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

Abstract

The invention discloses a dimethomorph hapten, a dimethomorph artificial antigen and a preparation method and application thereof. The dimethomorph artificial antigen and the anti-dimethomorph monoclonal antibody prepared from the dimethomorph hapten provided by the invention have strong redissolution specificity when being used for ELISA (enzyme-linked immunosorbent assay) detection, and the IC50 value is 0.075 g / L. The invention also establishes a fluorescent quantitative immunochromatography technology of the dimethomorph, the semi-quantitative detection of the dimethomorph can be quickly and conveniently realized, and the sensitivity reaches 0.3 g / L.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, and in particular to a dimethomorph hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Dimethomorph is a highly effective oomycete fungicide with systemic, therapeutic and antisporulogenic properties. It can be used to control diseases caused by downy mildew and phytophthora pathogens on a variety of crops. Dimethomorph is widely used in vegetables and fruits, so its residues have a high detection rate. "GB 2763-2021 Maximum Residue Limits of Pesticides in Food" stipulates that the maximum residue limit (MRL) of dimethomorph in pineapple is 0.01 mg / kg, the MRL for potatoes and strawberries is 0.05 mg / kg, and the minimum maximum residue limit (MRL) in other fruits and vegetables is 0.1 mg / kg. In order to ensure the safety of plant-derived food, it is of great significance to regularly monitor the residues of dimethomorph.

[0003] Currently, the detection method for dimethomorph is mainly based on instrumental methods such as high-performance liquid chromatography. Due to the expensive equipment and instruments required, the detection time is long, and professional personnel are required to operate, it is impossible to truly achieve on-site detection and rapid clinical inspection, which brings great inconvenience to daily dimethomorph detection work.

[0004] The key to immunoassay detection technology lies in the performance of the antigen and antibody, and the key to these two is the hapten. Therefore, to obtain antigens and antibodies with excellent performance, the structural design of the hapten is particularly important. Artificial antigens directly prepared from dimethomorph in the prior art suffer from poor sensitivity and low cross-reactivity, failing to meet the actual use needs of the existing market. Therefore, the development of highly specific dimethomorph haptens or artificial antigens is of vital importance for rapid, highly sensitive, and low-cost detection methods for dimethomorph. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a dimethomorph hapten, an artificial antigen, and a preparation method and application thereof.

[0006] The first object of the present invention is to provide a dimethomorph hapten.

[0007] The second object of the present invention is to provide a method for preparing the dimethomorph hapten.

[0008] The third object of the present invention is to provide the use of a compound having a structural formula (I) in the preparation of an artificial dimethomorph antigen.

[0009] Formula (I).

[0010] The fourth object of the present invention is to provide an artificial dimethomorph antigen.

[0011] The fifth object of the present invention is to provide a method for preparing the dimethomorph artificial antigen.

[0012] The sixth object of the present invention is to provide the use of the dimethomorph artificial antigen in the preparation of dimethomorph antibodies.

[0013] The seventh object of the present invention is to provide a dimethomorph artificial antigen combination.

[0014] The eighth object of the present invention is to provide the use of the dimethomorph artificial antigen combination in the preparation of a product for detecting dimethomorph.

[0015] The ninth object of the present invention is to provide a kit for detecting dimethomorph.

[0016] The tenth object of the present invention is to provide a method for detecting dimethomorph for purposes other than disease treatment and diagnosis.

[0017] In order to achieve the above object, the present invention is implemented through the following scheme: The dimethomorph hapten prepared by the present invention completely retains the characteristic structures of o-dimethyl ether phenyl, acylmorpholine and chlorophenyl, and the introduced active group is located at the end of the chlorophenyl group with weak recognition effect in the region. The electron cloud density between the hapten and the original drug is almost the same, so that the prepared dimethomorph artificial antigen has an excellent spatial structure and the immunogenicity of the antigen is improved.

[0018] A dimethomorph hapten, the structural formula of which is shown in formula (I),

[0019] Formula (I).

[0020] The preparation method of the dimetholine hapten is to fully react (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone and 4-acetylmorpholine under alkaline conditions to obtain the dimetholine hapten. The structural formula of the dimetholine hapten is The structural formula of the 4-acetylmorpholine is .

[0021] Preferably, the molar ratio of (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone to 4-acetylmorpholine is 1:(0.5-1.5).

[0022] More preferably, the molar ratio of (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone to 4-acetylmorpholine is 1:1.

[0023] Preferably, the preparation method of the dimethoxymorpholine hapten comprises the following steps: fully reacting (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone, xylene, sodium amide and 4-acetylmorpholine at 130-135° C., cooling, extracting with ethyl acetate, collecting the resulting organic phase, drying, concentrating, and purifying by silica gel column chromatography to obtain the product.

[0024] More preferably, the molar ratio of (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone, 4-acetylmorpholine and sodium amide is 1:(0.5-1.5):(0.5-1.5).

[0025] More preferably, the molar ratio of (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone to 4-acetylmorpholine is 1:1:1.07.

[0026] More preferably, the volume ratio of xylene to ethyl acetate is 2:(2-4).

[0027] More preferably, the volume ratio of xylene to ethyl acetate is 2:3.

[0028] In some specific embodiments, the method for preparing the dimethomorph hapten comprises the following steps: 1.00 g of 3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone is placed in a 50 ml round-bottom flask, followed by the addition of 10 ml of xylene and 0.14 g of sodium amide. The temperature is raised to 130-135°C, and at this temperature, 10 ml of xylene containing 0.44 g of 4-acetylmorpholine is added dropwise. After the addition is complete, the mixture is reacted at 130-135°C for 3-5 hours. After the reaction is complete, the mixture is cooled to room temperature, extracted with 30 ml of ethyl acetate, and the organic phase is collected and washed twice with 30 ml of pure water. The organic phase is then dried and concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography to yield 0.42 g of the dimethomorph hapten.

[0029] The use of a compound represented by the structural formula (I) in the preparation of an artificial dimethomorph antigen,

[0030] Formula (I).

[0031] An artificial dimethomorph antigen, the structural formula of which is shown in formula (II),

[0032] Formula (II); The protein is a carrier protein, and the carrier protein is bovine serum albumin or lactoferrin.

[0033] The preparation method of the dimethomorph artificial antigen comprises the following steps: coupling the dimethomorph hapten with a carrier protein under acidic conditions, and then reducing the hapten with sodium borohydride to obtain the artificial antigen.

[0034] Preferably, the method for preparing the dimethomorph artificial antigen comprises the following steps: coupling the dimethomorph hapten with a carrier protein activated by an activator under acidic conditions, and dialyzing the resultant.

[0035] More preferably, the activating agent is glutaraldehyde.

[0036] In some specific embodiments, the carrier protein is bovine serum albumin, and the method for preparing the dimethomorph artificial antigen comprises the following steps: 40 mg of bovine serum albumin was fully dissolved in 4 mL of MES buffer at pH 6.0, and then 40 µL of an aqueous solution containing 50% glutaraldehyde was added, and the mixture was stirred in the dark at room temperature for 16 to 24 h. The solution obtained in the previous step was ultrafiltered using an ultrafiltration tube with a pore size of 6 kD, and ultrafiltration was performed 6 times, each time with 4 mL of MES buffer to remove unreacted small molecules to obtain the carrier protein activated product. 10 mg of dimethomorph hapten was fully dissolved in 0.1 mL of dimethylformamide and added dropwise to the carrier protein activated product obtained in the previous step under stirring, and stirred in the dark at room temperature for 16 to 24 h. 10 mg of sodium borohydride was added to 1 mL of ice water to dissolve to obtain a NaBH4 solution, 0.11 mL of the NaBH4 solution was added to the solution obtained in the previous step, and stirred at room temperature for 1 to 1.5 h. 0.01 The solution obtained in the previous step was dialyzed against 1.5 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules. The resulting dialyzate was the dimethomorph artificial antigen with bovine serum albumin as the carrier protein.

[0037] In some specific embodiments, the carrier protein is lactoferrin, and the method for preparing the dimethomorph artificial antigen comprises the following steps: 40 mg of lactoferrin was fully dissolved in 4 mL of MES buffer at pH 6.0, and then 40 µL of an aqueous solution containing 50% glutaraldehyde was added, and the mixture was stirred at room temperature in the dark for 16 to 24 h. The solution obtained in the previous step was ultrafiltered using an ultrafiltration tube with a pore size of 6 kD, and ultrafiltration was performed 6 times, each time with 4 mL of MES buffer to remove unreacted small molecules to obtain the carrier protein activated product. 5 mg of ethylenediamine hapten was fully dissolved in 0.1 mL of dimethylformamide and added dropwise to the carrier protein activated product obtained in the previous step under stirring, and stirred at room temperature in the dark for 16 to 24 h. 10 mg of sodium borohydride was weighed and added to 1 mL of ice water to dissolve to obtain a NaBH4 solution. 0.11 mL of the NaBH4 solution was added to the solution obtained in the previous step and stirred at room temperature for 1 to 1.5 h. 0.01 The solution obtained in the previous step was dialyzed against 10 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules. The resulting dialyzate was the dimethomorph artificial antigen with lactoferrin as the carrier protein.

[0038] The use of any of the dimethomorph artificial antigens in the preparation of dimethomorph antibodies should also be within the scope of protection of the present invention.

[0039] The invention discloses a dimethomorph antibody, which is prepared by immunizing an animal with a dimethomorph artificial antigen whose carrier protein is lactoferrin as an immunogen.

[0040] Preferably, the dimethomorph antibody is a monoclonal antibody, which is obtained by immunizing animals with an artificial dimethomorph antigen whose carrier protein is lactoferrin as an immunogen to obtain hybridoma cells, culturing the obtained hybridoma cells and collecting the cells for animal immunization to obtain ascites, which is then identified and purified.

[0041] A dimethomorph artificial antigen combination comprises a coating agent and an immunogen, wherein the coating agent is obtained by coupling the dimethomorph hapten to bovine serum albumin; and the immunogen is obtained by coupling the dimethomorph hapten to lactoferrin.

[0042] The use of the dimethomorph artificial antigen combination in the preparation of products for detecting dimethomorph should also be within the scope of protection of the present invention.

[0043] A kit for detecting dimethomorph, comprising the dimethomorph artificial antigen combination.

[0044] Preferably, it comprises a fluorescent quantitative immunochromatography reagent strip and a fluorescent microsphere-labeled antibody; the fluorescent quantitative immunochromatography reagent strip comprises a base plate, on which a sample pad, a coated reaction membrane and a water-absorbing pad are sequentially overlapped, the reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area, the detection area is coated with the coating source, and the quality control area is coated with IgG; the fluorescent microsphere-labeled antibody is an enoylmorpholine antibody labeled with fluorescent microspheres, and the enoylmorpholine antibody is prepared by immunizing an animal with the immunogen.

[0045] Preferably, the sample pad is soaked in a sample treatment solution, which is a 0.01 M phosphate buffer solution containing 0.3 wt % Tween 20, 1 wt % sucrose, 0.5 wt % BSA and 0.05 wt % sodium azide (NaN 3 ).

[0046] Preferably, the IgG is rabbit IgG.

[0047] In some specific embodiments, the method for preparing the coated reaction membrane comprises the following steps: The concentration of an ethylenediamine morpholino artificial antigen with bovine serum albumin as the carrier protein was adjusted to 0.1 mg / mL to 0.5 mg / mL using a coating buffer to obtain a detection coating original solution; the concentration of rabbit IgG was adjusted to 0.1 mg / mL to 1 mg / mL using a coating buffer to obtain a quality control coating original solution; the coating original solution was sprayed onto the detection area corresponding to the nitrocellulose membrane at a membrane liquid volume of 0.8 μL / cm to 1.2 μL / cm, and the quality control coating original solution was sprayed onto the control area corresponding to the reaction membrane, with a spacing of 5 mm between the detection area and the control area. The membrane was placed in an oven at 40°C to 45°C for 30 to 35 hours to obtain a coated reaction membrane; the coating buffer was 0.01 M phosphate buffer containing 0.5 wt% PEG20000, 1 wt% sucrose, 0.5 wt% bovine serum albumin, and 0.05 wt% sodium azide.

[0048] In some specific embodiments, the method for preparing the fluorescent microsphere-labeled antibody comprises the following steps: Take 500 μL of an aqueous solution containing 5 mg of fluorescent microspheres, treat with ultrasound, and evenly disperse; add 5 mg of N-hydroxysuccinimide and 4 mg of diethyl carbonate respectively while stirring, control the temperature of the fluorescent microsphere solution to 4-10°C, and activate at this temperature for 20 minutes; after activation, adjust the pH to 8-9 with 0.1 M potassium carbonate solution, and control the temperature of the fluorescent microsphere solution to 4-10°C; add 0.25 mg of the anti-enoylmorpholine monoclonal antibody prepared in Example 3, stir evenly, remove the ice bath, let it naturally warm to room temperature, and stir at room temperature for coupling for 4-6 hours; after coupling is complete, centrifuge and remove the supernatant.

[0049] A method for detecting dimethomorph for the purpose of non-disease treatment and diagnosis, wherein the dimethomorph artificial antigen combination is combined with fluorescent quantitative immunochromatography for detection.

[0050] Compared with the prior art, the present invention has the following beneficial effects: The dimethomorph artificial antigen prepared from the dimethomorph hapten provided by the present invention and the anti-dimethomorph monoclonal antibody are used for ELISA detection with strong reconstitution specificity, IC 50 The present invention also establishes a fluorescence quantitative immunochromatographic technique for dimethomorph, which can quickly and conveniently achieve semi-quantitative detection of dimethomorph with a sensitivity of 0.3µg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a synthetic route for the dimethomorph hapten.

[0052] Figure 2 This is the mass spectrum of dimethomorph hapten.

[0053] Figure 3 This is the synthetic route of dimethomorph artificial antigen.

[0054] Figure 4 This is the ELISA standard curve.

[0055] Figure 5 The standard curve of fluorescence quantitative immunochromatography. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0057] Example 1 Synthesis and Identification of Dimethomorph Hapten 1. Synthesis of dimethomorph hapten The synthetic route of dimethomorph hapten is as follows Figure 1 The specific steps are as follows: To a 50-ml round-bottom flask, 1.00 g of compound a ((3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone, 3.43 mmol, CAS: 1082821-61-1) was added, followed by 10 ml of xylene and 0.14 g of sodium amide (3.66 mmol, CAS: 7782-92-5). The temperature was raised to 130-135°C, and 10 ml of xylene containing 0.44 g of compound b (3.43 mmol, CAS: 1696-20-4) was added dropwise. After the addition, the mixture was allowed to react at 130-135°C for 3-5 hours. After the reaction was complete, the mixture was cooled to room temperature and extracted with 30 ml of ethyl acetate. The organic phase was collected and washed twice with 30 ml of pure water. The organic phase was then dried and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to yield 0.42 g of the enoylmorpholine hapten.

[0058] 2. Identification of dimethomorph haptens The structural formula of dimethomorph hapten is shown in formula (I):

[0059] Formula (I).

[0060] The mass spectrometry results of dimethomorph hapten are as follows Figure 2 As shown, specifically: ESI- [M+H] - :403.

[0061] Example 2 Dimethomorph Artificial Antigen and Its Synthesis Method 1. Structural formula of dimethomorph artificial antigen The structural formula of the dimethomorph artificial antigen provided by the present invention is shown in formula (II):

[0062] Formula (II), The protein is a carrier protein, and the carrier protein is bovine serum albumin (BSA) or lactoferrin (LF).

[0063] 2. Synthesis of dimethomorph artificial antigen The synthetic route of dimethomorph artificial antigens is as follows Figure 3 shown.

[0064] (1) The specific steps for synthesizing the dimethomorph artificial antigen with BSA as the carrier protein are as follows: 1) Weigh 40 mg of BSA and dissolve it in 4 mL of MES buffer (pH 6.0). Add 40 µL of 50% glutaraldehyde in water and stir at room temperature in the dark for 16–24 h. 2) Ultrafiltration of the solution obtained in the previous step was performed using an ultrafiltration tube with a pore size of 6 kD. Ultrafiltration was performed 6 times, and each time the solution was reconstituted with 4 mL of MES buffer to remove unreacted small molecules to obtain the activated carrier protein. 3) Take 10 mg of the dimethomorph hapten prepared in Example 1, fully dissolve it in 0.1 mL of dimethylformamide (DMF), and add it dropwise to the activated carrier protein obtained in the previous step under stirring. Stir at room temperature in the dark for 16 to 24 hours; 4) Weigh 10 mg of sodium borohydride (NaBH4) and dissolve it in 1 mL of ice water to obtain a NaBH4 solution. Add 0.11 mL of the NaBH4 solution to the solution obtained in the previous step and stir at room temperature for 1–1.5 h. 5) The solution obtained in the previous step was dialyzed against 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times per day to remove unreacted small molecules. The resulting dialyzate was the enoylmorpholine artificial antigen with BSA as the carrier protein. 6) Aliquot and store at 4°C until use.

[0065] (2) The specific steps for synthesizing the olefine morphine artificial antigen with LF as the carrier protein are as follows: 1) Weigh 40 mg of FL and dissolve it in 4 mL of MES buffer (pH 6.0). Then add 40 µL of 50% glutaraldehyde in water and stir at room temperature in the dark for 16-24 h. 2) Ultrafiltration of the solution obtained in the previous step was performed using an ultrafiltration tube with a pore size of 6 kD. Ultrafiltration was performed 6 times, and each time the solution was reconstituted with 4 mL of MES buffer to remove unreacted small molecules to obtain the activated carrier protein. 3) 5 mg of the dimethomorph hapten prepared in Example 1 was fully dissolved in 0.1 mL of dimethylformamide (DMF) and added dropwise to the activated carrier protein obtained in the previous step under stirring. The mixture was stirred in the dark at room temperature for 16 to 24 hours. 4) Weigh 10 mg of sodium borohydride (NaBH4) and dissolve it in 1 mL of ice water to obtain a NaBH4 solution. Add 0.11 mL of the NaBH4 solution to the solution obtained in the previous step and stir at room temperature for 1–1.5 h. 5) The solution obtained in the previous step was dialyzed against 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times per day to remove unreacted small molecules. The resulting dialyzate was the olefin(e)morphine artificial antigen with LF as the carrier protein. 6) Aliquot and store at 4°C until use.

[0066] Example 3 Preparation of anti-dimethomorph monoclonal antibodies 1. Animal immunization BALB / C mice were immunized with the artificial dimethomorph antigen prepared in Example 2, using LF as the carrier protein, emulsified with an equal volume of Freund's adjuvant. Each mouse received a dose of 50-100 μg, with a two-week interval between immunizations. After three immunizations, serum titers were measured by collecting tail vein blood. If the antibody titer did not meet the required level, a booster immunization was performed. After the antibody titer stopped increasing, a subcutaneous booster immunization with 100 μg of the whole antigen was performed.

[0067] 2. Hybridoma Cell Preparation Five days after the last booster immunization, mouse spleen cells were harvested and fused with SP20 cells. The fused cells were selected in HAT medium and cultured five days later with complete medium replaced with HAT medium.

[0068] The cell supernatant was tested by ELISA, and the cells in the wells with strongly positive test results were cloned and cultured by limiting dilution method. After three clone culture tests, the cells in the wells that were positive were hybridoma cells that secreted monoclonal antibodies.

[0069] 3. Monoclonal Antibody Preparation After the hybridoma cells are expanded and cultured, they are inoculated into the peritoneal cavity of mice to produce ascites containing the antibody. The ascites is purified using the caprylic acid-ammonium sulfate precipitation method to obtain highly pure and specific anti-enoylmorpholine monoclonal antibodies.

[0070] Example 4 Application and Effect Evaluation of Dimethomorph Artificial Antigen and Anti-Dimethomorph Monoclonal Antibody in ELISA 1. Experimental methods The performance of the dimethomorph artificial antigen and anti-dimethomorph monoclonal antibody prepared by the present invention was evaluated by ELISA method, which includes the following steps: (1) Antigen coating The dimethomorph artificial antigen prepared in Example 2 with BSA as carrier protein was diluted to 0.1 μg / mL using carbonate buffer at pH 9.6 as coating diluent. 100 μL / well was added to a polystyrene microplate. The plate was coated overnight at 4°C, dried, and washed three times with PBST.

[0071] (2) Closed Add phosphate buffer containing 1% wt BSA at 280 μL / well, block at 37°C for 1 h, spin dry, wash three times with PBST, dry and vacuum pack for storage.

[0072] (3) Primary antibody dilution The anti-enoylmorpholine monoclonal antibody prepared in Example 3 was diluted to 0.1 μg / mL using phosphate buffer (pH 7.4) containing 0.05% wt sodium azide to obtain a primary antibody dilution solution, which was stored at 4° C. for later use.

[0073] (4) Preparation of standard solution The dimethomorph standard was dissolved in 0.01 M PBS to obtain dimethomorph standard solutions with concentrations of 0 μg / L, 0.05 μg / L, 0.1 μg / L, 0.2 μg / L, 0.4 μg / L, and 0.8 μg / L, respectively.

[0074] (5) Sample addition and primary antibody incubation Add 100 μL / well of dimethomorph standard solution of various concentrations to the blocked microwell ELISA plate, then add 20 μL / well of primary antibody dilution solution accordingly, and react at 37°C for 0.5 h. After drying, add 280 μL / well PBST, wash three times, and pat dry.

[0075] (6) Secondary antibody incubation Add 100 μL / well HRP enzyme-labeled goat anti-mouse IgG enzyme-labeled secondary antibody and react at 37°C for 0.5 h; add 280 μL / well PBST, wash three times and pat dry.

[0076] (7) Color development and absorbance determination Add 50 μL / well of TMB Color Developing Buffer A and 50 μL / well of TMB Color Developing Buffer B, respectively, and incubate at 37°C for 15 min. Stop color development by adding 50 μL / well of 1 M sulfuric acid. Place the microplate in a microplate reader and measure the OD value of each well at 450 nm.

[0077] 2. Experimental results Table 1 ELISA determination of OD values ​​of dimethomorph standard solutions at different concentrations

[0078] The OD value determination results of dimethomorph standard solutions with different concentrations are shown in Table 1. On this basis, ELISA Calc software was used to perform four-parameter Logistic curve fitting, and the following was obtained: Figure 4 The linear equation of the standard curve shown is: y = (AD) / [1+(x / C)^B]+D, r 2 =0.99985, where A=1.05966, B=1.28143, C=0.10003, D=0.10886, x represents the concentration of the analyte, and y represents the OD value. IC 50 The value was 0.075 μg / L, and it was linear in the range of 0.05 μg / L to 0.8 μg / L.

[0079] Example 5 Dimethomorph Fluorescence Quantitative Immunochromatographic Kit and Its Performance Evaluation 1. Composition of the kit (1) Fluorescence quantitative immunochromatographic reagent strips The fluorescent quantitative and qualitative immunochromatographic reagent strips contained in this kit are prepared by the following method: Preparation of coating reaction membrane: The concentration of the dimethomorph artificial antigen with BSA as carrier protein prepared in Example 2 was adjusted to 0.1 mg / mL to 0.5 mg / mL using coating buffer (0.01 M phosphate buffer (PBS) containing 0.5 wt% PEG20000, 1 wt% sucrose, 0.5 wt% BSA and 0.05 wt% sodium azide (NaN3)) to obtain the original solution for detection coating; the concentration of rabbit IgG was adjusted to 0.1 mg / mL~1mg / mL to obtain the quality control coating original solution; according to the membrane liquid volume of 0.8μL / cm~1.2μL / cm, spray the coating original solution onto the detection area corresponding to the reaction membrane (nitrocellulose membrane (NC membrane)), and spray the quality control coating original solution onto the control area corresponding to the reaction membrane, with a spacing of 5mm between the detection area and the control area. Place in a 40℃~45℃ oven for 30~35 hours to obtain the coated reaction membrane, and place it in a constant temperature and humidity storage box for use.

[0080] Preparation of sample pad: Soak a cut 0.3 cm × 2 cm blank sample pad in sample treatment solution (0.01 M phosphate buffer (PBS) containing 0.3 wt% Tween 20, 1 wt% sucrose, 0.5 wt% BSA, and 0.05 wt% sodium azide (NaN3)) for 5 minutes. Then, take it out and dry it at 37°C for 16 hours to obtain the sample pad. Place it in a constant temperature and humidity storage box until used.

[0081] To assemble a fluorescent quantitative immunochromatographic test strip, attach the sample pad, coated reaction membrane, and absorbent pad to the center of a PVC backing, with the absorbent pad adjacent to the control area of ​​the coated reaction membrane and the sample pad adjacent to the detection area. This creates a paper test strip. The paper strip is then cut into strips and loaded onto a test card to create a fluorescent quantitative immunochromatographic test strip.

[0082] (2) Fluorescent microsphere-labeled antibody detection solution The fluorescent microsphere-labeled antibody detection solution contained in this kit is prepared by the following method: Preparation of fluorescent microsphere-labeled antibody solution: Take 500 μL of fluorescent microsphere solution (i.e., an aqueous solution containing 5 mg of FITC polystyrene fluorescent microspheres) and use ultrasonic treatment to evenly disperse the fluorescent microspheres in the solution; add 5 mg of N-hydroxysuccinimide (NHS) and 4 mg of diethyl carbonate (DEC) while stirring, control the temperature of the fluorescent microsphere solution to 4-10°C, and activate at this temperature for 20 minutes; after activation, adjust the pH to 8-9 with 0.1 M potassium carbonate solution, and control the temperature of the fluorescent microsphere solution to 4-10°C; add 0.25 mg of the anti-enoylmorpholine monoclonal antibody prepared in Example 3, stir evenly, remove the ice bath, let it naturally warm to room temperature, and stir at room temperature for 4-6 hours; after coupling, centrifuge the resulting reaction solution at 10,000 rpm for 10 minutes, remove the supernatant, and add 1 mL of fluorescent buffer (containing 0.5 wt% PEG20000, 2 wt% sucrose, 0.1 wt% Tween 20, 0.5 wt% BSA and 0.05wt% sodium azide (NaN3) in 0.01M phosphate buffer (PBS) were added to the suspension, and the fluorescent microspheres were ultrasonically resuspended. The suspension was centrifuged again and the supernatant was removed. This operation was repeated three times. After the final centrifugation and supernatant removal, 0.5mL of fluorescent buffer was added to ultrasonically resuspend the fluorescent microspheres to obtain the fluorescent microsphere-labeled antibody solution, which was stored in a refrigerator at 2-8°C for later use.

[0083] Preparation of fluorescent microsphere-labeled antibody detection solution: Take out the fluorescent microsphere-labeled antibody solution and let it cool to room temperature. Dilute it 100-1000 times with fluorescent buffer to obtain fluorescent microsphere-labeled antibody detection solutions of different dilution multiples. Store it in a refrigerator at 2-8°C until use.

[0084] (3) Dimethomorph standard The dimethomorph standard contained in this kit is dissolved in 0.01 M PBS to obtain the dimethomorph standard solution.

[0085] 2. How to use the kit Detection method: After mixing 20 μL of fluorescent microsphere-labeled antibody detection solution and 100 μL of each concentration of dimethomorph standard solution for 1 minute, 100 μL was added dropwise to the fluorescent quantitative immunochromatographic test strip. After 15 minutes, the fluorescence signal value of the detection area and the fluorescence signal value of the control area were read using a fluorescence quantitative detector at an excitation wavelength of 470 nm and an emission wavelength of 525 nm. The ratio of the fluorescence signal value of the detection area to the fluorescence signal value of the control area (T / C value) was calculated. According to the concentration of the dimethomorph standard solution and the A four-parameter logistic curve was established according to the T / C value. In the quantitative curve setting interface of the fluorescence immunochromatographic analyzer, the four parameter values ​​obtained from the curve were entered into the calibration software of the fluorescence quantitative detector. After mixing 20 μL of fluorescent microsphere-labeled antibody detection solution and 100 μL of the test solution for 1 minute, 100 μL was added dropwise to the fluorescence quantitative immunochromatographic test strip. After 15 minutes, the fluorescence signal values ​​of the detection area and the control area were read using a fluorescence quantitative detector at the same wavelength, and the T / C value was calculated.

[0086] Result calculation: Input the T / C value of the solution to be tested into the calibration software of the fluorescence quantitative detector to obtain the concentration of dimethomorph in the solution to be tested.

[0087] 3. Performance evaluation of the kit The dimethomorph standard was dissolved in 0.01 M PBS to obtain dimethomorph standard solutions with concentrations of 0 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L, and 24.3 μg / L, respectively.

[0088] The T / C values ​​of the dimethomorph standard solutions at various concentrations were measured and shown in Table 2. Figure 5 The four-parameter logistic curve shown has a linear equation: y = (A - D) / [1 + (x / C)^B] + D, r 2 =0.99933, where A=5.14908, B=1.09641, C= 0.65964, D= 0.67482, x represents the concentration of the analyte, and y represents the T / C value.

[0089] Table 2 Fluorescence quantitative detection results of dimethomorph standard solutions at different concentrations

[0090] We also calculated B / B0 (the ratio of the T / C values ​​of dimethomorph standard solutions at different concentrations to the T / C value of a dimethomorph standard solution at zero concentration). The results are shown in Table 2. As can be seen, when the dimethomorph concentration was 0.3 μg / L, the B / B0 value was 75.10%, indicating that the T / C value detected at this concentration was significantly different from that of the dimethomorph derivative test card containing 0 μg / L. Therefore, the sensitivity of the fluorescence quantitative test strip for dimethomorph reached 0.3 μg / L. This kit enables rapid quantitative detection of dimethomorph.

[0091] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

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

2. The method for preparing the dimethomorph hapten according to claim 1, wherein: (3-Amino-4-chloro-phenyl)-(3,4-dimethoxy-phenyl)-methanone and 4-acetylmorpholine are fully reacted under alkaline conditions to obtain the product.

3. Use of the compound represented by formula (I) in the preparation of dimethomorph artificial antigens, Formula (I).

4. An artificial dimethomorph antigen, characterized in that Its structural formula is shown in formula (II). Formula (II); The protein is a carrier protein, and the carrier protein is bovine serum albumin or lactoferrin.

5. The method for preparing the dimethomorph artificial antigen according to claim 4, characterized in that: The olaquindox metabolite hapten according to claim 1 is coupled with a carrier protein under acidic conditions and then reduced with sodium borohydride to obtain the product.

6. Use of the dimethomorph artificial antigen according to claim 4 in the preparation of dimethomorph antibodies.

7. A dimethomorph artificial antigen combination, characterized in that: The invention comprises a coating agent and an immunogen, wherein the coating agent is obtained by coupling the dimethomorph hapten according to claim 1 with bovine serum albumin; and the immunogen is obtained by coupling the dimethomorph hapten according to claim 1 with lactoferrin.

8. Use of the dimethomorph artificial antigen combination according to claim 7 in the preparation of a product for detecting dimethomorph.

9. A kit for detecting dimethomorph, characterized in that: Comprising the dimethomorph artificial antigen combination according to claim 7.

10. A method for detecting dimethomorph for purposes other than disease diagnosis and treatment, characterized in that: Detection is performed using the dimethomorph artificial antigen combination described in claim 7 in combination with fluorescent quantitative immunochromatography.

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