A prohexadione-calcium coated antigen, immunogen, and monoclonal antibody and uses thereof

Abstract

This invention relates to the field of immunoassay technology, specifically to a chlorpheniramine-coated antigen, an immunoassay antigen, a monoclonal antibody, and their applications. The hybridoma cell line MXA9C3 obtained by immunizing a host with the immunoassay antigen can secrete a broad-spectrum, specific monoclonal antibody recognizing chlorpheniramine and its metabolites BTS44595 and BTS44596. Furthermore, the coating antigen provided by this invention enhances the sensitivity of the antibody. Therefore, this monoclonal antibody exhibits high specificity and detection sensitivity for chlorpheniramine precursor and its metabolites, with a high IC50 for chlorpheniramine, BTS44595, and BTS44596. 50 The values ​​were 8.075, 10.820, and 15.065 ng / mL, respectively, and there was no cross-reaction with other similar small molecules of pesticides, which is conducive to establishing an accurate immunological detection method for prochloraz parent compound and its metabolites.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to an imazalil-coated antigen, an immunoassay antigen, a monoclonal antibody, and their applications. Background Technology

[0002] Prochloraz, chemical formula C 15 H 16 C l3 N3O2 is an imidazole fungicide that mainly inhibits the activity of C-14 demethylase (CYP51) in the ergosterol biosynthesis pathway within fungal cells, thereby blocking the conversion of lanosterol to ergosterol and interfering with the synthesis of fungal cell membranes, thus inhibiting fungal growth and reproduction.

[0003] Imazalil is widely used in horticulture and agriculture to effectively prevent fungal diseases in grains, fruits, and vegetables, and is also used as a post-harvest preservative for most fruits. However, its increased use has exacerbated its residual risks in the environment, as imazalil has been detected in water bodies, aquatic organisms, and food.

[0004] Imazalil can be metabolized into various substances in plants and animals, including BTS44596, BTS44595, BTS44770, BTS9608, and BTS45186. Among these, BTS44595, BTS44596, and 2,4,6-trichlorophenol are the main metabolites, exhibiting relatively long stability in plants and animals. These metabolites are stable for 6 months at -18°C in high-starch plants and at least 3 months under frozen storage in animal-derived foods. 2,4,6-trichlorophenol is stable for up to 24 months in corn and 12 months in muscle, eggs, and milk.

[0005] Imazalil poses toxicological risks to humans and animals, particularly in terms of reproductive toxicity, significantly impacting men by potentially affecting male hormone secretion and increasing the risk of feminization in male infants. It also exhibits endocrine-disrupting and genotoxic properties, potentially damaging the human respiratory and nervous systems. In animals, imazalil can cause problems with sexual differentiation and impaired growth and development.

[0006] Given the stability of imazalil and its metabolites and their potential impact on human and animal health, establishing effective detection methods is crucial. Currently, the main detection methods for imazalil include liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography (GC), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS), as well as some colorimetric and electrochemical methods. While these methods offer high sensitivity and specificity, they are complex to operate, costly, and unsuitable for rapid on-site detection.

[0007] Existing studies have shown that antibodies against prochloraz can only detect prochloraz itself and cannot identify its metabolites, limiting its application in residue detection standards in many countries. Therefore, developing a broad-spectrum, specific antibody capable of simultaneously detecting prochloraz and its metabolites, and using this antibody to develop a reliable immunoassay method, is of great significance for improving detection efficiency and ensuring food safety. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a sulfadiazine-coated antigen, an immunogen, a monoclonal antibody, and their applications. By conjugating the sulfadiazine-coated hapten with a carrier protein to prepare a complete antigen, the antigen has good specificity and high sensitivity and can be used to establish an enzyme-linked immunosorbent assay (ELISA) method for detecting sulfadiazine metabolites.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an imazalil-coated hapten having the structure shown in Formula B: Formula B.

[0010] This invention provides a method for preparing the imazalil-coated hapten described in the above technical solution, comprising the following steps: Imazalil having the structure shown in Formula 1 was mixed with concentrated hydrochloric acid and subjected to an acid hydrolysis reaction to obtain a compound having the structure shown in Formula 2. Formula 1; Formula 2; Under the action of triethylamine, the compound having the structure shown in Formula 3 undergoes an isocyanate reaction with triphosgene to obtain the compound having the structure shown in Formula 4. Formula 3; Equation 4; Under the action of triethylamine, the compound having the structure shown in Formula 2 undergoes an addition reaction with the compound having the structure shown in Formula 4 to obtain the compound having the structure shown in Formula 5. Formula 5; Under alkaline conditions, the compound having the structure shown in Formula 5 undergoes a hydrolysis reaction, and after adjusting the pH value to acidic, an imazalil-coated hapten having the structure shown in Formula B is obtained.

[0011] This invention provides a prochloraz-coated antigen, obtained by coupling a prochloraz-coated hapten as described in the above-mentioned technical solution or a prochloraz-coated hapten prepared by the above-mentioned preparation method with a carrier protein; the prochloraz-coated antigen has the structure shown in Formula F: Formula F.

[0012] This invention provides an imazalil immunogen having the structure shown in Formula E: Formula E.

[0013] This invention provides a hybridoma cell line MXA 9C3, whose accession number is CGMCC No. 45936; The hybridoma cell line MXA 9C3 was obtained by immunizing the host with the imidacloprid immunosorbent antigen described in the above technical solution.

[0014] This invention provides an antibody secreted by the hybridoma cell line MXA 9C3 described in the above technical solution.

[0015] This invention provides a detection product comprising the imazalil-coated hapten described in the above technical solution, or the imazalil-coated hapten obtained by the preparation method, or the imazalil-coated antigen, or the imazalil immunoantigen, or the hybridoma cell line MXA 9C3, or the antibody.

[0016] Preferably, the product includes one or more of reagents, kits, and colloidal gold test strips.

[0017] The present invention provides the application of the imazalil-coated hapten described in the above technical solution, or the imazalil-coated hapten obtained by the preparation method, or the imazalil-coated antigen, or the imazalil immunoantigen, or the hybridoma cell line MXA 9C3, or the antibody, or the detection product in the detection of imazalil and its metabolites.

[0018] Preferably, the metabolites include metabolites BTS44595 and / or BTS44596.

[0019] Beneficial effects: This invention provides a prochloraz-coated hapten with the structure shown in Formula B. Using this hapten as a heterocoating agent enhances antibody affinity, and the prochloraz-coated hapten exhibits good solubility and stability.

[0020] The present invention provides the above-mentioned preparation method, which has a simple synthesis path, low synthesis cost, and is easy to realize industrial mass production.

[0021] This invention provides a prochloraz immunoantigen with the structure shown in Formula E. The hybridoma cell line MXA 9C3 obtained after immunizing a host with this immunoantigen can secrete a broad-spectrum, specific monoclonal antibody that recognizes prochloraz and its metabolites. This monoclonal antibody exhibits good specificity and detection sensitivity for prochloraz and its metabolites. Therefore, applying the prochloraz-coated antigen, immunoantigen, hybridoma cell line, or antibody provided by this invention to detection products is beneficial for establishing immunological detection methods for prochloraz and its metabolites, achieving accurate detection of prochloraz and its metabolites with high specificity and sensitivity.

[0022] Preservation of biological materials The hybridoma cell line MXA 9C3, classified as a mouse hybridoma cell line, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 45936, and the deposit date is June 4, 2024. Attached Figure Description

[0023] Figure 1 The synthetic route for imazalil-coated hapten B; Figure 2 For imazalil-coated hapten B 1 H NMR spectrum; Figure 3 For imazalil-coated hapten B 13 C NMR spectrum; Figure 4 The HPLC chromatogram of imazalil-coated hapten B is shown. Figure 5 MALDI-TOF-MS spectrum of BSA standard; Figure 6 The MALDI-TOF-MS spectrum of immunogen E; Figure 7 MALDI-TOF-MS spectrum of OVA standard; Figure 8 The MALDI-TOF-MS spectrum of prochloraz-coated antigen F; Figure 9 Standard curves for detecting imazalil parent compound and its metabolites BTS44595 and BTS44596 with 9C3 broad-spectrum specific antibody; Figure 10 The results show the antibody subtype detection results produced by the 9C3 cell line. Detailed Implementation

[0024] This invention provides an imazalil-coated hapten having the structure shown in Formula B: Formula B.

[0025] This invention provides a method for preparing the above-mentioned imazalil-coated hapten, comprising the following steps: Imazalil having the structure shown in Formula 1 was mixed with concentrated hydrochloric acid and subjected to an acid hydrolysis reaction to obtain a compound having the structure shown in Formula 2. Formula 1; Formula 2; Under the action of triethylamine, the compound having the structure shown in Formula 3 undergoes an isocyanate reaction with triphosgene to obtain the compound having the structure shown in Formula 4. Formula 3; Equation 4; Under the action of triethylamine, the compound having the structure shown in Formula 2 undergoes an addition reaction with the compound having the structure shown in Formula 4 to obtain the compound having the structure shown in Formula 5. Formula 5; Under alkaline conditions, the compound having the structure shown in Formula 5 undergoes a hydrolysis reaction, and after adjusting the pH value to acidic, an imazalil-coated hapten having the structure shown in Formula B is obtained.

[0026] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0027] In this invention, imazalil having the structure shown in Formula 1 is mixed with concentrated hydrochloric acid and subjected to an acid hydrolysis reaction to obtain a compound having the structure shown in Formula 2. In this invention, the mass concentration of the concentrated hydrochloric acid is preferably 36.5%. In this invention, the molar ratio of imazalil to the volume of concentrated hydrochloric acid is preferably 40 mmol: 100~200 mL.

[0028] In this invention, the acidolysis reaction is preferably carried out under stirring conditions; the acidolysis reaction temperature is preferably 100°C, and the time is preferably 24 hours.

[0029] After the acid hydrolysis reaction solution is obtained, the present invention preferably performs post-treatment on the obtained acid hydrolysis reaction solution, and the post-treatment preferably includes the following steps: The concentrated hydrochloric acid in the acidolysis reaction solution was removed under vacuum conditions. The resulting residue was mixed with water, and the pH value was adjusted to 8 using an alkaline reagent to obtain a mixture. The resulting mixture was extracted with ethyl acetate, the organic phases were combined, and the resulting organic phases were washed, dried, concentrated, and purified by column chromatography to obtain a pure compound having the structure shown in Formula 2.

[0030] In this invention, the alkaline reagent is preferably a NaOH solution with a concentration of 1 mol / L. In this invention, the washing reagent used is preferably water followed by a 1% (w / w) NaCl aqueous solution, and the washing is preferably performed twice. In this invention, the drying is preferably performed using anhydrous sodium sulfate. In this invention, the eluent for column chromatography purification is preferably petroleum ether (PE) and ethyl acetate (EtOAc), with a volume ratio of PE:EtOAc of 10:1 to 5:1.

[0031] In this invention, a compound having the structure shown in Formula 3 undergoes an isocyanate reaction with triphosgene under the action of triethylamine to obtain a compound having the structure shown in Formula 4. In this invention, the molar ratio of the compound having the structure shown in Formula 3 to triethylamine is preferably 1:2, and the molar ratio of the compound having the structure shown in Formula 3 to triphosgene is preferably 3:1.

[0032] In this invention, the isocyanate esterification reaction is preferably carried out in an organic solvent, preferably dichloromethane. Preferably, triethylamine, a compound having the structure shown in Formula 3, and an organic solvent are first mixed, cooled to 0°C, and then triphosgene is added to carry out the isocyanate esterification reaction.

[0033] In this invention, the isocyanate esterification reaction is preferably carried out under stirring conditions; the temperature of the isocyanate esterification reaction is preferably room temperature, and the time is preferably 2 hours.

[0034] Following the isocyanate esterification reaction, the present invention preferably performs post-treatment on the obtained isocyanate esterification reaction solution, the post-treatment preferably including the following steps: The isocyanate esterification reaction solution was mixed with water and extracted with ethyl acetate. The organic phases were combined, washed, dried, concentrated, and purified by column chromatography to obtain a pure compound having the structure shown in Formula 4.

[0035] In this invention, the washing reagents used are preferably water and saline solution in sequence, and the washing is preferably performed twice. In this invention, the drying is preferably performed using anhydrous sodium sulfate. In this invention, the eluent phase for column chromatography purification is preferably PE:EtOAc, with a volume ratio of PE:EtOAc = 10:1.

[0036] In this invention, under the action of triethylamine, the compound having the structure shown in Formula 2 undergoes an addition reaction with the compound having the structure shown in Formula 4 to obtain the compound having the structure shown in Formula 5. In this invention, the molar ratio of the compound having the structure shown in Formula 2 to triethylamine is preferably 1:2; the molar ratio of the compound having the structure shown in Formula 2 to the compound having the structure shown in Formula 5 is preferably 1:1.1.

[0037] In this invention, the addition reaction is preferably carried out in an organic solvent, preferably acetonitrile. Preferably, the compound having the structure shown in Formula 2, triethylamine, and the organic solvent are first mixed, cooled to 0°C, and then the compound having the structure shown in Formula 4 is added to carry out the addition reaction.

[0038] In this invention, the addition reaction is preferably carried out under stirring conditions; the temperature of the addition reaction is preferably room temperature, and the time is preferably 16 hours.

[0039] Following the addition reaction, the present invention preferably performs post-treatment on the resulting addition reaction solution, the post-treatment preferably including the following steps: The addition reaction solution was mixed with water and extracted with ethyl acetate. The organic phases were combined, washed, dried, concentrated, and purified by column chromatography to obtain a pure compound having the structure shown in Formula 5.

[0040] In this invention, the washing reagents used are preferably water and saline solution in sequence, and the washing is preferably performed twice. In this invention, the drying is preferably performed using anhydrous sodium sulfate. In this invention, the eluent phase for column chromatography purification is preferably PE:EtOAc, with a volume ratio of PE:EtOAc = 10:1.

[0041] In this invention, under alkaline conditions, a compound having the structure shown in Formula 5 undergoes a hydrolysis reaction, and after adjusting the pH to acidic, an imazalil-coated hapten having the structure shown in Formula B is obtained. In this invention, the alkaline reagent providing the alkaline environment is preferably an aqueous solution of LiOH. In this invention, the molar ratio of the compound having the structure shown in Formula 5 to LiOH is preferably 1:12.

[0042] The present invention preferably uses a solvent to dissolve the compound having the structure shown in Formula 5, wherein the solvent is preferably a mixture of THF and MeOH, and the volume ratio of THF to MeOH is preferably 1:1.

[0043] In this invention, the hydrolysis reaction is preferably carried out under stirring conditions; the hydrolysis reaction temperature is preferably room temperature, and the time is preferably 16 hours.

[0044] In this invention, the pH value adjusted to an acidic level is preferably 4, and the acidic reagent used to adjust the pH value is preferably an HCl solution, wherein the concentration of the HCl solution is preferably 1 mol / L.

[0045] This invention provides a prochloraz-coated antigen, obtained by conjugating the aforementioned prochloraz-coated hapten with a carrier protein. In this invention, the carrier protein is ovalbumin (OVA). In this invention, the prochloraz-coated antigen has the structure shown in Formula F: Formula F.

[0046] In this invention, -OVA in Formula F represents the remaining structure of ovalbumin except for -NH2.

[0047] In this invention, the method for preparing the imazalil-coated antigen preferably includes the following steps: The hapten coated with imazalil, the activator and the organic solvent were mixed and activated to obtain the activated hapten with the structure shown in Formula D. Formula D; The activated hapten with the structure shown in Formula D, the carrier protein, and the buffer solution were mixed and coupled to obtain the imazalil-coated antigen.

[0048] In this invention, the activator is preferably N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). In this invention, the molar ratio of the imazalil-coated hapten to NHS and EDC is preferably 1:2:2.

[0049] In this invention, the organic solvent is preferably N,N-dimethylformamide.

[0050] In this invention, the activation treatment is preferably carried out under stirring conditions; the activation treatment temperature is preferably 4°C, and the activation time is preferably 10 hours.

[0051] In this invention, the molar ratio of the activated hapten having the structure shown in Formula D to the carrier protein is preferably 10-80:1, more preferably 30-60:1. In this invention, the buffer solution preferably includes carbonate buffer, phosphate buffer (PBS buffer), borate buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer, more preferably PBS buffer; the amount of the buffer solution used as a solvent is sufficient to ensure the smooth progress of the coupling reaction, and this invention does not impose any special limitations on this.

[0052] In this invention, the preferred method for mixing the activated hapten with the structure shown in Formula D, the carrier protein, and the buffer is to mix the carrier protein with the buffer to obtain a buffer solution of the carrier protein, and then add the activated hapten; the pH value of the buffer solution of the carrier protein is preferably 5 to 9, more preferably 7.4.

[0053] In this invention, the temperature of the coupling reaction is preferably 0~50℃, more preferably 4~25℃; the time is preferably 4~36h, more preferably 4~12h; and the coupling reaction is preferably carried out under stirring conditions.

[0054] Following the coupling reaction, the present invention preferably dialyzes the resulting product system to obtain the imazalil-coated antigen. In an embodiment of the present invention, the coupling reaction is specifically carried out using PBS buffer as a solvent. The dialysate used for dialysis after the coupling reaction is preferably PBS buffer, with a pH of 7-10, more preferably 7.4; a concentration of 0.01-0.2 mol / L, more preferably 0.01 mol / L; a number of dialysis cycles of 5-7, more preferably 6; and a dialysis time of 3-5 hours, more preferably 4 hours.

[0055] This invention provides an imazalil immunogen having the structure shown in Formula E: Formula E.

[0056] In this invention, -BSA in Formula E represents the remaining structure of bovine serum albumin except for -NH2.

[0057] In this invention, the imazalil immunoantigen is obtained by coupling an imazalil hapten having the structure shown in Formula A with a carrier protein; the carrier protein is preferably bovine serum albumin (BSA).

[0058] Formula A.

[0059] In this invention, the imazalil hapten having the structure shown in Formula A is preferably sourced from commercially available sources. As a specific embodiment of this invention, the imazalil hapten having the structure shown in Formula A is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and its model number is T162192.

[0060] In this invention, the method for preparing the imazalil immunogen preferably includes the following steps: The imazalil hapten with the structure shown in Formula A, the activator, and the organic solvent were mixed and activated to obtain the activated hapten with the structure shown in Formula C. Formula C; The activated hapten with the structure shown in Formula C, the carrier protein, and the buffer solution are mixed and coupled to obtain the imazalil immunogen.

[0061] In this invention, the activator is preferably N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). In this invention, the molar ratio of the imazalil hapten having the structure shown in Formula A to NHS and EDC is preferably 1:2:2.

[0062] In this invention, the organic solvent is preferably N,N-dimethylformamide.

[0063] In this invention, the activation treatment is preferably carried out under stirring conditions; the activation treatment temperature is preferably 4°C, and the activation time is preferably 10 hours.

[0064] In this invention, the molar ratio of the activated hapten having the structure shown in Formula C to the carrier protein is preferably 10-80:1, more preferably 30-60:1. In this invention, the buffer solution preferably includes carbonate buffer, phosphate buffer (PBS buffer), borate buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer, more preferably PBS buffer; the amount of the buffer solution used as a solvent is sufficient to ensure the smooth progress of the coupling reaction, and this invention does not impose any special limitations on this.

[0065] In this invention, the preferred method for mixing the activated hapten having the structure shown in Formula C, the carrier protein, and the buffer is to mix the carrier protein with the buffer to obtain a buffer solution of the carrier protein, and then add the activated hapten having the structure shown in Formula C; the pH value of the buffer solution of the carrier protein is preferably 5 to 9, more preferably 7.4.

[0066] In this invention, the temperature of the coupling reaction is preferably 0~50℃, more preferably 4~25℃; the time is preferably 4~36h, more preferably 4~12h; and the coupling reaction is preferably carried out under stirring conditions.

[0067] Following the coupling reaction, the present invention preferably dialyzes the resulting product system to obtain the imazalil immunogenicity. In an embodiment of the present invention, the coupling reaction is specifically carried out using PBS buffer as a solvent. The dialysate used for dialysis after the coupling reaction is preferably PBS buffer, with a pH value of 7-10, more preferably 7.4; a concentration of 0.01-0.2 mol / L, more preferably 0.01 mol / L; a number of dialysis cycles of 5-7, more preferably 6; and a dialysis time of 3-5 hours per cycle, more preferably 4 hours.

[0068] This invention provides a hybridoma cell line MXA 9C3, with accession number CGMCC No. 45936; the hybridoma cell line MXA 9C3 is obtained by immunizing a host with the imazalil immune antigen described in the above technical solution; the immunization method has no special requirements and can adopt a well-known technical solution in the art; the hybridoma cell line can secrete antibodies to detect imazalil and its metabolites.

[0069] This invention provides an antibody secreted by the hybridoma cell line MXA 9C3 described in the above technical solution; the accession number of the hybridoma cell line has been described above and will not be repeated here; in a specific embodiment of this invention, the antibody is antibody 9C3, whose heavy chain subtype is IgG2b and light chain subtype is λ.

[0070] This invention provides a detection product comprising the imazalil-coated hapten described in the above technical solution, or the imazalil-coated hapten obtained by the preparation method, or the imazalil-coated antigen, or the imazalil immunoantigen, or the hybridoma cell line MXA 9C3, or the antibody; the product preferably comprises one or more of reagents, kits, and colloidal gold test strips.

[0071] This invention provides the application of the imazalil-coated hapten described in the above-mentioned technical solution, or the imazalil-coated hapten obtained by the preparation method, or the imazalil-coated antigen, or the imazalil immunoantigen, or the hybridoma cell line MXA 9C3, or the antibody, or the detection product in the detection of imazalil and its metabolites; preferably, for the detection of imazalil and its metabolites in food; the metabolites preferably include metabolite BTS44595 and / or metabolite BTS44596, more preferably including metabolite BTS44595 and metabolite BTS44596.

[0072] Experiments have shown that the technical solution provided by this invention is beneficial for establishing an immunological detection method for imazalil matrix and its metabolites, enabling accurate immunodetection of imazalil matrix and its metabolites with high specificity and sensitivity.

[0073] Example 1 Synthesis of imazalil-coated hapten B according to Figure 1 The route shown is for synthesizing imazalil-coated hapten B, and the method is as follows: Imazalil (15.07 g, 40 mmol) was dissolved in concentrated hydrochloric acid (200 mL) and stirred continuously at 100 °C for 24 hours. The concentrated hydrochloric acid was removed under vacuum, water (100 mL) was added to the reaction mixture, and the pH was adjusted to 8 with 1 M NaOH. The mixture was extracted with ethyl acetate (100 mL × 3), and the combined organic phases were washed with water (200 mL × 2) and brine (200 mL × 2), dried over anhydrous sodium sulfate, and concentrated. The product was purified by column chromatography (PE:EtOAc = 10:1~5:1) to give compound 2.

[0074] Compound 3 (15.36 g, 100 mmol) and Et3N (20.20 g, 200 mmol) were dissolved in DCM (300 mL), and the mixture was cooled to 0 °C. Triphosgene (9.79 g, 33 mmol) was slowly added, and the mixture was stirred continuously at room temperature for 2 h. At the end of the reaction, water (200 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with water (200 mL × 2) and brine (200 mL × 2), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography (PE:EtOAc = 10:1) to give compound 4 (12.51 g, 87.4%).

[0075] Compound 2 (10.15 g, 36 mmol) and triethylamine (7.29 g, 72 mmol) were dissolved in acetonitrile, and the mixture was cooled to 0 °C. Compound 4 (5.67 g, 39.6 mmol) was slowly added, and the mixture was stirred continuously at room temperature for 16 hours. At the end of the reaction, water (150 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (100 mL × 3). The bound organic phases were washed with water (200 mL × 2) and brine (200 mL × 2), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography (PE:EtOAc = 10:1) to give compound 5 (13.22 g, 86.3%).

[0076] Compound 5 (2.13 g, 5 mmol) was dissolved in a mixture of THF (15 mL) and MeOH (15 mL), and stirred continuously at room temperature for 16 h. Then, 30 mL of LiOH aqueous solution (2.52 g, 60 mmol) was added. Subsequently, THF and MeOH were removed by rotary evaporation under vacuum, water (20 mL) was added to the mixture, and the pH was adjusted to 4 using 1 M HCl. The aqueous solution was filtered several times to collect the precipitate, which was then dried under vacuum to obtain imazalil-coated hapten B.

[0077] use 1 H NMR and 13 The synthesized hapten B was characterized by 1H NMR, and the resulting imazalil-coated hapten B... 1 HNMR spectrum as follows Figure 2 As shown, 13 The C NMR spectrum is as follows Figure 3 As shown.

[0078] 1 H NMR results: 1H NMR (400MHz, DMSO-d6) δ 0.82 (t, J =8.0Hz, 3H), 1.49(m, J=8.0Hz, 3H), 2.37(t, J=6.8Hz, 2H), 3.22(q, J=8Hz, 2 H), 3.57(t, J=8Hz, 2H), 4.03(t, J=8.0Hz, 2H), 6.34(s, 1H), 7.69(s, 2H), and 12.16(s,1H). 13 C NMR results: 13 C NMR (100MHz, DMSO-d6) δ 173.16, 157.04, 149.99, 129.22, 128.81, 128.79, 72.46, 48.91, 46.283, 36.33, 34.70, 21.08, and 10.98. Based on this, it was confirmed that the structure of the imazalil-coated hapten B molecule was correct, indicating that the target hapten was successfully synthesized in this invention.

[0079] The purity of the hapten was analyzed by high-performance liquid chromatography (HPLC). The HPLC chromatogram of the imazalil-coated hapten B is shown below. Figure 4 As shown, the imazalil-coated hapten B exhibited two peaks at 7.35 min and 7.57 min, with peak area percentages of 99.71% and 0.29%, respectively, demonstrating that the purity of the hapten was 99.71%.

[0080] Example 2 Synthesis of prochlorazine immunogen E The synthesis method of imazalil immunogen E is as follows: The molar ratio of hapten A to bovine serum albumin (BSA) was set at 40:1. In the experiment, 2.75 mg of hapten A, 2.81 mg of N-hydroxysuccinimide (NHS), and 4.68 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were accurately weighed and completely dissolved in 0.5 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was then reacted with magnetic stirring at 4 °C for 10 h. This process yielded an activated product containing imazalil-containing immunohapten A, which could be directly used for subsequent coupling with the carrier protein. Next, 20 mg of BSA was dissolved in 2 mL of phosphate-buffered saline (PBS) at pH 7.4. The activated hapten A solution was then slowly added to the BSA solution, and the mixture was stirred at 25 °C for 4 h to complete the coupling reaction. After the reaction was completed, the product was dialyzed six times for 4 hours each time with 0.01 mol / L PBS at pH 7.4 to purify the product and obtain a solution containing imazalil immunogen E.

[0081] The obtained imazalil immunogen E was dialyzed with pure water and then measured by MALDI-TOF-MS. The MALDI-TOF-MS spectrum of bovine serum albumin (BSA) standard is shown below. Figure 5 As shown, the MALDI-TOF-MS spectrum of bovine serum albumin (BSA) conjugate with hapten A and immunogen E is as follows. Figure 6 As shown.

[0082] according to Figure 5 and Figure 6 It can be seen that the single-charge ion peaks of BSA and imazalil immunogen E are 67208.345 and 73067.484, respectively. Based on the formula: Coupling ratio = (Complete antigen molecular weight - BSA molecular weight) / Hapten A molecular weight, the coupling ratio of imazalil immunogen E is calculated to be 1:22.

[0083] Example 3 Synthesis of imazalil-coated antigen F The method for synthesizing prochloraz-coated antigen F is as follows: The molar ratio of hapten B to ovalbumin (OVA) was set at 40:1. In the experiment, 3.47 mg of hapten B, 2.10 mg of N-hydroxysuccinimide (NHS), and 3.50 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were accurately weighed and completely dissolved in 0.5 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was then reacted with magnetic stirring at 4 °C for 10 h. This process yielded an activated product containing imazalil-containing immunohapten B, which could be directly used for subsequent coupling with the carrier protein. Next, 10 mg of OVA was dissolved in 1 mL of phosphate-buffered saline (PBS) at pH 7.4. The activated hapten B solution was then slowly added to the OVA solution, and the mixture was stirred at 25 °C for 4 h to complete the coupling reaction. After the reaction was completed, the product was dialyzed six times with 0.01 mol / L PBS at pH 7.4 for 4 hours each time to purify the product and obtain a solution containing imazalil-coated antigen F.

[0084] The obtained imazalil-coated antigen F was dialyzed with pure water and then measured by MALDI-TOF-MS. The MALDI-TOF-MS spectrum of the ovalbumin (OVA) standard is shown below. Figure 7 As shown, the MALDI-TOF-MS spectrum of ovalbumin (OVA) conjugate with hapten B and immunocoated with antigen F is as follows. Figure 8 As shown.

[0085] according to Figure 7 and Figure 8 It can be seen that the single-charge ion peaks of OVA and coated antigen F are 44578.958 and 47379.876, respectively. According to the formula: Coupling ratio = (Molecular weight of coated antigen - molecular weight of OVA) / molecular weight of hapten B, the coupling ratio of coated antigen F is 1:7.

[0086] Example 4 Preparation of monoclonal antibodies Mouse immunization: (1) Balb / C mice aged 6-8 weeks were used as experimental animals (the weight of 8-week-old Balb / C mice was 23-25g).

[0087] (2) Primary immunization: The imazalil immunogen E (concentration of 1 mg / mL) prepared in Example 2 was used as the immunogen. After being filtered through a sterile filter, an equal volume of Freund's complete adjuvant was added and stirred thoroughly to emulsify until it did not diffuse when dropped into water, thus obtaining imazalil immunogen E emulsion. The immunization strategy of the imazalil immunogen E emulsion was to inject 0.05 mg into the peritoneum of mice and inject 0.05 mg subcutaneously at multiple points on the back, with a total injection dose of 0.1 mg imazalil immunogen E emulsion / mouse.

[0088] (3) Booster immunization: Two weeks after the initial immunization, take 1 mL of the above-mentioned imazalil immunogen E solution, then add an equal volume of Freund's incomplete adjuvant, stir thoroughly to emulsify until it does not diffuse when dropped into water, and obtain imazalil immunogen E emulsion; inject the imazalil immunogen E emulsion into the peritoneum of mice with 0.05 mg and into the subcutaneous tissue of the back with multiple injections of 0.05 mg, the total injection dose is 0.1 mg imazalil immunogen E emulsion / mouse; booster immunization is performed every 14 days. Starting from the third booster immunization, blood is collected from the orbital sinus of mice 3 days after each immunization to determine the antibody titer and the inhibition rate against imazalil to detect the immunogenicity of its complete antigen. If the serum titer is high and the inhibition rate is large, it indicates that the hapten structure is reasonable and the complete antigen synthesis is successful, with excellent immunogenicity; it can be used for the preparation of monoclonal antibodies.

[0089] 2. Screening of monoclonal cells (1) Resuscitation and expansion culture of myeloma cells: One week before cell fusion, SP2 / 0 myeloma cells were resuscitated. After being removed from the liquid nitrogen tank, the cells were quickly transferred to a 37°C water bath for thawing, and then transferred to centrifuge tubes containing preheated DMEM culture medium. After centrifugation and discarding the supernatant, the cells were resuspended and cultured at 37°C and 5% CO2 until the cells grew to a state suitable for fusion.

[0090] (2) Preparation of feeder cells: Feeder cells were collected from the peritoneal cavity of mice of the same strain one day before fusion. After appropriate euthanasia, the abdomen of the mice was carefully cut open with sterile tools, and DMEM culture medium was injected into the peritoneal cavity and collected. The collected cells were centrifuged and resuspended, and then cultured in 96-well plates.

[0091] (3) Preparation of spleen cells: 24 hours before fusion, feeding of mice was stopped to reduce surrounding fat. After euthanasia of mice, blood was collected and spleens were processed to remove fat and connective tissue, and then spleen cells were blown out in DMEM.

[0092] (4) Cell fusion: PEG 2000 was used as the fusion agent to mix spleen cells and myeloma cells at a ratio of 3:1 for fusion. The fused cells were resuspended in 2% HAT complete culture medium and cultured at 37°C and 5% CO2.

[0093] (5) Screening of specific hybridoma cells: The positivity and specificity of hybridoma cells were detected by icELISA. First, positive cells were screened by indirect ELISA, and then the inhibition rate of imazalil was detected by indirect competitive ELISA to screen out highly specific hybridoma cells.

[0094] (6) Cloning of hybridoma cells: Highly specific hybridoma cells were cloned using a limited gradient dilution method. The diluted cells were cultured in 96-well plates with feeder cells, and single clones were screened by microscopic observation and then expanded for further culture.

[0095] 3. Antibody purification (1) Preparation of monoclonal antibodies: The best monoclonal cell line 9C3 was selected, and ascites antibodies were prepared by in vivo preparation of ascites tumors. BALB / c female mice were selected, pretreated with paraffin oil, and then intraperitoneally injected with a suspension of monoclonal cells.

[0096] (2) Ascites fluid collection and preliminary treatment: After the mouse abdomen swells, collect the ascites fluid and centrifuge it. Take the clear liquid for subsequent purification.

[0097] (3) Salting out purification: Non-specific proteins in the ascites fluid were removed by salting out. First, the ascites fluid was mixed with PBS, and then saturated ammonium sulfate was added for precipitation. The precipitate was then dissolved and precipitated again.

[0098] (4) Dialysis and freeze drying: The purified antibody protein is dialyzed to remove excess salt and impurities, and then freeze-dried under vacuum.

[0099] (5) Antibody preservation: The lyophilized antibody powder was dissolved in PBS, 50% glycerol was added to prepare a stock solution of 1 mg / mL, and it was stored at -20℃.

[0100] 4. Detection of titer and inhibition rate in fusion mice After the blood of the fusion mice was incubated at 37°C for 30 minutes, it was then incubated at 4°C for 2 hours. Finally, it was centrifuged at 4°C and 10,000 rpm for 5 minutes to separate the polyclonal antibody serum of imazalil, which was used for the following experiments.

[0101] The buffer solutions used in the following experiments are as follows: (1) Coating buffer (pH=9.6, 0.05M carbonate buffer): Weigh 1.5g of Na2CO3 and 2.94g of NaHCO3, and bring the volume to 1000mL with ultrapure water; (2) Phosphate buffer (0.01M, pH=7.4): Weigh 0.2g KH2PO4, 8g NaCl and 2.92g NaH2PO4·12H2O, and bring the volume to 1000mL with ultrapure water; (3) Washing buffer: Add Tween-20 to the prepared phosphate buffer to make the volume fraction of Tween-20 0.1%; (4) Sample dilution solution: Add 10 mL of Tween-20 and 1 g of gelatin to the prepared phosphate buffer solution, heat in a microwave oven to melt, and then dilute with ultrapure water to 1 L; (5) Substrate buffer (pH=5.5): Weigh 9.22g of Na2HPO4·12H2O, 2.55g of citric acid monohydrate, measure 0.5mL of Tween-20, and bring the volume up to 1L with ultrapure water; (6) Termination solution (2M H2SO4): Measure 445.6mL of distilled water and add 54.4mL of 98% (volume / volume) concentrated sulfuric acid dropwise under stirring.

[0102] Preparation of prochloraz-coated complete antigen solution: After the prepared coated antigen F was lyophilized, the powder was dissolved in PBS, and 50% glycerol was added to prepare a 1 mg / mL stock solution. The stock solution was then serially diluted to the appropriate concentration at 1:1000, 1:2000, and 1:4000 for determination.

[0103] Preparation of antiserum and antibody diluent: The antiserum prepared in the above steps was diluted with the sample and the antibody stock solution. The antiserum and antibody stock solution were serially diluted to the appropriate concentration in the patterns of 1:1000, 1:2000 and 1:4000 for determination.

[0104] Checkerboard test with antigen and antiserum: (1) Coating: Add 100 μL of the coating antigen solution of imazalil complete antigen C1 prepared in step 1 to each well of a 96-well microplate, incubate at 37°C for 3 h, wash 3 times with washing buffer, and spin dry.

[0105] (2) Competition: Add 50 μL of sample diluent to each control well; add 50 μL of standard solution of imazalil or its metabolite prepared in step 2 to each inhibition well; add the antiserum diluent prepared in step 3 to the microplate (50 μL / well), incubate at 37°C for 30 min, wash the plate 3 times with washing buffer, and spin dry.

[0106] Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 1000 times. The diluent is 0.1M sample dilution buffer with a pH of 9.6. Add 100 μL to each well, incubate at 37°C for 30 min, wash the plate 3 times with washing buffer, and spin dry.

[0107] (3) Color development: The color development solution should be prepared fresh for use. Add 15~20mg of o-phenylenediamine (OPD) and 4μL of 30wt% hydrogen peroxide to each 10mL of substrate buffer. Add 100μL of the resulting mixed solution to each well and develop the color at room temperature in the dark for 15min.

[0108] (4) Termination: Add 50 μL of stop solution to each well and measure the OD value of each well at 492 nm using an ELISA reader.

[0109] The inhibition rate was calculated using the formula: IR = (CI) / C × 100%, where IR represents the inhibition rate, I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate. The antiserum titers after the third, fourth, and fusion mouse sprint immunizations are shown in Table 1.

[0110] Table 1. Detection of antiserum titer and inhibition rate in mice

[0111] Note: In Table 1, I represents the inhibition well in the ELISA plate, C represents the control well in the ELISA plate, IR represents the inhibition rate, K represents 1000-fold, the coating source concentration is 1 mg / mL, and the inhibition concentration is 1000 ng / mL (imazalil).

[0112] As shown in Table 1, the antiserum inhibition rate of the fusion mice after booster immunization was the best, reaching 60.64%, when the coated antigen was diluted 16,000 times and the antibody was diluted 128,000 times. This indicates that the imazalil immunogen E prepared in Example 2 can be used as an immunogen to prepare anti-imazalil antibodies.

[0113] Table 2. Antibody titers and inhibition rates (mAb 9C3) after purification from ascites fluid of monoclonal hybridoma 9C3.

[0114] Note: In Table 2, I represents the inhibition well in the ELISA plate, C represents the control well in the ELISA plate, IR represents the inhibition rate, K represents 1000-fold, the coating source concentration is 1 mg / mL, and the inhibition concentration is 100 ng / mL (imazalil).

[0115] Table 2 shows the titer and inhibition rate of the antibody purified from ascites fluid produced by the monoclonal hybridoma cell line 9C3. The antiserum showed the best inhibition rate when the coating antigen was diluted 1000 times and the antibody was diluted 32000 times. The inhibition rate reached 86.4% when the concentration of imazalil was 100 ng / mL, indicating that the purified antibody has high titer and affinity.

[0116] Establishment of standard curve Preparation of standards for imazalil and its metabolites: (1) Weigh 10 mg of imazalil, BTS44595, BTS44596 and 2,4,6-trichlorophenol standard respectively, and dissolve them completely in 10 mL of methanol to obtain their corresponding 1 mg / mL standard stock solution. (2) Prepare standard solutions of 1 μg / mL imazalil, BTS44595, and BTS44596 with the 1 mg / mL standard stock solution from step (1) using sample diluent. Dilute 1 μg / mL imazalil, BTS44595, and BTS44596 to concentrations of 0.003, 0.008, 0.025, 0.076, 0.229, 0.685, 2.058, 6.173, 18.519, 55.556, 166.667, and 500 ng / mL, respectively, and then perform ic-ELISA to obtain the corresponding OD values. 450 After setting the values, plot the B / B0 value on the ordinate, perform three replicate measurements for each concentration, and plot a standard curve. The results are as follows: Figure 9 As shown.

[0117] Depend on Figure 9 It can be seen that the IC50 values ​​of 9C3 for prochloraz, BTS44595, and BTS44596 were determined by ic-ELISA. 50 The values ​​were 8.075, 10.820, and 15.065 ng / mL, respectively.

[0118] Antibody specificity detection Eleven pesticides and imidazole fungicides with structures similar to the hapten, including 2,4,6-trichlorophenol, 2,4,6-trichlorobenzoic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), amitraz, tebuconazole, and imazalil, were analyzed using the ic-ELISA procedure described above, and their IC50 values ​​were calculated. 50 The cross-reactivity rate and specific results are shown in Table 3.

[0119] Table 3. Specificity detection of imazalil 9C3 monoclonal antibody

[0120] As shown in Table 3, the cross-reactivity of the 9C3 antibody with structurally similar haptens and imidazole fungicides, including 2,4,6-trichlorophenol, 2,4,6-trichlorobenzoic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), amitraz, tebuconazole, and imazalil, is all below 0.81%, demonstrating high specificity.

[0121] In addition, 9C3 ascites fluid was purified by saturated ammonium sulfate precipitation to obtain 9C3 monoclonal antibodies, which were then diluted to a concentration of 1 μg / mL using sample dilution buffer. Finally, the 9C3 monoclonal antibodies were identified using mouse monoclonal antibody subtype identification test strips. Specific results are shown in [link to results]. Figure 10 .

[0122] Depend on Figure 10 It can be seen that the heavy chain subtype of the 9C3 antibody is IgG2b, and the light chain subtype is λ.

[0123] In summary, the 9C3 antibody provided by this invention can detect imazalil, BTS44595, and BTS44596, which is beneficial for establishing an immunological detection method for imazalil precursor and its metabolites, and realizing accurate immunological detection of imazalil precursor and its metabolites.

[0124] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A prophenate-coated hapten, characterized in that, It has the structure shown in Equation B: Formula B.

2. The preparation of a proazulene-coated hapten according to claim 1, characterized in that Includes the following steps: Imazalil having the structure shown in Formula 1 was mixed with concentrated hydrochloric acid and subjected to an acid hydrolysis reaction to obtain a compound having the structure shown in Formula 2. Formula 1; Formula 2; Under the action of triethylamine, the compound having the structure shown in Formula 3 undergoes an isocyanate reaction with triphosgene to obtain the compound having the structure shown in Formula 4. Formula 3; Equation 4; Under the action of triethylamine, the compound having the structure shown in Formula 2 undergoes an addition reaction with the compound having the structure shown in Formula 4 to obtain the compound having the structure shown in Formula 5. Formula 5; Under alkaline conditions, the compound having the structure shown in Formula 5 undergoes a hydrolysis reaction, and after adjusting the pH value to acidic, an imazalil-coated hapten having the structure shown in Formula B is obtained.

3. A prochloraz-coated antigen, obtained by conjugating a prochloraz-coated hapten according to claim 1 or a prochloraz-coated hapten prepared by the method described in claim 2 with a carrier protein; the prochloraz-coated antigen has the structure shown in formula F: Formula F; In formula F, OVA represents the remaining structure of ovalbumin except for -NH2.

4. A prochlorazine immunogen, characterized in that, It has the structure shown in E: Formula E; In formula E, -BSA represents the remaining structure of bovine serum albumin except for -NH2.

5. A hybridoma cell line MXA 9C3, characterized in that, Its accession number is CGMCCNo.45936; The hybridoma cell line MXA 9C3 was obtained by immunizing the host with the imazalil immunogen described in claim 4.

6. An antibody, characterized in that, Produced by the hybridoma cell line MXA 9C3 as described in claim 5.

7. A testing product, characterized in that, The test product includes the imazalil-coated antigen as described in claim 3 or the antibody as described in claim 6.

8. The testing product according to claim 7, characterized in that, The product is one or more of the following: reagents, kits, and colloidal gold test strips.

9. The use of the imazalil-coated antigen of claim 3 or the antibody of claim 6 in the detection of imazalil and its metabolites; wherein the metabolites are metabolite BTS44595 and / or metabolite BTS44596; The structure of BTS44595 is as follows: The structure of BTS44596 is as follows: .

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