A hapten and a complete antigen of a nitroimidazole drug, a preparation method thereof, and applications thereof

Abstract

The present invention relates to the technical field of food safety, and specifically relates to a nitroimidazole drug hapten and complete antigen, their preparation methods and applications. In the present invention, 2-methyl-5-nitroimidazole is used as a raw material to carry out nucleophilic substitution with 4-bromo-1-butyne under alkaline conditions to synthesize intermediate 1, and intermediate 1 reacts with ethyl azidoacetate through a click reaction to synthesize intermediate 2; intermediate 2 is hydrolyzed under alkaline conditions to synthesize a nitroimidazole drug hapten. On the basis of the hapten, a carrier protein is coupled to synthesize a complete antigen. The complete antigen can expose the nitroimidazole structure as an antigenic determinant, and a broad-spectrum nitroimidazole drug monoclonal antibody is screened and obtained. The monoclonal antibody provided by the present invention is applied to a colloidal gold chromatography test strip, and a sample treatment scheme for extraction with ethyl acetate and salting out with sodium chloride and a rapid detection scheme for a colloidal gold chromatography test strip kit are established, which can be used for the rapid screening of nitroimidazole drugs in poultry eggs and egg products.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and particularly relates to a nitroimidazole drug hapten and antigen, and a preparation method and application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] The problem of veterinary drug residues in poultry eggs and egg products is one of the main safety risks of such products, which is mainly related to the excessive and out-of-range use of veterinary drugs in the poultry breeding process. Nitroimidazole antiprotozoal drugs, including metronidazole, dimetridazole, hydroxy metronidazole, hydroxydimetridazole, etc., can be used to treat avian histomoniasis, etc. The extensive use of this drug will lead to its residues in poultry eggs. After consumers eat poultry eggs containing nitroimidazole drug residues, the drugs will accumulate in the human body, thereby affecting the health of consumers. In response to the regulatory requirements for nitroimidazoles in animal-derived foods, relevant countries and organizations have formulated relevant regulatory standards. The "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Foods" (GB 31650-2019) in China stipulates that metronidazole, dimetridazole, etc. are allowed for therapeutic use, but shall not be detected in animal-derived foods. In order to further monitor the quality and safety of poultry egg products, since 2021, the national food safety sampling inspection tasks have detected nitroimidazole drugs in fresh eggs (chicken eggs, duck eggs, quail eggs, goose eggs, pigeon eggs), and included this indicator in the risk monitoring plan for reprocessed egg products in 2023.

[0004] At present, the relevant detection standards for nitroimidazole drugs in poultry eggs and egg products include "National Food Safety Standard - Determination Method for Residues of Nitroimidazole Drugs in Animal Foods" (GB 31658.23 - 2022), "Determination of Metronidazole, Dimetridazole and Their Metabolic Residues in Animal Foods by Liquid Chromatography - Tandem Mass Spectrometry" (Announcement No. 1025 - 2 - 2008 of the Ministry of Agriculture), "Determination Method for Residues of Multiple Alkaline Drugs in Animal - Derived Foods by Liquid Chromatography - Mass Spectrometry / Mass Spectrometry" (SN / T 2624 - 2010), etc. The above - mentioned standard methods all use liquid chromatography - tandem mass spectrometry for determination. Although they can ensure the detection accuracy, the detection efficiency is relatively low, including cumbersome pretreatment steps, long experimental periods, the need for large - scale instrument equipment and professional technical personnel, and relatively high detection costs. Compared with instrumental methods, the rapid method based on immunoassay is fast and simple to operate, does not require large - scale equipment, has low requirements for operators, saves time and effort, and has relatively low detection costs. It has the characteristics of rapidity, low cost, high throughput and strong specificity, and is suitable for the preliminary screening of a large number of samples and on - site detection. The design of antigens and the preparation of antibodies are the keys to the establishment of immunoassay methods. Therefore, it is particularly important to synthesize haptens of nitroimidazole drugs that can produce more sensitive antibodies and establish immunoassay methods. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a hapten and a complete antigen of nitroimidazole drugs, their preparation methods and applications.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a hapten of nitroimidazole drugs is provided, and its structural formula is shown as Formula I:

[0008]

[0009] In the second aspect of the present invention, a preparation method of the hapten of nitroimidazole drugs is provided, including:

[0010] (1) 2 - Methyl - 5 - nitroimidazole and 4 - bromo - 1 - butyne undergo nucleophilic substitution under alkaline conditions to synthesize Intermediate 1;

[0011] (2) Intermediate 1 and ethyl azidoacetate undergo a click reaction to synthesize Intermediate 2;

[0012] (3) Intermediate 2 is hydrolyzed under alkaline conditions to synthesize the hapten of nitroimidazole drugs;

[0013] The reaction route is as follows:

[0014]

[0015] In one or more embodiments, in step (1), the method for synthesizing intermediate 1 by nucleophilic substitution of 2-methyl-5-nitroimidazole and 4-bromobutyne under alkaline conditions includes:

[0016] Disperse 2-methyl-5-nitroimidazole, 4-bromobutyne, and potassium carbonate in N,N-dimethylformamide (DMF), and heat the reaction to synthesize intermediate 1.

[0017] Preferably, the mass ratio of 2-methyl-5-nitroimidazole, 4-bromobutyne, and potassium carbonate is 4.5 - 5.5:6 - 7:9 - 12, preferably 5:6.28:10.87.

[0018] Preferably, the concentration of 2-methyl-5-nitroimidazole in DMF is 45 - 55 g / L, preferably 50 g / L.

[0019] Preferably, the temperature of the heating reaction is 65 - 80 °C, preferably 70 °C, and the reaction time is 20 - 30 h, preferably 24 h.

[0020] In one or more embodiments, in step (2), the method for synthesizing intermediate 2 by click reaction of intermediate 1 and ethyl azidoacetate includes:

[0021] Disperse intermediate 1 and ethyl azidoacetate in a mixed solution of DMF and water, and then add sodium ascorbate and copper sulfate pentahydrate as catalysts, and heat the reaction to synthesize intermediate 2.

[0022] Preferably, the mass ratio of intermediate 1 and ethyl azidoacetate is 7.5 - 8.5:6.5 - 8, preferably 8:7.

[0023] Preferably, in the mixed solution of DMF and water, the volume ratio of DMF to water is 30:6 - 8, preferably 30:7.2.

[0024] Preferably, the concentration of intermediate 1 in the mixed solution of DMF and water is 0.2 - 0.23 g / L, preferably 0.215 g / L.

[0025] Preferably, the temperature of the heating reaction is 55 - 70 °C, preferably 60 °C, and the reaction time is 3 - 5 h, preferably 4 h.

[0026] In one or more embodiments, in step (3), the method for hydrolyzing intermediate 2 under alkaline conditions to synthesize a nitroimidazole drug hapten includes:

[0027] The intermediate is heated in an aqueous sodium hydroxide ethanol solution to synthesize a nitroimidazole drug hapten.

[0028] Preferably, the temperature of the heating reaction is 65-80°C, preferably 70°C, and the reaction time is 3-5 h, preferably 4 h.

[0029] In a third aspect of the present invention, there is provided a nitroimidazole drug complete antigen, which is obtained by conjugating the above-mentioned nitroimidazole drug hapten with a carrier protein.

[0030] In one or more embodiments, the carrier protein includes bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.

[0031] In a fourth aspect of the present invention, there is provided a nitroimidazole drug-specific antibody, which is a polyclonal antibody or a monoclonal antibody.

[0032] In one or more embodiments, the nitroimidazole drug-specific antibody is a monoclonal antibody prepared by immunizing an experimental animal (such as a mouse) with the nitroimidazole drug complete antigen as an immunogen and supplemented with an adjuvant.

[0033] In a fifth aspect of the present invention, there is provided a nitroimidazole drug hapten, or a nitroimidazole drug complete antigen or a nitroimidazole drug-specific antibody, or a nitroimidazole drug detection reagent or kit prepared therefrom.

[0034] In one or more embodiments, the nitroimidazole drugs include: metronidazole, dimetridazole, hydroxy metronidazole, hydroxy dimetridazole.

[0035] In a sixth aspect of the present invention, there is provided the use of a nitroimidazole drug hapten, or a nitroimidazole drug complete antigen or a nitroimidazole drug-specific antibody, or a detection reagent or kit in the ELISA immunoassay detection of nitroimidazole drugs.

[0036] In one or more embodiments, the ELISA immunoassay method is used to detect the residue of nitroimidazole drugs in actual samples.

[0037] Preferably, the actual samples include poultry eggs and egg products.

[0038] In a seventh aspect of the present invention, there is provided the use of a nitroimidazole drug hapten, or a nitroimidazole drug complete antigen or a nitroimidazole drug-specific antibody, or a detection reagent in the preparation of a colloidal gold test strip for nitroimidazole drugs.

[0039] The beneficial effects of the present invention:

[0040] (1) In the present invention, 2-methyl-5-nitroimidazole is used as a raw material to react with 4-bromo-1-butyne under alkaline conditions for nucleophilic substitution to synthesize intermediate 1. Intermediate 1 undergoes a click reaction with ethyl azidoacetate to synthesize intermediate 2. Intermediate 2 is hydrolyzed under alkaline conditions to synthesize a nitroimidazole drug hapten. Based on the hapten, a carrier protein is conjugated to synthesize a complete antigen, and the complete antigen can expose the nitroimidazole structure as an antigenic determinant, and a broad-spectrum nitroimidazole drug monoclonal antibody is screened and obtained.

[0041] (2) The monoclonal antibody provided by the present invention is applied to a colloidal gold chromatography test strip, and this colloidal gold chromatography test strip can be used for the rapid screening of nitroimidazole drugs in poultry eggs and egg products. The detection limit of this monoclonal antibody is 1.0 μg / kg, which meets the limit regulations of nitroimidazoles in the current national standard "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Foods" (GB 31650-2019). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0043] Figure 1 It is the mass spectrometry image of the nitroimidazole hapten shown in formula (I);

[0044] Figure 2 It is the indirect competitive ELISA standard curve of the antibody against nitroimidazole drugs detected during the animal immunization process (using metronidazole as a benchmark);

[0045] Figure 3 It is the schematic structural diagram of the nitroimidazole drug colloidal gold chromatography test strip prepared in Example 4;

[0046] Figure 4 It is a method for detecting the residue of nitroimidazole drugs in a sample using the nitroimidazole drug colloidal gold chromatography detection device prepared in Example 4, where A is the reaction of the sample extract with the nitroimidazole monoclonal antibody-colloidal gold label; B is the chromatographic reaction of the mixed test solution with the nitroimidazole drug colloidal gold chromatography test strip;

[0047] Figure 5 It is the schematic diagram of the possible results of the nitroimidazole drug chromatography test strip in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0049] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example 1

[0052] Preparation of the hapten of nitroimidazole drugs:

[0053] (1) Add 5 g of 2-methyl-5-nitroimidazole, 6.28 g of 4-bromo-1-butyne, 10.87 g of potassium carbonate, and 100 mL of DMF to a 250 mL clean single-necked flask in sequence, and react at 70 °C overnight. After the reaction is completed as detected by TLC, perform post-treatment. After the reaction is completed, rotary evaporate to remove the solvent, and purify by column chromatography to obtain the transparent crystal intermediate 1.

[0054] (2) Add intermediate 1 (800 mg), 700 mg of ethyl azidoacetate, 180 mg of copper sulfate pentahydrate, 250 mg of L-ascorbic acid, 7.2 mL of water, and dissolve in 30 mL of DMF to a 100 mL clean single-necked flask, and react at 60 °C for 4 h. After the reaction is completed as detected by TLC, perform post-treatment. Add water to the reaction solution, extract twice with ethyl acetate, combine the organic phases, and then purify by column chromatography to obtain intermediate 2.

[0055] (3) Add intermediate 2 (450 mg), 90 mg of sodium hydroxide, 10 mL of water, and 40 mL of ethanol to a 100 mL clean single-necked flask in sequence, and react at 70 °C for 4 h. After the reaction is completed as detected by TLC, perform post-treatment. Adjust the pH of the reaction solution to neutral, evaporate the solvent to dryness by rotary evaporation, and purify by column chromatography to obtain the light yellow powdery solid, which is the hapten of nitroimidazole drugs. The hapten was identified by mass spectrometry, and the obtained mass spectrum is shown in Figure 1 . It can be seen from the mass spectrum that the molecular ion peak of the hapten is ESI-[M-H] - : 279.24, and it is the highest peak, which is consistent with the molecular weight of the hapten of 280.24, indicating that the nitroimidazole hapten shown in formula (I) was successfully synthesized.

[0056] The reaction route is:

[0057]

[0058] Example 2

[0059] Preparation of nitroimidazole drugs complete antigen:

[0060] Weigh 50 mg of nitroimidazole drugs hapten, dissolve it in 2.5 mL of DMF, add 25 mg of N-hydroxysuccinimide (NHS) and 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), react at room temperature for 6 h to prepare an activation solution; take 45 mg of bovine serum albumin (BSA), dissolve it in 3 mL of 0.1 M boric acid buffer with pH 9.0, add 1 mL of DMF and 0.6 mL of the above activation solution, react at room temperature for 4 h, then dialyze with PBS (0.01 moL / L phosphate buffer with pH = 7.4), change the solution once every 4 h, change the solution 7 - 8 times, after dialysis, centrifuge at 4000 r / min for 5 min, take the supernatant to prepare the nitroimidazole drugs complete antigen, that is, nitroimidazole hapten-BSA conjugate, and store it at -20 °C.

[0061] Example 3

[0062] (1) Animal immunization

[0063] Select healthy BALB / c mice at 6 - 8 weeks old for immunization. Mix the nitroimidazole drugs complete antigen obtained in Example 2 with an equal amount of Freund's adjuvant and emulsify it, then perform multi-point subcutaneous injection immunization on the neck and back of BALB / c mice (except for the booster immunization). Use complete Freund's adjuvant for the first immunization, with a dose of 180 μg / mouse; perform booster immunization after 4 weeks, with a dose of 90 μg / mouse, emulsify it with incomplete Freund's adjuvant, and the time interval for subsequent booster immunizations is 3 weeks; the dose is halved again during the booster immunization, to 45 μg / mouse, dilute the complete antigen with physiological saline, and perform intraperitoneal injection on the mice. After the third immunization of the mice, tail bleeding can be performed for detection, and the titer and IC 50 of the mouse serum can be detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Select mice with high titer and low IC

[0064] The indirect competitive ELISA standard curve for detecting antibodies against nitroimidazole drugs (using metronidazole as the benchmark) is as Figure 2 shown. It can be seen from Figure 2 that the IC 50 value of the nitroimidazole antibody for detecting nitroimidazole is 2.81 ng / mL, the detection limit LOD (IC 10 ) is 0.27 ng / mL, and the linear range (IC 20 - IC 80 ) is 0.97 - 8.09 ng / mL, which can be used to establish a highly sensitive immunoassay method for detecting nitroimidazole.

[0065] (2) Cell fusion and cloning

[0066] Take the spleen cells of immunized BALB / c mice and fuse them with SP2 / 0 myeloma cells at a ratio of 10:1. Screen to obtain a nitroimidazole monoclonal hybridoma cell line that stably secretes the whole antigen of nitroimidazole drugs;

[0067] (3) Cell cryopreservation and resuscitation

[0068] Make the nitroimidazole monoclonal hybridoma cells into a cell suspension of 5×10 6 cells / mL with cryopreservation solution and store it in liquid nitrogen for a long time. When resuscitating, take out the cryopreservation tube, immediately put it into a 37°C water bath for rapid thawing, centrifuge to remove the cryopreservation solution, and then transfer it into a culture flask for culture;

[0069] (4) Preparation and purification of monoclonal antibodies

[0070] Incremental culture method: Place the nitroimidazole monoclonal hybridoma cells in a cell culture medium and culture them at 37°C. Purify the obtained culture solution by the octanoic acid-ammonium sulfate saturation method to obtain a nitroimidazole drug monoclonal antibody and store it at -20°C. Among them, the cell culture medium is obtained by adding calf serum and sodium bicarbonate to RPMI-1640 medium, so that the mass percentage of calf serum in the cell culture medium is 20%, and the mass percentage of sodium bicarbonate in the cell culture medium is 0.2%. The pH of the cell culture medium is 7.4.

[0071] (5) Preparation of goat anti-mouse antibody

[0072] Inject the mouse nitroimidazole monoclonal antibody prepared by the above operation into a sheep for an immune reaction to obtain a goat anti-mouse antibody.

[0073] Example 4

[0074] (1) Preparation of colloidal gold solution

[0075] Dilute a 1% chloroauric acid solution with double-distilled deionized water to 0.01% (mass fraction). Take 100 mL of the 0.01% chloroauric acid solution and place it in a conical flask. Heat it to boiling with a constant-temperature electromagnetic stirrer. Under continuous high temperature and continuous stirring, add 2.0 mL of 1% sodium citrate solution. Continue to stir evenly and heat until the solution turns into a bright red color and then stop. After cooling to room temperature, restore the volume to the original volume with deionized water to obtain a colloidal gold solution and store it at 4°C. The prepared colloidal gold solution has a pure, transparent appearance without precipitation and floating substances;

[0076] (2) Preparation of nitroimidazole monoclonal antibody-colloidal gold conjugate

[0077] Under magnetic stirring, the pH value of the colloidal gold solution was adjusted to 7.2 with 0.2 moL / L potassium carbonate. According to the standard of adding 20 - 60 μg of nitroimidazole monoclonal antibody per milliliter of the colloidal gold solution, the nitroimidazole monoclonal antibody prepared in Example 3 was added to the colloidal gold solution, and stirring was continued for 30 min. After standing for 10 min, 10% bovine serum albumin (BSA) solution was added to make its volume percentage content in the colloidal gold solution 1%, and then it was left standing for 10 min. Centrifugation was carried out at 12000 rpm for 40 min at 4 °C, the supernatant was discarded, and the precipitate was resuspended with a reconstitution buffer with a volume of 1 / 10 of the initial volume of the colloidal gold solution to prepare a nitroimidazole monoclonal antibody - colloidal gold conjugate, which was stored at 4 °C for later use;

[0078] Reconstitution buffer: 0.02 moL / L phosphate buffer containing 0.3% - 0.5% (v / v) bovine serum albumin, 0.1% - 0.3% (m / m) Tween - 20, 3% - 6% (m / m) trehalose, pH = 7.2;

[0079] (3) Preparation of microtiter reaction cups

[0080] 100 μL of the nitroimidazole monoclonal antibody - colloidal gold conjugate was added to the microtiter reaction cup, and it was placed in a freeze - dryer. After pre - freezing for 3 h at a cold trap temperature of - 50 °C, it was then vacuum - dried for 6 h, and then it could be taken out to obtain a microtiter reaction cup with freeze - dried nitroimidazole monoclonal antibody - colloidal gold conjugate, which was sealed and stored. The freeze - dried amount of the nitroimidazole monoclonal antibody - colloidal gold conjugate was 0.20 - 0.50 μg / mL.

[0081] (4) Preparation of sample loading pads

[0082] The sample loading pads were soaked in 0.02 moL / L phosphate buffer containing bovine serum albumin for 2 h and then dried at 50 °C for 2 h for later use. The pH of the 0.02 moL / L phosphate buffer was 7.2, and the volume percentage content of bovine serum albumin was 1.0%;

[0083] (5) Preparation of reaction membranes

[0084] Coating process: The coating antigen was diluted to a concentration of 10 mg / mL with phosphate buffer and coated on the test area (T area) of the nitrocellulose membrane using a gold - labeled membrane - spraying instrument at a coating concentration of 0.5 mg / mL; the goat anti - mouse antibody was diluted to a concentration of 10 mg / mL with 0.01 moL / L phosphate buffer with pH = 7.4 and coated on the control area (C area) of the nitrocellulose membrane using a gold - labeled membrane - spraying instrument at a coating concentration of 1.0 mg / mL. The coated reaction membrane was dried at 50 °C for 6 h.

[0085] (6) Preparation of Nitroimidazole Colloidal Gold Chromatographic Detection Device

[0086] The structure of the test strip is as Figure 3 shown. The sample loading pad, reaction pad, and water absorption pad are sequentially pasted on the bottom plate in order. Among them, the bottom plate is a PVC bottom plate, the sample loading pad is filter paper, the water absorption pad is absorbent filter paper, and the reaction pad is a nitrocellulose membrane. The end of the sample loading pad is connected to the start end of the reaction pad, the end of the reaction pad is connected to the start end of the water absorption pad, the start end of the sample loading pad is aligned with the start end of the bottom plate, and the end of the water absorption pad is aligned with the end of the bottom plate. Obtained. The distance from the C line to the end of the sample loading pad is 15.5 mm, and the distance from the T line to the end of the sample loading pad is 8.5 mm; the length of the sample loading pad is 15 mm, the length of the reaction pad is 2.5 cm, and the length of the water absorption pad is 43 mm.

[0087] The test strip obtained in the above steps is assembled with a micro reaction cup into a test strip box and stored in an environment of 2 - 8 °C, with a validity period of 12 months.

[0088] Example 5

[0089] Use the nitroimidazole colloidal gold chromatographic detection device prepared in Example 4 to detect the residue of nitroimidazole drugs in the sample.

[0090] Take about 500 g of the edible part of the sample, mix it evenly with a tissue homogenizer, take about 200 g of the prepared test sample, and put it into a clean container as the test sample and the retained sample, seal it, and mark it. The retained sample is stored at -18 °C. Accurately weigh 5 g of the test sample (accurate to 0.01 g), put it into a 50 mL centrifuge tube, add 7 mL of ethyl acetate and shake for extraction for 5 min. Add 3 g of anhydrous sodium sulfate to the test sample, vortex and mix for 1 min, then centrifuge at 4000 r / min for 5 min. Accurately pipette 4 mL of the supernatant into a 10 mL centrifuge tube, dry it at 60 °C with a sample concentrator, add 2 mL of n-hexane to completely dissolve it, then add 0.5 mL of sample diluent, vortex and mix for 1 min, centrifuge at 4000 r / min for 1 min, and then aspirate the lower layer liquid for detection.

[0091] (2) The test method is as follows: Pipette 200 μL of the test solution into the micro reaction cup, aspirate until the particles at the bottom of the hole are completely dissolved, and incubate at room temperature for 3 min. Insert the loading pad end of the test strip vertically downward into the micro reaction cup and react at room temperature for 5 min. The reaction operation process is as Figure 4 shown.

[0092] (3) The determination method is as follows: Take out the test strip from the micro reaction cup, discard the sample pad at the lower end of the test strip, and judge the result by comparing the color depth of the control line (C line) and the test line (T line), as Figure 5As shown. When the control line (C line) does not show color, regardless of whether the test line (T line) shows color or not, it indicates that the operation is incorrect or the test strip has expired, and the test result is invalid; when the control line (C line) shows color and the test line (T line) does not show color or the color is lighter than the control line (C line), it indicates that the content of metronidazole in the sample is higher than the method detection limit, and it is judged as positive; when the control line (C line) shows color and the test line (T line) is darker than the control line (C line) or the color is basically the same as the control line (C line), it indicates that the content of metronidazole in the sample is lower than the method detection limit, and it is judged as negative.

[0093] Example 6

[0094] To determine the optimal method and verify the method effect, various parameters and the method were optimized.

[0095] (1) Optimization of the extraction solvent for sample pretreatment:

[0096] In existing national and industry standards, to meet the needs of instrumental analysis, the main pretreatment method for the instrumental analysis of veterinary drug residues in fresh eggs and egg products is extraction with organic solvents. In methods such as "National Food Safety Standard - Determination of Nitroimidazole Drug Residues in Animal Foods" (GB 31658.23 - 2022), "Determination of Metronidazole, Dimetridazole and Their Metabolic Residues in Animal Foods by Liquid Chromatography - Tandem Mass Spectrometry" (Announcement No. 1025 - 2 - 2008 of the Ministry of Agriculture), and "Determination of Multiple Basic Drug Residues in Animal - Derived Foods by Liquid Chromatography - Mass Spectrometry / Mass Spectrometry" (SN / T 2624 - 2010), solvents such as ethyl acetate were used in the pretreatment process. Considering factors such as the specificity of the detection method, the characteristics of the sample matrix, and the convenience of rapid detection operation, ethyl acetate was used as the extraction solvent in this method. During the method development and optimization process, the standard addition recovery method of four target substances, namely metronidazole, dimetridazole, hydroxymetronidazole, and hydroxydimetridazole, combined with liquid chromatography - mass spectrometry detection means was used to test their extraction rates. The standard addition concentration was at the 5 μg / kg level, and the extraction rate results are shown in Table 1.

[0097] Table 1 Extraction rate data of each target substance in different matrices

[0098]

[0099] From the data in Table 1, it can be seen that the standard addition recovery extraction rates of the four target substances, metronidazole, dimetridazole, hydroxymetronidazole, and hydroxydimetridazole, in different matrices are in the range of 80.6% - 101.7%, showing ideal extraction rates, and the results also meet the recovery requirements for this concentration level in relevant national standards, indicating that ethyl acetate is an ideal extraction solvent.

[0100] (2) Optimization of the demulsifying and dehydrating agent for sample pretreatment

[0101] Due to the high protein content, the matrix of eggs and egg products is extremely prone to emulsification during the pretreatment process. Therefore, it is an important treatment process to use appropriate methods for demulsification, two-phase separation, and solvent dehydration. Referring to relevant research literature and standard methods, four dehydrating agents, namely saturated sodium chloride solution, sodium chloride, anhydrous sodium sulfate, and sodium chloride + anhydrous sodium sulfate, were selected for optimization testing. The test method was as follows: Weigh 5 g of egg and egg product samples and place them in 50 mL centrifuge tubes respectively. Add metronidazole standard product, mix well and let stand for 30 min. Then add 7 mL of ethyl acetate solvent for extraction, vortex for 5 min. Add dehydrating agents (3 mL, 5 mL, and 7 mL of saturated sodium chloride solution, 1 g, 3 g, 5 g of anhydrous sodium sulfate, 3 g, 5 g of sodium chloride, and a mixed salt of 2 g of anhydrous sodium sulfate + 1 g of sodium chloride) respectively and vortex for 1 min. Centrifuge at 4000 r / min for 5 min and observe the layering of the samples in the centrifuge tubes.

[0102] The test results are summarized in Table 2. Saturated sodium chloride solution is commonly used in the pretreatment of veterinary drug residues in meat and meat products. Since the water content of fresh egg matrix is larger than that of meat, adding saturated sodium chloride solution did not achieve the demulsification effect. Although clear layering could be seen with the naked eye, the supernatant was actually an emulsion layer, which became a jelly-like substance after air drying, and the effect was not good. Using sodium chloride for dehydration and demulsification had no obvious effect, and obvious layering could not occur. After treatment with anhydrous sodium sulfate + sodium chloride (2 g + 1 g), the mixed solution showed obvious layering, and the supernatant could meet the test requirements. Using anhydrous sodium sulfate alone for demulsification had an obvious effect, but the volume of the supernatant obtained with a small amount of addition was less than 4 mL, which had a great impact on subsequent analysis; when the usage amount was increased to 3 g and 5 g, it was found that the demulsification effect was obvious, and the volume of the supernatant could meet the requirements of subsequent tests. Considering the simplicity of operation and detection cost, 3 g of anhydrous sodium sulfate was determined as the demulsifying dehydrating agent in this invention.

[0103] Table 2 Demulsification and layering of dehydrating agents

[0104]

[0105] (3) Optimization of the incubation time of the test solution in the microplate

[0106] To investigate the effect of the incubation time of the test solution in the microplate wells on the antigen-antibody reaction, the measurement results of the incubation times of 1 min, 3 min, 5 min, and 7 min for egg matrix, duck egg matrix, goose egg matrix, quail egg matrix, marinated egg matrix, salted duck egg matrix, preserved egg matrix, and preserved sausage matrix were studied and compared respectively. The results showed that as the incubation time increased, the color development of the T line and the C line in the test groups of the eight matrices became gradually more obvious; when the incubation time was 1 min, the colors of the T line and the C line at the detection limit concentration were similar, and as time increased, the contrast between the T line and the C line became stable at 3 min and above, and the experimental results had good reproducibility and stability. Therefore, the optimal incubation time of the test solution in the microplate wells for all matrices in this method was set at 3 min, and the acceptable operation time was within the minute before or after it.

[0107] (4) Optimization of the reaction time between the test strip and the test solution

[0108] To investigate the effect of the reaction time between the test strip and the test solution on the antigen-antibody reaction, the measurement results of the reaction times of 1 min, 3 min, 5 min, and 7 min for egg matrix, duck egg matrix, goose egg matrix, quail egg matrix, marinated egg matrix, salted duck egg matrix, preserved egg matrix, and preserved sausage matrix were studied and compared respectively. The analysis found that for all matrices, when the reaction time was 1 min and 3 min, the colors of the T line and the C line were relatively light, which was not conducive to the interpretation of the results; as the reaction time increased, the color development of the T line and the C line became gradually more obvious, and the contrast between the T line and the C line became stable at 5 min and above, and both the positive and negative results of the test strip could be clearly identified, and the color development effect was significantly different; within 7 min, the positive and negative results could be accurately discriminated and did not change. To avoid the influence of long-term placement on the interpretation, therefore, the optimal reaction time between the test strip and the test solution was set at 5 min, and the acceptable operation time was within the two minutes before or after it.

[0109] (5) Verification of the applicability to multiple matrices

[0110] The applicability verification of the multi-factor method was carried out by using the spiked test method. The sample matrices selected were eggs, duck eggs, goose eggs, quail eggs, marinated eggs, salted duck eggs, preserved eggs and preserved egg sausage; the added drugs selected were the metronidazole group, the dimetridazole group, and the 1:1 mixed group of metronidazole and dimetridazole; the added concentrations selected were three content levels of negative matrix, 1.0 μg / kg (limit of determination), and 2.0 μg / kg (two times the limit of determination). The results are shown in Table 3. According to the method of the present invention for operation and testing, the color of the C line in the negative samples of each group was significantly darker than that of the T line, showing a negative result; the color of the C line in the samples at the limit of determination of each group was darker than that of the T line, showing a positive result; the color of the C line in the samples at the limit of determination of each matrix was significantly darker than that of the T line, also showing a positive result. The above results indicate that the method of the present invention has good applicability for the rapid detection of various nitroimidazole drugs in egg matrices such as egg matrix, duck egg matrix, goose egg matrix, quail egg matrix, marinated egg matrix, salted duck egg matrix, preserved egg matrix and preserved egg sausage matrix, and the sensitivity can fully meet the requirements of the national standard limit (GB 31650), with strong operability and practical significance.

[0111] Table 3 Results of consistency verification of multi-matrix nitroimidazole detection method

[0112]

[0113]

[0114] Compared with the existing laboratory detection method for detecting nitroimidazole veterinary drug residues such as metronidazole and dimetridazole in animal-derived foods based on liquid chromatography-mass spectrometry, the method of the present invention for rapid detection of nitroimidazole drugs in eggs and egg products has the following advantages: the site is flexible and convenient, the instrument is simple, the operation is easy to train and easy to master, and the requirements for the testing personnel are low. The detection time is shortened by more than 70%, and the detection cost is reduced by more than 90%.

[0115] Compared with the existing rapid detection methods for veterinary drug residues in eggs and egg products, the present invention can detect high-risk and key sampling and monitoring items such as metronidazole and dimetridazole. Compared with the existing rapid detection methods for metronidazole or dimetridazole, this method can realize the synchronous detection of multiple residues, greatly improving the efficiency of detection activities and reducing the detection economic cost and time cost; the positive detection limit of this method is consistent with the national standard limit, with higher practical promotion value, and the optimized sample pretreatment conditions have higher extraction efficiency and wider matrix applicability.

[0116] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nitroimidazole drug hapten, characterized in that, Its structural formula is shown in Formula I: Formula I.

2. The preparation method of the nitroimidazole drug hapten according to claim 1, characterized in that, It includes: (1) 2-Methyl-5-nitroimidazole and 4-bromobut-1-yne undergo nucleophilic substitution under alkaline conditions to synthesize Intermediate 1; (2) Intermediate 1 undergoes a click reaction with ethyl azidoacetate to synthesize Intermediate 2; (3) Intermediate 2 is hydrolyzed under alkaline conditions to synthesize the nitroimidazole drug hapten; The reaction route is: 。 3. The complete antigen of nitroimidazole drugs, characterized in that, It is obtained by conjugating the nitroimidazole drug hapten described in Claim 1 with a carrier protein.

4. The nitroimidazole drug complete antigen according to claim 3, characterized in that, The carrier protein includes bovine serum albumin, human serum albumin, chicken ovalbumin, or hemocyanin.

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