0006]

[0007] The second object of the present invention is to provide the use of the picoxystrobin hapten shown in formula II in the preparation of picoxystrobin artificial antigens. The structural formula of formula II is as follows:

[0008]

[0009] The third object of the present invention is to provide an artificial picoxystrobin antigen.

[0010] The fourth object of the present invention is to provide the use of the picoxystrobin artificial antigen in the preparation of picoxystrobin artificial antibody.

[0011] The fifth object of the present invention is to provide a picoxystrobin monoclonal antibody.

[0012] The sixth object of the present invention is to provide a composition of an immunogen and a coating agent for enzyme-linked immunosorbent assay of picoxystrobin.

[0013] The seventh objective of the present invention is to provide the use of the picoxystrobin artificial antigen or the picoxystrobin monoclonal antibody in the preparation and detection of picoxystrobin products.

[0014] The eighth object of the present invention is to provide a kit for detecting picoxystrobin.

[0015] In order to achieve the above object, the present invention is implemented by the following scheme:

[0016] The compound of formula II is used as a picoxystrobin hapten, and the structural formula of formula II is as follows:

[0017]

[0018] The use of the picoxystrobin hapten shown in formula II in the preparation of picoxystrobin artificial antigen, the structural formula of formula II is as follows:

[0019]

[0020] A picoxystrobin artificial antigen is obtained by coupling the picoxystrobin hapten shown in formula II with a carrier protein, wherein the carrier protein is any one of hemocyanin, bovine serum albumin, and ovalbumin, and the structural formula of the picoxystrobin artificial antigen is shown in formula III:

[0021]

[0022]

[0023] The use of the picoxystrobin artificial antigen in the preparation of picoxystrobin artificial antibodies.

[0024] A picoxystrobin monoclonal antibody is prepared by taking the picoxystrobin artificial antigen as an immunogen.

[0025] Preferably, the picoxystrobin artificial antigen for preparing the picoxystrobin monoclonal antibody is obtained by coupling the picoxystrobin hapten with hemocyanin.

[0026] A composition of an immunogen and a coating source for enzyme-linked immunosorbent assay of picoxystrobin, wherein the immunogen and the coating source are artificial antigens; the carrier protein on the immunogen is hemocyanin, and the carrier protein on the coating source is bovine serum albumin.

[0027] Application of the picoxystrobin artificial antigen or the picoxystrobin monoclonal antibody in the preparation and detection of picoxystrobin products.

[0028] A kit for detecting picoxystrobin, comprising a picoxystrobin fluorescent quantitative immunochromatographic test strip and a detection solution, wherein the picoxystrobin fluorescent quantitative immunochromatographic test strip comprises a chromatography membrane, a sample pad, a water absorbent pad and a PVC plate; the detection area of ​​the chromatography membrane is coated with the picoxystrobin artificial antigen as a T line, and the quality control area of ​​the chromatography membrane is coated with goat anti-rabbit IgG as a C line; the detection solution contains an antibody labeled with fluorescent microspheres, and the antibody is the picoxystrobin monoclonal antibody.

[0029] Preferably, the coating concentration of the picoxystrobin artificial antigen is 0.2-0.8 mg / mL, the coating concentration of goat anti-rabbit IgG is 0.1-0.5 mg / mL, and the dilution multiple of the fluorescent microsphere-labeled antibody in the detection solution is 80-200 times.

[0030] Preferably, the treatment solution of the sample pad contains 0.01-0.05 M PBS buffer solution with a mass concentration of 0.48-0.52% BSA, 2.8-3.2% sucrose, and 0.05-0.10% Triton 100.

[0031] Preferably, the picoxystrobin artificial antigen coated on the detection area of ​​the chromatographic membrane is obtained by coupling picoxystrobin hapten with bovine serum albumin.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] Fluorescence quantitative immunochromatography technology is a rapid detection and quantitative method developed based on immunological principles and colloidal gold immunochromatography technology. This technology has the high sensitivity of ELISA method and the rapidity of colloidal gold method. The detection time is only 10 to 15 minutes, and the sensitivity is 3 to 5 times that of colloidal gold method. It is easy to operate, highly sensitive, high throughput and rapid.

[0034] (1) The picoxystrobin hapten prepared by the present invention retains the characteristic structure of picoxystrobin to the greatest extent, so that the immunogenicity of picoxystrobin hapten is significantly enhanced; the picoxystrobin hapten has a carboxyl group that can be coupled with a carrier protein, and the picoxystrobin artificial antigen obtained by coupling the picoxystrobin hapten with the carrier protein is used to immunize animals, which is more conducive to stimulating the immune response of animals and producing antibodies with stronger specificity and higher sensitivity.

[0035] (2) The method for preparing the picoxystrobin hapten disclosed in the present invention has readily available raw materials, simple reaction operation, and easily controllable reaction conditions. The purity and yield of the prepared picoxystrobin hapten are relatively high.

[0036] (3) The picoxystrobin artificial antigen of the present invention is used to immunize animals to obtain a picoxystrobin monoclonal antibody with high sensitivity. The minimum detection limit of picoxystrobin is 0.5 μg / L using ELISA detection technology.

[0037] (4) The picoxystrobin artificial antigen of the present invention is used in the fluorescent quantitative immunochromatographic technology to quickly and conveniently detect picoxystrobin. The picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in the present invention has a detection sensitivity of up to 1 μg / L for picoxystrobin. In addition, the test strip has high precision and can be stably stored at room temperature for more than one year, which is conducive to storage and transportation, and can realize rapid quantitative detection of picoxystrobin. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the synthetic route of picoxystrobin hapten.

[0039] Figure 2 This is the mass spectrum of picoxystrobin hapten.

[0040] Figure 3 These are the ultraviolet absorption spectra of picoxystrobin hapten, immunogen and carrier protein, wherein A is the ultraviolet spectrum of KLH, picoxystrobin hapten and picoxystrobin artificial antigen-KLH; and B is the ultraviolet spectrum of BSA, picoxystrobin hapten and picoxystrobin artificial antigen-BSA.

[0041] Figure 4 This is the standard curve of picoxystrobin ELISA detection results.

[0042] Figure 5 This is the standard curve for quantitative detection of picoxystrobin fluorescent quantitative immunochromatographic test strips.

[0043] Figure 6 These are the stability test results of the fluorescent quantitative immunochromatographic test strips, where A is the stability of the test strips at 4°C, and B is the stability of the test strips at 45°C.

[0044] Figure 7 The figure shows the effect of EDC dosage on picoxystrobin monoclonal antibody labeled with fluorescent microspheres, where A is the result of UV light detection at different EDC dosages, and B is the result of fluorescence instrument detection at different EDC dosages.

[0045] Figure 8 Figure 3 is the effect of activation time on picoxystrobin monoclonal antibody labeled with fluorescent microspheres, where A is the UV light detection result at different activation times, and B is the fluorescence instrument detection result at different activation times.

[0046] Fig. 9 These are the inhibition test results of the test strips at different antigen-antibody concentrations under ultraviolet light, where A is the test result of different antibody dilution multiples at an antigen concentration of 1 mg / mL; B is the test result of different antibody dilution multiples at an antigen concentration of 0.8 mg / mL; C is the test result of different antibody dilution multiples at an antigen concentration of 0.4 mg / mL; D is the test result of different antibody dilution multiples at an antigen concentration of 0.2 mg / mL; (-) indicates that the concentration of the picoxystrobin standard solution is 0 μg / L; (+) indicates that the concentration of the picoxystrobin standard solution is 20 μg / L.

[0047] Fig.10 These are the UV light test results for different sample pads, where (-) indicates the negative sample L, and (+) indicates the spiked sample concentration is 100 μg / kg. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] Example 1 Preparation method of picoxystrobin hapten

[0050] The synthetic route of picoxystrobin hapten is shown in Figure 1 , the specific preparation method is as follows:

[0051] 1. (1) Take 1.0 g (2.72 mmol) of picoxystrobin of the structural formula shown in Formula I, add 8 mL of methanol into a 50 mL round-bottom flask, stir at room temperature to dissolve, then add 4 mL of lithium hydroxide (1 mol / L) and react at room temperature (20°C to 30°C) for 72 h;

[0052] (2) After heating and evaporating the solvent in step 1, the obtained residue was directly added with 10 mL of pure water to dissolve, extracted with ethyl acetate to remove impurities twice, and the pH was adjusted to 3-5 with hydrochloric acid. A large amount of white precipitate was produced, which was filtered and dried to obtain 0.53 g of the product, which was the picoxystrobin hapten with the structural formula shown in Formula II.

[0053] 2. (1) Take 1.0 g (2.72 mmol) of picoxystrobin of the structural formula shown in Formula I, add 10 mL of methanol into a 50 mL round-bottom flask, stir and dissolve at 20°C to 30°C, then add 6 mL of lithium hydroxide (1 mol / L) and react at room temperature (20°C to 30°C) for 72 h;

[0054] (2) After heating and evaporating the solvent in step 1, the obtained residue was directly added with 10 mL of pure water to dissolve, extracted and removed with dichloromethane twice, and the pH was adjusted to 3-5 with hydrochloric acid. A large amount of white precipitate was produced, which was filtered and dried to obtain 0.53 g of the product, which was the picoxystrobin hapten with the structural formula shown in Formula II.

[0055] 3. (1) Take 1.0 g (2.72 mmol) of picoxystrobin of the structural formula shown in Formula I, add 10 mL of methanol into a 50 mL round-bottom flask, stir and dissolve at 20°C to 30°C, then add 4 mL of lithium hydroxide (1 mol / L) and react at room temperature (20°C to 30°C) for 48 h;

[0056] (2) After heating and evaporating the solvent in step 1, the obtained residue was directly added with 8 mL of pure water to dissolve, extracted and removed with dichloromethane twice, and the pH was adjusted to 3-5 with hydrochloric acid. A large amount of white precipitate was produced, which was filtered and dried to obtain 0.48 g of the product, which was the picoxystrobin hapten with the structural formula shown in Formula II.

[0057] The structural formulas of Formula I and Formula II are shown below:

[0058]

[0059] The mass spectrum of the obtained picoxystrobin hapten is as follows: Figure 2 As shown, judging from the mass spectrum results, there are relatively few impurity peaks. Currently, 0.53 g of hapten can be obtained from 1 g of raw material, with high purity and yield.

[0060] Example 2 Synthesis of picoxystrobin artificial antigen

[0061] 1. Synthesis of picoxystrobin artificial antigen with bovine serum albumin as carrier protein

[0062] (1) 5 mg of the picoxystrobin hapten in Example 1 was dissolved in 0.25 mL of dimethylformamide (DMF), and after sufficient stirring, 5 mg of carbodiimide hydrochloride (EDC) and 4 mg of N-hydroxysuccinimide (NHS) were added, and stirred at room temperature (20° C. to 30° C.) for 5 h to obtain the picoxystrobin hapten activated ester;

[0063] (2) Weigh 24 mg of bovine serum albumin (BSA) and fully dissolve it in 2.5 mL of 0.01 mol / L PBS solution to form a carrier protein solution. The picoxystrobin hapten activated ester prepared in step 1 is slowly added dropwise to the carrier protein solution under stirring, and stirred at room temperature (20° C. to 30° C.) for 16 to 24 hours;

[0064] (3) The solution obtained in step (2) was dialyzed with 0.01 mol / L PBS at room temperature (20°C-30°C) for 3 days, with the dialyzate being changed 3 times a day to remove unreacted small molecules, thereby obtaining an artificial picoxystrobin antigen with bovine serum albumin as the carrier protein, named picoxystrobin artificial antigen-BSA;

[0065] (4) Aliquot and store at 4°C until needed.

[0066] 2. Synthesis of picoxystrobin artificial antigen with hemocyanin as carrier protein

[0067] 5 mg of hemocyanin (KLH) was selected as the carrier protein, and the other preparation methods were the same as the synthesis method of picoxystrobin artificial antigen-BSA, and the picoxystrobin artificial antigen with hemocyanin as the carrier protein was obtained, which was named picoxystrobin artificial antigen-KLH.

[0068] 3. Identification of artificial antigen synthesis results

[0069] like Figure 3 As shown, Figure 3A shows that the hapten has a strong characteristic absorption at 260nm, and the artificial antigen-BSA coated with original picoxystrobin has a strong characteristic absorption peak at 273nm, which has a significant migration relative to the hapten absorption peak. Figure 3 B shows that the immunogen picoxystrobin artificial antigen-KLH has a broad absorption peak at around 265nm, which has an obvious migration relative to the hapten absorption peak. Therefore, it can be said that the coated original picoxystrobin artificial antigen-BSA and the immunogen picoxystrobin artificial antigen-KLH were successfully synthesized.

[0070] Example 3 Preparation of picoxystrobin monoclonal antibody

[0071] The picoxystrobin artificial antigen-KLH prepared in Example 2 was used as an immunogen, and after being emulsified 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. Seven days after the second booster immunization, 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 boost the immunization. After the antibody titer no longer increases, 100 μg of the whole antigen is used for subcutaneous booster immunization. After 3 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 strong 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 mouse peritoneal cavity to produce ascites containing antibodies. By purifying ascites with the caprylic acid-ammonium sulfate precipitation method, high-purity and high-specificity picoxystrobin monoclonal antibodies can be obtained.

[0072] Example 4 Application and effect evaluation of picoxystrobin artificial antigen in ELISA

[0073] 1. The picoxystrobin artificial antigen used in the ELISA test was the picoxystrobin artificial antigen-BSA prepared in Example 2.

[0074] Using carbonate buffer at pH 9.6 as coating diluent, picoxystrobin artificial antigen-BSA was diluted to 0.5 μg / mL, added to a polystyrene microplate at 100 μL / well, coated overnight at 4°C, spun dry, added 1% BSA at 250 μL / well, blocked in phosphate buffer at 37°C for 1 h, spun dry, and vacuum packed for storage after drying to obtain a microplate coated with picoxystrobin artificial antigen-BSA.

[0075] 2. Add 50 μL / well of picoxystrobin series standard solution to the microporous ELISA plate coated with picoxystrobin artificial antigen-BSA in step 1, and then add 50 μL / well of 0.2 μg / mL picoxystrobin monoclonal antibody solution prepared in Example 3, and react at 37°C for 0.5h; after drying, add 300 μL / well of washing solution, wash 3 times and pat dry; then add 100 μL / well of enzyme-labeled secondary antibody, react at 37°C for 0.5h; wash again 3 times and pat dry, add 50 μL / well of color developing solution A and color developing solution B respectively, and react at 37°C for 15min; add 2M sulfuric acid 50 μL / well to stop, measure the OD value of each well at a wavelength of 450nm with an ELISA instrument, and calculate the ratio B / B0 of the OD value of the negative control respectively. The negative control is set to a concentration of 0mg / L of the picoxystrobin standard solution, and the conditions of other steps remain unchanged. The washing solution is a PBS solution (pH 7.4, 0.02M) containing 0.05% Tween-20; the preparation method of the color developing solution A is as follows: 10 mg of 3,3',5,5'-tetramethylbenzidine (TMB) is taken, dissolved in 5 mL of anhydrous ethanol, and the volume is made up to 50 mL with double distilled water; the preparation method of the color developing solution B is as follows: 1.46 g of disodium hydrogen phosphate, 0.93 g of citric acid, 0.64 mL of urea hydrogen peroxide (0.75%), the volume is made up to 100 mL with double distilled water, and the pH is adjusted to 5.0-5.4.

[0076] The results are shown in Table 1.

[0077] Table 1 OD values ​​and B / B0 values ​​of different concentrations of picoxystrobin standards tested by icELISA

[0078]

[0079] As shown in Table 1, when the concentration of picoxystrobin is 0.5 μg / L, its B / B0 value is 73.19%, indicating that the OD value at this concentration is significantly different from the OD value of picoxystrobin at a concentration of 0 μg / L.

[0080] Based on the data in Table 1, the standard curve was drawn by using ELISA Calc software for logistic curve fitting ( Figure 4 ), and the linear equation was: y=(AD) / [1+(x / C)^B]+D, r2=0.99944, A=1.92316, B=0.72878, C=1.74497, D=0.06780, x represents the concentration of the analyte, y represents the OD value, and the calculated IC50 value is 1.94μg / L, which is linear in the range of 0.5μg / L to 40.5μg / L.

[0081] Example 5 Preparation method of picoxystrobin monoclonal antibody labeled with fluorescent microspheres

[0082] 1. Take 500 μL of fluorescent microsphere solution (solid content of fluorescent microspheres is 1%, i.e., containing 5 mg of fluorescent microspheres); add 5 mg of N-hydroxysuccinimide (NHS) and 5 mg of carbodiimide hydrochloride (EDC) respectively under stirring in an ice bath, control the temperature of the fluorescent microsphere solution to 4°C, and activate it at this temperature for 20 minutes to obtain an activated fluorescent microsphere solution.

[0083] 2. After activation in step 1, the pH of the activated fluorescent microsphere solution was adjusted to 9 with 0.1 M potassium carbonate solution, and the temperature of the fluorescent microsphere solution was controlled to 4°C; 0.4 mg of the picoxystrobin monoclonal antibody prepared in Example 3 was added, stirred evenly, and the ice bath was removed, and the mixture was allowed to rise to room temperature (20°C to 30°C) naturally, and stirred at room temperature (20°C to 30°C) for 4 hours to couple the fluorescent microspheres and the picoxystrobin monoclonal antibody.

[0084] 3. After the coupling of step 2 is completed, the reaction solution of the coupled fluorescent microspheres and picoxystrobin monoclonal antibody is centrifuged at 10000rpm for 10min, the supernatant is removed, 1mL of fluorescent buffer is added, and after ultrasonic resuspending, centrifugation and supernatant removal are performed. The operation is repeated 3 times, and after the last centrifugation, 0.5mL of fluorescent buffer is added to ultrasonically resuspend the fluorescent microspheres after removing the supernatant, and the fluorescent microspheres are stored in a refrigerator at 2-8°C for later use. The fluorescent microsphere-labeled picoxystrobin monoclonal antibody is obtained, and the fluorescent buffer used is 0.01M PBS buffer containing 0.5% PVP, 3% sucrose, 2% BSA, and 0.05% sodium azide by mass concentration.

[0085] Example 6 Kit for detecting picoxystrobin

[0086] 1. Preparation method

[0087] 1. Preparation method of picoxystrobin fluorescent quantitative immunochromatographic test strip

[0088] (1) Preparation of a chromatographic membrane coated with artificial antigen and goat anti-rabbit IgG:

[0089] A nitrocellulose membrane (NC membrane) was used as a chromatographic membrane, and the picoxystrobin artificial antigen-BSA prepared in Example 2 was diluted to 0.4 mg / mL with a coating buffer, and the goat anti-rabbit IgG was diluted to a concentration of 0.2 mg / mL with a coating buffer, wherein the coating buffer contained 2% sucrose in PBS (pH 7.4). The antigen and goat anti-rabbit IgG were sprayed onto the detection area and control area corresponding to the reaction membrane at a spraying rate of 1 μL / cm, respectively, with an interval of 4 mm between the detection area and the control area. The membrane was placed in a 45° C. oven for 24 h and placed in a constant temperature and humidity storage box for use. The coating buffer used was a 0.01 M PBS buffer containing 0.5% sucrose, 0.5% BSA, and 0.05% sodium azide at a mass concentration.

[0090] (2) Preparation of sample pad:

[0091] The cut blank sample pad was soaked in the sample pad treatment solution for 5 minutes, then taken out and dried at 37°C for 16 hours, and placed in a constant temperature and humidity storage box for use. The sample pad treatment solution used was 0.05M PBS buffer containing 0.05% Triton100, 3% sucrose, and 0.5% BSA, with a pH of 7.4.

[0092] (3) Assembly of fluorescent quantitative immunochromatographic test strips:

[0093] The chromatographic membrane prepared in step 1 is stacked in the middle of the PVC board backing, and the sample pad and absorbent pad prepared in step 2 are stacked at both ends respectively. The chromatographic membrane is connected with the absorbent pad and the sample pad, with the detection area close to the sample pad and the control area close to the absorbent pad to obtain a test paper board, which is cut into test strips, and the test strips are loaded into a test paper card to obtain a fluorescent quantitative immunochromatographic test strip.

[0094] 2. Preparation method of fluorescent microsphere-labeled picoxystrobin monoclonal antibody detection solution:

[0095] The fluorescent microsphere-labeled picoxystrobin monoclonal antibody prepared in step 1 is taken out and placed at room temperature (20°C to 30°C), and diluted 100 times with a fluorescent buffer to obtain a fluorescent microsphere-labeled picoxystrobin monoclonal antibody detection solution, the fluorescent buffer composition is the same as that in step 1, and then stored in a refrigerator at 2 to 8°C for later use.

[0096] 2. Composition of the kit for detecting picoxystrobin

[0097] The kit for detecting picoxystrobin is prepared by the preparation method of the kit for detecting picoxystrobin.

[0098] Example 7 Sensitivity of Picoxystrobin Fluorescence Quantitative Immunochromatographic Test Strip

[0099] 1. Experimental Methods

[0100] A series of standard solutions of picoxystrobin with different concentrations were prepared with PBS containing 0.01M phosphate buffer, which were 0, 1, 3, 9, 27 and 81 μg / L respectively. Then 50 μL of the fluorescent microsphere-labeled picoxystrobin monoclonal antibody detection solution prepared in Example 6 was taken, and mixed with 50 μL of the prepared series of standard solutions for 1 min to obtain the fluorescent sample test solution. 80 μL of the fluorescent sample test solution was added to the sample area of ​​the fluorescent quantitative immunochromatographic test strip prepared in Example 5, and after 15 min, the fluorescent signal value ratio of T / C was read with a fluorescent quantitative detector, and 3 groups of repetitions were set for the detection test. Then a four-parameter Logistic curve was established through the T / C values ​​corresponding to the concentrations of the series of standard products, and the 4 parameter values ​​obtained from the curve were entered into the calibration software of the fluorescent quantitative detector, so that the rapid quantitative detection of the fluorescent quantitative immunochromatographic test strip on the fluorescent immunochromatographic analyzer can be realized.

[0101] 2. Experimental Results

[0102] Table 2 T / C value and B / B0 value of different concentrations of picoxystrobin standard tested by test strips

[0103]

[0104] The determination results are shown in Table 2. The T / C values ​​of picoxystrobin standards of different concentrations were tested using picoxystrobin fluorescence quantitative test strips, and the ratio of the T / C value to the negative control, B / B0, was calculated. It can be seen from Table 2 that when the picoxystrobin content was 1 μg / L, its B / B0 value was 73.93%, indicating that at this concentration point, there was a significant difference in the detection result with the negative control. The detection sensitivity of the picoxystrobin fluorescence quantitative immunochromatographic test strip to picoxystrobin can be set to 1 μg / L.

[0105] According to the data in Table 2, the standard curve was drawn by using ELISA Calc software for logistic curve fitting ( Figure 5 ), the linear equation is: y = (AD) / [1 + (x / C) ^ B] + D, r 2 =0.99910, A=3.53680, B=0.76975, C=3.95840, D=0.25989, the x value represents the concentration of the analyte, and the y value represents the T / C value. The standard curve is input into the matching fluorescence quantitative immunoassay analyzer, and combined with the fluorescence quantitative immunochromatographic test strip, the rapid quantitative detection of picoxystrobin can be achieved.

[0106] Example 8 Actual sample addition recovery experiment

[0107] 1. Pretreatment method

[0108] (1) Vegetables and fruits: Select representative vegetable and fruit samples, cut them into 1 cm square pieces, weigh 2.0 g into a 15 mL centrifuge tube, add 4 mL of sample extract, shake upside down and shake for more than 50 times, and the supernatant is the test solution. The sample extract is PBS buffer at pH 7.2;

[0109] (2) Dairy products: Pipette 500 μL of milk sample into a 2 mL centrifuge tube, add 500 μL of sample extract, and mix well to obtain the test solution, wherein the sample extract is a PBS buffer solution at pH 7.2;

[0110] (3) Grains and cereals: After the grain sample is crushed and homogenized, 0.5 g of the grain sample is weighed into a 15 mL centrifuge tube, and 2.5 mL of sample extract (hexane: acetone = 1:2) is added. The sample is shaken for 1 min, and centrifuged at 4000 r / min for 2 min. 500 μL of the supernatant is transferred to a 2 mL centrifuge tube and blown dry with cold air (the drying temperature is within 45°C). After drying, 200 μL of the sample extract is taken for re-dissolution, and vortexed for 30 s to mix evenly. The re-dissolved solution is the test solution. The sample extract is a PBS buffer solution with a pH of 7.2.

[0111] 2. Recovery rate experiment

[0112] Samples verified as negative were selected, and recovery rate tests were carried out on three categories of samples: vegetables, fruits, and grains. The specific samples were: vegetables (cucumbers, baby cabbages, peppers, and bok choy), fruits (grapes, watermelons, strawberries, and pears), and grains (rice, wheat, corn, and soybeans). A certain concentration of picoxystrobin standard solution was prepared for sample spike-in. The spike-in concentrations of vegetable and fruit samples were 0μg / kg, 5μg / kg, 10μg / kg, and 20μg / kg, respectively; the spike-in concentrations of grain samples were 0μg / kg, 10μg / kg, 20μg / kg, and 50μg / kg, respectively.

[0113] After the sample is pre-treated according to the method in step 1, the test solution is obtained. Take a 1.5mL centrifuge tube, add 50μL of the detection solution prepared in Example 6 and 50μL of different sample test solutions and mix for 1 minute to obtain the fluorescent sample test solution. 80μL of fluorescent sample test solutions of different concentrations are respectively aspirated and dripped onto the fluorescent quantitative immunochromatographic test strip prepared in Example 5. After 15 minutes, the standard curve is entered using a fluorescent quantitative immunochromatographic analyzer (see Example 7) for testing, and the ratio of the fluorescent signal value of T / C is read and the results are recorded. Among them, the added recovery rates of vegetable samples and fruit samples are 80.2% to 120.1%; the added recovery rate of grain samples is 70.1% to 120.9%.

[0114] Example 9 Stability of Picoxystrobin Fluorescence Quantitative Immunochromatographic Test Strip

[0115] 1. Experimental Methods

[0116] In order to ensure the stability of the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5, an accelerated destructive test was performed on the test strip. The picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5 was placed continuously at 4°C and 45°C for 60 days, and the fluorescence signal value was detected on the 1st day, 2nd day, 4th day, 7th day, 14th day, 30th day and 60th day, respectively. The detection method is shown in Example 8, and the T value is the fluorescence signal value of the detection area, and the C value is the fluorescence signal value of the control area.

[0117] 2. Experimental Results

[0118] Depend on Figure 6 A and Figure 6 B It can be seen that under the temperature environment of 4°C and 45°C, the T-line fluorescence signal value, C-line fluorescence signal value and T / C value of the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5 did not change significantly after being placed for 60 days. In the accelerated stability test at 45°C, it can be stably stored for at least 60 days, indicating that the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5 can be stored at room temperature for more than one year.

[0119] Example 10 Precision of Fluorescence Quantitative Immunochromatographic Test Strips

[0120] 1. Experimental Methods

[0121] The precision of the method was represented by the intra-batch error and inter-batch error of the detection area, control area and T / C value of the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5, and the intra-batch error and inter-batch error were reflected by the intra-batch coefficient of variation and inter-batch coefficient of variation, respectively.

[0122] The data of the signal values ​​of the detection area and the control area and the T / C value of 10 picoxystrobin fluorescent quantitative immunochromatographic test strips prepared in Example 5 from the same batch were measured, and the data were subjected to a discrete degree analysis to obtain the intra-batch coefficient of variation.

[0123] The data of the signal values ​​of the detection area and the control area and the T / C value of 10 picoxystrobin fluorescent quantitative immunochromatographic test strips prepared in Example 5 from different batches were measured, and the data were subjected to a discrete degree analysis to obtain the inter-batch variation coefficient.

[0124] According to the coefficient of variation formula CV%=[standard deviation SD value / average value]×100%, the intra-batch coefficient of variation and the inter-batch coefficient of variation of the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5 were calculated respectively.

[0125] See Example 8 for the detection method.

[0126] 2. Experimental Results

[0127] As shown in Table 3, when the intra-batch coefficient of variation was determined, the C-line fluorescence signal value test mean was 3480.56, and the coefficient of variation CV value was 1.46%; the T-line fluorescence signal value test mean was 12092, and the coefficient of variation CV value was 2.67%; the T / C value test mean was 3.47, and the coefficient of variation CV value was 2.61%, indicating that the intra-batch coefficient of variation of the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5 was small and the precision was high.

[0128] As shown in Table 4, when the inter-batch coefficient of variation was determined, the C-line fluorescence signal value test mean was 3481.33, and the coefficient of variation CV value was 1.82%; the T-line fluorescence signal value test mean was 12070.89, and the coefficient of variation CV value was 2.56%; the T / C value test mean was 3.47, and the coefficient of variation CV value was 3.06%, indicating that the inter-batch coefficient of variation of the picoxystrobin fluorescent quantitative immunochromatographic test strip prepared in Example 5 was small and the precision was high.

[0129] Table 3 Intra-batch precision and coefficient of variation of fluorescent quantitative immunochromatographic test strips (n=10)

[0130]

[0131] Table 4 Inter-batch precision and coefficient of variation of fluorescent quantitative immunochromatographic test strips (n=10)

[0132]

[0133] Example 11 Effect of EDC dosage on labeling of picoxystrobin monoclonal antibody labeled with fluorescent microspheres

[0134] 1. Experimental Methods

[0135] 1 mg, 3 mg, 5 mg, 7 mg, 9 mg and 12 mg of EDC were added to the fluorescent microspheres respectively. The preparation methods of the other fluorescent microsphere-labeled picoxystrobin monoclonal antibodies were the same as in Example 5, and the detection method was as shown in Example 8.

[0136] 2. Experimental Results

[0137] The test results under ultraviolet light are as follows Figure 7 As shown in A, the fluorescence signal value is detected by fluorescence quantitative immunoassay analyzer. Figure 7 As shown in B, Figure 7 As shown in A, the fluorescence intensity will be weakened if the amount of EDC added is too low or too high. Figure 7 It can be seen from the curve B that when the amount of EDC is 4 mg to 10 mg, the fluorescence signal value exceeds 10000, and reaches the maximum value when the added amount of EDC is 5 mg. Therefore, the amount of EDC can be determined to be 5 mg.

[0138] Example 12 Effect of activation time on picoxystrobin monoclonal antibody labeled with fluorescent microspheres

[0139] 1. Experimental Methods

[0140] The activation time of the fluorescent microspheres was controlled to be 5 min, 10 min, 20 min, 30 min, 40 min, 60 min, 90 min and 120 min, respectively. The preparation methods of the picoxystrobin monoclonal antibodies labeled with other fluorescent microspheres were the same as in Example 5, and the detection method was as shown in Example 8.

[0141] 2. Experimental Results

[0142] The test results under ultraviolet light are as follows Figure 8 As shown in A, the fluorescence signal value is detected by fluorescence quantitative immunoassay analyzer. Figure 8 As shown in B, Figure 8 A and B show that when the activation time is 5min to 20min, the fluorescence signal value increases rapidly. When the activation time reaches between 20min and 120min, the fluorescence signal value is relatively stable. The signal values ​​detected by the fluorescence instrument are all above 11000. When the activation time reaches more than 20min, the activation of the fluorescent microspheres is in a saturated state. Therefore, the activation time can be determined to be 20min.

[0143] Example 13 Effect of the coating original concentration of the immunochromatographic test strip and the dilution multiple of the fluorescent microsphere-labeled picoxystrobin monoclonal antibody in the detection solution on the detection effect

[0144] 1. Experimental Methods

[0145] According to the method of Example 5, the coating source (the picoxystrobin artificial antigen-BSA prepared in Example 2) was diluted to 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL and 1 mg / mL 4 concentrations with antigen coating buffer to prepare picoxystrobin fluorescence quantitative immunochromatographic test strips. The picoxystrobin monoclonal antibody labeled with fluorescent microspheres prepared in Example 5 was diluted 50, 80, 100, 200 and 300 times with fluorescent buffer to prepare a picoxystrobin monoclonal antibody detection solution labeled with fluorescent microspheres. The concentration of the picoxystrobin standard solution was 20 μg / L, and the inhibition rate of the picoxystrobin standard solution under different antigen-antibody combination conditions was tested to determine the best antigen-antibody combination, and the detection was performed with a fluorescent quantitative immunoassay analyzer, and its T / C value was calculated. The detection method is shown in Example 8.

[0146] 2. Experimental Results

[0147] The test results under ultraviolet light are as follows Fig. 9As shown, A is the test result of different antibody dilution multiples when the antigen concentration is 1 mg / mL; B is the test result of different antibody dilution multiples when the antigen concentration is 0.8 mg / mL; C is the test result of different antibody dilution multiples when the antigen concentration is 0.4 mg / mL; D is the test result of different antibody dilution multiples when the antigen concentration is 0.2 mg / mL; (-) indicates that the concentration of the picoxystrobin standard solution is 0 μg / L; (+) indicates that the concentration of the picoxystrobin standard solution is 20 μg / L. The fluorescence intensity of the test strip is positively correlated with the coating concentration and the dilution multiple of the fluorescent microspheres. It is necessary to calculate the inhibition rate in combination with the instrument detection data for comprehensive screening. The inhibition test results are shown in Table 5.

[0148] When the coating concentration of the coating source is 0.4 mg / mL and the dilution multiple of the detection solution of picoxystrobin monoclonal antibody labeled with fluorescent microspheres is 100 times, the inhibition rate of the picoxystrobin standard solution with a concentration of 20 μg / L can reach 67.94%. Therefore, the coating concentration of the coating source is 0.4 mg / mL, and the optimal dilution multiple of the picoxystrobin monoclonal antibody labeled with fluorescent microspheres is 100 times.

[0149] Table 5 Inhibition test results of test strips at different antigen-antibody concentrations

[0150]

[0151] Example 14 Effect of sample pad treatment solution on detection effect

[0152] 1. Experimental Methods

[0153] Four sample pad treatment solution formulas were designed according to the different ionic strengths of the sample pads and the concentrations of the surfactants, as shown in Table 2. Sample pads were prepared according to the method of Example 5 to obtain picoxystrobin fluorescence quantitative immunochromatographic test strips. Tomatoes were selected as samples, picoxystrobin standard solution was added to a final sample concentration of 100 μg / kg, and the samples were tested using a fluorescence quantitative immunoassay analyzer. The detection method is shown in Example 8.

[0154] 2. Experimental Results

[0155] The results are as follows Fig.10 As shown in the figure, the fluorescence signals of the three negative samples of sample pads B, C and D are the strongest, and the fluorescence signals of the positive sample results of groups A and C are weak, so from the perspective of inhibition effect, group C is better. The fluorescence quantitative immunoassay was used for detection, and the inhibition rate was calculated according to the T / C value. The results are shown in Table 6.

[0156] The inhibitory effect of the sample pad in group C was the best, and it could be used as the best solution for the sample pad formulation, that is, the sample pad treatment solution was a PBS buffer (pH=7.4, 0.05M) containing 0.5% BSA, 3.0% sucrose, and 0.05% Triton 100.

[0157] Table 6 Inhibition rate test results of different sample pad formulas

[0158]

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The use of the compound of formula II as a picoxystrobin hapten, the structural formula of formula II is as follows:

2. The use of the picoxystrobin hapten shown in formula II in the preparation of picoxystrobin artificial antigens, the structural formula of formula II is as follows:

3. A picoxystrobin artificial antigen, characterized in that: It is obtained by coupling the picoxystrobin hapten shown in formula II with a carrier protein, the carrier protein is hemocyanin, and the structural formula of the picoxystrobin artificial antigen is shown in formula III:

4. Use of the picoxystrobin artificial antigen according to claim 3 in the preparation of picoxystrobin artificial antibodies.

5. A composition of an immunogen and a coating agent for enzyme-linked immunosorbent assay of picoxystrobin, characterized in that: The immunogen and coating source are the artificial antigens according to claim 3; the carrier protein on the immunogen is hemocyanin, and the carrier protein on the coating source is bovine serum albumin.

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