Fluopicolide hapten, fluopicolide artificial antigen and preparation method and application of fluopicolide hapten and fluopicolide artificial antigen

Abstract

The invention belongs to the field of fluopicolide detection, and relates to a fluopicolide hapten, a fluopicolide artificial antigen, and a preparation method and an application of the fluopicolide hapten and the fluopicolide artificial antigen. The structural formula of the hapten is as shown in formula (I). The fluopicolide structure is modified to obtain the fluopicolide hapten which is not reported in the prior art, and the fluopicolide hapten is coupled with carrier protein to prepare the antigen. By using the prepared antigen to immunize BALB / c mice, a monoclonal antibody with high sensitivity and strong specificity can be obtained, and the blank at home and abroad is filled. The fluopicolide antibody is prepared by using the conjugate of the hapten and the carrier protein provided by the invention, the titer of the antibody is increased quickly after immunization is started, the sensitivity is gradually improved, and finally the stability is achieved. The fluopicolide hapten and the fluopicolide antigen provided by the invention are simple and economic in preparation process and high in practical value, and have a good application prospect in pesticide residue detection.

Description

Technical Field

[0001] The present invention belongs to the field of fluopicolide detection, and specifically relates to a fluopicolide hapten, an artificial antigen, a preparation method thereof and an application thereof. Background Art

[0002] Fluopicolide, also known as fluazinam, has the chemical name of 2,6-dichloro-N-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methylbenzamide. Fluopicolide is a novel benzamide fungicide. The technical material appears as fine beige powder crystals and has no cross-resistance with other fungicides. However, its mechanism of action is different from all currently known fungicides for controlling oomycete diseases, mainly by acting on spectrin-like proteins between cell membranes and cytoskeletons. Since its registration was approved by the Ministry of Agriculture and Rural Affairs of the People's Republic of China in 2005, fluopicolide has been widely used in grapes and various vegetables due to its protective and broad-spectrum therapeutic effects, mainly for controlling common oomycete diseases such as downy mildew and phytophthora blight, and showing excellent control effects.

[0003] With the increase in the amount of drug used, the expansion of the application range and the need for repeated application. Once such drugs remain in various plant-derived foods such as fruits and vegetables, two problems will arise. On the one hand, the popularization and use of such compounds on crops will lead to the generation of drug resistance in pathogenic bacteria; on the other hand, the resulting new chemical pollution risk poses a new threat to the food safety of cross-border foods, thus causing many negative impacts on human health. In 2021, the "National Food Safety Standard Maximum Residue Limits of Pesticides in Foods" GB2763-2021 in China stipulates that the temporary MRLs in plant-derived foods are 0.05 mg / kg to 30 mg / kg. Except for the temporary MRLs in raw milk in animal-derived foods being 0.02 mg / kg, the rest are 0.01 mg / kg. Compared with foreign countries, China's MRLs for fluopicolide in animal-derived foods are more comprehensive and stricter.

[0004] Currently, instrumental analysis methods are the most commonly used methods for fluopicolide residue detection. However, this method has disadvantages such as high cost, high operation threshold, throughput bottleneck, and complex maintenance. In comparison, immunoassay methods, due to their simple development, low cost, simple operation, and high-throughput rapid screening, are the main technical supports for rapid detection methods. The performance of the core reagent antibody in immunoassay methods is largely determined by the structures of haptens and antigens. Therefore, it is particularly important to develop fluopicolide haptens and antigens that can prepare monoclonal antibodies with high sensitivity and high specificity. Summary of the Invention

[0005] The object of the present invention is to provide a fluopicolide hapten, a preparation method and application thereof. The antibody obtained after immunizing an animal with the antigen further prepared from the hapten has a high titer and strong specificity, and the subsequent established detection method can well meet the current needs of trace detection.

[0006] To achieve the above object, a first aspect of the present invention provides a fluopicolide hapten, and the structural formula of the hapten is shown in formula (I): Formula (I).

[0007] A second aspect of the present invention provides a preparation method of the fluopicolide hapten, comprising the following steps: (1) In the presence of a first solvent and a first catalyst, 2,6-dichloro-4-hydroxybenzoic acid is subjected to an acyl chlorination reaction with oxalyl chloride to obtain a first reaction product; (2) In the presence of a second solvent and a first base, the first reaction product is subjected to an amidation reaction with 2-(aminomethyl)-3-chloro-5-trifluoromethylpyridine hydrochloride to obtain a second reaction product; (3) In the presence of a third solvent and a second base, the second reaction product is subjected to a substitution reaction with tert-butyl bromomethylbenzoate. After the reaction is completed, filtration, concentration under reduced pressure and purification by column chromatography are carried out to obtain a third reaction product; (4) In the presence of a fourth solvent and an acid, the third reaction product is subjected to a hydrolysis reaction. After the reaction is completed, concentration under reduced pressure and purification by column chromatography are carried out to obtain the compound shown in formula (I).

[0008] A third aspect of the present invention provides a fluopicolide artificial antigen, and the artificial antigen is a conjugate obtained by conjugating the fluopicolide hapten with a carrier protein BSA, and its structural formula is shown in formula (II): Formula (II) Among them, on average, 15-20 compounds shown in formula (I) are conjugated to each carrier protein BSA.

[0009] A fourth aspect of the present invention provides a preparation method of the fluopicolide artificial antigen, comprising the following steps: The carrier protein BSA is conjugated to the carboxyl group of the compound shown in formula (I) by the active ester method to obtain the artificial antigen.

[0010] A fifth aspect of the present invention provides a fluopicolide antibody, and the fluopicolide antibody is obtained by immunizing an animal with the fluopicolide artificial antigen.

[0011] A sixth aspect of the present invention provides any one of the following uses of the fluopicolide hapten, the fluopicolide artificial antigen or the fluopicolide antibody: (1) Use in the preparation of a fluopicolide detection reagent; (2) Use in the preparation of fluopicolide immunochromatographic test strips; (3) Use in the detection of fluopicolide.

[0012] The present invention has the following beneficial effects: (1) The structure of fluopicolide was modified in the present invention to obtain a fluopicolide hapten not reported in the prior art, and the hapten was conjugated with a carrier protein to prepare an antigen. Immunizing BALB / c mice with the prepared antigen can obtain monoclonal antibodies with high sensitivity and strong specificity, filling the domestic and foreign gaps.

[0013] (2) Using the conjugate of the hapten and the carrier protein provided by the present invention to prepare fluopicolide antibodies, the antibody titer rises rapidly after the start of immunization, the sensitivity gradually increases, and finally reaches stability.

[0014] (3) The preparation process of the fluopicolide hapten and antigen provided by the present invention is simple, economical, and has high practical value, and has good application prospects in the detection of pesticide residues.

[0015] Other features and advantages of the present invention will be described in detail in the following specific implementation manner section. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] By describing the exemplary embodiments of the present invention in more detail in conjunction with the drawings, the above and other objects, features, and advantages of the present invention will become more apparent.

[0017] Figure 1 It is the mass spectrum of the fluopicolide hapten shown in formula (I).

[0018] Figure 2 It is the 1H NMR spectrum of the fluopicolide hapten shown in formula (I).

[0019] Figure 3 It is the matrix-assisted laser desorption / ionization time-of-flight mass spectrum of the antigen.

[0020] Figure 4 It is the standard curve of fluopicolide. SPECIFIC EMBODIMENTS

[0021] The following will describe the preferred embodiments of the present invention in more detail. Although the following describes the preferred embodiments of the present invention, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein.

[0022] To achieve the above object, a first aspect of the present invention provides a fluopicolide hapten, and the structural formula of the hapten is shown in formula (I): Formula (I).

[0023] The second aspect of the present invention provides a method for preparing the fluopicolide hapten as described above, comprising the following steps: (1) In the presence of a first solvent and a first catalyst, 2,6-dichloro-4-hydroxybenzoic acid is subjected to an acyl chlorination reaction with oxalyl chloride to obtain a first reaction product; (2) In the presence of a second solvent and a first base, the first reaction product is subjected to an amidation reaction with 2-(aminomethyl)-3-chloro-5-trifluoromethylpyridine hydrochloride to obtain a second reaction product; (3) In the presence of a third solvent and a second base, the second reaction product is subjected to a substitution reaction with tert-butyl bromomethylbenzoate. After the reaction is completed, filtration, concentration under reduced pressure, and purification by column chromatography are carried out to obtain a third reaction product; (4) In the presence of a fourth solvent and an acid, the third reaction product is subjected to a hydrolysis reaction. After the reaction is completed, concentration under reduced pressure and purification by column chromatography are carried out to obtain the compound shown in formula (I).

[0024] CN116854628A discloses a fluopicolide hapten, which mainly focuses on reaction selectivity, side reaction control, protecting group strategy, yield, etc. For example, when 2,6-dichloro-4-aminobenzoic acid reacts with Boc-anhydride, the amino group is the main reaction site, but Boc-anhydride may also react with other potential active sites (such as carboxyl groups) to cause side reactions, resulting in a decrease in product purity; the reaction of 2-(aminomethyl)-3-chloro-5-trifluoromethylpyridine with intermediate 1 may produce by-products. For example, the nitrogen atom on the pyridine ring may participate in the reaction, or the aminomethyl part may undergo self-condensation reaction. In addition, introducing a Boc protecting group in step (1) and removing the Boc protecting group in step (3) increases the reaction steps and complexity, and the removal of the Boc protecting group requires strong acidic conditions (85% phosphoric acid), which may affect other sensitive functional groups in the molecule.

[0025] Compared with CN116854628A, while retaining all the characteristic groups of fluopicolide, the hapten designed and synthesized in the present invention introduces a spacer arm with a benzene ring on the benzene ring of fluopicolide, which can increase the structural rigidity and is beneficial to stimulating animals to produce antibodies with high sensitivity and strong specificity. This new structure and preparation method have significant advantages in reaction design, step simplification, selectivity control, and yield. Only four reaction steps are required, and there is no need to introduce and remove protecting groups, which simplifies the reaction process, reduces the operation steps and time costs. The target compound is directly synthesized through acyl chlorination, amidation, substitution, and hydrolysis reactions without using protecting groups, which simplifies the reaction process and reduces the possibility of side reactions. Finally, a target hapten product with high yield and purity can be obtained and used for coupling a large number of different carrier proteins according to requirements.

[0026] According to a specific embodiment of the present invention, the reaction formula is as follows: .

[0027] According to the present invention, preferably, in step (1), the first solvent is dichloromethane; the first catalyst is N,N-dimethylformamide; based on 1 g of 2,6-dichloro-4-hydroxybenzoic acid, the dosage of the first solvent is 75 - 112.5 mL, the dosage of the first catalyst is 10 - 15 μL, and the dosage of oxalyl chloride is 5 - 7.5 mL; the conditions for the acylation reaction include: the reaction temperature is 20 - 25 °C, and the reaction time is 2 - 4 h.

[0028] According to the present invention, preferably, in step (2), the second solvent is dichloromethane; the first base is triethylamine and / or diisopropylethylamine (DIEA); the mass ratio of 2-(aminomethyl)-3-chloro-5-(trifluoromethyl)pyridine hydrochloride to the first reaction product is 1.0 - 1.3:1; the conditions for the amidation reaction include: the reaction temperature is -10 °C to 10 °C, and the reaction time is 2 - 4 h.

[0029] According to the present invention, preferably, in step (3), the third solvent is N,N-dimethylformamide; the second base is potassium carbonate; the mass ratio of the second product to tert-butyl bromomethylbenzoate is 1 - 1.2:1 - 1.1; the column chromatography eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 2 - 5:1 - 2; the conditions for the substitution reaction include: the reaction temperature is 70 - 90 °C, and the reaction time is 4 - 6 h.

[0030] According to the present invention, preferably, in step (4), the fourth solvent is dichloromethane; the acid is trifluoroacetic acid; based on 1 g of the third product, the dosage of the fourth solvent is 15 - 17 mL, and the dosage of the acid is 11 - 13 mL; the column chromatography eluent is a mixture of dichloromethane and methanol in a volume ratio of 40 - 60:1; the conditions for the hydrolysis reaction include: the reaction temperature is 0 - 40 °C, and the reaction time is 0.5 - 2 h.

[0031] The third aspect of the present invention provides a fluopicolide artificial antigen, which is a conjugate obtained by conjugating the fluopicolide hapten as described above with the carrier protein BSA, and its structural formula is shown as formula (Ⅱ): Formula (Ⅱ) Among them, on average, 15 - 20 compounds shown in formula (Ⅰ) are conjugated to each carrier protein BSA.

[0032] In the present invention, considering factors such as the success rate of antigen preparation, immunogenicity, and cost, a conjugate obtained by conjugating with the carrier protein BSA is finally selected.

[0033] The fourth aspect of the present invention provides a method for preparing the fluopicolide artificial antigen, comprising the following steps: coupling the carrier protein BSA to the carboxyl group of the compound shown in formula (I) by the active ester method to obtain the artificial antigen.

[0034] According to the present invention, preferably, it includes the following steps: (a) First, dissolve the compound shown in formula (I) in a solvent, then add EDC and NHS to react to obtain a liquid phase; (b) Dissolve the carrier protein BSA in a PBS buffer solution containing N,N-dimethylformamide to obtain a carrier protein solution; (c) React the liquid phase of step (a) with the carrier protein solution prepared in step (b) to obtain a conjugate solution; (d) Dialyze the conjugate solution obtained in step (c) to obtain the artificial antigen.

[0035] According to the present invention, preferably, in step (a), the solvent is N,N-dimethylformamide; the carboxyl activating agent is selected from at least one of a combination of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, a combination of isobutyl chloroformate and n-butylamine, and dicyclohexylcarbodiimide; the concentration of the compound shown in formula (I) is 20-30 mg / mL; the molar ratio of the carboxyl activating agent to the compound shown in formula (I) is 1-1.5:1; the reaction conditions include: temperature is 0-40 °C, and time is 1-5 h.

[0036] According to the present invention, preferably, in step (b), the volume content of DMF in the PBS buffer solution is 10-25%, and the mass-volume ratio of the carrier protein to the PBS buffer solution is 50-70 mg:8-10 mL.

[0037] According to the present invention, preferably, in step (c), the mass ratio of the compound shown in formula (I) to BSA in the protein solution is 2-3:4-7.

[0038] According to the present invention, preferably, in step (d), the dialysis is carried out in a 7-10 KDa dialysis bag, using a PBS buffer solution at 4-6 °C for dialysis for 48-72 h, and the PBS buffer solution is replaced every 12-14 h.

[0039] The fifth aspect of the present invention provides a fluopicolide antibody, which is obtained by immunizing an animal with the fluopicolide artificial antigen described above.

[0040] According to the present invention, preferably, the antibody is a monoclonal antibody.

[0041] The sixth aspect of the present invention provides any of the following uses of the fluopyram hapten, the fluopyram artificial antigen or the fluopyram antibody: (1) Use in the preparation of fluopyram detection reagents; (2) Use in the preparation of fluopyram immunochromatographic test paper; (3) Use in the detection of fluopyram.

[0042] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0043] Among them, FLC is the abbreviation of fluopyram. NHS is the abbreviation of N-hydroxysuccinimide. EDC is the abbreviation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. DMF is the abbreviation of N,N-dimethylformamide. Fluopyram was purchased from Beijing Bailingwei Technology Co., Ltd., and the product catalog number is F111293. Tert-butyl bromomethylbenzoate was purchased from Bid Pharmaceutical Technology Co., Ltd., and the product number is DRE-C13740000. N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were purchased from Sigma. Anhydrous sodium carbonate, petroleum ether, ethyl acetate, N,N-dimethylformamide, sodium hydroxide, hydrochloric acid, methanol, etc. were purchased from Sinopharm Group. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma, and the product catalog numbers were F-5881 and F-5506, respectively. Goat anti-mouse IgG enzyme-labeled antibody was purchased from Jackson Immunoresearch, product catalog number 115-035-003. The 96-well ELISA plate was purchased from Costar, product catalog number 2592.

[0044] Bovine serum albumin (BSA) was purchased from Sigma with the product catalog number 9048-46-18.

[0045] BALB / c mice were purchased from Sibeifu Biotechnology Co., Ltd. (Beijing).

[0046] Unless otherwise specified, the PBS buffer used in the examples is a PBS buffer of pH 7.4 and 0.01 M.

[0047] PBST solution: PBS buffer (pH 7.2) containing 0.05% (volume percentage) Tween-20. Example 1. Synthesis and identification of haptens

[0048] 1. Synthesis of haptens 1. Charge 400 mg of 2,6-dichloro-4-hydroxybenzoic acid and 30 mL of dichloromethane into a 50 mL round-bottom flask. After stirring evenly, add 4 μL of DMF, and then dropwise add 1700 μL of oxalyl chloride. Stir and react at 20 - 25 °C for 3 hours, then concentrate under reduced pressure. After adding 10 mL of dichloromethane to dissolve the residue, set it aside for use.

[0049] 2. Charge 494 mg of 2-(aminomethyl)-3-chloro-5-trifluoromethylpyridine hydrochloride and 22 mL of dichloromethane into a 50 mL round-bottom flask. After stirring evenly, add 834 μL of triethylamine, cool the temperature to 0 - 5 °C in an ice bath, and dropwise add the dichloromethane solution of 2,6-dichloro-4-hydroxybenzoyl chloride prepared in step 1. Under nitrogen protection, stir and react for 3 hours. After the reaction, concentrate under reduced pressure. Add 20 mL of water to the concentrated solution to precipitate a solid, filter and collect the solid, and dry it in a forced-air drying oven at 40 °C to obtain 694 mg of white solid powder.

[0050] 3. Take a 100 mL conical flask, add 694 mg of the product generated in step 2 and 10 mL of DMF, stir until dissolved, add 720.12 mg of K 2 CO 3 , charge 565 mg of tert-butyl bromomethylbenzoate, stir and react at 80 °C for 5 hours. After the reaction solution cools to 20 - 25 °C, filter to remove K 2 CO 3 . After concentrating the filtrate under reduced pressure, obtain 1.43 g of an oily substance, dissolve it with dichloromethane, add 2860 mg of silica gel with a mesh size of 100 - 200 for sample mixing, pack a column with silica gel of 200 - 300 mesh, elute with petroleum ether:ethyl acetate = 5:1, and collect the main product to obtain 495 mg of tert-butyl bromomethylbenzoate-substituted product.

[0051] 4. Take the product obtained in step 3, add 16 mL of dichloromethane to dissolve it, add 6 mL of trifluoroacetic acid, react at room temperature for 1 hour. After the reaction, concentrate under reduced pressure. After adding dichloromethane to dissolve it, add 1000 mg of silica gel with a mesh size of 100 - 200 for sample mixing, pack a column with silica gel of 200 - 300 mesh, elute with dichloromethane:methanol = 50:1, and collect the main product, which is the fluopyram hapten.

[0052] II. Identification of the Hapten Perform mass spectrometry and nuclear magnetic resonance identification on the obtained fluopyram hapten.

[0053] The mass spectrometry results are shown in Figure 1 , and it can be clearly seen that there is a peak at m / z 533 (M + H+), which is consistent with the target molecular weight (533.71). The 1H nuclear magnetic resonance spectrum is shown in Figure 2 , indicating that the synthesis of the fluopyram hapten is successful.

[0054] The results showed that the obtained fluopicolide hapten had the structure shown in the following formula (I).

[0055] Formula (I). Example 2. Preparation of antigen (active ester method)

[0056] The conjugate of the hapten and BSA was named FLC-BSA and used as the antigen, as shown in formula (II).

[0057] Formula (II) I. Preparation of immunogen 1. Weigh 24 mg of the fluopicolide hapten, dissolve it in 1 mL of DMF, then add 15 mg of EDC and 8 mg of NHS, place it on a magnetic stirrer, and react for 2 h at 400 rpm and room temperature to obtain a reaction product; 2. Take 50 mg of BSA and dissolve it in 10 mL of PBS buffer containing 10% (volume percentage) of DMF to obtain a protein solution; 3. Slowly add dropwise the liquid phase completed in step 1 to the protein solution prepared in step 2, then transfer it to a dialysis bag with a molecular weight cut-off of 7 KDa, and then place the dialysis bag in PBS buffer and dialyze at 4 °C for 72 h (change the solution every 12 - 14 h).

[0058] 4. After completing step 3, take the dialysis bag, take out the liquid phase therein, centrifuge at 5000 rpm for 3 min, collect the supernatant, which is the solution containing FLC-BSA, and name it FLC-BSA solution.

[0059] 5. Use matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to determine the binding ratio of BSA to the hapten in the FLC-BSA solution. The results are shown in Figure 3 .

[0060] Binding ratio = {M(conjugate) - M(protein)} / M(hapten) The molecular weight of BSA is 66131.86, and the molecular weight of the hapten is 533.71. By analyzing the highest peak mass spectrum, the molecular weight of the conjugate is 76093.25. After calculation, the binding ratio of BSA to the hapten is 18.7, that is, an average of 18.7 haptens of formula (I) are conjugated to one BSA molecule.

[0061] II. Preparation of coating antigen OVA was used instead of BSA to obtain the FLC-OVA solution. The feeding ratios of hapten, activator, and OVA and the reaction conditions involved in the preparation of the coating antigen were the same as those in the preparation of the immunogen, and the FLC-OVA solution was obtained. Example 3. Preparation of Monoclonal Antibodies

[0062] The addition amount of FLC-BSA, calculated based on the total protein amount, was determined by detecting the total protein concentration of the FLC-BSA solution using the Bradford method. The addition amount of FLC-BSA was the total protein concentration multiplied by the added volume.

[0063] I. Animal Immunization Preparation method of the primary immunization preparation: Take the FLC-BSA antigen solution prepared in Example 2, dilute it with PBS buffer, and then mix and emulsify it with an equal volume of Freund's complete adjuvant.

[0064] Preparation method of the booster immunization preparation: Take the FLC-BSA solution prepared in Example 2, dilute it with PBS buffer, and then mix and emulsify it with an equal volume of Freund's incomplete adjuvant.

[0065] Eight BALB / c mice were taken and immunized four times. The specific steps were as follows (the immunization method was intradermal immunization at 4-8 points on the back of the neck): The first immunization (primary immunization): Each mouse was immunized with 1 mL of the primary immunization preparation (each 1 mL of the primary immunization preparation contained 1 mg of FLC-BSA).

[0066] The second to fourth immunizations (booster immunizations): Starting from the first immunization, the booster immunizations were carried out once every 21 days for a total of 3 times; each time for the booster immunization, each mouse was immunized with 200 μL of the booster immunization preparation (each 200 μL of the booster immunization preparation contained 150 μg of FLC-BSA).

[0067] Starting from the second immunization, one week after the immunization was completed, blood was collected (from the orbital venous plexus, 0.30 mL per mouse), and the serum was collected by centrifugation.

[0068] II. Serum Detection The polyclonal antibody to be detected was the serum obtained in Step I (the sera 1 week after the second immunization, the third immunization, and the fourth immunization respectively).

[0069] Take a 96-well enzyme-linked immunosorbent assay (ELISA) plate and perform the following steps in sequence: 1. Coating Add 100 μL of coating solution to each well, incubate at 37 °C for 2 h, and then wash the plate once with PBST solution.

[0070] Coating solution: Dilute the FLC-OVA solution prepared in Example 2 with a carbonate buffer solution at pH 9.6 and 0.05 M so that the concentration of FLC-OVA is 0.18 μg / mL (based on the total protein concentration).

[0071] 2. Blocking Add 150 μL of blocking solution to each well, incubate at 37 °C for 1 h, then wash 3 times with PBST solution and pat dry.

[0072] Blocking solution: 0.4 g / 100 mL aqueous solution of skim milk.

[0073] 3. Add the antibody to be tested Test wells: Add 50 μL of PBS buffer and 50 μL of the dilution of the polyclonal antibody to be tested to each well; Control wells: Add 50 μL of PBS buffer and 50 μL of the serum before the first immunization to each well; Incubate at 37 °C for 30 min, then wash 4 times with PBST solution and pat dry.

[0074] Diluent: Starting from a dilution of 1:5000, with an initial concentration of 3.6 mg / mL, a total of 8 dilutions; PBS buffer is used as the solvent.

[0075] 4. Add the enzyme-labeled secondary antibody Add 100 μL of the enzyme-labeled secondary antibody dilution to each well, incubate at 37 °C for 30 min, then wash 4 times with PBST solution and pat dry.

[0076] Enzyme-labeled secondary antibody diluent: The goat anti-mouse IgG enzyme-labeled antibody is diluted to 5000 times its volume.

[0077] 5. Color development Add 100 μL of the color development solution to each well and incubate at 37 °C for 15 min.

[0078] Color development solution: Mix equal volumes of 2% 3,3’,5,5’-tetramethylbenzidine solution and 30% hydrogen peroxide.

[0079] 6. Termination Add 50 μL of 2 mol / L concentrated sulfuric acid to each well.

[0080] 7. Reading Measure the OD value of each well at a wavelength of OD 450 nm. The negative OD value should not be greater than 0.15, and the antibody titer is the antibody dilution corresponding to the maximum OD value between 1.5 - 2.0.

[0081] From the second immunization to the fourth immunization, the titer of fluopicolide polyclonal antibody showed a trend of gradually increasing, then tending to be stable or slightly decreasing. After the fourth immunization, the serum titer was 20,000.

[0082] III. Preparation and purification of monoclonal antibody (Protein A affinity column antibody purification method) According to the results of Step II, select mice with low serum inhibition rate (B / Bo), low IC 50 low, and high titer. After booster immunization, boost immunize on the 21st day, and perform cell fusion 4 days after boost immunization. After four to five rounds of subcloning and screening, finally obtain a monoclonal cell line that can secrete high-affinity antibodies. Select blank Balb / c mice over 10 weeks old and prepare a large amount of monoclonal antibody by the in vivo induction method. Collect ascites, separate the supernatant and store it in aliquots at -20 °C.

[0083] 1. Preparation: Filter all solutions with a 0.22 μm or 0.45 μm filter membrane before use. The average antibody purification yield of ascites is 4 mg / mL. Ensure that the amount of antibody to be purified is less than the total loading capacity of the affinity column.

[0084] 2. Sample filtration: Dilute the ascites or polyclonal antiserum from which the precipitate has been removed by centrifugation 4-5 times with the binding solution, filter and collect with a 0.22 μm filter, and label it as the sample.

[0085] 3. Column equilibration: Take out the Protein A column from the refrigerator and equilibrate it at room temperature for 30 min. Take 20 mL of the binding solution with a disposable syringe to equilibrate the column, and control the flow rate at 1 mL - 2 mL / min, that is, 20 drops - 40 drops / min.

[0086] 4. Loading: Pass the sample through the column with a disposable syringe, control the flow rate at 1 mL - 2 mL / min, collect the flow-through and record the volume.

[0087] 5. Washing: Pass 20 mL of the binding solution through the column to elute the miscellaneous proteins adsorbed on the column material, and discard the washings.

[0088] 6. Elution: Label multiple small centrifuge tubes with serial numbers, pre-add a specific volume of neutralizing solution to each tube, and collect a specific volume of eluate in each tube until the baseline is reached (OD280 value is less than 0.03). The volume of the neutralizing solution and eluate in each tube can be increased proportionally. Detect the concentration of the antibody in each tube with Nanodrop, and combine the collection tubes with an antibody concentration greater than 0.1 mg / mL.

[0089] 7. Replacement buffer: Replace the antibody buffer with the required solution (usually 10 mM PBS pH 7.2) by dialysis or ultrafiltration. Dialyze three times with an intact dialysis membrane (8 kMWCO), 2 L each time for more than 3 h. When the amount of antibody is less than 2 mg, ultrafiltration can be used to replace the buffer.

[0090] 8. Column packing cleaning: a) For alkali-resistant columns, they can be cleaned by passing 2 column volumes of equilibration buffer, 0.1 M NaOH, and equilibration buffer through the column in the forward or reverse direction successively.

[0091] b) To remove some precipitates or denatured substances, pass 2 column volumes of 6 M guanidine hydrochloride solution and 5 column volumes of equilibration buffer through the column in the forward or reverse direction successively.

[0092] c) To remove some non-specific adsorption substances caused by hydrophobic adsorption. Clean with 3 - 4 column volumes of 70% ethanol or 2 column volumes of 1% Triton™ X-100, and then immediately clean with 5 column volumes of equilibration buffer.

[0093] 9. Column storage: Pass 2 column volumes of 20% ethanol through the column, cover the upper and lower sealing caps, and store at 4°C.

[0094] IV. Dilution of the purified monoclonal antibody Use the purified monoclonal antibody prepared in Step 3 to replace the "test polyclonal antibody" in Step 2 and operate according to Step 2. Obtain the antibody dilution corresponding to the maximum OD value between 1.5 - 1.8.

[0095] Use PBS buffer as the solvent and dilute the purified monoclonal antibody prepared in Step 3 according to the obtained antibody dilution to obtain an antibody dilution solution.

[0096] V. Determination of the sensitivity of the monoclonal antibody 1. Coating Add 100 μL of coating solution to each well, incubate at 37°C for 2 h, and then wash the plate once with PBST solution.

[0097] 2. Blocking Add 150 μL of blocking solution to each well, incubate at 37°C for 1 h, and then wash 3 times with PBST solution and pat dry.

[0098] 3. Adding standard and antibody Add 50 μL of fluopicolide standard solution and 50 μL of the antibody dilution solution prepared in Step 4 to each well, incubate at 37°C for 30 min, and then wash 4 times with PBST solution and pat dry.

[0099] The solvent of the fluopicolide standard solution is PBS buffer solution, and the concentrations of the fluopicolide standard are 0, 0.011, 0.033, 0.1, 0.3, 0.9, 2.7, 8.1 ng / mL solutions, with three parallels for each concentration.

[0100] 4. Add enzyme-labeled secondary antibody Add 100 μL of enzyme-labeled secondary antibody dilution solution to each well, incubate at 37 °C for 30 min, then wash 4 times with PBST solution and pat dry.

[0101] 5. Color development Add 100 μL of color development solution to each well and incubate at 37 °C for 15 min.

[0102] 6. Termination Add 50 μL of 2 mol / L concentrated sulfuric acid to each well.

[0103] 7. Reading Measure the OD value of each well at a wavelength of OD 450 nm.

[0104] Using the value of -log10 (fluopicolide concentration) as the abscissa and the OD value (measuring OD 450 nm) as the ordinate, fit with the four-parameter equation of Origin 8.0 to establish a standard curve to obtain the IC 50 value.

[0105] The standard curve is shown in Figure 4 , and the IC 50 value of the purified monoclonal antibody is 0.11 ng / mL, and the linear range is 0.04 - 0.29 ng / mL. Comparative example

[0106] This comparative example is used to illustrate the detection results of the hapten prepared by CN116854628A.

[0107] The difference in the preparation of the antigen in this comparative example from that in Example 1 is that the hapten is prepared by the preparation method of CN116854628A, and the structure is as follows.

[0108]

[0109] To further prove the advantages of the antigen of the present invention, the coating antigen and immunogen of CN116854628A are used to prepare antibodies and detect sensitivity under the same conditions as in Example 3 of the present invention. Finally, the IC50 value of the obtained monoclonal antibody is 18.82 ng / mL, the linear range is 3.36 - 62.3 ng / mL, and the serum titer is 60000.

[0110] The IC50 value of the monoclonal antibody prepared from the novel hapten structure designed by the present invention is 0.11 ng / mL, the linear range is 0.04 - 0.29 ng / mL, and the serum titer is 64000. By comparison, it can be seen that the monoclonal antibody prepared from the novel hapten structure designed by the present invention has better affinity and detection performance.

[0111] The embodiments of the present invention have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A fluopyram hapten, characterized in that: The structural formula of the hapten is shown in formula (I):

2. The method for preparing the fluopyram hapten according to claim 1, characterized in that: The steps include: (1) in the presence of a first solvent and a first catalyst, subjecting 2,6-dichloro-4-hydroxybenzoic acid to an acyl chlorination reaction with oxalyl chloride to obtain a first reaction product; (2) subjecting the first reaction product to an amidation reaction with 2-(aminomethyl)-3-chloro-5-trifluoromethylpyridine hydrochloride in the presence of a second solvent and a first base to obtain a second reaction product; (3) in the presence of a third solvent and a second base, subjecting the second reaction product to a substitution reaction with tert-butyl bromomethylbenzoate, and filtering, concentrating under reduced pressure, and purifying by column chromatography after the reaction is completed to obtain a third reaction product; (4) In the presence of a fourth solvent and an acid, the third reaction product is subjected to a hydrolysis reaction, and after completion of the reaction, the product is concentrated under reduced pressure and purified by column chromatography to obtain a compound represented by formula (I).

3. The preparation method according to claim 2, wherein In step (1), the first solvent is dichloromethane; the first catalyst is N,N-dimethylformamide; based on 1 g of 2,6-dichloro-4-hydroxybenzoic acid, the amount of the first solvent is 75-112.5 mL, the amount of the first catalyst is 10-15 μL, and the amount of oxalyl chloride is 5-7.5 mL; the conditions of the chlorination reaction include: the reaction temperature is 20-25°C, and the reaction time is 2-4 hours; In step (2), the second solvent is dichloromethane; the first base is triethylamine and / or diisopropylethylamine; the mass ratio of the 2-(aminomethyl)-3-chloro-5-trifluoromethylpyridine hydrochloride to the first reaction product is 1.0-1.3:1; the conditions of the amidation reaction include: the reaction temperature is -10°C to 10°C, and the reaction time is 2-4h; In step (3), the third solvent is N,N-dimethylformamide; the second base is potassium carbonate; the mass ratio of the second product to tert-butyl bromomethylbenzoate is 1-1.2:1-1.1; the column chromatography eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 2-5:1-2; the conditions of the substitution reaction include: the reaction temperature is 70-90°C and the reaction time is 4-6h. In step (4), the fourth solvent is dichloromethane; the acid is trifluoroacetic acid; based on 1g of the third product, the amount of the fourth solvent is 15-17mL, and the amount of the acid is 11-13mL; the column chromatography eluent is a mixture of dichloromethane and methanol in a volume ratio of 40-60:1; the conditions of the hydrolysis reaction include: the reaction temperature is 0-40°C and the reaction time is 0.5-2h.

4. An artificial fluopyram antigen, characterized in that: The artificial antigen is a conjugate obtained by coupling the fluopyram hapten described in claim 1 with the carrier protein BSA, and its structural formula is shown in formula (II): Formula (II) Among them, an average of 15 to 20 compounds represented by formula (I) are coupled to each carrier protein BSA.

5. The method for preparing the fluopyram artificial antigen according to claim 4, characterized in that: The method comprises the following steps: using the active ester method to couple the carrier protein BSA to the carboxyl group of the compound shown in formula (I) to obtain the artificial antigen.

6. The preparation method according to claim 5, wherein: The following steps are involved: (a) first dissolving the compound represented by formula (I) in a solvent, then adding EDC and NHS to react to obtain a liquid phase; (b) dissolving the carrier protein BSA in a PBS buffer containing N,N-dimethylformamide to obtain a carrier protein solution; (c) reacting the liquid phase of step (a) with the carrier protein solution prepared in step (b) to obtain a conjugate solution; (d) dialyzing the conjugate solution obtained in step (c) to obtain the artificial antigen.

7. The preparation method according to claim 5, wherein: In step (a), the solvent is N,N-dimethylformamide; the carboxyl activator is selected from at least one of a composition of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, a composition of isobutyl chloroformate and mono-n-butylamine, and dicyclohexylcarbodiimide; the concentration of the compound represented by formula (I) is 20-30 mg / mL; the molar ratio of the carboxyl activator to the compound represented by formula (I) is 1-1.5:1; the reaction conditions include: a temperature of 0-40°C and a reaction time of 1-5 hours; In step (b), the volume content of DMF in the PBS buffer is 10-25%, and the mass volume ratio of the carrier protein to the PBS buffer is 50-70 mg:8-10 mL; In step (c), the liquid phase of step (a) is added dropwise to the carrier protein solution prepared in step (b), wherein the mass ratio of the compound represented by formula (I) to BSA in the protein solution is 2-3:4-7; In step (d), the dialysis is performed in a 7-10 KDa dialysis bag using a 4-6° C. PBS buffer for 48-72 h, and the PBS buffer is replaced every 12-14 h.

8. A fluopyram antibody, characterized in that: The fluopyram antibody is obtained by immunizing an animal with the fluopyram artificial antigen according to claim 7.

9. The fluopyram antibody according to claim 8, wherein The antibody is a monoclonal antibody.

10. Any of the following uses of the fluopyram hapten according to claim 1, the fluopyram artificial antigen according to claim 4, or the fluopyram antibody according to claim 8 or 9: (1) Use in the preparation of fluopyram detection reagents; (2) Use in the preparation of fluopyram immunochromatographic test paper; (3) Use in the detection of fluopyram.

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