Buqualquate hapten and artificial antigen, and preparation method and application of buqualquate hapten and artificial antigen

Abstract

The invention relates to the technical field of biochemistry, and discloses a buquantel hapten and artificial antigen as well as a preparation method and application of the buquantel hapten and artificial antigen. The structural formula of the buquantel hapten is shown as a formula (I), a formula (II), a formula (III) or a formula (IV). The buquantel artificial antigen is obtained by coupling the buquantel hapten and carrier protein. When the artificial antigen of the buquantel is used for immunizing animals, a specific antibody with high titer and high affinity can be obtained, and a foundation is laid for establishing a quick, simple, convenient, sensitive and reliable buquantel detection method; the process for preparing the buquantel antibody by using the conjugate of the hapten and the carrier protein provided by the invention is simple and economic, the established analysis method is high in sensitivity and practical value, and a method reference is provided for the work of residue monitoring, pollution prevention and control, risk assessment and the like of buquantel in food and agricultural products.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, in particular to butaquinate hapten and artificial antigen and preparation methods and applications thereof. Background Art

[0002] Buquinolate (BQ) is a quinoline-based chemically synthesized anticoccidial drug that was developed in 1966 for the prevention and treatment of coccidiosis in poultry. It has a broad-spectrum anticoccidial activity and has a good inhibitory effect on various coccidia of the genus Eimeria. The site of action is the parasite cytochrome bC1 complex, which can block electron transfer in the cytochrome system in the mitochondria of the coccidia, inhibiting the development of coccidian sporozoites and first-generation schizonts, thereby achieving the effect of preventing and treating coccidiosis. It plays an important role in the prevention and treatment of coccidiosis.

[0003] However, quinoline anticoccidial drugs have been observed to be fetotoxic at high doses, potentially causing fetal skeletal malformations. Furthermore, their use can easily lead to the development of drug resistance in coccidia, leading to a tendency to increase their use in livestock production, posing a potential threat to food safety and consumer health. Consequently, Canada has established a maximum residue limit (MRL) of 100-400 μg / kg for butaquinate in animal foods, and Japan has included it on the positive list of antimicrobial drugs, with a unified limit of 10 μg / kg. To ensure the smooth production, consumption, import, and export of animal foods in my country, there is an urgent need to establish a method for detecting butaquinate residues in food matrices for strict monitoring.

[0004] Currently, the main detection method for butaquinate is instrumental analysis methods such as liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, these instruments are bulky and expensive, require complex sample pretreatment, and place high demands on researchers' detection skills, which greatly limits their application in on-site detection of butaquinate.

[0005] Immunoassays based on antigen-antibody specific recognition are currently one of the main technologies for veterinary drug residue detection. They offer advantages such as ease of development, low cost, and simple operation, enabling high-throughput rapid screening and widespread application in on-site detection of anticoccidial drugs. However, butaquinoxaline is a weakly polar, hydrophobic compound insoluble in water or ethanol, making hapten design and antibody preparation difficult. Currently, no immunoassay methods have been reported for this compound. Therefore, it is crucial to improve the molecular hydrophilicity, rationally design haptens, and prepare high-affinity antibodies specific for butaquinoxaline, thereby developing a rapid, sensitive, and low-cost immunoassay for the detection of butaquinoxaline residues in animal foods. Summary of the Invention

[0006] The purpose of the present invention is to provide a butaquinate hapten and an artificial antigen as well as a preparation method and application thereof.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides four buquinoc haptens, whose structural formulas are shown in formula (I), formula (II), formula (III) or formula (IV): (I) (II) (III) (IV) Among them, the compound of formula (III) is based on the molecular structure of butaquinate and connected to a spacer arm containing an ether bond to increase the hydrophilicity of the hapten, and the compound of formula (IV) is based on the molecular structure of butaquinate and connected to a spacer arm containing an amide group to increase the hydrophilicity of the hapten.

[0008] In a second aspect, the present invention provides a method for preparing the butaquinate hapten, which, when the butaquinate hapten is a compound represented by formula (I), specifically comprises the following steps: S1. To 500 mL of N,N-dimethylformamide as solvent, add 25-35 g (preferably 31 g) of 4-nitro-1,2-diphenol and 100-120 g (preferably 110 g) of potassium carbonate, mix and stir, add 70-90 g (preferably 82 g) of isobutyl bromide, stir the system at 80°C for 5 hours, cool to room temperature, filter, wash the filter cake with ethyl acetate, concentrate the filtrate under reduced pressure, and then separate by silica gel column chromatography; S2. Add 300 mL of 2:1 ethanol-water solution to the product of step S1 (30-50 g, preferably 40 g) and 15-20 g (preferably 16.1 g) of ammonium chloride, stirring. Slowly add 15-20 g (preferably 16.8 g) of iron powder in batches, reflux with stirring, and monitor the reaction by TLC until the reaction is complete. Filter, concentrate the filtrate, add 1000 mL of water, extract with 2000 mL of ethyl acetate, and combine the organic phases. Wash once with 1000 mL of saturated sodium chloride, concentrate under reduced pressure, and separate by silica gel column chromatography. S3. Mix 5-9 g (preferably 7.12 g) of the product from step S2 with 5-8 g (preferably 6.49 g) of diethyl ethoxymethylene malonate, and stir at 120° C. until the reaction is complete as monitored by TLC. Cool the system to room temperature and purify by column chromatography. S4. Add 5-8 g (preferably 7 g) of the product from step S3 to 30 mL of diphenyl ether as solvent, heat the system to 280°C, and react until the reaction is complete as monitored by TLC. Cool to room temperature to precipitate crystals to obtain compound 1. S5. Add 0.3-0.4 g (preferably 0.36 g) of compound 1 and 0.25-0.3 g (preferably 0.28 g) of potassium carbonate to 10 mL of acetonitrile as solvent, and add 0.3-0.5 g (preferably 0.41 g) of tert-butyl p-bromomethylbenzoate under stirring. Stir the system at 80°C overnight until the reaction is complete as detected by TLC. Cool the system to room temperature, filter, concentrate, and purify using silica gel column chromatography. S6. Using 10 mL of dichloromethane as solvent, add 0.3-0.4 g (preferably 0.35 g) of the product of step S5 and 1 mL of trifluoroacetic acid, stir at room temperature until the reaction is complete as monitored by TLC, and concentrate to obtain the butaquinate hapten.

[0009] When the butaquinate hapten is a compound represented by formula (II), the preparation method thereof specifically comprises the following steps: S1. To 10 mL of acetonitrile as solvent, add 0.3-0.4 g (preferably 0.36 g) of compound 1, 0.25-0.3 g (preferably 0.28 g) of potassium carbonate, and 0.025-0.04 g (preferably 0.03 g) of potassium iodide. Add 0.3-0.45 g (preferably 0.38 g) of tert-butyl 6-bromohexanoate under stirring. The system is placed at 80°C to react overnight until the reaction is complete as monitored by TLC. The mixture is cooled to room temperature, filtered, concentrated, and purified by silica gel column chromatography. S2. Add 0.3-0.4 g (preferably 0.35 g) of the product from step S1 to 10 mL of dichloromethane as solvent, followed by 1 mL of trifluoroacetic acid, and stir at room temperature. After the reaction is complete as monitored by LC-MS, concentrate to obtain the butaquinate hapten.

[0010] When the butaquinate hapten is a compound represented by formula (III), the preparation method thereof specifically comprises the following steps: S1. Add 0.3-0.4 g (preferably 0.36 g) of compound 1, 0.25-0.3 g (preferably 0.28 g) of potassium carbonate, and 0.025-0.04 g (preferably 0.03 g) of potassium iodide to 10 mL of acetonitrile as solvent, and add 0.35-0.5 g (preferably 0.42 g) of tert-butyl bromodiethylene glycol acetate under stirring. The system is placed at 80°C to react overnight until the reaction is complete as monitored by TLC. The mixture is cooled to room temperature, filtered, concentrated, and purified by silica gel column chromatography. S2. Add 0.3-0.4 g (preferably 0.36 g) of the product from step S1 to 10 mL of dichloromethane as solvent, followed by 1 mL of trifluoroacetic acid, and stir at room temperature. After the reaction is complete as monitored by LC-MS, concentrate to obtain the butaquinate hapten.

[0011] When the butaquinate hapten is a compound represented by formula (IV), the preparation method thereof specifically comprises the following steps: S1. Add 0.6-0.8 g (preferably 0.72 g) of compound 1 to 15 mL of anhydrous methanol as solvent, and add 4 mol L -1 1 mL of sodium hydroxide aqueous solution was stirred at room temperature for 2 hours. Methanol was removed by rotary evaporation, 10 mL of deionized water was added, and 2 mol L -1 Adjust pH to 3-4 with dilute hydrochloric acid and filter; S2. Add 0.4-0.6 g (preferably 0.5 g) of the product from step S1, 0.3-0.5 g (preferably 0.41 g) of β-alanine tert-butyl ester hydrochloride, 0.7-1.0 g (preferably 0.86 g) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 0.55-0.62 g (preferably 0.58 g) of N,N-diisopropylethylamine to 10 mL of N,N-dimethylformamide as solvent. React at room temperature overnight under nitrogen. After the reaction is complete as monitored by TLC, add 50 mL of deionized water to the system until solids precipitate. S3. Using 10 mL of dichloromethane as solvent, add 0.25-0.35 g (preferably 0.3 g) of the product from step S2, followed by 1 mL of trifluoroacetic acid. Stir at room temperature until the reaction is complete as monitored by TLC, and then concentrate to obtain the butaquinate hapten.

[0012] In a third aspect, the present invention provides an artificial butaquinate antigen, which is obtained by coupling the butaquinate hapten with a carrier protein. The artificial butaquinate antigen can be used as an immunogen or a coating agent.

[0013] Specifically, in the above technical solution, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, bovine thyroglobulin, and human serum albumin; preferably, it is bovine serum albumin or bovine thyroglobulin.

[0014] In a fourth aspect, the present invention provides a method for preparing the butaquinate artificial antigen, wherein the carrier protein is coupled to the carboxyl carbon of the butaquinate hapten using an activated ester method to prepare the butaquinate artificial antigen.

[0015] Preferably, in the above technical solution, the coupling molar ratio of the butaquinate hapten represented by formula (I) to the carrier protein is 14.8:1.

[0016] Preferably, in the above technical solution, the coupling molar ratio of the butaquinate hapten represented by formula (II) to the carrier protein is 14.1:1.

[0017] Preferably, in the above technical solution, the coupling molar ratio of the butaquinate hapten represented by formula (II) to the carrier protein is 18.8:1.

[0018] Preferably, in the above technical solution, the coupling molar ratio of the butaquinate hapten represented by formula (II) to the carrier protein is 15.4:1.

[0019] In a fifth aspect, the present invention provides specific antibodies prepared from the butaquinate artificial antigen, including polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies.

[0020] More preferably, the amino acid sequences of the heavy and light chains of the monoclonal antibody are shown in SEQ ID NOs: 1 and 2, respectively.

[0021] In a sixth aspect, the present invention provides any of the following uses of the butaquinate hapten or the butaquinate artificial antigen: ① Application in the preparation of anti-buquinocet specific antibodies; ②Application in detecting anti-buquinocet specific antibodies.

[0022] In a seventh aspect, the present invention provides an anti-buquinoc monoclonal antibody obtained by immunizing experimental animals with the said artificial butaquinoc antigen.

[0023] Preferably, in the above technical solution, the butaquinate artificial antigen is obtained by coupling the butaquinate hapten with bovine thyroglobulin.

[0024] In an eighth aspect, the present invention provides a butaquinate detection reagent or kit prepared using the specific antibody or the monoclonal antibody.

[0025] In a ninth aspect, the present invention provides any of the following uses of the specific antibody or the monoclonal antibody: (1) Application in the design of highly hydrophobic haptens; (2) Application in the detection of butaquinate; (3) Application in the preparation of ELISA detection kit for butaquinate; (4) Application in the preparation of immunochromatographic test strips for butaquinate.

[0026] Preferably, in the above technical solution, the coating agent is obtained by coupling the butaquinate hapten with bovine serum albumin.

[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (1) The present invention discloses for the first time four novel butaquinate haptens, artificial antigens, and methods for their preparation. Immunizing animals with the butaquinate artificial antigens can produce specific antibodies with high titer and affinity. The butaquinate haptens and antibodies prepared therefrom provided by the present invention lay the foundation for establishing a rapid, simple, sensitive, and reliable method for detecting butaquinate. (2) The use of the artificial antigen provided by the present invention to prepare monoclonal antibodies against butaquinate is a simple and economical process. The established analytical method has high detection sensitivity and high practical value, providing a methodological reference for residue monitoring, pollution prevention and control, and risk assessment of butaquinate in food and agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flow chart for the preparation of the butaquinate hapten represented by formula (I) in Example 1 of the present invention; Figure 2 This is a flow chart for the preparation of the butaquinate hapten represented by formula (II) in Example 1 of the present invention; Figure 3 This is a flow chart for the preparation of the butaquinate hapten represented by formula (III) in Example 1 of the present invention; Figure 4 This is a flow chart for the preparation of the butaquinate hapten represented by formula (IV) in Example 1 of the present invention; Figure 5 1H NMR spectrum of the butaquinate hapten represented by formula (I) in Example 1 of the present invention; Figure 6 This is the 1H NMR spectrum of the butaquinate hapten represented by formula (II) in Example 1 of the present invention; Figure 7 1H NMR spectrum of the butaquinate hapten represented by formula (III) in Example 1 of the present invention; Figure 8 1H NMR spectrum of the butaquinate hapten represented by formula (IV) in Example 1 of the present invention; Figure 9 This is a MALDI-TOF-MS image of BSA in Example 2 of the present invention; Figure 10 This is a MALDI-TOF-MS image of BQ-1-BSA in Example 2 of the present invention; Figure 11 This is a MALDI-TOF-MS image of BQ-2-BSA in Example 2 of the present invention; Figure 12 This is a MALDI-TOF-MS image of BQ-3-BSA in Example 2 of the present invention; Figure 13 This is a MALDI-TOF-MS image of BQ-4-BSA in Example 2 of the present invention; Figure 14 UV-vis graphs of BQ-1-BTG, BQ-2-BTG, BQ-3-BTG, and BQ-4-BTG in Example 2 of the present invention; Figure 15 This is a standard curve diagram for detecting butaquinate using a monoclonal antibody in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] The quantitative tests in the examples of the present invention were repeated three times, and the results were averaged. The PBS buffer used in the examples of the present invention was a PBS buffer with a pH of 7.4 and a value of 0.01 M. The carbonate buffer used in the examples of the present invention was a sodium carbonate buffer with a pH of 9.6 and a value of 0.05 mol / L.

[0031] 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; NHS and EDC were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; bovine thyroglobulin (BTG) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; bovine serum albumin (BSA), Freund's complete adjuvant, and Freund's incomplete adjuvant were all purchased from Sigma.

[0032] The stationary phase used in column chromatography was 200-300 mesh silica gel.

[0033] Example 1 Preparation and Characterization of Butaquinate Hapten 1. Preparation of Butaquinoxaline Hapten 1. Preparation of the hapten of butaquinate represented by formula (I) like Figure 1 As shown, the specific steps include: S1. Using DMF as solvent, add 31 g of 4-nitro-1,2-diphenol and preferably 110 g of potassium carbonate, mix and stir, add 82 g of isobutyl bromide, stir the system at 80°C for 5 hours, cool, filter, wash with ethyl acetate, concentrate and purify. The yield is 81%; S2. Dissolve 40 g of the product from step S1 and 16.1 g of ammonium chloride in a 2:1 ethanol-water solution with stirring. Slowly add 16.8 g of iron powder in batches, reflux with stirring, and monitor the reaction by TLC. Filter, concentrate, add water, extract with ethyl acetate, and combine the organic phases. Wash with saturated sodium chloride, concentrate, and purify. The yield is 71%. S3. Mix 7.12 g of the product from step S2 with 6.49 g of diethyl ethoxymethylene malonate, stir at 120° C. until the reaction is complete as monitored by TLC, cool, and purify. The yield is 91%; S4. Dissolve 7 g of the product from step S3 in diphenyl ether, heat the system to 280°C for reaction until the reaction is complete as monitored by TLC, cool, and precipitate crystals to obtain compound 1 in a yield of 73%; S5. Add 0.36 g of the compound and 0.28 g of potassium carbonate to acetonitrile, add 0.41 g of tert-butyl p-bromomethylbenzoate while stirring, and stir the system at 80°C overnight until the reaction is complete as detected by TLC. Cool, filter, concentrate, and purify. The yield is 75%; S6. Dissolve 0.35 g of the product from step S5 in dichloromethane, then add 1 mL of trifluoroacetic acid, and stir at room temperature until the reaction is complete as monitored by TLC. Concentrate to obtain the butaquinate hapten represented by formula (I), named BQ-1, with a yield of 48%.

[0034] 2. Preparation of the hapten of butaquinate represented by formula (II) like Figure 2 As shown, the specific steps include: S1. Add 0.36 g of compound 1, 0.28 g of potassium carbonate, and 0.03 g of potassium iodide to acetonitrile, add 0.38 g of tert-butyl 6-bromohexanoate while stirring, and react the system at 80°C overnight until the reaction is complete as monitored by TLC. Cool, filter, concentrate, and purify. The yield is 72%; S2. Dissolve 0.35 g of the product from step S1 in dichloromethane, then add 1 mL of trifluoroacetic acid and stir at room temperature. After the reaction is complete, as monitored by LC-MS, concentrate the mixture to obtain the butaquinate hapten of formula (II), designated BQ-2, in a yield of 52%.

[0035] 3. Preparation of the hapten of butaquinate represented by formula (III) like Figure 3 As shown, the specific steps include: S1. Add 0.36 g of compound 1, 0.28 g of potassium carbonate, and 0.03 g of potassium iodide to acetonitrile, add 0.42 g of tert-butyl bromodiethylene glycol acetate while stirring, and place the system at 80°C to react overnight until the reaction is complete as monitored by TLC. Cool, filter, concentrate, and purify. The yield is 79%; S2. Dissolve 0.36 g of the product from step S1 in dichloromethane, then add 1 mL of trifluoroacetic acid and stir at room temperature. After the reaction is complete, as monitored by LC-MS, concentrate the solution to obtain the butaquinate hapten of formula (III), designated BQ-3, in a yield of 52%.

[0036] 4. Preparation of the hapten of butaquinate represented by formula (IV) like Figure 4 As shown, the specific steps include: S1. Dissolve 0.72 g of compound 1 in anhydrous methanol and add 4 mol L -1 1 mL of sodium hydroxide aqueous solution was stirred at room temperature for 2 hours. Methanol was removed by rotary evaporation, 10 mL of deionized water was added, and 2 mol L -1 Adjust the pH to 3-4 with dilute hydrochloric acid and filter. The yield is 92%; S2. Dissolve 0.5 g of the product from step S1, 0.41 g of β-alanine tert-butyl ester hydrochloride, 0.86 g of HATU, and 0.58 g of N,N-diisopropylethylamine in DMF and react overnight at room temperature under nitrogen. After the reaction is complete as monitored by TLC, add 50 mL of deionized water until a solid precipitates. The yield is 84%. S3. Dissolve 0.3 g of the product from step S2 in dichloromethane, then add 1 mL of trifluoroacetic acid, stir at room temperature, and complete the reaction as monitored by TLC. Then concentrate to obtain the butaquinate hapten represented by formula (IV), named BQ-4, with a yield of 58%.

[0037] II. Characterization of the Butaquinate Hapten 1. Nuclear magnetic resonance identification The nuclear magnetic resonance detection results of the hapten of butaquinate shown in the above formula (I) are as follows Figure 5 The results are analyzed as follows: 1HNMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 8.77 (s, 1H), 7.87 (d, J=8.0 Hz, 2H), 7.54 (s, 1H), 7.31 (d, J=8.0 Hz, 2H), 6.87 (s, 1H), 5.72 (s, 2H), 4.18 (q, J=7.1 Hz, 2H), 3.75 (d, J=8.0 Hz, 2H), 3.64 (d, J=4.0 Hz, 2H), 2.01-1.98 (m,1H), 1.82-1.79 (m,1H), 1.24 (t, J=7.1 Hz, 3H), 0.94 (d, J=6.7 Hz, 6H), 0.85(d, J=6.7 Hz, 6H); analysis of the above data showed that the synthesized product was the target product.

[0038] The nuclear magnetic resonance detection results of the hapten of butaquinate shown in the above formula (II) are as follows Figure 6 The results are analyzed as follows: 1 HNMR (600 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.54 (s, 1H), 7.61 (s, 1H), 7.08 (s,1H), 4.37 (t, J=7.3 Hz, 2H), 4.21 (q, J=7.1 Hz, 2H), 3.96 (d, J=6.3 Hz, 2H),3.84 (d, J=6.3 Hz, 2H), 2.21 (t, J=7.3 Hz, 2H), 2.10 (m, 2H), 1.81-1.72 (m,2H), 1.60-1.52 (m, 2H), 1.37-1.31 (m, 2H), 1.28 (t, J=7.1 Hz, 3H), 1.04 (2d, J=6.0Hz, 12H); analysis of the above data showed that the synthesized product was the target product.

[0039] The results of the nuclear magnetic resonance detection of the hapten of the above-mentioned formula (III) are as follows: Figure 7 The results are analyzed as follows: 1HNMR (500 MHz, DMSO-d6) δ 12.55 (s, 1H), 8.48 (s, 1H), 7.60 (s, 1H), 7.16 (s,1H), 4.56 (t, J=5.0 Hz, 2H), 4.20 (2d, J=5.0 Hz, 4H), 1.03 (2d, J=5.0 Hz, 12H); analysis of the above data showed that the synthesized product was the target product.

[0040] The results of nuclear magnetic resonance detection of the hapten of the above formula (IV) are as follows: Figure 8 The results are analyzed as follows: 1 HNMR (400 MHz, DMSO-d6) δ 12.30 (d, J=6.0 Hz, 1H), 12.21 (s, 1H), 10.25 (t, J=5.8 Hz, 1H), 8.55 (d, J=6.2 Hz, 1H), 7.51 (s, 1H), 7.05 (s, 1H), 3.81 (2d, J=8.0 Hz, 4H), 3.47 (m, 2H), 2.05 (m, 2H), 0.98 (2d, =3.7 Hz, 12H); analysis of the above data showed that the synthesized product was the target product.

[0041] Example 2 Preparation and Characterization of Butaquinoxaline Artificial Antigen The difference between the preparation methods of the immunogen and the coating agent lies in the type of carrier protein used. The immunogen carrier protein mainly uses BTG, and the coating agent carrier protein mainly uses BSA. The coupling method used is the activated ester method.

[0042] 1. Synthesis and identification of butaquinate coating 1. Preparation of Butaquinate Coating S1. Dissolve 9 mg of the compound of formula (I) prepared in Example 1 in 1 mL of DMF, add 3.3 mg of NHS and 5.3 mg of EDC, and stir at room temperature overnight to obtain Solution I; S2. Add 20 mg of BSA to 10 mL of CB buffer and dissolve thoroughly to obtain Solution II. S3. Slowly add solution I dropwise to solution II, stir slowly at 4°C for 16 h, then place into a dialysis bag and dialyze in PBS buffer at 4°C for 72 h (change the water 6 times in between) to obtain the original solution of butaquinate coating, which was stored at -20°C.

[0043] The butaquinate coating synthesized from the compound represented by the above formula (I) was originally designated as BQ-1-BSA.

[0044] Similarly, the above method was used to prepare the butaquinate coating source of the compound represented by formula (II), which was recorded as BQ-2-BSA; the butaquinate coating source of the compound represented by formula (III) was prepared, which was recorded as BQ-3-BSA; and the butaquinate coating source of the compound represented by formula (IV) was prepared, which was recorded as BQ-4-BSA.

[0045] 2. Identification of the Butaquinoxaline Coating Source The binding ratios of BSA to hapten in BQ-1-BSA, BQ-2-BSA, BQ-3-BSA, and BQ-4-BSA solutions were determined by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS).

[0046] Binding ratio = [M(conjugate) - M(protein)] / M(hapten).

[0047] The molecular weight of the hapten in formula (I) is 495.57, and the MALDI-TOF-MS of the carrier BSA (results are shown in Figure 9 The molecular weight of the conjugate BQ-1-BSA was 65761.23, and the MALDI-TOF-MS results were as follows: Figure 10 The molecular weight of the conjugate is higher than that of the carrier protein BSA, indicating that BQ-1-BSA was successfully synthesized.

[0048] Calculations showed that the binding ratio of BSA to hapten was 14.8:1, meaning that an average of 14.8 haptens were coupled to one BSA molecule in BQ-1-BSA.

[0049] The molecular weight of the hapten of formula (II) is 475.58, and the MALDI-TOF-MS of the conjugate BQ-2-BSA (results are shown in Figure 11 The molecular weight of the conjugate is 72448.48, which is higher than that of the carrier protein BSA, indicating that BQ-2-BSA was successfully synthesized.

[0050] Calculations showed that the binding ratio of BSA to hapten was 14.1:1, meaning that an average of 14.1 haptens were coupled to one BSA molecule in BQ-2-BSA.

[0051] The molecular weight of the hapten of formula (III) is 507.58. The MALDI-TOF-MS of the conjugate BQ-3-BSA (results are shown in Figure 12 The molecular weight of the conjugate is higher than that of the carrier protein BSA, indicating that BQ-3-BSA was successfully synthesized.

[0052] Calculations showed that the binding ratio of BSA to hapten was 18.8:1, meaning that an average of 18.8 haptens were coupled to one BSA molecule in BQ-3-BSA.

[0053] The molecular weight of the hapten of formula (IV) is 404.46. The MALDI-TOF-MS of the conjugate BQ-4-BSA (results are shown in Figure 13 The molecular weight of the conjugate is higher than that of the carrier protein BSA, indicating that BQ-4-BSA was successfully synthesized.

[0054] Calculations showed that the binding ratio of BSA to hapten was 15.4:1, meaning that an average of 15.4 haptens were coupled to one BSA molecule in BQ-2-BSA.

[0055] 2. Synthesis and identification of butaquinate immunogen 1. Preparation of Immunogen The preparation method is similar to that of the aforementioned butaquinate coating agent, except that BTG is used instead of BSA. The butaquinate immunogens synthesized from the compounds represented by Formula (I) to Formula (IV) are designated BQ-1-BTG, BQ-2-BTG, BQ-3-BTG, and BQ-4-BTG, respectively.

[0056] 2. Identification of immunogens Ultraviolet-visible spectroscopy (UV-vis) was used to identify the BQ-BTG conjugate. Figure 14 As shown, compared with the carrier protein BTG and the hapten, the four immunogens produced characteristic absorption peaks at 257-261 nm, indicating that the BQ-1-BTG, BQ-2-BTG, BQ-3-BTG, and BQ-4-BTG immunogens were all successfully synthesized.

[0057] Example 3 Preparation of monoclonal antibody against butaquinate 1. Animal Immunization 1. Eight SPF-grade Balb / c female mice aged 6-8 weeks and weighing 30-40 g were immunized with the BQ-1-BTG, BQ-2-BTG, BQ-3-BTG, and BQ-4-BTG solutions prepared in Example 2, respectively.

[0058] 2. The first vaccination uses Freund's complete adjuvant, which is injected subcutaneously at multiple points on the back of the neck, with an immunization dose of 100 μg / animal; additional vaccination is performed once every 3 weeks, for a total of 2 times, and the adjuvant is changed to Freund's incomplete adjuvant. The immunization dose and injection method remain unchanged; shock immunization is performed 4 days before fusion, and the injection method is changed to intraperitoneal injection.

[0059] 3. Blood was collected from the orbital venous plexus of mice within 7-10 days after each immunization. The titer of antiserum was detected by indirect ELISA, and the inhibition level of antiserum was detected by indirect competitive ELISA.

[0060] 2. Preparation of Monoclonal Antibodies 1. Select mice with the best antiserum performance in each immunization group for cell fusion. Fusion of splenocytes from mice that were immunized 4 days prior with myeloma cell suspension at a 1:1 volume ratio. After 7 days of culture, the cell supernatant was tested for positivity and inhibition using an indirect competitive ELISA.

[0061] 2. Cell wells with high positive values and inhibition rates were screened, and the cell clusters in the wells were subcloned for 3-5 rounds using the limiting dilution method to obtain a hybridoma cell line that can stably secrete butaquinate-specific antibodies. This cell line was named 10E7.

[0062] 3. Prepare monoclonal antibodies in large quantities through in vivo induction method. Collect mouse ascites, centrifuge and separate the supernatant. Purify using Protein A immunoaffinity column method. Store the purified antibodies at -20℃.

[0063] The amino acid sequences of the heavy and light chains of the monoclonal antibody secreted by 10E7 are shown in SEQ ID NOs: 1 and 2, respectively.

[0064] Example 4 Affinity determination of butaquinate monoclonal antibody 1. Screening of coating materials BQ-1-BSA, BQ-2-BSA, BQ-3-BSA and BQ-4-BSA were used as coating agents, and the titer of monoclonal antibody against butaquinate was determined by homologous and heterologous coating methods, respectively.

[0065] 2. Screening of the best coating agent-antibody pair The monoclonal antibody and its corresponding best-reacting coating agent were serially diluted and plated using the checkerboard method. The butaquinate standard was substituted into the competitive reaction to preliminarily determine the affinity of the monoclonal antibody. The best-performing coating agent-antibody pair was selected and its optimal working concentration was determined.

[0066] 3. IC 50 Determination of value According to the screening results, add the optimal working concentration of antibody dilution to the system, substitute different concentrations of standard solution, establish a standard curve, and determine the optimal IC value of the monoclonal antibody. 50 value.

[0067] The specific steps are as follows: S1. Coating: The antigen in Example 2 was serially diluted starting from 1 μg / mL in 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37°C for 2 h. S2, washing, pouring off the solution in the plate, drying, washing 3 times, 3 minutes each time, drying; S3, blocking, add 150 μL / well blocking solution, react at 37℃ for 1 h, spin dry and set aside; S4. Prepare the butaquinate standard solution: prepare the butaquinate standard into a 2 μg / mL stock solution with 0.01 mol / L, pH 7.4 PBS solution. Before adding the sample, dilute the stock solution serially with PBS solution to the working concentrations of 0.1 ng / mL, 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. S5. Add samples. Add 50 μL of each concentration of butaquinate standard working solution to each well. Dilute the purified monoclonal antibody to the optimal working concentration and add 50 μL to each well. Incubate at 37°C for 30 min. After thorough washing, add 100 μL / well of 1:5000 diluted HRP-goat anti-mouse IgG and incubate at 37°C for 30 min. Wash and spin dry. S6, color development: remove the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at room temperature in the dark for 15 minutes; S7, stop and measure, add 50 μL stop solution to each well to stop the reaction, and then measure the OD of each well with a microplate reader. 450 value; S8. Data processing: The logarithm of the concentration of the butaquinate standard is used as the horizontal axis, and the corresponding OD values after the competition reaction of different concentrations of the standard are recorded as the vertical axis. The competition standard curve is fitted using a four-parameter equation to calculate the IC 50 value, IC 50 The lower the value, the higher the affinity of the antibody for the standard.

[0068] The results of affinity determination of monoclonal antibodies obtained by hybridoma technology are as follows Figure 15 As shown, the IC of monoclonal antibody to butaquinate was calculated from the standard curve. 50 The value is 0.99 ng / mL.

[0069] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Butaquinoxaline hapten, characterized in that Its structural formula is shown in formula (I), formula (II), formula (III) or formula (IV): （I） （Ⅱ） （Ⅲ） （Ⅳ）。 2. The method for preparing the butaquinate hapten according to claim 1, wherein When the butaquinate hapten is a compound represented by formula (I), the method specifically comprises the following steps: S1. To 500 mL of N,N-dimethylformamide as solvent, add 25-35 g of 4-nitro-1,2-benzenediol and 100-120 g of potassium carbonate, mix and stir, add 70-90 g of isobutyl bromide, stir the system at 80°C for 5 hours, cool to room temperature, filter, wash the filter cake with ethyl acetate, concentrate the filtrate under reduced pressure, and then separate by silica gel column chromatography; S2. Add 30-50 g of the product from step S1 and 15-20 g of ammonium chloride to 300 mL of ethanol-water solution as solvent, stir, slowly add 15-20 g of iron powder in batches, reflux with stirring, and monitor by thin layer chromatography until the reaction is complete; filter, concentrate the filtrate, add 1000 mL of water, extract with 2000 mL of ethyl acetate, and combine the organic phases; wash once with 1000 mL of saturated sodium chloride, concentrate under reduced pressure, and separate by silica gel column chromatography; The volume ratio of ethanol to water in the ethanol-water solution is 2:1; S3. Mix 5-9 g of the product from step S2 with 5-8 g of diethyl ethoxymethylene malonate, and stir at 120° C. until the reaction is complete as monitored by TLC. Cool the system to room temperature and purify by column chromatography; S4. Add 5-8 g of the product from step S3 to 30 mL of diphenyl ether as solvent, heat the system to 280°C, react until the reaction is complete as monitored by TLC, and cool to room temperature; S5. Add 0.3-0.4 g of the product from step S4 and 0.25-0.3 g of potassium carbonate to 10 mL of acetonitrile as solvent, and add 0.3-0.5 g of tert-butyl p-bromomethylbenzoate under stirring. Stir the system at 80° C. overnight until the reaction is complete as detected by TLC. Cool the system to room temperature, filter, concentrate, and purify using silica gel column chromatography. S6. Add 0.3-0.4 g of the product from step S5 and 1 mL of trifluoroacetic acid to 10 mL of dichloromethane as solvent, stir at room temperature until the reaction is complete as monitored by TLC, and concentrate to obtain the butaquinate hapten represented by formula (I); When the butaquinate hapten is a compound represented by formula (II), the method specifically comprises the following steps: S1. Using 10 mL of acetonitrile as solvent, add 0.3-0.4 g of the product of step S4 in claim 2, 0.25-0.3 g of potassium carbonate, and 0.025-0.04 g of potassium iodide. Add 0.3-0.45 g of tert-butyl 6-bromohexanoate while stirring. Place the system at 80°C to react overnight until the reaction is complete as monitored by TLC. Cool to room temperature, filter and concentrate, and purify using silica gel column chromatography; S2. Add 0.3-0.4 g of the product from step S1 to 10 mL of dichloromethane as a solvent, followed by 1 mL of trifluoroacetic acid, and stir at room temperature; after monitoring the completion of the reaction by liquid chromatography-mass spectrometry, concentrate to obtain the butaquinate hapten represented by formula (II); When the butaquinate hapten is a compound represented by formula (III), the method specifically comprises the following steps: S1. Use 10 mL of acetonitrile as solvent, add 0.3-0.4 g of the product of step S4 in claim 2, 0.25-0.3 g of potassium carbonate, and 0.025-0.04 g of potassium iodide, add 0.35-0.5 g of tert-butyl bromodiethylene glycol acetate under stirring, place the system at 80°C to react overnight, and react until the reaction is complete as monitored by TLC. Cool to room temperature, filter and concentrate, and purify using silica gel column chromatography; S2. Add 0.3-0.4 g of the product from step S1 to 10 mL of dichloromethane as solvent, followed by 1 mL of trifluoroacetic acid, and stir at room temperature. After the reaction is complete as monitored by LC-MS, concentrate to obtain the butaquinate hapten represented by formula (III); When the butaquinate hapten is a compound represented by formula (IV), the method specifically comprises the following steps: S1, using 15 mL of anhydrous methanol as solvent, add 0.6-0.8 g of the product of step S4 in claim 2, and add 4 mol L -1 Sodium hydroxide aqueous solution, stirred at room temperature for 2 hours; methanol was removed by rotary evaporation, 10 mL of deionized water was added, and 2 molL -1 Adjust pH to 3-4 with dilute hydrochloric acid and filter; S2. Add 0.4-0.6 g of the product from step S1, 0.3-0.5 g of β-alanine tert-butyl ester hydrochloride, 0.7-1.0 g of HATU, and 0.55-0.62 g of N,N-diisopropylethylamine to 10 mL of N,N-dimethylformamide as solvent, and react at room temperature overnight under nitrogen protection. After the reaction is complete as monitored by TLC, add 50 mL of deionized water to the system until solid precipitates. S3. Using 10 mL of dichloromethane as solvent, add 0.25-0.35 g of the product from step S2, followed by 1 mL of trifluoroacetic acid. Stir at room temperature until the reaction is complete as monitored by TLC, and then concentrate to obtain the butaquinate hapten represented by formula (IV).

3. Butylquinate artificial antigen, characterized in that Obtained by coupling the butaquinate hapten according to claim 1 with a carrier protein; Wherein, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, bovine thyroglobulin, and human serum albumin; preferably bovine serum albumin or bovine thyroglobulin.

4. The method for preparing the butaquinate artificial antigen according to claim 3, characterized in that: The carrier protein is coupled to the carboxyl carbon of the butaquinate hapten according to claim 1 by using an activated ester method.

5. The method according to claim 4, characterized in that The coupling molar ratios of the butaquinate hapten represented by formula (I) to formula (IV) and the carrier protein are 14.8:1, 14.1:1, 18.8:1 and 15.4:1, respectively.

6. The specific antibody against butaquinoxaline prepared from the butaquinoxaline artificial antigen according to claim 3, characterized in that: including polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies; More preferably, the amino acid sequences of the heavy and light chains of the monoclonal antibody are shown in SEQ ID NOs: 1 and 2, respectively.

7. Any of the following uses of the butaquinate hapten according to claim 1 or the butaquinate artificial antigen according to claim 3: ① Application in the preparation of anti-buquinocet specific antibodies; ②Application in detecting anti-buquinocet specific antibodies.

8. Anti-buquinocet monoclonal antibody, characterized in that Obtained by immunizing experimental animals with the artificial butaquinate antigen according to claim 3; Preferably, the butaquinate artificial antigen is obtained by coupling the butaquinate hapten with bovine thyroid protein.

9. A butaquinate detection reagent or kit prepared from the specific antibody according to claim 6 or the monoclonal antibody according to claim 8.

10. Any of the following uses of the specific antibody according to claim 6 or the monoclonal antibody according to claim 8: (1) Application in the design of highly hydrophobic haptens; (2) Application in the detection of butaquinate; (3) Application in the preparation of ELISA detection kit for butaquinate; (4) Application in the preparation of immunochromatographic test strips for butaquinate.

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