A metalaxyl hapten, artificial antigen and its application

Abstract

The present invention discloses a propamocarb hapten, an artificial antigen thereof and applications thereof. The propamocarb artificial antigen and monoclonal antibody prepared from the propamocarb hapten prepared in the present invention have strong specificity for ELISA detection, and the IC<subgt;50< / subgt> value is 21.29 μg / L. The present invention also provides an immunochromatographic test strip for detecting propamocarb, which is coated with the coating antigen of the propamocarb artificial antigen prepared in the present invention and a monoclonal antibody. The detection limit for the propamocarb standard solution is 25 μg / kg, and the detection sensitivity in fruit and vegetable samples can reach 100 μg / kg, realizing rapid and accurate qualitative detection of propamocarb, and having broad promotion and application value.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and specifically, to a propamocarb hapten, an artificial antigen and their applications. Background Art

[0002] Propamocarb is an aliphatic fungicide with local systemic action, which is highly effective against oomycete fungi. It can inhibit the formation of the pathogen cell membrane, reduce the formation of sporangia and the number of zoospores, thereby achieving the purpose of preventing and controlling diseases. It has excellent effects on downy mildew, phytophthora blight and damping-off of fruit trees, and is suitable for foliar spraying and soil treatment. Propamocarb also has the effect of promoting crop growth. The residue limits of propamocarb in China have been clearly defined on different samples, with the lowest being 0.2 mg / kg for cauliflower samples, and the residue amount on fruits and vegetables in the United States is 2 mg / kg. Studying a rapid detection method for propamocarb drugs is of great significance.

[0003] The detection methods of propamocarb mainly rely on instrumental detection, and there are few simple methods suitable for on-site detection, which brings great inconvenience to daily detection work. Immunoassay detection technology is a commonly used detection method, which is simple to operate and takes less time. However, its detection effect is determined by the performance of antigens and antibodies, and the key to antigens and antibodies lies in haptens. Therefore, to obtain antigens and antibodies with excellent performance, the structural design of haptens is particularly important. The propamocarb hapten disclosed in the prior art CN113759120A introduces a characteristic structure with an amide bond and a six-membered ring, which causes a large change in the electron cloud density of the entire propamocarb molecular structure, and is not conducive to the preparation of antigens and antibodies with high sensitivity and high specificity. There is a lack of a highly sensitive propamocarb immunoassay detection technology, which cannot meet the actual use needs of the existing market. Summary of the Invention

[0004] To solve the problem of the lack of a rapid, sensitive and simple immunoassay method in the prior art, the present invention provides a propamocarb hapten, an artificial antigen and their applications.

[0005] The first object of the present invention is to provide a propamocarb hapten.

[0006] The second object of the present invention is to provide a preparation method for the compound shown in the structural formula (I),

[0007]

[0008] The third object of the present invention is to provide the application of the compound shown in the structural formula (I) in the preparation of a propamocarb artificial antigen,

[0009]

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

[0011] The fifth object of the present invention is to provide a preparation method of the above-mentioned propamocarb artificial antigen.

[0012] The sixth object of the present invention is to provide the use of a compound represented by the structural formula (Ⅱ) in the preparation of a propamocarb antibody.

[0013]

[0014] The seventh object of the present invention is to provide a propamocarb artificial antigen combination.

[0015] The eighth object of the present invention is to provide the use of the above-mentioned propamocarb artificial antigen combination in the preparation of a kit for detecting propamocarb.

[0016] The ninth object of the present invention is to provide a kit for detecting propamocarb.

[0017] In order to achieve the above objects, the present invention is achieved by the following solutions:

[0018] The hapten prepared by the present invention completely retains the amide ester structural characteristic groups of propamocarb, and the designed coupling arm is a straight chain with active groups, so that the small molecule structure on the prepared artificial antigen completely retains the original structural characteristics, and the immunogenicity of the antigen is improved.

[0019] A propamocarb hapten, characterized in that its structural formula is as shown in formula (I).

[0020]

[0021] A preparation method of a compound represented by the structural formula (I), propamocarb and bromoacetic acid are fully reacted, and the obtained reactant is purified to obtain it.

[0022]

[0023] The structural formula of the propamocarb is:

[0024] The structural formula of the bromoacetic acid is:

[0025] Preferably, the molar ratio of the propamocarb to the bromoacetic acid is 1:(1-3).

[0026] More preferably, the molar ratio of the propamocarb to the bromoacetic acid is 1:2.

[0027] Preferably, the solvent of the reaction system of the propamocarb and the bromoacetic acid contains water.

[0028] Preferably, the purification method comprises: drying and washing.

[0029] More preferably, the drying method comprises evaporation under reduced pressure.

[0030] More preferably, the washing method comprises washing with ethyl acetate.

[0031] Further preferably, the washing method comprises slurry washing with ethyl acetate.

[0032] Specifically, the preparation method comprises the following steps:

[0033] 1.0 g of propamocarb (CAS: 24579-73-5, 5.31 mmol) was placed in a 50 mL round-bottom flask, and 10 mL of purified water and 1.48 g of bromoacetic acid (CAS: 79-08-3, 10.62 mmol) were added in sequence. The mixture was thoroughly mixed and reacted at 60°C for 6 to 8 h. After the reaction was completed, the mixture was evaporated to dryness under reduced pressure, and the precipitate was washed three times with 50 mL of ethyl acetate to obtain 0.94 g of propamocarb hapten.

[0034] A propamocarb artificial antigen is obtained by coupling the propamocarb hapten with a carrier protein, and its structural formula is shown in formula (II).

[0035]

[0036] Wherein, Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

[0037] The method for preparing the above-mentioned propamocarb artificial antigen is characterized in that the above-mentioned propamocarb hapten is coupled to a carrier protein through an active ester method.

[0038] Preferably, the active ester method comprises the following steps:

[0039] The above-mentioned propamocarb hapten, dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are fully reacted to obtain a hapten-activated ester; the carrier protein is dissolved in phosphate buffer to obtain a carrier protein solution; the hapten-activated ester and the carrier protein solution are fully reacted and then dialyzed to obtain the product.

[0040] Specifically, the active ester method comprises the following steps:

[0041] Take 10 mg of propamocarb hapten, dissolve it in 0.2 mL of dimethylformamide, and after sufficient stirring, add 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 5 mg of N-hydroxysuccinimide, and stir at room temperature for 4 h to obtain the activated ester of the hapten; take 40 mg of carrier protein and dissolve it fully in 4 mL of PBS solution with a concentration of 0.01 mol / L to form a carrier protein solution; slowly dropwise add the activated ester of the hapten to the carrier protein solution drop by drop, stir while adding, and stir at room temperature for 16 - 24 h; dialyze the obtained reaction solution with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecule substances, and thus obtain the propamocarb artificial antigen.

[0042] The application of the compound shown in formula (Ⅱ) in the preparation of propamocarb antibody should also be within the protection scope of the present invention.

[0043]

[0044] Among them, Protein is the carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

[0045] A propamocarb antibody is prepared by immunizing an animal with the above-mentioned propamocarb artificial antigen with hemocyanin as the carrier protein as the immunogen.

[0046] Preferably, the propamocarb antibody is a monoclonal antibody, which is obtained by immunizing an animal with the above-mentioned propamocarb artificial antigen with hemocyanin as the carrier protein as the immunogen to obtain hybridoma cells, culturing the obtained hybridoma cells, collecting the cells for animal immunization to obtain ascites, and identifying and purifying it.

[0047] A propamocarb artificial antigen combination includes an immunogen and a coating antigen. The immunogen is obtained by coupling the above-mentioned propamocarb hapten with hemocyanin, that is, the above-mentioned propamocarb artificial antigen with hemocyanin as the carrier protein; the coating antigen is obtained by coupling the above-mentioned propamocarb hapten with bovine serum albumin, that is, the above-mentioned propamocarb artificial antigen with bovine serum albumin as the carrier protein.

[0048] The application of the above-mentioned propamocarb artificial antigen combination in the preparation of a kit for detecting propamocarb should also be within the protection scope of the present invention.

[0049] An immunochromatographic test strip for detecting propamocarb, comprising a base plate, on which a gold pad, a sample pad, a coated reaction membrane and a water absorption pad are sequentially lapped; the gold pad is a glass fiber pad containing an antibody against propamocarb, and the antibody against propamocarb is prepared by immunizing an animal with an immunogen in the above propamocarb artificial antigen combination and is labeled with colloidal gold; 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 antigen in the propamocarb artificial antigen combination, and the quality control area is coated with IgG.

[0050] Preferably, the sample pad is soaked with a sample treatment solution, and the sample treatment solution is a 0.1M PB buffer solution containing 0.3%wt Tween 20, 1%wt sucrose, 0.5%wt BSA and 0.05%wt sodium azide.

[0051] Preferably, the IgG is murine IgG.

[0052] Preferably, the antibody against propamocarb is a monoclonal antibody, which is obtained by immunizing an animal with an immunogen in the above propamocarb artificial antigen combination to obtain hybridoma cells, culturing the obtained hybridoma cells, collecting the cells for animal immunization to obtain ascites, and identifying and purifying.

[0053] More preferably, the antibody against propamocarb is obtained by fully reacting the above monoclonal antibody with colloidal gold at a mass-to-volume ratio of (5 μg - 10 μg): 1 mL.

[0054] Further preferably, the antibody against propamocarb is obtained by fully reacting the above monoclonal antibody with colloidal gold at a mass-to-volume ratio of 8 μg: 1 mL.

[0055] Specifically, the preparation method of the immunochromatographic test strip is as follows:

[0056] Using the propamocarb artificial antigen with bovine serum albumin as the carrier protein as the coating antigen, diluting the concentration to 0.1 mg / mL - 0.5 mg / mL with a coating buffer solution (0.01M PBS buffer solution containing 1%wt sucrose and 0.05%wt sodium azide, pH = 7.6); adjusting the concentration of IgG to 0.1 mg / mL - 0.5 mg / mL with the coating buffer solution;

[0057] Using a nitrocellulose membrane as the reaction membrane, spraying the diluted coating antigen onto the detection area of the reaction membrane at a membrane liquid volume of 0.8 μL / cm - 1.2 μL / cm, spraying the diluted IgG onto the control area of the reaction membrane, with a 2.5 mm interval between the detection area and the control area, and placing it in an oven at 45 °C for 12 h - 16 h to obtain the coated reaction membrane;

[0058] Soak the cut 30 cm × 30 cm blank sample pad in the sample pad treatment solution (0.01 M PBT buffer containing 0.5% wt polyethylene glycol (PEG), 0.1% wt BSA, 0.1% wt Triton X-100, 1% wt sucrose, and 0.05% wt sodium azide). After soaking for 5 minutes, take it out and dry it at 37°C for 16 hours to obtain the sample pad.

[0059] Take 1 g of chloroauric acid, dissolve it with pure water and ultrasound, and make up the volume to 100 mL to obtain the chloroauric acid solution, which is stored in the dark at 4°C for later use. Take 1 mL of the chloroauric acid solution and add it to 100 mL of pure water. Heat it to boiling, add 0.5 mL of 0.06% wt sodium citrate solution, continue heating for 10 minutes, cool it to room temperature, and then add pure water to make up the volume to 100 mL to obtain the colloidal gold solution. Take 1 mL of the colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the added amount is the minimum amount when there is no color change in the subsequent labeling process), add 8 μg of the monoclonal antibody to the colloidal gold solution, react at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking. After mixing well, centrifuge at 12,000 rpm for 10 minutes and discard all the supernatant to obtain the antibody for detecting propamocarb. Re-dissolve the antibody for detecting propamocarb obtained in the previous step with 1 mL of gold diluent (aqueous solution containing 2% wt Tris, 5% wt bovine serum albumin, 0.05% wt thimerosal, and 5% wt sucrose), and evenly apply it on a glass fiber pad with an area of 5 cm × 5 cm. Dry it at 37°C for 16 hours to obtain the gold pad.

[0060] Paste the gold pad, sample pad, coated reaction membrane, and absorbent pad in the middle of the PVC board. The gold pad, sample pad, coated reaction membrane, and absorbent pad are overlapped in sequence, where the absorbent pad is adjacent to the control area of the coated reaction membrane, and the sample pad is adjacent to the detection area of the coated reaction membrane to obtain the test strip board. Cut the test strip board into test strips with a width of 3 mm, load the test strips into the test strip card, with the sample pad adjacent to the sample addition hole.

[0061] The application of any of the above immunochromatographic test strips in the preparation of a kit for detecting propamocarb should also be within the protection scope of the present invention.

[0062] A kit for detecting propamocarb, comprising the above propamocarb artificial antigen combination.

[0063] Preferably, the kit contains an immunochromatographic test strip, which includes a base plate. A gold pad, a sample pad, a coated reaction membrane, and a water absorption pad are sequentially overlapped on the base plate. The gold pad is a glass fiber pad containing an antibody against propamocarb. The antibody against propamocarb is prepared by immunizing an animal with the immunogen in the above-mentioned propamocarb artificial antigen combination and is labeled with colloidal gold. 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 antigen in the propamocarb artificial antigen combination, and the quality control area is coated with IgG.

[0064] Preferably, the sample pad is soaked in a sample treatment solution, which is a 0.1M PB buffer solution containing 0.3%wt Tween 20, 1%wt sucrose, 0.5%wt BSA, and 0.05%wt sodium azide.

[0065] Preferably, the IgG is murine IgG.

[0066] Preferably, the antibody against propamocarb is a monoclonal antibody. Hybridoma cells are obtained by immunizing an animal with the immunogen in the above-mentioned propamocarb artificial antigen combination. The obtained hybridoma cells are cultured, and the cells are collected for animal immunization to obtain ascites. After identification and purification, the monoclonal antibody is obtained.

[0067] More preferably, the antibody against propamocarb is obtained by fully reacting the above-mentioned monoclonal antibody with colloidal gold at a mass-to-volume ratio of (5 μg - 10 μg):1 mL.

[0068] Further preferably, the antibody against propamocarb is obtained by fully reacting the above-mentioned monoclonal antibody with colloidal gold at a mass-to-volume ratio of 8 μg:1 mL.

[0069] Specifically, the preparation method of the immunochromatographic test strip is as follows:

[0070] Using the propamocarb artificial antigen with bovine serum albumin as the carrier protein as the coating antigen, dilute the concentration to 0.1 mg / mL - 0.5 mg / mL with a coating buffer solution (a 0.01M PBS buffer solution containing 1%wt sucrose and 0.05%wt sodium azide, pH = 7.6); adjust the concentration of IgG to 0.1 mg / mL - 0.5 mg / mL with the coating buffer solution.

[0071] Using the nitrocellulose membrane as the reaction membrane, spray the diluted coating antigen onto the detection area of the reaction membrane at a membrane liquid volume of 0.8 μL / cm - 1.2 μL / cm, and spray the diluted IgG onto the control area of the reaction membrane. The distance between the detection area and the control area is 2.5 mm. Place it in an oven at 45°C for 12 h - 16 h to obtain the coated reaction membrane.

[0072] Soak the cut 30 cm × 30 cm blank sample pad in the sample pad treatment solution (0.01 M PBT buffer containing 0.5% wt polyethylene glycol (PEG), 0.1% wt BSA, 0.1% wt Triton X-100, 1% wt sucrose, and 0.05% wt sodium azide). After soaking for 5 minutes, take it out and dry it at 37°C for 16 hours to obtain the sample pad.

[0073] Take 1 g of chloroauric acid, dissolve it with pure water and ultrasonic waves, and then make up the volume to 100 mL to obtain the chloroauric acid solution, which is stored in the dark at 4°C for standby. Take 1 mL of the chloroauric acid solution into 100 mL of pure water, heat it to boiling, add 0.5 mL of 0.06% wt sodium citrate solution, continue to heat for 10 minutes, cool it to room temperature, and then make up the volume to 100 mL with pure water to obtain the colloidal gold solution. Take 1 mL of the colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the added amount is the minimum amount when there is no color change in the subsequent labeling process), add 8 μg of the monoclonal antibody to the colloidal gold solution, react at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking, mix well, centrifuge at 12,000 rpm for 10 minutes, and discard all the supernatant to obtain the antibody for detecting propamocarb. Redissolve the antibody for detecting propamocarb obtained in the previous step with 1 mL of gold dilution solution (aqueous solution containing 2% wt Tris, 5% wt bovine serum albumin, 0.05% wt thimerosal, and 5% wt sucrose), and evenly apply it on a glass fiber pad with an area of 5 cm × 5 cm, and dry it at 37°C for 16 hours to obtain the gold pad.

[0074] Paste the gold pad, sample pad, coated reaction membrane, and absorbent pad in the middle of the PVC board. The gold pad, sample pad, coated reaction membrane, and absorbent pad are overlapped in sequence, where the absorbent pad is adjacent to the control area of the coated reaction membrane, and the sample pad is adjacent to the detection area of the coated reaction membrane to obtain the test strip board. Cut the test strip board into test strips with a width of 3 mm, load the test strips into the test strip card, with the sample pad adjacent to the sample addition hole. [[ID=?]] [[ID=?]]

[0075] Preferably, the sample to be tested is a fruit and vegetable sample. The fruit and vegetable samples that can be detected by the present invention include but are not limited to leafy vegetables, root vegetables, and fruits.

[0076] Preferably, the crushed sample to be tested is fully mixed with the buffer solution, and solid-liquid separation is carried out, and the liquid is collected to obtain the test solution.

[0077] More preferably, the buffer solution is a phosphate buffer solution.

[0078] More preferably, the method of solid-liquid separation includes standing still.

[0079] Further preferably, the method of solid-liquid separation also includes centrifugation.

[0080] Compared with the prior art, the present invention has the following beneficial effects:

[0081] The artificial antigen and monoclonal antibody of propamocarb prepared from the propamocarb hapten prepared by the present invention are used for ELISA detection with strong specificity, and the IC 50 value is 21.29 μg / L. The present invention also provides an immunochromatographic test strip for detecting propamocarb, which is coated with the coating antigen of the propamocarb artificial antigen prepared by the present invention and a monoclonal antibody. The detection limit for the propamocarb standard solution is 25 μg / kg, and the detection sensitivity in fruit and vegetable samples can reach 100 μg / kg, realizing the rapid and accurate qualitative detection of propamocarb, and having broad popularization and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 is the synthesis route of the propamocarb hapten.

[0083] Figure 2 is the mass spectrometry result of the propamocarb hapten.

[0084] Figure 3 is the synthesis route of the propamocarb artificial antigen.

[0085] Figure 4 is the standard curve of the propamocarb standard solution. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0086] The present invention will be further described in detail below with reference to the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc., used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0087] Example 1 Synthesis and Identification of Propamocarb Hapten

[0088] 1. Synthesis of Propamocarb Hapten

[0089] The synthesis route of the propamocarb hapten is as Figure 1 shown, and the specific steps are as follows:

[0090] Take 1.0 g of compound a (i.e., propamocarb, CAS: 24579-73-5, 5.31 mmol) in a 50 mL round-bottom flask, and then sequentially add 10 mL of purified water and 1.48 g of compound b (i.e., bromoacetic acid, CAS: 79-08-3, 10.62 mmol), mix well, and react at 60 °C for 6-8 h. After the reaction is completed, evaporate to dryness under reduced pressure, and wash the precipitate with 50 mL of ethyl acetate by pulping 3 times to obtain 0.94 g of the propamocarb hapten.

[0091] 2. Identification of Propamocarb Hapten

[0092] The structural formula of the propamocarb hapten is shown in Formula (I):

[0093]

[0094] The mass spectrometry results of the propamocarb hapten are as Figure 2 shown, specifically: ESI-MS: 246 [M-1].

[0095] Example 2 Synthesis of the propamocarb artificial antigen

[0096] The structural formula of the propamocarb artificial antigen provided by the present invention is shown in Formula (II),

[0097]

[0098] where Protein is the carrier protein, and the carrier protein is bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).

[0099] The synthesis route of the above propamocarb artificial antigen is as Figure 3 shown. Taking the propamocarb artificial antigen with BSA as the carrier protein as an example, the specific preparation steps are as follows:

[0100] Take 10 mg of the propamocarb hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), stir well, add 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; weigh 40 mg of BSA and dissolve it fully in 4 mL of PBS solution with a concentration of 0.01 mol / L to form a carrier protein solution; slowly dropwise add the hapten activated ester to the carrier protein solution drop by drop, stir while adding, and stir at room temperature for 16 - 24 h; dialyze the obtained reaction solution with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecule substances, and obtain the propamocarb artificial antigen with BSA as the carrier protein, aliquot and store it at 4°C for standby.

[0101] According to the above steps, the only difference is using KLH instead of BSA, and the propamocarb artificial antigen with KLH as the carrier protein can be obtained.

[0102] Example 3 Preparation of the propamocarb monoclonal antibody

[0103] 1. Animal immunization

[0104] Using the propamocarb artificial antigen with hemocyanin as the carrier protein prepared in Example 2 as an immunogen, emulsify it with an equal volume of Freund's adjuvant and immunize BALB / C mice. The immunization dose for each mouse is 50 μg - 100 μg. The immunization interval is 2 weeks. After immunizing 3 times, collect the tail vein blood of the mice to detect the serum titer. According to the detection results, boost the immunization until the antibody titer no longer increases, and perform a subcutaneous boost immunization with 100 μg of the whole antigen.

[0105] 2. Preparation of hybridoma cells

[0106] Five days after the last boost immunization, take the spleen cells of the mice and fuse them with human osteosarcoma cells SP20 to obtain fused cells. Screen the fused cells with HAT medium. After 5 days of screening, replace the HAT medium with complete medium and continue to culture the fused cells. Detect the cell supernatant by ELISA, and clone and culture the cells in the wells with strongly positive detection results by the limiting dilution method. After 3 times of clone culture detection, the cells in the wells that are all positive are the hybridoma cells secreting monoclonal antibodies.

[0107] 3. Preparation of monoclonal antibodies

[0108] Amplify and culture the hybridoma cells, inoculate them into the abdominal cavity of mice to produce ascites containing antibodies. Purify the ascites by the caprylic acid-ammonium sulfate precipitation method to obtain a high-purity and high-specificity propamocarb monoclonal antibody.

[0109] Example 4 ELISA performance evaluation

[0110] 1. Experimental method

[0111] Use the ELISA method to evaluate the performance of the artificial antigen and monoclonal antibody prepared in the present invention, including the following steps:

[0112] (1) Antigen coating

[0113] Using the propamocarb artificial antigen with bovine serum albumin as the carrier protein prepared in Example 2 as the coating antigen, dilute the coating antigen with a coating dilution solution (carbonate buffer, pH 9.6) to obtain a coating antigen dilution solution with a concentration of 0.3 μg / mL. Add 100 μL / well to a polystyrene microplate and coat overnight at 4°C. Discard the liquid by centrifugation, wash three times with PBST to obtain a coated plate.

[0114] (2) Blocking

[0115] Add 280 μL / well to the blocking solution (phosphate buffer containing 1% wt BSA), block at 37°C for 1 h, discard the liquid by centrifugation, wash three times with PBST, and store in a vacuum package after drying.

[0116] (3) Primary antibody dilution

[0117] Using the propamocarb monoclonal antibody prepared in Example 3 as the primary antibody, dilute the primary antibody with an antibody diluent (phosphate buffer containing 0.05% wt sodium azide, pH 7.4) to obtain a primary antibody diluent with a concentration of 0.1 μg / mL, and store it at 4 °C for later use.

[0118] (4) Preparation of standard solutions

[0119] Dissolve the propamocarb standard in 0.01 M PBS to obtain propamocarb standard solutions with concentrations of 0 μg / L, 5 μg / L, 15 μg / L, 45 μg / L, 135 μg / L, and 405 μg / L respectively.

[0120] (5) Sample addition and primary antibody incubation

[0121] Add the propamocarb standard solution to the coated plate at 100 μL / well, and then add the 0.1 μg / mL primary antibody diluent correspondingly at 20 μL / well, react at 37 °C for 0.5 h, and drain.

[0122] (6) Wash the primary antibody

[0123] Add PBST at 280 μL / well, wash 3 times, and pat dry.

[0124] (7) Secondary antibody incubation

[0125] Add the HRP-labeled goat anti-mouse IgG secondary antibody at 100 μL / well and react at 37 °C for 0.5 h.

[0126] (8) Wash the secondary antibody

[0127] Add PBST at 280 μL / well, wash 3 times, and pat dry.

[0128] (9) Color development

[0129] Add the TMB color development solution at 100 μL / well and react at 37 °C for 15 min; add 1 M sulfuric acid at 50 μL / well to terminate the color development.

[0130] (10) Absorbance measurement

[0131] Put it into an enzyme-linked immunosorbent assay (ELISA) reader and measure the OD value of each well at a wavelength of 450 nm.

[0132] 2. Experimental results

[0133] Table 1 OD values of ELISA tests for propamocarb standard solutions at different concentrations

[0134]

[0135] The OD value measurement results of propamocarb standard solutions with different concentrations are shown in Table 1. The data shown in Table 1 was subjected to four-parameter Logistic curve fitting using ELISA Calc software, and the standard curve was plotted as shown in Figure 4 follows, and its linear equation is as follows:

[0136] y = (A - D) / [1 + (x / C)^B] + D, r 2 = 0.99941; where A = 2.40612, B = 0.88191,

[0137] C = 16.94755, D = 0.22014, x represents the concentration of the analyte, and y represents the OD value.

[0138] The IC 50 value was calculated to be 21.29 μg / L, showing a linear relationship in the range of 5 μg / L to 405 μg / L.

[0139] Example 5 A colloidal gold qualitative immunochromatographic kit for detecting propamocarb and its effect evaluation

[0140] I. Composition of the colloidal gold qualitative immunochromatographic kit

[0141] This kit contains an immunochromatographic test strip, and its preparation method is as follows:

[0142] 1. Preparation of the coated reaction membrane

[0143] Using the propamocarb artificial antigen with bovine serum albumin as the carrier protein prepared in Example 2 as the coating antigen, dilute the concentration to 0.1 mg / mL to 0.5 mg / mL with the coating buffer (0.01 M PBS buffer containing 1% wt sucrose and 0.05% wt sodium azide, pH = 7.6); adjust the concentration of mouse IgG to 0.1 mg / mL to 0.5 mg / mL with the coating buffer.

[0144] Using a nitrocellulose membrane (NC membrane) as the reaction membrane, spray the diluted coating antigen onto the test area (T line) of the reaction membrane at a membrane liquid volume of 0.8 μL / cm to 1.2 μL / cm, and spray the diluted mouse IgG onto the control area (C line) of the reaction membrane. The distance between the test area and the control area is 2.5 mm, and place it in an oven at 45°C for 12 h to 16 h to obtain the coated reaction membrane, and store it in a constant temperature and humidity storage box for later use.

[0145] 2. Preparation of the sample pad

[0146] Soak the cut 30 cm × 30 cm blank sample pad in the sample pad treatment solution (0.01 M PBT buffer containing 0.5% wt polyethylene glycol (PEG), 0.1% wt BSA, 0.1% wt Triton X-100, 1% wt sucrose, and 0.05% wt sodium azide). After soaking for 5 minutes, take it out and dry it at 37°C for 16 hours to obtain the sample pad, which is placed in a constant temperature and humidity storage box for later use.

[0147] 3. Preparation of the gold pad

[0148] (1) Preparation of the colloidal gold solution

[0149] Take 1 g of chloroauric acid, dissolve it with pure water and ultrasonic waves, and make up the volume to 100 mL to obtain the chloroauric acid solution, which is stored in the dark at 4°C for later use. Take 1 mL of the chloroauric acid solution into 100 mL of pure water, heat it to boiling, add 0.5 mL of 0.06% wt sodium citrate solution, continue to heat for 10 minutes, cool to room temperature, and add pure water to make up the volume to 100 mL to obtain the colloidal gold solution, which is placed in the dark at room temperature for later use. All glassware used needs to be soaked overnight in a mixed solution of potassium permanganate and sulfuric acid, and then washed and dried before use.

[0150] (2) Labeling of the propamocarb monoclonal antibody

[0151] Take 1 mL of the colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the added amount is the minimum amount when the color does not change in the subsequent labeling process), add 5 μg of the propamocarb monoclonal antibody prepared in Example 3 to the colloidal gold solution, react at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking. After mixing well, centrifuge at 12,000 rpm for 10 minutes, and discard all the supernatant. The obtained precipitate is the colloidal gold-labeled propamocarb monoclonal antibody.

[0152] (3) Preparation of the gold pad

[0153] Redissolve the colloidal gold-labeled propamocarb monoclonal antibody obtained in the previous step with 1 mL of gold diluent (aqueous solution containing 2% wt Tris, 5% wt bovine serum albumin, 0.05% wt thimerosal, and 5% wt sucrose), and evenly apply it on a 5 cm × 5 cm glass fiber pad. Dry it at 37°C for 16 hours to obtain the gold pad, which is stored for later use.

[0154] 4. Assembly

[0155] Paste the sample pad, gold pad, coated reaction membrane, and absorbent pad in the middle of the PVC board. The sample pad, gold pad, coated reaction membrane, and absorbent pad are overlapped in sequence. Among them, the absorbent pad is adjacent to the C line of the coated reaction membrane, and the gold pad is adjacent to the T line of the coated reaction membrane, thus obtaining the test strip board. Cut the test strip board into test strips with a width of 3 mm, and load the test strips into the test strip card with the sample pad adjacent to the sample adding hole, thus obtaining the colloidal gold qualitative immunochromatographic test strip card.

[0156] II. Usage Method

[0157] (1) Sample Treatment

[0158] Take 2 g of fruit and vegetable samples (cut leafy vegetables into fragments about 1 cm 3 in length, take cross-sectional samples or the epidermis of tuberous vegetables), put them into a 50 mL centrifuge tube, add 6 mL of 0.01 M PBS, shake vigorously and mix well for 2 min. After leaving the centrifuge tube to stand for 2 min, collect the supernatant, thus obtaining the test solution. If turbidity appears or there are too many impurities after adding PBS, it can be filtered or centrifuged at 4000 revolutions for 3 min and then collect the supernatant as the test solution.

[0159] (2) Detection

[0160] Lay the colloidal gold qualitative immunochromatographic test strip card flat, take 100 μL of the test solution and add it to the sample adding hole. The test solution passes through the sample pad, gold pad, T line of the reaction membrane, C line of the reaction membrane, and absorbent paper in sequence. Start timing after adding the sample, and observe the result after standing for 5 min - 8 min. Interpretation is invalid if it exceeds 8 min. Set 3 groups of replicates for the detection experiment.

[0161] (3) Result Interpretation

[0162] The interpretation method is as follows:

[0163] When the C line does not show color, it indicates that the detection result is invalid, the operation process is incorrect, or the test strip has expired; when the C line shows color, and the T line shows color stronger than the C line or there is no obvious difference in color between the T line and the C line, the detection result is negative (-), indicating that there is no propamocarb in the test sample; when the C line shows color, and the T line shows color significantly weaker than the C line or the T line does not show color, the detection result is positive (+), indicating that there is propamocarb in the test sample.

[0164] III. Performance Evaluation

[0165] 1. Sensitivity

[0166] Dilute the propamocarb standard product with 0.01 M PBS buffer solution to concentrations of 0 μg / L, 12.5 μg / L, 25 μg / L, 50 μg / L, and 100 μg / L, and use the colloidal gold qualitative immunochromatographic kit of this example for detection.

[0167] Table 2 Results of sensitivity determination

[0168]

[0169] As shown in Table 2, the colloidal gold qualitative immunochromatographic kit prepared by the present invention has high sensitivity for the detection of propamocarb, and the lower limit of detection is 25 μg / L.

[0170] 2. Specificity

[0171] The cross-reaction rates of other fungicides (carbendazim, cymoxanil, pyraclostrobin, imidacloprid, and procymidone) were determined using the propamocarb kit. The above-mentioned detection targets were prepared into a solution with a concentration of 2500 μg / L using 0.01M PBS, spotted according to the detection steps, and 3 test cards were repeated for each target. The results are shown in Table 3.

[0172] Table 3 Results of specificity determination

[0173]

[0174] As shown in Table 3, the detection results of the colloidal gold qualitative immunochromatographic kit prepared by the present invention for carbendazim, cymoxanil, pyraclostrobin, imidacloprid, and procymidone at a concentration of 2500 μg / L were all negative, and the cross-reaction rate was <1%.

[0175] 3. Stability

[0176] The storage condition of the colloidal gold qualitative immunochromatographic test strip in this example is room temperature. To determine the stability of the test strip, an accelerated destructive experiment was carried out on the test strip. It was continuously placed at 45°C for 60 days, and on the 0th day, 5th day, 10th day, 20th day, 30th day, 40th day, 50th day, and 60th day, the propamocarb standard was diluted to concentrations of 0 μg / L, 12.5 μg / L, 25 μg / L, and 50 μg / L with 0.01M PBS buffer, and the colloidal gold qualitative immunochromatographic kit prepared by the present invention was used for detection. The experiment was set up with 3 replicates.

[0177] Table 4 Results of stability determination

[0178]

[0179] As shown in Table 4, for the colloidal gold qualitative immunochromatographic test strip stored sealed at 45°C, after 60 days of storage, the color development depths of the T line and C line of the test strip showed no obvious change, indicating that the colloidal gold qualitative immunochromatographic test strip can be stably stored for at least 60 days at 45°C in the accelerated experiment. Accelerating for 37.5 days at 45°C is equivalent to storing at room temperature for one year. Therefore, the propamocarb colloidal gold qualitative immunochromatographic test strip prepared by the present invention can be stably stored at room temperature for more than one year, fully meeting the market's requirements during storage and transportation.

[0180] 4. Detection Limit of Spiked Fruits and Vegetables Samples

[0181] Using cauliflower, eggplant, Brussels sprouts, chicory, Chinese cabbage, grapes and plums as the samples to be tested, and according to the "sample treatment" method in this embodiment, the sample solutions of each sample to be tested are obtained. The sample solutions of each sample to be tested are subjected to gradient spiking detection with propamocarb standard, and the spiking gradients are 0 μg / kg, 50 μg / kg, 100 μg / kg and 200 μg / kg, and the colloidal gold qualitative immunochromatographic kit of this embodiment is used for detection.

[0182] Table 5 Determination Results of Detection Limit of Spiked Fruits and Vegetables Samples

[0183]

[0184] As shown in Table 5, the detection results of the colloidal gold qualitative immunochromatographic kit prepared by the present invention in 7 kinds of samples have good repeatability. When the content of propamocarb in the sample is lower than 100 μg / kg, they are all negative; when it is higher than 100 μg / kg, they are all positive. Therefore, the detection limit of the propamocarb colloidal gold immunochromatographic reagent card prepared by the present invention in fruit and vegetable samples is 100 μg / kg.

[0185] 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 those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

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

2. A method for preparing a compound with a structural formula as shown in formula (I), characterized in that, Propamocarb and bromoacetic acid react fully, and the obtained reactant is purified to obtain it; Formula (I).

3. Application of the compound with the structural formula shown in Formula (I) in the preparation of a propamocarb artificial antigen, Formula (I).

4. A propamocarb artificial antigen, characterized in that, It is obtained by conjugating the propamocarb hapten described in Claim 1 with a carrier protein, and its structural formula is shown in Formula (II), Formula (II), wherein Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

5. The preparation method of the propamocarb artificial antigen according to claim 4, characterized in that, Conjugate the propamocarb hapten described in Claim 1 with a carrier protein by the active ester method.

6. Application of the compound with the structural formula shown in Formula (II) in the preparation of a propamocarb antibody, Formula (II), Among them, Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

7. A combination of propamocarb artificial antigens, characterized in that, It includes a coating antigen and an immunogen. The coating antigen is obtained by conjugating the propamocarb hapten in Claim 1 with bovine serum albumin; the immunogen is obtained by conjugating the propamocarb hapten described in Claim 1 with hemocyanin.

8. Application of the propamocarb artificial antigen combination described in Claim 7 in the preparation of a kit for detecting propamocarb.

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