Pyridine bacteria amide hapten as well as preparation method and application thereof

Abstract

The invention relates to a picolinamide hapten of which the structural formula is shown as a formula (I), the structure of the picolinamide is modified, a spacer arm is introduced away from the characteristic structure of the picolinamide, the picolinamide hapten is obtained, and the antigen is prepared by coupling the picolinamide hapten and carrier protein through an active ester method. By using the prepared immune mouse, the murine monoclonal antibody with high sensitivity and strong specificity can be obtained. When the conjugate of the hapten and the carrier protein provided by the invention is used for preparing a picolinamide antibody, along with the increase of immunization times, the titer of antiserum is gradually increased, the affinity of the antibody is gradually improved, and finally, the stability is achieved. The preparation process of the picolinamide hapten and antigen provided by the invention is low in production cost and high in practical value, and has a good application prospect in pesticide residue detection.

Description

Technical Field

[0001] The present invention relates to the field of pyridobactamide detection, and in particular to a pyridobactamide hapten and a preparation method and application thereof. Background Art

[0002] Pyridine (Florylpicoxamid), also known as fluoropyrazoline, has the chemical name (1S)-2,2-bis(4-fluorophenyl)-1-methylethyl N-[[3-(acetyloxy)-4-methoxy-2-pyridyl]carbonyl]-L-alaninate. Pyridine is a second-generation pyridineamide fungicide developed by Corteva. Its mechanism of action is similar to that of the first-generation product, fenpyrazoline, targeting the Qi site of the cytochrome bc1 complex in the mitochondrial respiratory system of pathogenic fungi, known as Qi inhibitors (QiIs). This strongly inhibits mycelial growth, conidia production, and germination. Currently registered in Canada and Australia, pyridine has a broad spectrum of control, primarily used against cereals, grapes, and vegetables, including powdery mildew, anthracnose, scab, and fungal diseases caused by Septoria, Botrytis cinerea, Alternaria alternata, and Sclerotinia sclerotiorum. Pyridoxamide has favorable toxicological and environmental profiles and can be used at multiple crop growth stages to improve crop yield and quality. It demonstrates effective disease control at low application rates, reducing crop losses and improving agricultural product quality.

[0003] However, the use of relatively safe fungicides in agricultural production is different from the use of drugs to control fungal infections in humans. The former, when used at higher doses, can leave residues in various plant-based foods, such as fruits and vegetables, potentially causing human poisoning. This emerging chemical contamination risk poses a new threat to the safety of cross-border food safety, leading to numerous negative impacts on human health. In January 2022, Australia announced that it would set the maximum residue limit (MRL) for pyridinamide fungicides at 0.02 mg / kg for mammalian meat and edible offal, and 0.01 mg / kg for poultry, eggs, dairy products, and edible offal. Health Canada issued Notice PMRL2022-19, announcing that the Pest Control Agency is proposing to revise the maximum residue limit (MRL) for pyridinamide in certain foods. The MRL for rapeseed (crop subgroup 20A) will be 0.015 mg / kg, for dry shelled beans, peas, wheat, and beetroot will be 0.01 mg / kg, and for animal-derived foods (including metabolites) will be 0.02 mg / kg.

[0004] Instrumental analysis is currently the most commonly used method for pesticide residue detection. However, no rapid in situ detection method for pyridoxamide has been identified. However, immunoassays offer advantages over conventional methods, such as ease of development, low cost, and simple operation. The performance of the core reagent antibody is largely determined by its hapten structure. Therefore, the development of pyridoxamide haptens and antigens that can be used to generate highly sensitive and specific monoclonal antibodies is crucial. Summary of the Invention

[0005] The antigen prepared from the pyridobactamide hapten used in the present invention has high antibody titer and strong specificity after immunizing animals, and the detection method established subsequently can well meet the current needs of trace detection.

[0006] The first object of the present invention is to provide a pyridinamide hapten, the structural formula of which is shown in formula (I):

[0007]

[0008] The second object of the present invention is to provide a method for preparing the compound represented by formula (I). The synthetic route is as follows:

[0009]

[0010] Furthermore, the preparation method of the compound represented by formula (I) comprises the following steps:

[0011] (S1) Under nitrogen protection, an affinity addition reaction is carried out between a phenyl Grignard reagent and (S)-ethyl lactate. After the reaction is completed, the reaction is quenched to obtain an intermediate product, a white solid F1;

[0012] (S2) (S)-2-[(tert-Butoxycarbonyl)aminopropionic acid] was dissolved in an organic solvent, stirred evenly, and cooled to 0-5°C. Triethylamine and pivaloyl chloride were added, and the mixture was stirred at 0-5°C. Intermediate product F1 and 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature. The mixture was quenched and post-treated to obtain an oily product F2.

[0013] (S3) Under an inert atmosphere, the oil F2, an organic solvent, and triethylsilane are mixed at low temperature, trifluoroacetic acid is slowly added while maintaining the system temperature at 0-8°C, and the temperature is raised to room temperature with stirring to react. The intermediate oil F3 is obtained after post-treatment;

[0014] (S4) Under an inert atmosphere, 5-hydroxypicolinic acid and ethyl chloroformate are reacted at low temperature to form a mixed anhydride. The temperature is maintained, and the oily substance F3 is added. The reaction is carried out at low temperature, and the reaction is quenched. After post-treatment, a white solid F4 is obtained.

[0015] (S5) The intermediate product F4 is reacted with tert-butyl 4-bromobutyrate under alkaline conditions at 50-80° C. with stirring for 4-10 hours, the temperature is lowered, the mixture is filtered, and the filtrate is concentrated under reduced pressure; the concentrate is purified by column chromatography to obtain the tert-butyl 4-bromobutyrate-substituted product;

[0016] (S6) The tert-butyl 4-bromobutyrate-substituted product is reacted with trifluoroacetic acid at room temperature, and the product is purified by column chromatography to obtain the pyridamidine hapten represented by formula (I).

[0017] Furthermore, in step (S1), the low-temperature reaction is controlled at 0-4°C for 3-5 hours; the phenyl Grignard reagent is selected from at least one of (4-fluorophenyl)magnesium bromide and 4-fluorophenylmagnesium chloride; a saturated NH4Cl aqueous solution is used to quench the reaction; the ratio of the phenyl Grignard reagent to (S)-ethyl lactate is 1 mmol:20-50 mg, preferably 1 mmol:32-36 mg.

[0018] Furthermore, in step (S2), the organic solvent is selected from at least one of dichloromethane, ethyl acetate, and chloroform; preferably dichloromethane; the mass ratio of (S)-2-[(tert-butoxycarbonyl)aminopropionic acid and the intermediate product F1 obtained in step (S1) is 1:1-1.2; the ratio of (S)-2-[(tert-butoxycarbonyl)aminopropionic acid, triethylamine, pivaloyl chloride, and 4-dimethylaminopyridine is 1 mg:1.3-1.6 μL:0.5-0.7 μL:0.02-0.03 mg; and the post-treatment is liquid separation, extraction, drying the combined organic layer, filtering and concentrating, and purification by column chromatography.

[0019] Furthermore, in step (S3), the inert atmosphere is nitrogen and / or argon; the ratio of oil F2, triethylsilane, and trifluoroacetic acid is 1 g: 1300-1700 μL: 3000-3500 μL; post-treatment involves removing the solvent by distillation under reduced pressure and co-evaporating with dichloromethane. The resulting oil is dissolved in dichloromethane, a saturated solution of NaHCO₃ is added portionwise, the aqueous layer is extracted with dichloromethane, and the combined organic layers are washed with brine and dried over Na₂SO₄. The mixture is concentrated under reduced pressure, dissolved in dichloromethane, and purified by column chromatography to obtain the oil F3.

[0020] Furthermore, in step (S4), the low temperature is 0-5°C; the mass ratio of the oil F3 and 5-hydroxypicolinic acid is 1:0.4-0.5; further, the ratio of the oil F3, triethylamine and ethyl chloroformate is 1:1.2-1.5μL:0.55-0.65μL.

[0021] Further, in step (S5), the ratio of the intermediate product F4 and tert-butyl bromomethylbenzoate is 1 mg: 0.9-1 μL;

[0022] Furthermore, in step (S6), the ratio of the tert-butyl 4-bromobutyrate substitution product and the trifluoroacetic acid compound is 1 mg: 3-4 μL.

[0023] The third object of the present invention is to provide a pyridobactamide artificial antigen, which is a conjugate obtained by coupling the compound represented by formula (I) with the carrier protein BSA, and the structure is shown in formula (II).

[0024]

[0025] Furthermore, the pyridobactamide artificial antigen has an average of 5.9-6.9 compounds of formula (I) coupled to one carrier protein molecule.

[0026] The fourth object of the present invention is to provide a method for preparing the conjugate shown in formula (II), comprising the following steps: coupling the carrier protein to the carboxyl group of the compound shown in formula (I) using the active ester method.

[0027] Specifically, the method for preparing the pyridobactamide artificial antigen represented by formula (II) comprises the following steps:

[0028] (T1) The compound represented by formula (I) is dissolved in DMF, and a carboxyl group activating agent is added to obtain a hapten solution;

[0029] (T2) dissolving the carrier protein in PBS buffer to obtain a carrier protein solution;

[0030] (T3) The hapten solution and the carrier protein solution are mixed and dialyzed in a dialysis bag with a molecular weight cut-off of 7-10 kDa using PBS buffer at 4-6° C. for 48-72 hours, with the PBS buffer being replaced every 12-14 hours.

[0031] Furthermore, in step (T1), the concentration of the compound represented by formula (I) in the DMF solution is 20-30 mg / mL; in step (2), the carboxyl activator micro EDC and NHS; the mass ratio of the compound of formula (I), EDC and NHS is 1:0.6-0.7:0.3-0.35; in step (T2), the concentration of BSA in the carrier protein solution is 1-10 mg / mL, preferably 3-5 mg / mL; in step (T3), the mass ratio of the compound of formula (I) in the hapten solution to BSA in the carrier protein solution is 1:1.9-2.5; the reaction is stirred at room temperature and 400-600 rpm for 2-5 hours.

[0032] A fifth object of the present invention is to provide a pyridobactamide antibody, which is obtained by immunizing an animal with the artificial pyridobactamide antigen represented by formula (II). Furthermore, the antibody is a monoclonal antibody.

[0033] A sixth object of the present invention is to provide any of the following uses of the pyridobactamide hapten represented by formula (I), and / or the pyridobactamide artificial antigen represented by formula (II), and / or the pyridobactamide antibody represented by formula (II):

[0034] (1) Use in the preparation of pyridobactamide-specific antibodies;

[0035] (2) Use in the preparation of pyridobactamide detection reagents;

[0036] (3) Use in the preparation of pyridobactamide immunochromatographic test paper;

[0037] (4) Use in the detection of pyridoxamide.

[0038] The present invention modifies the structure of pyridobactamide to produce a pyridobactamide hapten not previously reported in the prior art, and couples it to a carrier protein to prepare an antigen. Immunizing BALB / c mice with the prepared antigen yields highly sensitive and specific monoclonal antibodies. Previously, no information on the use of the hapten and antigen described in this study for antibody production has been available. Using the conjugate of the hapten and carrier protein provided by the present invention to prepare pyridobactamide antibodies, antibody titers rose rapidly after initial immunization, sensitivity gradually increased, and ultimately stabilized. The preparation process for the pyridobactamide hapten and antigen provided by the present invention is simple, economical, and highly practical, and has promising application prospects in pesticide residue detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The mass spectrum of pyridinamide hapten represented by formula (I) is shown.

[0040] Figure 2 The figure is the hydrogen nuclear magnetic resonance spectrum of the pyridobactamide hapten represented by formula (I).

[0041] Figure 3 Matrix-assisted laser desorption ionization time-of-flight mass spectrum of the antigen.

[0042] Figure 4 This is the standard curve of pyridobactamide. DETAILED DESCRIPTION

[0043] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were performed in four replicates, and the results were averaged.

[0044] FLCD is the abbreviation for pyridoxal. NHS is the abbreviation for N-hydroxysuccinimide. EDC is the abbreviation for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. DMF is the abbreviation for N,N-dimethylformamide. Pyridoxal was purchased from Shanghai Bidex Pharmaceutical Technology Co., Ltd., product catalog number BD01755393. 4′-Bromomethylbiphenyl-2-carboxylic acid methyl ester was purchased from Bidex Pharmaceutical Technology Co., Ltd., product catalog number BD8268. 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, and methanol were purchased from Sinopharm Group. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma, product catalog numbers F-5881 and F-5506, respectively. Goat anti-mouse IgG enzyme-labeled antibody was purchased from Jackson Immunoresearch, product catalog number 115-035-003. 96-well ELISA plate was purchased from Costar, product catalog number 2592.

[0045] Bovine serum albumin (BSA) was purchased from Sigma, with the product catalog number being 9048-46-18.

[0046] BALB / c mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0047] Unless otherwise specified, the PBS buffer used in the examples is a 0.01 M PBS buffer at pH 7.4.

[0048] PBST solution: PBS buffer (pH 7.2) containing 0.05% (volume percentage) Tween-20.

[0049] Example 1. Synthesis and identification of haptens

[0050] 1. Synthesis of haptens

[0051] 1. Under nitrogen, add (4-fluorophenyl)magnesium bromide (20 mL, 20 mmol, 1 M THF solution) to a three-necked flask. Cool the mixture to 0°C and slowly add 713 mg of (S)-ethyl lactate dissolved in 6 mL of THF solution via syringe. The reaction temperature is controlled at 0-4°C for 3 hours. Quench the reaction with 2-3 mL of saturated aqueous NH4Cl solution and stir until the reaction reaches room temperature. The liquid layer is decanted from the white solid. The white solid is suspended in EtOAc, filtered, and the filter cake is washed with EtOAc. The combined organic phases are concentrated under vacuum. The residue is dissolved in EtOAc, transferred to a separatory funnel, and washed with water. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain 2 g of a yellow oil, which was dissolved in dichloromethane and mixed with 4 g of 100-200 mesh silica gel. The product was then loaded onto a 200-300 mesh silica gel column for chromatography, eluting with petroleum ether:ethyl acetate = 10:1. The main product was collected to obtain 1.36 g of a white solid F1.

[0052] 2. Add 1135.2 mg of (S)-2-[(tert-Butoxycarbonyl)aminopropionic acid] and 20 ml of dichloromethane to a 100 mL flask and cool to 0°C. Add 1739 μL of triethylamine to the reaction flask and dropwise add 709 μL of pivaloyl chloride to produce a white precipitate. After stirring at 0°C for 15 min, add 1320 mg of solid F1 and then 25 mg of 4-dimethylaminopyridine. Stir and react at room temperature for 2 h. Quench the reaction with water and separate the layers. Extract the aqueous layer with dichloromethane. Dry the combined organic layers over Na2SO4, filter, and concentrate to obtain 2350 mg of an oil. Add 4.7 g of 100-200 mesh silica gel and mix the sample. Apply 200-300 mesh silica gel to a column for chromatography, eluting with petroleum ether:ethyl acetate = 5:1. Collect the main product to obtain 2005 mg of oily F2.

[0053] 3. Under nitrogen, add 2000 mg of the F2 oil, 20 mL of dichloromethane, and 2930 μL of triethylsilane to a 100 mL three-necked flask equipped with a thermometer. Maintain the internal temperature of the flask at 4°C with an ice bath. Add 6666 μL of trifluoroacetic acid over 15 minutes. The internal temperature should not exceed 8°C during the addition. Warm the reaction to room temperature and stir for 4 hours. Remove the solvent under reduced pressure and co-evaporate with 200 mL of dichloromethane. Dissolve the resulting oil in 50 mL of dichloromethane and add 70 mL of a saturated NaHCO3 solution in portions. Extract the aqueous layer with 50 mL of dichloromethane. Wash the combined organic layers with brine and dry over Na2SO4. The mixture was concentrated under reduced pressure to obtain 4500 mg of an oily substance, which was dissolved in dichloromethane and mixed with 9000 mg of 100-200 mesh silica gel. The mixture was packed with 200-300 mesh silica gel and subjected to column chromatography using petroleum ether:ethyl acetate = 1:1 as the eluent. The main product was collected to obtain 1240 mg of an oily substance F3.

[0054] 4. Under nitrogen protection, add 540.7 mg of 5-hydroxypicolinic acid and 25 ml of dichloromethane to a 100 ml three-necked flask equipped with a thermometer. After stirring evenly, cool to 0°C, add 1622 μL of triethylamine, stir vigorously for 10 minutes, and slowly add 740 μL of ethyl chloroformate dropwise until a white precipitate begins to form. After stirring at 0°C for 15 minutes, add 1240 mg of oily F3 in 10 ml of dichloromethane solution dropwise. The reaction mixture is stirred at 0°C for 3 minutes, and the reaction is quenched with 20 mL of water and 5 mL of 2N HCl. The two-phase mixture is diluted with dichloromethane and transferred to a separatory funnel. Separate the liquids and extract, dry the organic phase over anhydrous Na2SO4, filter, and concentrate to obtain a light yellow oil. The crude product is purified by silica gel chromatography using an ethyl acetate / petroleum ether gradient elution to obtain 1625 mg of white solid F4.

[0055] 5. Place 440.45 mg of intermediate F4 in a 50 ml flask, add 15 ml of N-N-dimethylformamide, stir until dissolved, then add 690 mg of anhydrous potassium carbonate. Stir at room temperature for 30 min, then add 399 μL of tert-butyl 4-bromobutyrate and stir at 60°C for 6 hours. Filter to remove salts, concentrate the filtrate under reduced pressure, dissolve in dichloromethane, add 2000 mg of 100-200 mesh silica gel, mix the sample, and apply column chromatography on 200-300 mesh silica gel with a ratio of petroleum ether:ethyl acetate of 1:1. Collect the main product to obtain 555 mg of the tert-butyl bromobutyrate-substituted product. Dissolve in 10 ml of dichloromethane, add 2 ml of trifluoroacetic acid, react at room temperature for 3 hours, and concentrate under reduced pressure. Dissolve in dichloromethane, add 1000 mg of 100-200 mesh silica gel, mix the sample, and apply column chromatography on 200-300 mesh silica gel with a ratio of petroleum ether:ethyl acetate of 1:2. Collect the main product, which is the pyridoxamide hapten.

[0056] 2. Identification of Haptens

[0057] The obtained product, pyridobactamide hapten, was identified by mass spectrometry and nuclear magnetic resonance.

[0058] Mass spectrometry results are shown in Figure 1 , the peak of m / z 527 (M+H+) can be clearly seen, which is consistent with the target molecular weight (526.54). Figure 2 , indicating that the pyridinamide hapten was successfully synthesized.

[0059] The results showed that the structure of the obtained pyridinamide hapten was as shown in the following formula (I).

[0060]

[0061] Example 2. Preparation of Antigen (Active Ester Method)

[0062] The conjugate of hapten and BSA was named FLCD-BSA as the artificial antigen of pyridobactamide, which is shown in formula (II):

[0063]

[0064] 1. Preparation of Immunogen

[0065] 1. Weigh 24 mg of pyridobactamide hapten, add 15 mg of EDC and 8 mg of NHS, place on a magnetic stirrer, activate at 400 rpm in the dark for 24 h, and dissolve in 1 mL of DMF;

[0066] 2. Take 50 mg of BSA and dissolve it in 10 mL of PBS buffer containing 10% (volume percentage) DMF to obtain a protein solution;

[0067] 3. Add the liquid phase from step 1 dropwise to the protein solution prepared in step 2, then transfer to a dialysis bag with a molecular weight cutoff of 7 kDa. Place the dialysis bag in PBS buffer and dialyze at 4 °C for 72 h (change the solution every 12-14 h).

[0068] 5. After completing step 3, remove the dialysis bag, take out the liquid phase therein, centrifuge at 5000 rpm for 3 minutes, and collect the supernatant, which is the solution containing FLCD-BSA, and name it FLCD-BSA solution.

[0069] 6. The binding ratio of BSA to hapten in FLCD-BSA solution was determined by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS). Figure 3 .

[0070] Binding ratio = {M(conjugate) - M(protein)} / M(hapten)

[0071] The molecular weight of BSA is 66131.86, and the molecular weight of the hapten is 526.54. The molecular weight of the conjugate was 69249.09 according to the highest peak of the mass spectrometry. The calculated binding ratio of BSA to hapten was 5.9, i.e., an average of 5.9 haptens of formula (I) were conjugated to one BSA molecule.

[0072] 2. Preparation of Coating Agent

[0073] OVA was used instead of BSA to obtain FLCD-OVA solution. The preparation of coating source involved the same feed ratio of hapten, activator and OVA as that of immunogen and the same reaction conditions to obtain FLCD-OVA solution.

[0074] Example 3. Preparation of monoclonal antibodies

[0075] The amount of FLCD-BSA added is calculated based on the total protein amount. That is, the total protein concentration of the FLCD-BSA solution is detected by the Bradford method. The total protein concentration multiplied by the added volume is the amount of FLCD-BSA added.

[0076] 1. Animal Immunization

[0077] Preparation of the first immunization preparation: Dilute the FLCD-BSA antigen solution prepared in Example 2 with PBS buffer, then mix and emulsify with an equal volume of Freund's complete adjuvant. Preparation of the booster immunization preparation: Dilute the FLCD-BSA solution prepared in Example 2 with PBS buffer, then mix and emulsify with an equal volume of Freund's incomplete adjuvant.

[0078] Eight BALB / c mice were immunized four times. The specific steps are as follows (immunization method is immunization at points 4-8 on the back of the neck):

[0079] First immunization (primary immunization): 1 ml of the first immunization preparation was administered to each animal (each 1 ml of the first immunization preparation contained 1 mg of FLCD-BSA).

[0080] The second to fifth immunizations (boost immunizations): Count the days from the first immunization, and perform a booster immunization every 21 days, for a total of 4 booster immunizations; each booster immunization uses 200 μL of the booster preparation (each 200 μL of the booster preparation contains 100 μg FLCD-BSA) per immunization.

[0081] Starting from the third immunization, blood was collected one week after immunization (0.25 ml from each orbital venous plexus) and serum was collected by centrifugation.

[0082] 2. Serological testing

[0083] The polyclonal antibodies to be tested are the serum obtained in step 1 (the serum obtained one week after the third immunization, the serum obtained one week after the fourth immunization, and the serum obtained one week after the fifth immunization).

[0084] Take a 96-well ELISA plate and perform the following steps in sequence:

[0085] 1. Packing

[0086] 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.

[0087] Coating solution: The FLCD-OVA solution prepared in Example 2 was diluted with a 0.05 M carbonate buffer solution at pH 9.6 to a concentration of 0.2 μg / ml (based on total protein concentration) of FLCD-OVA.

[0088] 2. Closed

[0089] 150 μL of blocking solution was added to each well, incubated at 37°C for 1 h, and then washed three times with PBST solution and patted dry.

[0090] Blocking solution: 0.4g / 100ml skim milk aqueous solution.

[0091] 3. Add the antibody to be tested

[0092] Test wells: Add 50 μL PBS buffer and 50 μL dilution of the polyclonal antibody to be tested to each well;

[0093] Control wells: add 50 μL PBS buffer and 50 μL serum before the first immunization to each well;

[0094] Incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry.

[0095] Dilution: Starting from 1:5000 dilution, with a gradient of 2, for a total of 8 dilutions; PBS buffer was used as the solvent.

[0096] 4. Add enzyme-labeled secondary antibody

[0097] Add 100 μL of enzyme-labeled secondary antibody dilution to each well, incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry.

[0098] Enzyme-labeled secondary antibody diluent: Dilute goat anti-mouse IgG enzyme-labeled antibody to 5000 times volume.

[0099] 5. Color rendering

[0100] Add 100 μL of color development solution to each well and incubate at 37°C for 15 min.

[0101] Color development solution: Mix equal volumes of 2% 3,3',5,5'-tetramethylbenzidine solution and 30% hydrogen peroxide.

[0102] 6. Termination

[0103] Add 50 μL of 2 mol / L concentrated sulfuric acid to each well.

[0104] 7. Reading

[0105] The OD value of each well was measured at a wavelength of 450 nm. The antibody titer was determined by taking the antibody dilution corresponding to a negative OD value of no more than 0.15 and a maximum OD value between 1.5 and 2.0.

[0106] From the third to the fifth immunization, the titer of pyridobactamide polyclonal antibodies showed a trend of gradual increase, then stabilization or slight decrease. After the fifth immunization, the serum titer was 20,000.

[0107] 3. Preparation and purification of monoclonal antibodies (protein A affinity column antibody purification method)

[0108] According to the results of step 2, select the serum inhibition rate (B / Bo) with low IC 50 Mice with low titers and high titers were given a boost immunization 21 days after the boost immunization. Cell fusion was performed 4 days after the boost immunization. After four to five rounds of subcloning screening, a monoclonal cell line was obtained that secreted high-affinity antibodies. Balb / c mice aged 10 weeks or older were selected for large-scale production of monoclonal antibodies using an in vivo induction method. Ascites was collected, the supernatant was separated, and stored in aliquots at -20°C.

[0109] 1. Preparation: All solutions should be filtered with a 0.22μm or 0.45μm filter membrane before use. The average antibody purification yield of ascites is 4mg / ml. Ensure that the amount of purified antibody is less than the total capacity of the affinity column.

[0110] 2. Sample filtration: Dilute the ascites or polyantibody serum after centrifugation to remove the precipitate 4-5 times with binding buffer, collect it through 0.22μm filter, and mark it as sample.

[0111] 3. Column equilibration: Remove the Protein A column from the refrigerator and equilibrate it at room temperature for 30 minutes. Use a disposable syringe to dispense 20 ml of the binding solution through the column to equilibrate it. Control the flow rate to 1 ml to 2 ml / min, i.e., 20 to 40 drops / min.

[0112] 4. Sample loading: Use a disposable syringe to pass the sample through the column, control the flow rate at 1ml-2ml / min, collect the liquid passing through the column and record the volume.

[0113] 5. Eluent: Use 20 ml of binding solution to pass through the column to elute the impurities adsorbed in the column material, and discard the eluent.

[0114] 6. Elution: Take several small centrifuge tubes and label them. Pre-add a specific volume of neutralizing solution to each tube. Collect a specific volume of eluate in each tube until the baseline is reached (OD280 value is less than 0.03). The volume of neutralizing solution and eluate in each tube can be increased proportionally. Use nanodrop to detect the concentration of the antibody in each tube. Combine the collection tubes with an antibody concentration greater than 0.1 mg / ml.

[0115] 7. Replacement buffer: Use dialysis or ultrafiltration to replace the antibody buffer with the desired solution (usually 10mM PBS pH

[0116] 7.2) Use an intact dialysis membrane (8kMWCO) to dialyze three times, 2L for more than 3 hours each time. If the antibody volume is less than 2mg, the buffer can be replaced by ultrafiltration.

[0117] 8. Filler cleaning:

[0118] a) For alkali-resistant columns, wash the column with 2 column volumes of equilibration solution, 0.1 M NaOH, and equilibration solution in the forward or reverse direction.

[0119] b) To remove some precipitates or denatured substances, use 2 column volumes of 6M guanidine hydrochloride solution, 5 column volumes of

[0120] Volume of equilibrium solution is used for forward or reverse column cleaning.

[0121] c) Remove some non-specific adsorption substances caused by hydrophobic adsorption. Use 3-4 column volumes of 70% ethanol or 2 column volumes of 1% Triton TM X-100, and then immediately wash with 5 column volumes of equilibration buffer.

[0122] 9. Column storage: Pass 2 times the column volume of 20% ethanol through the column, cover the upper and lower sealing caps, and store at 4°C.

[0123] 4. Dilution of purified monoclonal antibodies

[0124] Replace the "test polyclonal antibody" in step 2 with the purified monoclonal antibody prepared in step 3 and proceed as in step 2. Obtain the antibody dilution that results in a maximum OD value between 1.5 and 1.8.

[0125] The purified monoclonal antibody prepared in step 3 was diluted according to the antibody dilution obtained above using PBS buffer as a solvent to obtain an antibody dilution solution.

[0126] 5. Determination of Monoclonal Antibody Sensitivity

[0127] 1. Packing

[0128] 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.

[0129] 2. Closed

[0130] 150 μL of blocking solution was added to each well, incubated at 37°C for 1 h, and then washed three times with PBST solution and patted dry.

[0131] 3. Add standards and antibodies

[0132] Add 50 μL of pyridobactamide standard solution and 50 μL of antibody dilution prepared in step 4 to each well, incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry.

[0133] The solvent of the pyridobactamide standard solution is PBS buffer, and the concentrations of the pyridobactamide standard solution are 0, 0.1, 0.3, 0.9, 2.7, 8.1, and 24.3 ng / mL, respectively.

[0134] 4. Add enzyme-labeled secondary antibody

[0135] Add 100 μL of enzyme-labeled secondary antibody dilution to each well, incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry.

[0136] 5. Color rendering

[0137] Add 100 μL of color development solution to each well and incubate at 37°C for 15 min.

[0138] 6. Termination

[0139] Add 50 μL of 2 mol / L concentrated sulfuric acid to each well.

[0140] 7. Reading

[0141] The OD value of each well was measured at a wavelength of 450 nm.

[0142] -log 10 The (pyridobactamide concentration) value was taken as the horizontal axis, and the OD value (measured at OD 450nm) was taken as the vertical axis. The four-parameter equation of Origin 8.0 was used for fitting to establish a standard curve to obtain the IC 50 value.

[0143] Standard curve see Figure 4 , IC of the purified monoclonal antibody 50 The value is 1.62ng / mL.

[0144] VI. Determination of the cross-reactivity rate between pyridamyl monoclonal antibodies and fluopyram

[0145] According to the operation of step 5, the cross-reaction rate of the prepared pyridamyl monoclonal antibody to fluopyram was determined.

[0146] The cross-reaction rate between the prepared monoclonal antibody and fluopyram was determined to be less than 0.1%.

Claims

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

2. The method for preparing the compound represented by formula (I) according to claim 1, characterized in that: The synthetic route is as follows:

3. The preparation method according to claim 2, characterized in that The following steps are involved: (S1) Under nitrogen protection, an affinity addition reaction is carried out between a phenyl Grignard reagent and (S)-ethyl lactate. After the reaction is completed, the reaction is quenched to obtain an intermediate product, a white solid F1; (S2) (S)-2-[(tert-Butoxycarbonyl)aminopropionic acid] was dissolved in an organic solvent, stirred evenly, and cooled to 0-5°C. Triethylamine and pivaloyl chloride were added, and the mixture was stirred at 0-5°C. Intermediate product F1 and 4-dimethylaminopyridine were added, and the reaction was stirred at room temperature. The mixture was quenched and post-treated to obtain an oily product F2. (S3) Under an inert atmosphere, the oil F2, an organic solvent, and triethylsilane are mixed at low temperature, trifluoroacetic acid is slowly added while maintaining the system temperature at 0-8°C, and the temperature is raised to room temperature with stirring to react. The intermediate oil F3 is obtained after post-treatment; (S4) Under an inert atmosphere, 5-hydroxypicolinic acid and ethyl chloroformate are reacted at low temperature to form a mixed anhydride. The temperature is maintained, and the oily substance F3 is added. The reaction is carried out at low temperature, and the reaction is quenched. After post-treatment, a white solid F4 is obtained. (S5) The intermediate product F4 is reacted with tert-butyl 4-bromobutyrate under alkaline conditions at 50-80° C. with stirring for 4-10 hours, the temperature is lowered, the mixture is filtered, and the filtrate is concentrated under reduced pressure; the concentrate is purified by column chromatography to obtain the tert-butyl 4-bromobutyrate-substituted product; (S6) The tert-butyl 4-bromobutyrate-substituted product is reacted with trifluoroacetic acid at room temperature, and the product is purified by column chromatography to obtain the pyridamidine hapten represented by formula (I).

4. The preparation method according to claim 3, characterized in that In step (S1), the low-temperature reaction is carried out at a temperature of 0-4°C for 3-5 hours; the phenyl Grignard reagent is selected from at least one of (4-fluorophenyl)magnesium bromide and 4-fluorophenylmagnesium chloride; the reaction is quenched with a saturated aqueous NH4Cl solution; the ratio of the phenyl Grignard reagent to (S)-ethyl lactate is 1 mmol:20-50 mg, preferably 1 mmol:32-36 mg; and / or In step (S2), the organic solvent is selected from at least one of dichloromethane, ethyl acetate, and chloroform; preferably dichloromethane; the mass ratio of (S)-2-[(tert-butoxycarbonyl)aminopropionic acid and the intermediate product F1 obtained in step (S1) is 1:1-1.2; the ratio of (S)-2-[(tert-butoxycarbonyl)aminopropionic acid, triethylamine, pivaloyl chloride, and 4-dimethylaminopyridine is 1 mg:1.3-1.6 μL:0.5-0.7 μL:0.02-0.03 mg; the post-treatment is separation, extraction, drying the combined organic layer, filtration and concentration, and purification by column chromatography; and / or In step (S3), the inert atmosphere is nitrogen and / or argon; the ratio of oil F2, triethylsilane, and trifluoroacetic acid is 1 g: 1300-1700 μL: 3000-3500 μL; the post-treatment is to remove the solvent by distillation under reduced pressure and co-evaporate with dichloromethane, dissolve the resulting oil in dichloromethane, add a saturated solution of NaHCO3 in portions, extract the aqueous layer with dichloromethane, combine the organic layers, wash with brine, and dry with Na2SO4; and / or In step (S4), the low temperature is 0-5°C; the mass ratio of the oil F3 to 5-hydroxypicolinic acid is 1:0.4-0.5; further, the ratio of the oil F3, triethylamine, and ethyl chloroformate is 1:1.2-1.5 μL:0.55-0.65 μL; and / or in step (S5), the ratio of the intermediate product F4 to tert-butyl bromomethylbenzoate is 1 mg:0.9-1 μL; and / or In step (S6), the ratio of tert-butyl 4-bromobutyrate substitution product and trifluoroacetic acid compound is 1 mg:3-4 μL.

5. A pyridobactamide artificial antigen, which is a conjugate obtained by coupling the compound represented by formula (I) with the carrier protein BSA, and its structure is shown in formula (II).

6. The pyridobactamide artificial antigen according to claim 5, characterized in that An average of 5.9-6.9 compounds represented by formula (I) are coupled to one carrier protein molecule.

7. The method for preparing the pyridobactamide artificial antigen according to claim 6, characterized in that: The method comprises the following steps: coupling the carrier protein to the carboxyl group of the compound represented by formula (I) by using an active ester method; Furthermore, the preparation method comprises the following steps: (T1) The compound represented by formula (I) is dissolved in DMF, and a carboxyl group activating agent is added to obtain a hapten solution; (T2) dissolving the carrier protein in PBS buffer to obtain a carrier protein solution; (T3) The hapten solution and the carrier protein solution are mixed and dialyzed in a dialysis bag with a molecular weight cut-off of 7-10 kDa using PBS buffer at 4-6° C. for 48-72 hours, with the PBS buffer being replaced every 12-14 hours.

8. The preparation method according to claim 7, characterized in that In step (T1), the concentration of the compound represented by formula (I) in the DMF solution is 20-30 mg / mL; and / or in step (2), the carboxyl activators micro EDC and NHS; the mass ratio of the compound of formula (I), EDC and NHS is 1:0.6-0.7:0.3-0.35; and / or in step (T2), the concentration of BSA in the carrier protein solution is 1-10 mg / mL, preferably 3-5 mg / mL; and / or in step (T3), the mass ratio of the compound of formula (I) in the hapten solution to BSA in the carrier protein solution is 1:1.9-2.5; the reaction is stirred at room temperature and 400-600 rpm for 2-5 hours.

9. A pyridobactamide antibody, obtained by immunizing an animal with the pyridobactamide artificial antigen according to claim 5; further, the antibody is a monoclonal antibody.

10. Any of the following uses of the pyridobactamide hapten according to claim 1, or the pyridobactamide artificial antigen according to claim 5 or 6, or the pyridobactamide antibody according to claim 9: (1) Use in the preparation of pyridobactamide-specific antibodies; (2) Use in the preparation of pyridobactamide detection reagents; (3) Use in the preparation of pyridobactamide immunochromatographic test paper; (4) Use in the detection of pyridoxamide.

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