Metronidazole hapten, artificial antigen and application thereof

Abstract

The application discloses a metronidazole hapten, an artificial antigen and application thereof. The metronidazole hapten prepared by the application, the metronidazole artificial antigen prepared from the metronidazole hapten and the monoclonal antibody are used for ELISA detection, and the specificity is high, and the IC50 value is 0.068 ug / L. The metronidazole artificial antigen and the monoclonal antibody in the application are applied to colloidal gold immunochromatography technology, qualitative detection of metronidazole can be realized quickly and conveniently, the detection lower limit in a standard solution is 2 ug / kg, the detection lower limit in an egg sample is 0.5 ug / kg, the sensitivity is high, and the practical requirements of on-site detection and rapid clinical detection of metronidazole can be met.

Description

Technical Field

[0001] This invention relates to the field of biochemical engineering, specifically to a metronidazole hapten, an artificial antigen, and their applications. Background Technology

[0002] Metronidazole is an antibacterial and antiprotozoal drug that kills and prevents anaerobic bacteria and pathogens. It is commonly used to prevent and treat trichomoniasis and coccidiosis in poultry, swine spirochetal dysentery, and various anaerobic infections in animals, and is widely used in agricultural production. my country's National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food (GB 31650—2019) stipulates that metronidazole is permitted for therapeutic use in veterinary drugs, but it must not be detected in animal-derived foods. Japan stipulates that metronidazole must not be detected in food, while the European Union and the United States have designated metronidazole as a prohibited veterinary drug.

[0003] The main methods for detecting metronidazole are instrumental detection and immunoassay. The former suffers from drawbacks such as expensive equipment, long processing times, and the need for specialized personnel, failing to meet the practical needs of on-site testing and rapid clinical trials, and causing significant inconvenience to routine testing work. The latter suffers from the shortcomings of artificial antigens directly prepared from metronidazole, which have poor sensitivity and low cross-reactivity rates, failing to meet the actual needs of the current market. Therefore, there is a need to develop highly specific metronidazole haptens or artificial antigens to improve the detection efficiency and sensitivity of metronidazole. Summary of the Invention

[0004] To address the lack of a rapid, sensitive, and convenient immunoassay method for metronidazole in existing technologies, this invention provides a metronidazole hapten, an artificial antigen, and their applications.

[0005] The first objective of this invention is to provide a metronidazole hapten.

[0006] A second object of the present invention is to provide a method for preparing a compound with the structural formula shown in formula (I).

[0007]

[0008] A third object of the present invention is to provide the use of compounds with the structural formula shown in formula (I) in the preparation of metronidazole artificial antigens.

[0009]

[0010] The fourth objective of this invention is to provide a metronidazole artificial antigen.

[0011] The fifth objective of this invention is to provide a method for preparing the above-mentioned metronidazole artificial antigen.

[0012] The sixth object of this invention is to provide the use of compounds with the structural formula shown in formula (II) in the preparation of metronidazole antibodies.

[0013]

[0014] The seventh objective of this invention is to provide a metronidazole artificial antigen combination.

[0015] The eighth object of the present invention is to provide the application of the above-described metronidazole artificial antigen combination in the preparation of a kit for detecting metronidazole.

[0016] The ninth object of the present invention is to provide a kit for detecting metronidazole.

[0017] To achieve the above objectives, the present invention is implemented through the following solution:

[0018] The metronidazole hapten prepared by this invention completely retains the nitroimidazole characteristic group of metronidazole, as well as the hydroxyl group. Furthermore, the designed coupling arm is a straight chain with an active group, which ensures that the small molecule structure of the prepared metronidazole artificial antigen retains its original characteristics, making it more conducive to presenting the structural characteristics of metronidazole and improving the immunogenicity of the antigen.

[0019] A metronidazole hapten, the structural formula of which is shown in formula (I),

[0020]

[0021] The preparation method of the compound with the structural formula shown in formula (I) involves reacting metronidazole and ethyl 4-bromobutyrate to a complete extent, followed by extraction with ethyl acetate to remove impurities and obtain an intermediate product; the intermediate product is then fully hydrolyzed under alkaline conditions, and the pH of the resulting hydrolysate is adjusted to acidic to obtain the final product.

[0022]

[0023] The structural formula of metronidazole is:

[0024] The structural formula of the ethyl 4-bromobutyrate is:

[0025] The structural formula of the intermediate product is:

[0026] Preferably, the molar ratio of metronidazole to ethyl 4-bromobutyrate is 1:(1-3).

[0027] More preferably, the molar ratio of metronidazole to ethyl 4-bromobutyrate is 1:2.

[0028] Preferably, the solvent of the reaction system of metronidazole and ethyl 4-bromobutyrate comprises acetonitrile and water in a volume ratio of 1:(1-2).

[0029] More preferably, the solvent for the reaction system of metronidazole and ethyl 4-bromobutyrate is acetonitrile and water in a volume ratio of 1:1.

[0030] Preferably, the alkaline condition for hydrolyzing the intermediate product is to add lithium hydroxide to the reaction system at a final concentration of 1 mol / L to 2 mol / L.

[0031] More preferably, the alkaline condition for hydrolyzing the intermediate product is to add lithium hydroxide to the reaction system at a final concentration of 1 mol / L.

[0032] Preferably, the pH of the resulting hydrolysis product is adjusted to acidic using hydrochloric acid.

[0033] More preferably, the pH of the resulting hydrolysis product is adjusted to 4-5 using hydrochloric acid.

[0034] More preferably, the pH of the obtained hydrolysis product is adjusted to 4-5 using 4M-5M hydrochloric acid.

[0035] More preferably, the pH of the resulting hydrolysis product is adjusted to 4-5 using 4M hydrochloric acid.

[0036] Preferably, the pH of the obtained hydrolysis product is adjusted to acidic before purification.

[0037] More preferably, the purification method includes drying and washing.

[0038] More preferably, the drying method includes vacuum evaporation.

[0039] More preferably, the washing method includes washing with methanol and ethyl acetate.

[0040] More preferably, the washing is performed with a mixture obtained by thoroughly mixing 70% (v / v) methanol and ethyl acetate at a volume ratio of (7-8):(2-3).

[0041] More preferably, the washing is performed with a mixture obtained by thoroughly mixing 70% (v / v) methanol and ethyl acetate at a volume ratio of 7:3.

[0042] Specifically, the preparation method includes the following steps:

[0043] Take 1.0 g of metronidazole (CAS: 443-48-1, 5.84 mmol) into a 50 mL round-bottom flask, then add 10 mL of acetonitrile, 10 mL of purified water, and 2.27 g of [unspecified ingredient]. Ethyl 4-bromobutyrate (CAS: 2969-81-5, 11.68 mmol) was thoroughly mixed and reacted at 60 °C for 3–5 h. After the reaction was complete, 10 mL of purified water was added to the reaction product, and the mixture was extracted twice with ethyl acetate to obtain an aqueous solution containing compound c. The aqueous solution containing compound c was diluted to 20 mL with purified water, and then 0.48 g of lithium hydroxide was added to make the molar concentration of lithium hydroxide in the solution 1 mol / L. The mixture was thoroughly mixed and reacted at room temperature for 8–12 h. After the reaction was complete, the pH was adjusted to 4–5 with 4 M hydrochloric acid, and the water was evaporated under reduced pressure. The precipitate was washed three times with a 70% methanol / ethyl acetate mixture (i.e., 70% (v / v) methanol and ethyl acetate were mixed thoroughly at a volume ratio of 7:3). The washing liquid was collected and evaporated under reduced pressure to obtain 0.76 g of metronidazole hapten.

[0044] The use of compounds with the structural formula shown in formula (I) in the preparation of metronidazole artificial antigens should also be within the scope of protection of this invention.

[0045]

[0046] A metronidazole artificial antigen, obtained by conjugating the above-mentioned metronidazole hapten with a carrier protein, has the structural formula shown in formula (II).

[0047]

[0048] Protein is a carrier protein, which is bovine serum albumin or hemocyanin.

[0049] The method for preparing the above-mentioned metronidazole artificial antigen is characterized in that the above-mentioned metronidazole hapten is coupled to a carrier protein via an active ester method.

[0050] Preferably, the active ester method includes the following steps:

[0051] The metronidazole hapten, dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were reacted thoroughly to obtain solution A; the carrier protein was dissolved in phosphate buffer to obtain solution B; after solution A and solution B were reacted thoroughly, they were dialyzed to obtain the final product.

[0052] Specifically, the method for preparing the metronidazole artificial antigen, in which the carrier protein is bovine serum albumin, is as follows:

[0053] Take 10 mg of the above metronidazole hapten, dissolve it in 0.2 mL of dimethylformamide, stir thoroughly, then add 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 5 mg of N-hydroxysuccinimide, stir at room temperature for 4 h to obtain solution A; weigh 40 mg of bovine serum albumin, dissolve it thoroughly in 4 mL of 0.01 mol / L PBS solution to form solution B, slowly add solution A dropwise to solution B while stirring, and stir at room temperature for 16–24 h; dialyze the obtained reaction solution with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysate 3 times a day, and the obtained dialysate is the metronidazole artificial antigen with bovine serum albumin as the carrier protein.

[0054] Specifically, the method for preparing the metronidazole artificial antigen, in which the carrier protein is hemocyanin, is as follows:

[0055] Take 10 mg of the above metronidazole hapten, dissolve it in 0.2 mL of dimethylformamide, stir thoroughly, then add 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 5 mg of N-hydroxysuccinimide, stir at room temperature for 4 h to obtain solution A; weigh 40 mg of hemocyanin, dissolve it thoroughly in 4 mL of 0.01 mol / L PBS solution to form solution B, slowly add solution A dropwise to solution B while stirring, and stir at room temperature for 16–24 h; dialyze the obtained reaction solution with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysate 3 times a day, and the obtained dialysate is the metronidazole artificial antigen with hemocyanin as the carrier protein.

[0056] The application of compounds with the structural formula shown in formula (II) in the preparation of metronidazole antibodies should also be within the scope of protection of this invention.

[0057]

[0058] Protein is a carrier protein, which is bovine serum albumin or hemocyanin.

[0059] A metronidazole antibody is prepared by immunizing animals with the aforementioned metronidazole artificial antigen, whose carrier protein is hemocyanin, as an immunogen.

[0060] Preferably, the metronidazole antibody is a monoclonal antibody, obtained by immunizing animals with the above-mentioned metronidazole artificial antigen, whose carrier protein is hemocyanin, to obtain hybridoma cells, culturing the obtained hybridoma cells, collecting the cells for animal immunization to obtain ascites, and then identifying and purifying them to obtain the antibody.

[0061] A metronidazole artificial antigen combination includes an immunogen and a coating antigen, wherein the immunogen is obtained by conjugating the above-mentioned metronidazole hapten with hemocyanin, that is, the above-mentioned metronidazole artificial antigen with hemocyanin as the carrier protein; and the coating antigen is obtained by conjugating the above-mentioned metronidazole hapten with bovine serum albumin, that is, the above-mentioned metronidazole artificial antigen with bovine serum albumin as the carrier protein.

[0062] The application of the above-mentioned metronidazole artificial antigen combination in the preparation of a kit for detecting metronidazole should also be within the scope of protection of this invention.

[0063] An immunochromatographic test strip for detecting metronidazole includes a base plate on which a sample pad, a reaction membrane, and an absorbent pad are sequentially overlapped. The reaction membrane is a nitrocellulose membrane with a detection zone and a control zone. The detection zone is coated with a coating antigen obtained by conjugating metronidazole hapten with hemocyanin, and the control zone is coated with IgG. The detection zone is a T line, and the control zone is a C line.

[0064] Preferably, the sample pad is soaked in a sample processing solution, which is a 0.1M PB buffer containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide.

[0065] Specifically, the preparation method of the immunochromatographic test strip is as follows:

[0066] Metronidazole artificial antigen with bovine serum albumin as the carrier protein was used as the antigen. The concentration was adjusted to 0.05 mg / mL to 0.2 mg / mL with coating buffer (0.01 M PBS buffer containing 1% wt sucrose and 0.05% wt sodium azide, pH=7.6). Mouse IgG was adjusted to 0.1 mg / mL to 0.5 mg / mL with coating buffer. Nitrocellulose membrane was used as the reaction membrane. The diluted antigen was sprayed onto the T line of the reaction membrane at a membrane volume of 0.8 μL / cm to 1.2 μL / cm, and the diluted mouse IgG was sprayed onto the C line of the reaction membrane. The T line and C line were spaced 2.5 mm apart. The membrane was placed in an oven at 45℃ for 12 h to 16 h to obtain the antigen-coated reaction membrane.

[0067] The cut 30cm×30cm blank sample pads were immersed in the sample preparation solution (0.1M PB buffer containing 0.3%wt Tween 20, 1%wt sucrose, 0.5%wt BSA and 0.05%wt sodium azide) for 5 minutes, then removed and dried at 37℃ for 16 hours to obtain the sample pads.

[0068] Using a PVC board as the base, the above-mentioned antigen-coated reaction membrane is pasted in the middle of the PVC board. The absorbent pad and the above-mentioned sample pad are pasted on both ends of the reaction membrane, with the absorbent pad near the C line and the sample pad near the T line, thus obtaining the test strip. The test strip is cut into test strips with a width of 3mm, and the test strips are loaded into the test strip card, with the sample pad near the sample application hole, thus obtaining the test strip.

[0069] A kit for detecting metronidazole, comprising the above-mentioned combination of metronidazole artificial antigens.

[0070] Preferably, the kit comprises an immunochromatographic test strip and an antibody for detecting metronidazole; the immunochromatographic test strip includes a base plate, on which a sample pad, a reaction membrane, and an absorbent pad are sequentially overlapped; the reaction membrane is a nitrocellulose membrane with a detection zone and a control zone; the detection zone is coated with the coating antigen from the metronidazole artificial antigen combination, and the control zone is coated with IgG; the antibody for detecting metronidazole is prepared by immunizing animals with the immunogen from the aforementioned metronidazole artificial antigen combination.

[0071] More preferably, the antibody for detecting metronidazole is labeled with colloidal gold.

[0072] More preferably, the antibody is a monoclonal antibody, obtained by immunizing animals with the immunogen in the above-mentioned metronidazole artificial antigen combination to obtain hybridoma cells, culturing the obtained hybridoma cells and collecting the cells to immunize animals to obtain ascites, and then identifying and purifying them to obtain the antibody.

[0073] More preferably, the antibody for detecting metronidazole is obtained by reacting the above-mentioned monoclonal antibody with colloidal gold at a mass-volume ratio of (5 μg to 10 μg): 1 mL.

[0074] More preferably, the antibody for detecting metronidazole is obtained by fully reacting the above-mentioned monoclonal antibody with colloidal gold at a mass-volume ratio of 8 μg: 1 mL.

[0075] Specifically, the method for preparing the antibody for detecting metronidazole is as follows:

[0076] Take 1g of chloroauric acid, dissolve it in pure water and sonicate, and then bring the volume to 100mL to obtain a chloroauric acid solution. Store it at 4℃ protected from light for later use. Take 1ml of chloroauric acid solution into 100mL of pure water, heat to boiling, add 0.5mL of 0.06%wt sodium citrate solution, continue heating for 10min, cool to room temperature, and then add pure water to bring the volume to 100mL to obtain a colloidal gold solution.

[0077] Take 1 mL of colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the amount added is the minimum amount required to prevent discoloration in the subsequent labeling process), add 8 μg of the monoclonal antibody to the colloidal gold solution, react at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking, mix thoroughly, centrifuge at 12,000 rpm for 10 minutes, discard all supernatant, and the product is obtained.

[0078] A method for detecting metronidazole for non-disease treatment and diagnosis purposes, using any of the above-described kits to detect the sample to be tested.

[0079] Preferably, the method includes the following steps:

[0080] After the sample to be tested is fully reacted with the colloidal gold antibody, it is then fully contacted with the sample pad of the immunochromatographic test strip. The result is determined based on the color development of the immunochromatographic test strip. The specific determination method is as follows:

[0081] When the C line does not develop color, the test result is invalid because the operation was incorrect or the test strip has expired. When both the C and T lines develop color, with the T line showing stronger color than the C line, or when there is no significant difference between the T and C lines, the test result is negative (-), indicating that there is no metronidazole in the sample. When both the C and T lines develop color, with the T line showing significantly weaker color than the C line, or when the C line develops color but the T line does not, the test result is positive (+), indicating that there is metronidazole in the sample.

[0082] Preferably, the sample to be tested is an egg sample, including egg yolk and / or egg white.

[0083] The egg samples that this invention can detect include, but are not limited to, free-range chicken eggs, black-boned chicken eggs, duck eggs, goose eggs, and quail eggs.

[0084] Preferably, the sample to be tested is mixed evenly and extracted to obtain the test solution.

[0085] More preferably, the method of mixing uniformly includes stirring and homogenizing.

[0086] More preferably, the extraction method includes: extracting the homogeneously mixed sample to be tested with ethyl acetate and n-hexane.

[0087] More preferably, the extraction method includes: first extracting the well-mixed sample with ethyl acetate, centrifuging, collecting the supernatant, drying, and further extracting with n-hexane.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] The metronidazole hapten prepared in this invention yields metronidazole artificial antigen and monoclonal antibody, which exhibit high specificity for ELISA detection, with an IC50 value of 0.068 μg / L. When applied to colloidal gold immunochromatography, the metronidazole artificial antigen and monoclonal antibody of this invention enable rapid and convenient qualitative detection of metronidazole. The detection limit is 2 μg / kg in standard solutions and 0.5 μg / kg in egg samples, demonstrating high sensitivity that meets the practical needs of on-site detection and rapid clinical testing of metronidazole. Attached Figure Description

[0090] Figure 1 This is the synthetic route for metronidazole hapten.

[0091] Figure 2 The mass spectrometry results are for metronidazole hapten.

[0092] Figure 3 This describes the synthetic route for metronidazole artificial antigen.

[0093] Figure 4 This is the standard curve for metronidazole standard solution. Detailed Implementation

[0094] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0095] Example 1: Synthesis of Metronidazole Hapten

[0096] 1. Synthesis of metronidazole hapten

[0097] The synthetic route of metronidazole hapten is as follows: Figure 1 As shown, the specific steps are as follows:

[0098] (1) Take 1.0 g of compound a (i.e., metronidazole, CAS: 443-48-1, 5.84 mmol) into a 50 mL round-bottom flask, then add 10 mL of acetonitrile, 10 mL of purified water and 2.27 g of compound b (i.e., ethyl 4-bromobutyrate, CAS: 2969-81-5, 11.68 mmol) in sequence, mix thoroughly, and react at 60 °C for 3–5 h. After the reaction is complete, add 10 mL of purified water to the reaction product, and extract twice with ethyl acetate to obtain an aqueous solution containing compound c.

[0099] (2) The aqueous solution containing compound c was diluted to 20 mL with purified water, and then 0.48 g of lithium hydroxide was added to make the molar concentration of lithium hydroxide in the solution 1 mol / L. The mixture was thoroughly mixed and reacted at room temperature for 8–12 h. After the reaction was completed, the pH was adjusted to 4–5 with 4 M hydrochloric acid, and the water was evaporated under reduced pressure. The precipitate was washed three times with a 70% methanol / ethyl acetate mixture (i.e., 70% (v / v) methanol and ethyl acetate were mixed evenly at a volume ratio of 7:3). The washing liquid was collected and evaporated under reduced pressure to obtain 0.76 g of metronidazole hapten.

[0100] 2. Identification of metronidazole hapten

[0101] The structural formula of the metronidazole hapten is shown in formula (Ⅰ):

[0102]

[0103] Mass spectrometry results of metronidazole hapten are as follows: Figure 2 As shown, specifically: ESI-MS: 257[M-1].

[0104] Example 2: Synthesis and Identification of Metronidazole Artificial Antigen

[0105] The structural formula of the metronidazole artificial antigen of this invention is shown in formula (II).

[0106]

[0107] Protein is a carrier protein, which is bovine serum albumin (BSA) or hemocyanin (KLH).

[0108] The synthetic route of metronidazole artificial antigen is as follows: Figure 3 As shown, the specific steps are as follows:

[0109] (1) Metronidazole artificial antigen with bovine serum albumin as the carrier protein

[0110] Take 10 mg of the metronidazole hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), stir thoroughly, add 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain hapten activated ester 1;

[0111] Weigh 40 mg of bovine serum albumin (BSA) and dissolve it completely in 4 mL of 0.01 mol / L PBS solution to form carrier protein solution 1. Add hapten activated ester 1 dropwise slowly to the carrier protein solution 1 while stirring, and stir at room temperature for 16–24 h.

[0112] The reaction solution was dialyzed with 0.01 mol / L PBS solution for 3 days at room temperature, with the dialysate changed 3 times a day to remove unreacted small molecules. The resulting dialysate was metronidazole artificial antigen with bovine serum albumin as the carrier protein. It was aliquoted and stored at 4°C for later use.

[0113] (2) Metronidazole artificial antigen with hemocyanin as the carrier protein

[0114] Take 10 mg of the metronidazole hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), stir thoroughly, add 10 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain hapten activated ester 2;

[0115] Weigh 40 mg of hemocyanin (KLH) and dissolve it completely in 4 mL of 0.01 mol / L PBS solution to form carrier protein solution 2. Add the hapten activated ester 2 dropwise slowly to the carrier protein solution 2 while stirring, and stir at room temperature for 16-24 h.

[0116] The reaction solution was dialyzed with 0.01 mol / L PBS solution for 3 days at room temperature, with the dialysate changed 3 times a day to remove unreacted small molecules. The resulting dialysate was metronidazole artificial antigen with hemocyanin as the carrier protein. It was aliquoted and stored at 4°C for later use.

[0117] Example 3: Preparation of Metronidazole Monoclonal Antibody

[0118] 1. Animal immunization

[0119] Using the metronidazole artificial antigen of hemocyanin prepared in Example 2 as the carrier protein, it was emulsified with an equal volume of Freund's adjuvant and immunized in BALB / c mice at a dose of 50 μg to 100 μg per mouse. Immunization was performed three times at two-week intervals, and serum titers were measured by collecting tail vein blood from the mice. If the antibody titer did not meet the requirements, booster immunizations were necessary.

[0120] Once the antibody titer stops rising, subcutaneous booster immunization is performed with 100 μg of whole antigen.

[0121] 2. Hybridoma cell preparation

[0122] Five days after the final booster immunization, mouse spleen cells were fused with human osteosarcoma SP20 cells. The fused cells were screened in HAT medium, and after 5 days, the medium was replaced with complete medium for further culture.

[0123] Cell supernatant was analyzed using ELISA. Cells in wells showing strong positive results were then subjected to limiting dilution clonal culture. Cells in wells showing positive results after three clonal culture assays were identified as hybridoma cells secreting monoclonal antibodies.

[0124] 3. Monoclonal antibody preparation

[0125] After amplification and culture of hybridoma cells, they were inoculated into the peritoneal cavity of mice to produce ascites containing antibodies. The ascites was purified using the caprylic acid-ammonium sulfate precipitation method to obtain high-purity and highly specific metronidazole monoclonal antibodies.

[0126] Example 4: ELISA Performance Evaluation

[0127] 1. Experimental Methods

[0128] The performance of the artificial antigens and monoclonal antibodies prepared in this invention was evaluated using the ELISA method, including the following steps:

[0129] (1) Antigen coating

[0130] Using carbonate buffer at pH 9.6 as the coating diluent, the metronidazole artificial antigen with bovine serum albumin as the carrier protein prepared in Example 2 was diluted to 0.2 μg / mL, and added to polystyrene microplates at 100 μL / well. The plates were coated overnight at 4°C, dried, and washed three times with PBST.

[0131] (2) Closed

[0132] Add 280 μL / well to phosphate buffer containing 1% wt BSA, block at 37°C for 1 h, spin dry, wash three times with PBST, dry and vacuum package for storage.

[0133] (3) Primary antibody dilution

[0134] The metronidazole monoclonal antibody prepared in Example 3 was diluted to 0.1 μg / mL with phosphate buffer containing 0.05% wt sodium azide (pH 7.4) and stored at 4°C for later use.

[0135] (4) Preparation of standard solutions

[0136] Metronidazole standard was dissolved in 0.01M PBS to obtain metronidazole standard solutions with concentrations of 0 μg / L, 0.03 μg / L, 0.06 μg / L, 0.12 μg / L, 0.24 μg / L and 0.48 μg / L.

[0137] (5) Sample addition and primary antibody incubation

[0138] Add 100 μL of metronidazole standard solution to each well of an ELISA plate coated with metronidazole artificial antigen, and then add 20 μL of 0.1 μg / mL metronidazole monoclonal antibody dilution solution to each well. Incubate at 37°C for 0.5 h, then dry.

[0139] (6) Washing antibody

[0140] Add 280 μL of PBST per well, wash 3 times, and then pat dry.

[0141] (7) Second antibody incubation

[0142] Add 100 μL / well of HRP-labeled goat anti-mouse IgG enzyme-labeled secondary antibody and react at 37°C for 0.5 h.

[0143] (8) Washing secondary antibody

[0144] Add 280 μL of PBST per well, wash three times, and then pat dry.

[0145] (9) Color development

[0146] Add 100 μL of TMB colorimetric solution per well and react at 37 °C for 15 min; then add 50 μL of 1M sulfuric acid per well to stop the colorimetric reaction.

[0147] (10) Absorbance measurement

[0148] Place the microplate obtained in the previous step into the microplate reader and set the reader to a wavelength of 450 nm to measure the OD value of each well.

[0149] 2. Experimental Results

[0150] Table 1. OD values ​​of metronidazole standard solutions of different concentrations tested by ELISA

[0151]

[0152] The OD values ​​of metronidazole standard solutions at different concentrations are shown in Table 1. Using the data in Table 1, a four-parameter logistic curve was fitted using ELISA Calc software, and the results are shown below. Figure 4 The standard curve shown has the following linear equation:

[0153] y = (AD) / [1 + (x / C)^B] + D, r 2 =0.998905; where A = 1.17520, B = 1.09598, C = 0.05801, D = 0.09335, x represents the concentration of the analyte, and y represents the OD value.

[0154] IC is obtained through calculation 50The value was 0.068 μg / L, and it showed a linear relationship in the range of 0.03 μg / L to 0.48 μg / L.

[0155] Example 5: A colloidal gold qualitative immunochromatographic kit for detecting metronidazole and its efficacy evaluation.

[0156] I. Composition of the Colloidal Gold Qualitative Immunochromatographic Reagent Kit

[0157] 1. Immunochromatographic test strips

[0158] The preparation method of the immunoassay strips included in this kit is as follows:

[0159] (1) Preparation of reaction membranes coated with artificial antigens and goat anti-mouse IgG

[0160] Using the metronidazole artificial antigen with bovine serum albumin as the carrier protein obtained in Example 2 as the antigen, the concentration was adjusted to 0.1 mg / mL to 0.5 mg / mL with coating buffer (0.01 M PBS buffer containing 1% wt sucrose and 0.05% wt sodium azide, pH = 7.6); and the concentration of mouse IgG was adjusted to 0.1 mg / mL to 0.5 mg / mL with coating buffer. Using a nitrocellulose membrane (NC membrane) as the reaction membrane, the diluted antigen was sprayed onto the detection area (T line) of the reaction membrane at a membrane solution volume of 0.8 μL / cm to 1.2 μL / cm, and the diluted mouse IgG was sprayed onto the control area (C line) of the reaction membrane. The interval between the detection area and the control area was 2.5 mm. The membrane was placed in an oven at 45°C for 12 h to 16 h and then stored in a constant temperature and humidity incubator for later use.

[0161] (2) Preparation of sample pad

[0162] The cut 30cm×30cm blank sample pads were immersed in the sample preparation solution (0.01M PBT buffer containing 0.1%wt Tween 20, 1%wt sucrose and 0.05%wt sodium azide) for 5 minutes, then removed and dried at 37℃ for 16 hours. They were then placed in a constant temperature and humidity storage box for later use.

[0163] (3) Assembly

[0164] The reaction membrane prepared in step 1 is adhered to the middle of a PVC board. The absorbent pad and the sample pad prepared in step 2 are respectively adhered to both ends of the reaction membrane, with the absorbent pad adjacent to the control area (C line) and the sample pad adjacent to the detection area (T line), thus obtaining the test strip. The test strip is cut into test strips with a width of 3 mm. The test strips are loaded into the test strip card, with the sample pad adjacent to the sample application well, thus obtaining the colloidal gold qualitative immunochromatographic test strip card.

[0165] 2. Gold Label Micropores

[0166] The method for preparing the gold nanoparticle micropores is as follows:

[0167] (1) Preparation of colloidal gold solution

[0168] Dissolve 1g of chloroauric acid in pure water and sonicate, then bring the volume to 100mL to obtain a chloroauric acid solution. Store at 4℃ protected from light for later use. Add 1ml of the chloroauric acid solution to 100mL of pure water, heat to boiling, add 0.5mL of 0.06% wt sodium citrate solution, continue heating for 10 minutes, cool to room temperature, and then add pure water to bring the volume to 100mL to obtain a colloidal gold solution. Store at room temperature protected from light for later use. All glassware used must be soaked overnight in a mixture of potassium permanganate and sulfuric acid, then rinsed and dried before use.

[0169] (2) Labeling of metronidazole monoclonal antibody

[0170] Take 1 mL of colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the amount added is the minimum amount required to prevent discoloration in the subsequent labeling process), add 8 μg of metronidazole monoclonal antibody prepared in Example 3 to the colloidal gold solution, react at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking, mix thoroughly, centrifuge at 12,000 rpm for 10 minutes, discard all supernatant, and the resulting precipitate is the colloidal gold-labeled metronidazole monoclonal antibody.

[0171] (3) Drying of the micropores of the gold label

[0172] The colloidal gold-labeled metronidazole monoclonal antibody obtained in the previous step was reconstituted with 1 mL of gold diluent (an aqueous solution containing 2% wt Tris, 5% wt bovine serum albumin, 0.05% wt thimerosal, and 5% wt sucrose). The solution was then aliquoted into microwells at 15 μL / well and dried at 45 °C for 16 hours to obtain gold-labeled microwells, which were then stored for later use.

[0173] II. Instructions for use of the colloidal gold qualitative immunochromatographic assay kit

[0174] (1) Sample processing

[0175] A representative sample (such as egg yolk or egg white; note: the sample used for testing must be fresh, odorless, and unspoiled) is homogenized. Weigh 5.0 ± 0.1 g of the homogenized sample into a 15 mL centrifuge tube, add 5 mL of ethyl acetate, tighten the cap, and vortex or manually shake at 120 rpm for 1-2 minutes until the tissue is evenly dispersed. Centrifuge at room temperature at 4000 rpm or higher for 3 minutes, and transfer all the supernatant to a 10 mL centrifuge tube. Dry the tube at 70°C. Add 1 mL of n-hexane and 0.5 mL of 0.01 M PBS solution to the dried centrifuge tube, vortex for 30 seconds, discard the supernatant, and collect the lower layer to obtain the test solution (if the lower layer is turbid, it can be purified by centrifuging at 4000 rpm for 1 minute and then discarding the supernatant).

[0176] (2) Detection

[0177] Add 100 μL of the test solution to the gold-labeled microwell, repeatedly pipet to reconstitute the solution, let it stand for 3 minutes, then transfer the solution from the gold-labeled microwell to the sample well of the immunochromatographic test strip. Start timing after adding the sample, and observe the results after standing for 5 to 8 minutes. Results exceeding 8 minutes are invalid. The test should be performed in triplicate.

[0178] (3) Result Interpretation

[0179] The test results of the sample are interpreted based on the color development of the immunochromatographic test strip. The specific method is as follows:

[0180] When the C line does not develop color, the test result is invalid because the operation was incorrect or the test strip has expired. When both the C and T lines develop color, with the T line showing stronger color than the C line, or when there is no significant difference between the T and C lines, the test result is negative (-), indicating that there is no metronidazole in the sample. When both the C and T lines develop color, with the T line showing significantly weaker color than the C line, or when the C line develops color but the T line does not, the test result is positive (+), indicating that there is metronidazole in the sample.

[0181] III. Performance Evaluation of Colloidal Gold Qualitative Immunochromatographic Reagent Kit

[0182] 1. Sensitivity

[0183] Metronidazole standards were diluted to concentrations of 0 μg / L, 1 μg / L, 2 μg / L, 4 μg / L, and 8 μg / L using 0.01 M PBS buffer, and detected using the colloidal gold qualitative immunochromatographic kit of this embodiment.

[0184] Table 2 Sensitivity Measurement Results

[0185]

[0186] As shown in Table 2, the colloidal gold qualitative immunochromatographic kit prepared in this invention has high sensitivity for the detection of metronidazole, with a detection limit as low as 2 μg / L.

[0187] 2. Stability

[0188] The colloidal gold qualitative immunochromatographic test strips included in this kit should be stored at room temperature. To determine their stability, accelerated destructive testing was performed.

[0189] The colloidal gold qualitative immunochromatographic test strips were placed at 45°C for 60 consecutive days. On days 0, 5, 10, 20, 30, 40, 50, and 60, metronidazole standard was diluted with 0.01M PBS buffer to concentrations of 0 μg / L, 1 μg / L, 2 μg / L, and 4 μg / L, respectively. The results were then analyzed using the colloidal gold qualitative immunochromatographic kit described in this embodiment. The experiment was performed in triplicate.

[0190] Table 3. Results of stability tests

[0191]

[0192] As shown in Table 3, after 60 days of storage at 45℃, the color depth of the T and C lines on the colloidal gold qualitative immunochromatographic test strips showed no significant change, indicating that the colloidal gold qualitative immunochromatographic test strips can be stably stored for at least 60 days at 45℃ under accelerated testing. Accelerated storage at 45℃ for 37.5 days is equivalent to one year of storage at room temperature. Therefore, the metronidazole colloidal gold qualitative immunochromatographic test strips prepared in this invention can be stably stored at room temperature for more than one year, fully meeting market demands for storage and transportation.

[0193] 3. Limit of detection for spiked egg samples

[0194] Chicken eggs, free-range chicken eggs, black-bone chicken eggs, duck eggs, goose eggs, and quail eggs were used as test samples. Sample solutions for each test sample were obtained according to the "Sample Processing" method described in this embodiment. Metronidazole standard was used to perform gradient spiking detection on the sample solutions of each test sample, with spiking increments of 0 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 1 μg / kg, and 2 μg / kg. The colloidal gold qualitative immunochromatographic assay kit of this embodiment was used for detection.

[0195] Table 4 Results of Detection Limits for Spiked Fruit and Vegetable Samples

[0196]

[0197] As shown in Table 4, the colloidal gold qualitative immunochromatographic reagent kit prepared in this invention showed good repeatability in the detection results of 10 egg samples. When the metronidazole content in the samples was below 0.5 μg / kg, all results were negative; when it was above 0.5 μg / kg, all results were positive. Therefore, the detection limit of the metronidazole colloidal gold immunochromatographic reagent kit prepared in this invention in egg samples is 0.5 μg / kg.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A metronidazole hapten, characterized in that, Its structural formula is shown in equation (I).

2. A method for preparing a compound with the structural formula shown in formula (I), characterized in that, Metronidazole and ethyl 4-bromobutyrate were reacted completely, and then extracted with ethyl acetate to remove impurities to obtain an intermediate product. The intermediate product was then fully hydrolyzed under alkaline conditions, and the pH of the hydrolysate was adjusted to acidic to obtain the final product. The structural formula of the intermediate product is:

3. Application of compounds with the structural formula shown in formula (I) in the preparation of metronidazole artificial antigen.

4. A metronidazole artificial antigen, characterized in that, The metronidazole hapten conjugate carrier protein described in claim 1 has the structural formula shown in formula (II). Protein is a carrier protein, which is bovine serum albumin or hemocyanin.

5. The method for preparing the metronidazole artificial antigen according to claim 4, characterized in that, The metronidazole hapten of claim 1 is coupled to a carrier protein via an active ester method.

6. Application of compounds with the structural formula shown in formula (II) in the preparation of metronidazole antibodies. in, The protein is a carrier protein, which is bovine serum albumin or hemocyanin.

7. A metronidazole artificial antigen combination, characterized in that, It includes a coating antigen and an immunogen, wherein the coating antigen is obtained by conjugating bovine serum albumin with the metronidazole hapten of claim 1; and the immunogen is obtained by conjugating hemocyanin with the metronidazole hapten of claim 1.

8. The use of the metronidazole artificial antigen combination according to claim 7 in the preparation of a kit for detecting metronidazole.

9. A kit for detecting metronidazole, characterized in that, It includes the metronidazole artificial antigen combination as described in claim 7.

10. The reagent kit according to claim 9, characterized in that, The invention comprises an immunochromatographic test strip and an antibody for detecting metronidazole; the immunochromatographic test strip includes a base plate, on which a sample pad, a reaction membrane, and an absorbent pad are sequentially overlapped; the reaction membrane is a nitrocellulose membrane having a detection zone and a control zone; the detection zone is coated with the coating antigen as described in claim 7, and the control zone is coated with IgG; the antibody for detecting metronidazole is prepared by immunizing animals with the immunogen as described in claim 7.

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