Oxicam compound hapten, complete antigen and application thereof

Abstract

The application relates to a detection method of oxicams for non-diagnostic purposes, and the application obtains a new oxicam hapten by optimizing the design of spatial configuration and chemical characteristics, and significantly improves the immunization efficiency of an antibody and the performance of an immunodetection device. The universal oxicam immunodetection device developed by the application integrates high sensitivity, wide detection range, anti-interference capability and rapid portability, overcomes the defects of the prior art, provides an efficient and reliable solution for rapid screening of oxicams in samples, and has significant technical progress and wide application prospect.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to an oxacin-like compound hapten, a complete antigen, and their applications. Background Technology

[0002] Oxicams are a class of nonsteroidal anti-inflammatory drugs (NSAIDs) belonging to the enol class of 4-hydroxy-1,2-benzothiazide 3-carboxamine derivatives. They include piroxicam (PIR), meloxicam (MLX), tenoxicam (TNX), and lornoxicam (LOR). Most oxicam NSAIDs are non-selective COX inhibitors, while meloxicam is a selective COX-2 inhibitor. Meloxicam's selectivity for COX-2 is approximately six times that for COX-1, which helps avoid the typical gastrointestinal, renal, and cardiovascular side effects common with NSAIDs.

[0003] Although oxacin-type nonsteroidal anti-inflammatory drugs (NSAIDs) have a wide range of therapeutic effects, a number of serious adverse reactions have been increasingly discovered, including cardiovascular risks, cerebral complications, gastrointestinal toxicity, hypertension, hepatotoxicity, and kidney damage. With the increasing prevalence of diseases caused by NSAID overdose, there is an urgent need to develop new analytical methods to monitor oxacin-type NSAID levels in food, preventing consumers from unknowingly consuming excessive amounts over a long period, causing unnecessary physical harm and threatening public health.

[0004] Currently, methods for detecting oxacin derivatives include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and immunoassay. GC-MS and LC-MS / MS methods are sensitive and accurate, but require expensive equipment and time-consuming sample pretreatment.

[0005] Immunoassay has become an important technique for detecting hazardous substances in food due to its simplicity, low cost, and high specificity. Its core lies in the preparation of specific antibodies, which in turn depends on the design of highly efficient haptens. Currently, there are few reports on the preparation of monoclonal antibodies against broad-spectrum oxacinoids, indicating significant potential for further development. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an oxacin-like compound hapten, a complete antigen, and their applications.

[0007] This invention develops a novel universal oxacin-like drug hapten. By optimizing the hapten design, a complete antigen and a broad-spectrum antibody are prepared, improving detection sensitivity and reducing detection costs and time, which is currently in demand. Based on this, a high-performance immunoassay device can be developed, which can improve practicality, detection efficiency, and accuracy.

[0008] The first objective of this invention is to provide a hapten of an oxacin-like compound.

[0009] A second object of the present invention is to provide a method for preparing a compound with the structure shown in formula (I).

[0010] A third objective of this invention is to provide the use of the aforementioned oxacin hapten in the preparation of oxacin hapten complete antigens.

[0011] The fourth objective of this invention is to provide a complete antigen of an oxacin-like compound.

[0012] A fifth object of the present invention is to provide a composition of an immunogen and a coating antigen.

[0013] A sixth object of the present invention is to provide a composition for detecting antibodies and detecting antigens.

[0014] A seventh object of the present invention is to provide the hapten of the said oxacin compound, the complete antigen of the said oxacin compound, and the use of the said composition in the preparation of immunoassay kits or immunoassay devices for oxacin compounds, or in the establishment of non-diagnostic methods for detecting oxacin compounds.

[0015] The eighth object of the present invention is to provide an immunoassay kit or immunoassay device for oxacin-like compounds.

[0016] The ninth objective of this invention is a method for detecting non-diagnostic oxacin compounds.

[0017] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for an oxacin-like compound hapten, the structural formula of which is shown in formula (I). .

[0018] The hapten prepared in this invention retains the two core structures of oxacin-like drugs: 2-methyl-4-hydroxy-2H-1,2-benzothiazine-3-carboxylamine and the aminopyridine ring. Furthermore, the designed coupling arm is a straight chain with an active group, ensuring that the small molecule structure of the prepared artificial antigen retains its original characteristics. This is more conducive to presenting the structural characteristics of universal oxacin-like drugs and improves the immunogenicity of the antigen. Its function is achieved through coupling with proteins. (1) Ortho configuration optimization: By introducing a spacer arm with 4 carbons, the present invention avoids shielding the core structure of the oxacin drug. Moreover, the arm of the present invention is located in the middle of the oxacin drug structure, which significantly exposes the core structure of the oxacin drug (2-methyl-4-hydroxy-2H-1, 2-benzothiazine-3-carboxamine and aminopyridine ring) while ensuring the stability of the universal oxacin drug hapten structure.

[0019] (2) Spacer arm length and carboxyl modification: This invention provides higher reactivity and more efficient coupling with carrier proteins by incorporating a carboxylic acid arm. This design introduces a 4-carbon spacer arm at the arm to avoid the folding of the hapten molecule conformation caused by the long spacer arm, so that the hapten molecule is close to the surface of the carrier protein and is shielded, thereby improving the immune recognition efficiency of the antigen epitope.

[0020] This invention exposes the core structure of hapten compounds and enhances immunoreaction efficiency by optimizing their spatial configuration and chemical properties. This design not only simplifies the synthesis pathway of haptens but also significantly improves the broad-spectrum and sensitivity of antibodies while maintaining chemical stability, laying the foundation for improved performance in immunoassays.

[0021] It also claims a method for preparing a compound with the structural formula shown in formula (I), comprising the following steps: Enolate compound a was hydrolyzed under light-protected, sodium ethoxide, and alkaline conditions to give the first intermediate compound b. S2. An aminopyridine compound c and a haloester compound d are subjected to a substitution reaction under alkaline conditions to obtain a second intermediate compound e; S3. The first intermediate compound b and the second intermediate compound e are reacted with an amide in an organic solvent to obtain the third intermediate compound f; S4. Hydrolyze the third intermediate compound f in an alkaline solution, adjust the pH to precipitate a solid, and obtain a compound with the structural formula shown in formula (I); The compound a is ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide, with CAS: 24683-26-9; Compound c is 2-aminopyridine, with CAS number 504-29-0; The haloester compound d is ethyl 4-bromobutyrate, with CAS number 2969-81-5.

[0022] Preferably, the substitution reaction in step S2 is carried out in a polar aprotic solvent.

[0023] More preferably, the polar aprotic solvent is N,N-dimethylformamide (DMF).

[0024] Preferably, in step S2, the ratio of compound c to compound d is (300-700) mg : (1000-2000) mg.

[0025] More preferably, in step S2, the ratio of compound c to compound d is 500 mg : 1243 mg.

[0026] Preferably, the alkaline conditions in step S1 are pH = 8.5 to 10.5.

[0027] More preferably, the alkaline condition described in step S1 is pH = 9.

[0028] Optionally, the alkaline conditions described in step S1 can be controlled by adjusting the amount of lithium hydroxide added.

[0029] Preferably, the reaction in step S1 is carried out in the dark.

[0030] More preferably, the reaction in step S1 is carried out in the dark at 40°C.

[0031] Preferably, the amide reaction in step S3 is carried out in a nonpolar to moderately polar solvent environment.

[0032] More preferably, the nonpolar to moderately polar solvent environment is provided by a mixed solvent of toluene and thionyl chloride.

[0033] Preferably, the alkaline solution in step S4 is an aqueous solution of lithium hydroxide.

[0034] More preferably, the alkaline solution in step S4 is a 4-8 mol / L lithium hydroxide aqueous solution (most preferably 6 mol / L).

[0035] Preferably, in step S5, adjusting the pH to precipitate solids involves adjusting the pH to 5-6.

[0036] As an alternative implementation method, the preparation method of the above-mentioned universal oxacin hapten includes the following steps: S1. Dissolve compound a in ethanol, then add lithium hydroxide solid, react at 40°C in the dark for 10-12 hours, add purified water, and extract twice with ethyl acetate. Discard the ethyl acetate layer, adjust the pH of the aqueous phase to 5-6 with 4 M hydrochloric acid, and the solid precipitates out. Filter and dry to obtain compound b.

[0037] S2. Compound c is thoroughly mixed with N,N-dimethylformamide, potassium carbonate and compound d, and reacted at 80℃ for 2 h to 8 h. After the reaction is complete, purified water is added, the aqueous phase is extracted twice with n-hexane, the n-hexane layer is discarded, the aqueous phase is extracted three times with ethyl acetate, and the organic phase is removed under reduced pressure to obtain compound e.

[0038] S2. Compound b was dissolved in a mixed solution of toluene and thionyl chloride, and compound e was added and stirred at room temperature for 4–8 h. After the reaction was complete, purified water was added, the aqueous phase was extracted three times with ethyl acetate, and the organic phase was washed with saturated NaHCO3 solution. The organic phase was desolventized under reduced pressure to obtain compound f.

[0039] S3. Dissolve compound f in methanol, then add 6 M lithium hydroxide aqueous solution, react at room temperature for 10-12 h, add purified water, and extract twice with ethyl acetate. Discard the ethyl acetate layer, adjust the pH of the aqueous phase to 5-6 with 4 M hydrochloric acid, and the solid precipitates. Filter and dry to obtain a universal oxacin hapten.

[0040] More preferably, in step S2, the ratio of compound c, N,N-dimethylformamide, potassium carbonate and compound d is (700-900) mg : (3.5-5.5) mL : (880-1480) mg : (1000-1500) mg.

[0041] Most preferably, in step S2, the ratio of compound a, N,N-dimethylformamide, potassium carbonate, and compound b is 500 mg : 4 mL : 1099 mg : 1243 mg.

[0042] More preferably, the ratio of toluene to thionyl chloride in step S3 is (0.5-2):(0.5-4).

[0043] Most preferably, the ratio of toluene to thionyl chloride in step S3 is 1:1.

[0044] This invention also claims the use of the described oxacin hapten in the preparation of oxacin complete antigens.

[0045] The present invention also claims protection for a complete antigen of an oxacin-like compound, which is a compound with the structural formula shown in formula (I) coupled with a carrier protein.

[0046] Preferably, it has a structural formula as shown in formula (II). .

[0047] As an alternative implementation, the method for preparing the universal oxacin-type drug complete antigen includes the following steps: S1. Dissolve the above-mentioned universal oxacin hapten in dimethylformamide (DMF), add EDC and N-hydroxysuccinimide (NHS) to react and obtain the activated hapten ester; S2. Add the hapten activated ester to the carrier protein solution; S3. Dialyze the solution obtained in step S2 to obtain the universal oxacin complete antigen.

[0048] More preferably, the carrier protein is one of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin, and hemocyanin.

[0049] A composition of an immunogen and a coating antigen, wherein the coating antigen is a complete antigen of the oxicam-like compound, and the immunogen is the complete antigen of the oxicam-like compound as an immunogen.

[0050] Preferably, the carrier protein of the coating antigen is bovine serum albumin, and the carrier protein of the immunogen is lactoferrin.

[0051] A composition for detecting an antibody and a detecting antigen, wherein the detecting antigen is a complete antigen of the oxacin-like compound, and the detecting antibody is an antibody obtained by immunizing animals with the complete antigen of the oxacin-like compound.

[0052] Preferably, the carrier protein of the detection antigen is bovine serum albumin, and the detection antibody is obtained by immunizing animals with the complete antigen of the oxacin-like compound with lactoferrin as the carrier protein.

[0053] The present invention also claims protection for the said oxacin hapten, the said oxacin complete antigen, and the use of the said composition in the preparation of immunoassay kits or immunoassay devices for oxacin compounds or in the establishment of non-diagnostic methods for the detection of oxacin compounds.

[0054] Preferably, the oxacone compound is one or more of meloxicam, lornoxicam, sudoxicam, piroxicam, droxicam, and tenoxacone.

[0055] The present invention also claims an immunoassay kit or immunoassay device for an oxacin-like compound, comprising or prepared using said composition.

[0056] The present invention also claims a method for detecting non-diagnostic oxacin compounds using the aforementioned composition.

[0057] The immunoassay kit or immunoassay device includes, but is not limited to, one or more of enzyme immunoassay, immunochromatography, immunosensing, and immunogold assays.

[0058] As an alternative implementation, the preparation method of the kit includes the following steps: S1. Preparation of reaction membrane: Spray a solution containing the above-mentioned universal oxacin-type drug complete antigen onto the detection zone (T line) of the nitrocellulose membrane (NC membrane), and spray sheep-derived immunoglobulin (sheep anti-mouse IgG) onto the control zone (C line). S2. Preparation of colloidal gold-labeled microwells: Colloidal gold was prepared by sodium citrate reduction method. The above-mentioned broad-spectrum oxacin monoclonal antibody was bound to colloidal gold, bovine serum albumin was added for blocking, the precipitate was collected by centrifugation, and then reconstituted and dispensed into microwells. S3. Preparation of sample pads: Soak blank sample pads in buffer solution and then dry them for later use; S4. Assemble the test card: Stack the sample pad obtained in step S3, the reaction membrane obtained in step S1, and the absorbent pad on the PVC board in sequence, cut them into test strips, and insert them into the test card case.

[0059] In this process, a 0.1–0.6 mg / mL solution of a universal oxacin-type drug complete antigen is sprayed in the detection area (T line), and a 0.05–0.2 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) is sprayed in the control area (C line).

[0060] Preferably, a 0.4 mg / mL universal oxacin-type drug complete antigen solution is sprayed in the detection area (T line), and a 0.1 mg / mL sheep-derived immunoglobulin (sheep anti-mouse IgG) solution is sprayed in the control area (C line).

[0061] The method of using the colloidal gold immunochromatographic assay device for oxacin-like compounds in the above-mentioned optional implementation scheme includes the following steps: S1. Preparation of test solution: Add 5g of test sample to 5mL of ethyl acetate solution containing 1% acetic acid, mix well and centrifuge, take 4.5mL of supernatant, blow dry with nitrogen gas and add 150μL of phosphate buffer to mix, and prepare test solution; S2. Detection: Add the test solution to the colloidal gold-labeled microwell of the universal oxacin-type colloidal gold immunochromatographic assay device, mix to reconstitute the colloidal gold-labeled antibody, let stand for 3 minutes, then transfer the solution from the colloidal gold-labeled microwell to the sample well of the universal oxacin-type colloidal gold immunochromatographic assay device card; start timing after sample addition, observe the results within 5-8 minutes and determine the presence or absence of universal oxacin-type drug residues based on the colorimetric results; results after 8 minutes are invalid; the result interpretation criteria are as follows: (1) Negative (-): The T line shows stronger color than the C line or shows no significant difference in color between the two lines; (2) Positive (+): The T line is significantly weaker than the C line or the T line is not visible; (3) Invalid: No C line appears, the operation process is incorrect or the test strip has expired.

[0062] Sensitivity tests were conducted on universal oxacin standard solutions and test samples. The limits of detection (LODs) for oxacin-like drugs in the standard solutions using the optional implementation scheme described above were: meloxicam 0.7 μg / L, lornoxicam 1.0 μg / L, sudoxicam 2.8 μg / L, piroxicam 4.0 μg / L, droxicam 8.0 μg / L, and tenoxacin 20.0 μg / L. The LOD for oxacin-like drugs in the test samples reached 1 µg / kg. The universal oxacin-like drug colloidal gold immunochromatographic detection device of this invention exhibits low LODs for oxacin-like drugs in samples, meeting the requirements for trace detection and demonstrating excellent detection performance.

[0063] Through stability testing, the colloidal gold immunochromatographic detection device for universal oxacin drugs of the above-mentioned optional implementation schemes showed good stability under storage conditions. It can still accurately detect universal oxacin drug residues after storage and can be stably stored at room temperature for more than one year, which can meet the market requirements in terms of storage and transportation.

[0064] Compared with the prior art, the present invention has the following beneficial effects: (1) Broad spectrum. This invention prepares a complete antigen and a broad-spectrum antibody based on a novel universal meloxicam hapten. The antibody has a high affinity for meloxicam, and its IC50 value is high. 50 The value was 0.169 μg / L. The colloidal gold immunochromatographic test strip prepared in this invention exhibited sensitivities of 0.7 µg / kg, 1.0 µg / kg, 2.8 µg / kg, 4.0 µg / kg, 8.0 µg / kg, and 20.0 µg / kg for meloxicam, lornoxicam, sudoxicam, piroxicam, droxicam, and tenoxicam in standard solutions, respectively, with cross-reactivity rates of 100%, 70%, 25%, 17.5%, 8.75%, and 3.5%, respectively. The resulting universal immunoassay device for meloxicam-like drugs improves detection efficiency, shortens detection time, and reduces detection costs.

[0065] (2) High sensitivity. The universal meloxicam immunoassay device based on the preparation of the novel universal meloxicam hapten of this invention has a detection limit of 0.7 μg / L for meloxicam in standard solution and a detection limit of 1 µg / kg for test sample. The sensitivity is greatly improved, which significantly enhances the detection capability of low concentration meloxicam drug residues.

[0066] (3) Strong anti-interference ability. Through the optimized design of the universal oxacin hapten structure and the high affinity of the antibody, the universal oxacin immunoassay device maintains stable performance under the test sample environment. The invention significantly reduces the influence of matrix effect, and the detection limit of the test sample reaches 1µg / kg, ensuring the reliability of practical application.

[0067] (4) Good stability. The universal oxacin immunoassay device prepared based on the novel universal oxacin hapten of the present invention can be stably stored at room temperature for more than one year, and can still accurately detect universal oxacin residues after long-term storage.

[0068] (5) Fast and portable. The universal oxacin immunoassay device based on the novel universal oxacin hapten prepared according to the present invention has a detection time of only 30 minutes, is easy to operate, and is suitable for use by grassroots testing personnel.

[0069] In summary, this invention significantly improves antibody immunoassay efficiency and the performance of immunoassay devices by optimizing the spatial configuration and chemical properties of a novel universal oxacin hapten structure. The resulting universal oxacin immunoassay device integrates high sensitivity, wide detection range, anti-interference capability, and rapid portability, overcoming the shortcomings of existing technologies and providing an efficient and reliable solution for rapid screening of oxacin drugs in samples. It demonstrates significant technological advancement and broad application prospects. Attached Figure Description

[0070] Figure 1 This is a synthetic route diagram of the universal oxacin hapten used in the examples.

[0071] Figure 2 The mass spectrum of the universal oxacin hapten of the present invention.

[0072] Figure 3 The synthetic route diagram of the universal oxacin-type drug complete antigen of the present invention.

[0073] Figure 4 The universal oxacin hapten, universal oxacin complete antigen-BSA, and BSA ultraviolet scan images in Example 2 of the present invention.

[0074] Figure 5 The universal oxacin hapten, universal oxacin complete antigen-LF, and LF ultraviolet scan images in Example 2 of the present invention.

[0075] Figure 6 Standard curve for meloxicam detection by monoclonal antibody ELISA.

[0076] Figure 7 Standard curve for monoclonal antibody ELISA detection of lornoxicam.

[0077] Figure 8 Standard curve for monoclonal antibody ELISA detection of sudoxicam.

[0078] Figure 9 Standard curve for ELISA detection of piroxicam using monoclonal antibody.

[0079] Figure 10 The standard curve for detecting draloxiconazole using monoclonal antibody ELISA.

[0080] Figure 11 Standard curve for the detection of tenoxicam by monoclonal antibody ELISA.

[0081] Figure 12 This is a schematic diagram of the result judgment criteria for the universal colloidal gold immunochromatographic detection device for oxacin drugs in Example 11. Detailed Implementation

[0082] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. 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.

[0083] Example 1: Preparation and Identification of Universal Oxicam Hapten I. Experimental Methods 1. Preparation of universal oxacin hapten See the chorus route for oxacin haptens. Figure 1 , The specific synthesis method is as follows: S1. Dissolve 2.0 g of compound a (7.0 mmol, CAS: 24683-26-9) in 4 mL of ethanol, then add 576 mg of sodium ethoxide (8.47 mmol) and 339 mg of lithium hydroxide (14.12 mmol) in sequence. React at 40 °C in the dark for 10–12 h. Add 70 mL of purified water and extract twice with 30 mL of dichloromethane. Adjust the pH of the aqueous phase to 5–6 with 4 M hydrochloric acid. The precipitated solid is filtered, dried, and 341 mg of crude compound b is obtained.

[0084] S2. Take 500.0 mg of compound c (5.31 mmol, CAS: 504-29-0) and place it in a 50 mL round-bottom flask. Then add 4 mL of DMF (N,N-dimethylformamide), 1100 mg of potassium carbonate (7.96 mmol), and 1243 mg of compound d (6.37 mmol, CAS: 2969-81-5) in sequence. After stirring and mixing thoroughly, react at 80 °C for 2 h to 8 h. After the reaction is complete, add 50 mL of purified water. Extract the aqueous phase twice with n-hexane and discard the n-hexane layer. Extract the aqueous phase three times with ethyl acetate. Remove the solvent from the organic phase under reduced pressure to obtain 312 mg of crude compound e.

[0085] S3. 295 mg of crude compound b was dissolved in 4 mL of a 1:1 mixture of toluene and thionyl chloride, 10 µL of LDMF was added, and the reaction was carried out at 60 °C for 1–2 h. After the reaction was complete, the solution was evaporated to dryness under reduced pressure. The residue was dissolved in 10 mL of dichloromethane and slowly added dropwise under ice bath to a mixed solution containing 304 mg of triethylamine (4.77 mmol, CAS: 121-44-8), 200 mg of crude compound e, and 10 mL of dichloromethane. After the addition was complete, the mixture was brought to room temperature and stirred at room temperature for 4–8 h. After the reaction was complete, purified water was added, the aqueous phase was extracted three times with ethyl acetate, and the organic phase was washed with saturated NaHCO3 solution. The organic phase was desolventized under reduced pressure, and the residue was purified by column chromatography to give 213 mg of compound f.

[0086] S4. Dissolve 200 mg of compound f in 3 mL of methanol, then add 3 mL of 6 mol / L lithium hydroxide aqueous solution, react at room temperature in the dark for 10–12 h, add 70 mL of purified water, extract twice with 30 mL of dichloromethane, adjust the pH of the aqueous phase to 5–6 with 4 M hydrochloric acid, precipitate the solid, filter and dry to obtain 118 mg of universal oxacin hapten.

[0087] 2. Identification of haptens from oxacin-like drugs Mass spectrometry and nuclear magnetic resonance were used to detect the universal oxacin hapten.

[0088] II. Experimental Results Mass spectrum detection results as follows Figure 2 The molecular ion peak of the universal oxacin hapten in ESI negative ion mode is [MH]. -The m / z value is 415.9 (the theoretical molecular weight of the universal oxacin hapten is 417 Da), consistent with the theoretical value. The 1H NMR results are shown below: 1H NMR (500 MHz, CDCl3) δ 12.40 (s, 1H), 11.79 (s, 1H), 8.44 (dd, J = 3.5, 1.6 Hz, 1H), 8.29 – 8.24 (m, 1H), 8.01 – 7.96 (m, 3H), 7.61 (td, J = 7.7, 1.6 Hz, 1H), 7.09 (dd, J = 7.8, 1.3 Hz, 1H), 7.02 (ddd, J = 7.3, 3.4, 1.3 Hz, 1H), 4.53 (t, J = 6.4 Hz, 2H), 2.86 (s, 3H), 2.39 (t, J = 7.0 Hz, 2H), 2.14 – 2.04 (m, 2H). This demonstrates the synthesis of the universal oxacin hapten shown in formula (I). .

[0089] Example 2: Preparation and identification of complete antigens for universal oxacin drugs I. Experimental Methods The synthetic route for the complete antigen of universal oxacin drugs is shown in [link to relevant documentation]. Figure 3 Based on different carrier proteins, haptens 1 to haptens 5 of type oxacin were synthesized.

[0090] 1. Preparation of universal oxacin hapten (1) Preparation of complete antigen 1 of universal oxacin class drugs S1. Take 5 mg of the universal oxacin hapten of formula (I) prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; S2. Weigh 30 mg of bovine serum albumin (BSA) and dissolve it completely in 5 mL of 0.1 mol / L CB solution to form a bovine serum albumin carrier solution. Under stirring, slowly add the hapten activated ester obtained in step S1 dropwise to the bovine serum albumin carrier solution and stir at room temperature for 16–24 h. S3. Dialyze the solution obtained in step S2 with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules, and obtain the universal oxacin complete antigen (i.e., universal oxacin hapten-BSA conjugate), aliquot and store at 4°C for later use.

[0091] (2) Preparation of universal oxacin complete antigen 2 The preparation of the same complete antigen 1 as the universal oxacin class drugs differs in that "30 mg bovine serum albumin (BSA)" is changed to "50 mg ovalbumin (OVA)".

[0092] (3) Preparation of universal oxacin complete antigen 3 The preparation of the same complete antigen 1 as the universal oxacin class drugs differs in that "30 mg bovine serum albumin (BSA)" is changed to "100 mg lactoferrin (LF)".

[0093] (4) Preparation of complete antigen 4 of universal oxacin drugs The preparation of the same complete antigen 1 as the universal oxacin class drugs differs in that "30 mg bovine serum albumin (BSA)" is changed to "240 mg human serum albumin (HSA)".

[0094] (5) Preparation of complete antigen 5 of universal oxacin drugs The preparation of the same complete antigen 1 as the universal oxacin class drugs differs in that "30 mg bovine serum albumin (BSA)" is changed to "240 mg hemocyanin (KLH)".

[0095] 2. Identification of complete antigens of universal oxacin drugs Taking universal coxicam complete antigen 1 and universal coxicam complete antigen 3 as examples, ultraviolet scanning (190-400 nm) was used to measure the two carrier proteins, universal coxicam hapten, universal coxicam complete antigen 1 and universal coxicam complete antigen 3.

[0096] II. Experimental Results The results are as follows Figure 4 and Figure 5 As shown. The UV characteristic absorption peak of the universal oxacin complete antigen is shifted to varying degrees relative to both the universal oxacin hapten and the carrier protein. Furthermore, the universal oxacin complete antigen exhibits characteristic absorption peaks of both the universal oxacin hapten and the carrier protein, indicating successful coupling between the universal oxacin hapten and the carrier protein. Thus, the universal oxacin complete antigen with the structural formula shown in (II) was successfully prepared. .

[0097] Example 3 Preparation of a universal monoclonal antibody against oxacin drugs I. Experimental Methods Broad-spectrum oxacin monoclonal antibodies were prepared using the universal oxacin complete antigen 3 (i.e., universal oxacin hapten-LF conjugate) prepared in Example 2 as the immunization antigen.

[0098] The specific method is as follows: S1. After emulsifying the universal oxacin complete antigen with an equal volume of Freund's adjuvant, BALB / C mice were subcutaneously immunized. Each mouse received 100 μg of immunization per immunization, with an interval of 2 weeks between immunizations, for a total of 3 immunizations. S2. After the third immunization, take tail vein blood from mice to test serum antibody titer. If the antibody titer is lower than 1:8000, administer a booster immunization of 100 μg every 7 days until the titer stabilizes above 1:32000. S3. Subcutaneous booster immunization was performed using 0.01 M PBS solution containing 100 μg of universal oxacin complete antigen. Five days later, mouse spleen cells were collected and fused with SP20 mouse myeloma cells. S4. The fused cells were screened in HAT medium, and after 5 days they were cultured in complete medium (fetal bovine serum, basal medium, streptomycin-penicillin). S5. Using the universal oxacin complete antigen 1 (universal oxacin hapten-BSA conjugate) prepared in Example 2 as the coating antigen, the titer and inhibition of the cell supernatant were tested by enzyme-linked immunosorbent assay (ELISA) to screen OD. 450 Positive cells with an inhibition rate of over 85% when the concentration of the six oxacin drugs is 10 μg / mL were cultured three times using the limiting dilution method to confirm that the positive cells were hybridoma cells secreting monoclonal antibodies against broad-spectrum oxacin drugs. S6. After expanding and culturing hybridoma cells, use 1×10 6 Injecting one cell / mouse into the peritoneal cavity of mice to induce ascites, the ascites was purified by octanoic acid-ammonium sulfate precipitation to obtain a universal oxacin monoclonal antibody.

[0099] Example 4: Performance of a Universal Oxacon-like Monoclonal Antibody I. Experimental Methods Using the universal oxacin complete antigen 1 (universal oxacin hapten-BSA conjugate) prepared in Example 2 as the coating antigen, and taking the broad-spectrum oxacin monoclonal antibody prepared in Example 3 as an example, the performance of the broad-spectrum oxacin monoclonal antibody was evaluated by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: S1. Using carbonate buffer (pH = 9.6) as the coating diluent, dilute universal oxacin complete antigen 1 to 0.1 μg / mL, add 100 μL / well to polystyrene microplates, coat overnight at 4°C, spin dry, add 280 μL / well of 1% BSA solution, block in phosphate buffer containing 1% BSA at 37°C for 1 h, spin dry, and vacuum package for storage. S2. Dilute the broad-spectrum oxacin monoclonal antibody to 0.3 μg / mL with phosphate buffer containing 0.05% sodium azide (pH = 7.4) and store at 4°C for later use; S3. Add 50 μL / well of standard solutions of six coxicams (meroxicam, lornoxicam, sudoxicam, piroxicam, droxicam, and tenoxicam, 0–100 μg / L) to the microplate coated with the complete antigen of universal coxicam drugs in step S1, and then add 50 μL / well of the broad-spectrum coxicam monoclonal antibody solution from step S2. Incubate at 37°C for 0.5 h, and then dry. S4. Add 280 μL of washing buffer to each well, wash three times, pat dry, then add 100 μL of enzyme-labeled secondary antibody to each well, and incubate at 37°C for 0.5 h. S5. After washing three times, pat dry. Add 100 μL of TMB chromogenic solution to each well and react at 37°C for 15 min. Stop the reaction by adding 50 μL of 2 M sulfuric acid solution to each well. Set the microplate reader to 450 nm and measure the OD value of each well. II. Experimental Results The results are shown in Table 1. Table 1:

[0100] Based on the data in Table 1, the standard curve was plotted using OriginPro 2024 software for logistic curve fitting. Figures 6 to 11 The linear equations for the standard curves of various drugs are indicated in the respective figures, where X represents the concentration of the standard solutions of the six oxacin drugs, and y represents the B / B0 value. The IC50 values ​​of the monoclonal antibodies against the six oxacin drugs were calculated. 50 The values ​​were 0.169 μg / L for meloxicam, 0.243 μg / L for lornoxicam, 0.694 μg / L for sudoxicam, 0.97 μg / L for piroxicam, 2.0 μg / L for droxicam, and 5.0 μg / L for tenoxicam. This demonstrates that the monoclonal antibodies against oxicams obtained by immunizing mice with the universal oxicam hapten prepared in Example 1 have broad-spectrum activity, high affinity, and multi-recognition capability, significantly improving the detection performance of various oxicams.

[0101] Example 5: Preparation of a universal colloidal gold immunochromatographic detection device for oxacin-like drugs S1. Preparation of reaction membranes coated with universal oxacin-type drug complete antigen and mouse IgG. Using a nitrocellulose membrane (NC membrane) as the substrate, a 0.4 mg / mL solution of the universal oxacin-type drug complete antigen 1 prepared in Example 2 was sprayed onto the detection zone (T line), and a 0.1 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH = 7.6), with a spraying volume of 1.0 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity. S2. Preparation of microporous monoclonal antibodies containing colloidal gold-labeled universal oxacin monoclonal antibodies S2.1 Preparation of Colloidal Gold Take 1g of chloroauric acid, dissolve it in pure water by sonication, and then bring the volume to 100 mL. Take 4 mL of the above solution into 100 mL of pure water, heat to boiling, add 1.2 mL of 0.06% sodium citrate solution for reduction, and continue heating for 10 minutes. After heating, cool to 25℃ and then add pure water to the original volume to obtain a colloidal gold solution. Store it away from light. All glassware used should be soaked overnight in a mixture of potassium permanganate and sulfuric acid, and then washed and dried before use. S2.2 Labeling of Universal Oxacon Monoclonal Antibodies Colloidal gold solution was dispensed in 1 mL vials, and the pH was adjusted dropwise to different values ​​within the range of 7.0–9.0 using 0.1 mol / L K₂CO₃ solution to optimize the binding of the antibody to the colloidal gold. 5 μg of the universal oxacin monoclonal antibody prepared in Example 3 was added to the colloidal gold solution at each pH, ​​and the reaction was carried out at 25°C for 5 minutes. The color change of the solution was observed, and the lowest pH value that maintained a red color was selected as the optimal pH. The optimal pH was approximately pH 6.8 when 5 μL of 0.1 mol / L K₂CO₃ solution was added. 10 μL of 10% bovine serum albumin (BSA) solution was added for blocking, and the mixture was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was collected. S2.3 Preparation of colloidal gold labeled micropores The precipitate (i.e. labeled antibody) was reconstituted with colloidal gold diluent containing 0.02 M Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose. The solution was dispensed into microwells at 10 μL / well and dried at 37°C for 16 hours before storage. S3. Prepare the sample pad The cut 30×30 cm blank sample pads were immersed in 0.05 M PBS buffer containing 0.05% Tween 20, 1% sucrose, 0.5% PVP40, and 0.05% sodium azide for 5 min. After immersion, the pads were removed and dried at 37℃ for 16 h. They were then stored at 20-25℃ and ≤ 35% humidity. S4. Assemble the colloidal gold chromatography detection card The reaction membrane prepared in step S1 is stacked in the middle of the PVC board backing; the sample pad prepared in step S3 is stacked on the side of the reaction membrane near the detection area, and the absorbent pad is stacked on the side near the control area; the two ends of the reaction membrane are connected to the sample pad and the absorbent pad respectively, and the connection parts of the sample pad, reaction membrane and absorbent pad overlap by 1-2 mm, and are attached to the PVC board; the base plate is cut into 3 mm wide test strips using a strip cutter and inserted into the detection card with a sample application hole, with the sample application hole aligned with the sample pad area of ​​the test strip.

[0102] Example 6: Preparation of a universal colloidal gold immunochromatographic detection device for oxacin-like drugs S1. Preparation of reaction membranes coated with universal oxacin-type drug complete antigen and mouse IgG. Using a nitrocellulose membrane (NC membrane) as the substrate, a 0.1 mg / mL solution of the universal oxacin-type drug complete antigen 1 prepared in Example 2 was sprayed onto the detection zone (T line), and a 0.1 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH = 7.6), with a spraying volume of 0.8 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity. The other steps (S2 to S4) are the same as S2 to S4 in Example 5.

[0103] Example 7: Preparation of a universal colloidal gold immunochromatographic detection device for oxacin-like drugs S1. Preparation of reaction membranes coated with universal oxacin-type drug complete antigen and mouse IgG. Using a nitrocellulose membrane (NC membrane) as the substrate, a 0.2 mg / mL solution of the universal oxacin-type drug complete antigen 1 prepared in Example 2 was sprayed onto the detection zone (T line), and a 0.08 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH = 7.6), with a spraying volume of 1.0 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity. The other steps (S2 to S4) are the same as S2 to S4 in Example 5.

[0104] Example 8: Preparation of a universal colloidal gold immunochromatographic detection device for oxacin-like drugs S1. Preparation of reaction membranes coated with universal oxacin-type drug complete antigen and mouse IgG. Using a nitrocellulose membrane (NC membrane) as the substrate, a 0.3 mg / mL solution of the universal oxacin-type drug complete antigen 1 prepared in Example 2 was sprayed onto the detection zone (T line), and a 0.12 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH = 7.6), with a spraying volume of 1.2 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity. The other steps (S2 to S4) are the same as S2 to S4 in Example 5.

[0105] Example 9: Preparation of a universal colloidal gold immunochromatographic detection device for oxacin-type drugs S1. Preparation of reaction membranes coated with universal oxacin-type drug complete antigen and mouse IgG. Using a nitrocellulose membrane (NC membrane) as the substrate, a 0.4 mg / mL solution of the universal oxacin-type drug complete antigen 1 prepared in Example 2 was sprayed onto the detection zone (T line), and a 0.15 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH = 7.6), with a spraying volume of 0.9 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity. The other steps (S2 to S4) are the same as S2 to S4 in Example 5.

[0106] Example 10: Preparation of a universal colloidal gold immunochromatographic detection device for oxacin-like drugs S1. Preparation of reaction membranes coated with universal oxacin-type drug complete antigen and mouse IgG. Using a nitrocellulose membrane (NC membrane) as the substrate, a 0.4 mg / mL solution of the universal oxacin-type drug complete antigen 1 prepared in Example 2 was sprayed onto the detection zone (T line), and a 0.2 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH = 7.6), with a spraying volume of 0.9 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity. The other steps (S2 to S4) are the same as S2 to S4 in Example 5.

[0107] Example 11: Detection method, sensitivity, and specificity of a universal colloidal gold immunochromatographic assay device for oxacin-like drugs. I. Experimental Methods 1. Detection of standard solution samples of universal oxacin drugs A series of standard solutions of oxacin drugs with different concentrations (meroxacin, lornoxicam, sudoxicam, piroxicam, droxicam, and tenoxacin, 0–20 μg / L) were prepared using 0.01 M PBS buffer. Then, 100 μL of each standard solution was added to the colloidal gold-labeled microwells of the universal oxacin drug colloidal gold immunochromatographic detection device prepared in Example 5, and the solution was repeatedly reconstituted by pipetting. After standing for 5 min, the solution in the colloidal gold-labeled microwells was transferred to the sample wells of the detection card of the universal oxacin drug colloidal gold immunochromatographic detection device of this invention. Timing was started after sample addition, and the results were observed after 5–8 min (the signal intensity was uniform during this time period; the specific reading time could be adjusted according to the ambient temperature, for example, reading the result at 6 minutes). Results after 8 min were considered invalid. The detection experiment was performed in triplicate.

[0108] The criteria for determining the results are as follows: Figure 12 As shown, specifically: (1) Negative (-): The T line shows stronger color than the C line or shows no significant difference in color between the two lines; (2) Positive (+): The T line is significantly weaker than the C line or the T line is not visible; (3) Invalid: No C line appears, the operation process is incorrect or the test strip has expired.

[0109] II. Experimental Results The detection results are shown in Table 2. Table 2 shows that the universal colloidal gold immunochromatographic detection device for oxacin analogues prepared in this invention has a sensitivity of 0.7 μg / L for meloxicam; 1.4 μg / L for lornoxicam; 2.8 μg / L for sudoxicam; 4.0 μg / L for piroxicam; 8.0 μg / L for droxicam; and 20.0 μg / L for tenoxacin. The cross-reactivity rates to lornoxicam, sudoxicam, piroxicam, droxicam, and tenoxacin, relative to meloxicam, are 70%, 25%, 17.5%, 8.75%, and 3.5%, respectively. This product exhibits high cross-reactivity to oxacin analogues and strong broad-spectrum activity.

[0110] Table 2. Determination results of standard solutions of different concentrations of oxacin derivatives.

[0111] Example 12 Detection of spiked milk and herbal tea samples using a universal colloidal gold immunochromatographic assay device for oxacin-like drugs. I. Experimental Methods Milk and herbal tea were selected as blank samples. Standard solutions of six oxacin derivatives (meloxacin, lornoxicam, sudoxicam, piroxicam, droxicam, and tenoxacin) were used to perform gradient spiking assays on the two blank samples, with spiking gradients of 0, 0.125, 0.25, 0.5, 1, and 2 μg / kg, respectively. The assay was performed in triplicate.

[0112] Take 5g of the liquid sample to be tested and place it in a 15mL centrifuge tube. Add 5mL of ethyl acetate solution containing 1% acetic acid, mix well, and centrifuge. Take 4.5mL of the supernatant, dry it under nitrogen, concentrate it, and then add 150μL of phosphate buffer and 500μL of n-hexane. Mix vigorously for 2 minutes. After the centrifuge tube has stood for 2 minutes, the lower layer of the solution is the test solution. Add 100μL of the test solution to the colloidal gold-labeled microwell of the universal colloidal gold immunochromatographic detection device for oxacin drugs of the present invention. Repeatedly blow and mix the solution to reconstitute it evenly. After standing for 5 minutes, transfer the solution in the colloidal gold-labeled microwell to the sample well of the detection card of the universal colloidal gold immunochromatographic detection device for oxacin drugs of the present invention. Start timing after adding the sample. After 5-8 minutes, observe the results according to the judgment criteria (same as Example 11).

[0113] II. Experimental Results The test results are shown in Table 3. The results show that a negative result was obtained when the concentration of the six oxacinoids in the sample was 0 μg / kg; positive results were obtained when the concentration was 1 μg / kg or higher. Therefore, the detection limit of the universal colloidal gold immunochromatographic detection device for oxacinoids prepared in this invention is 1 μg / kg.

[0114] Table 3. Detection Limits for Spiked Samples

Claims

1. A hapten of an oxacinoid compound, 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, Includes the following steps: Enolate compound a was hydrolyzed under light-protected, sodium ethoxide, and alkaline conditions to give the first intermediate compound b. S2. An aminopyridine compound c and a haloester compound d are subjected to a substitution reaction under alkaline conditions to obtain a second intermediate compound e; S3. The first intermediate compound b and the second intermediate compound e are reacted with an amide in an organic solvent to obtain the third intermediate compound f; S4. Hydrolyze the third intermediate compound f in an alkaline solution, adjust the pH to precipitate a solid, and obtain a compound with the structural formula shown in formula (I); The compound a is ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide, with CAS: 24683-26-9; Compound c is 2-aminopyridine, with CAS number 504-29-0; The haloester compound d is ethyl 4-bromobutyrate, with CAS number 2969-81-5.

3. The use of the hapten of the oxacin class compound according to claim 1 in the preparation of the complete antigen of the oxacin class compound.

4. A complete antigen of an oxacin-like compound, characterized in that, It is a compound with the structural formula shown in formula (I) coupled with a carrier protein.

5. The complete antigen of the oxacinoid compound according to claim 4, characterized in that, Its structural formula is as shown in formula (II). 。 6. A composition of an immunogen and a coating antigen, characterized in that, The coating is a complete antigen of the oxacin-like compound as described in claim 4 or 5, and the immunogen is a complete antigen of the oxacin-like compound as described in claim 4 or 5.

7. A composition for detecting antibodies and detecting antigens, characterized in that, The detection antigen is the complete antigen of the oxacin-like compound as described in claim 4 or 5, and the detection antibody is the antibody obtained by immunizing animals with the complete antigen of the oxacin-like compound as described in claim 4 or 5.

8. The use of the hapten of the oxacin class compound according to claim 1, the complete antigen of the oxacin class compound according to claim 4 or 5, and the composition according to claim 6 or 7 in the preparation of an immunoassay kit or immunoassay device for oxacin class compounds, or in the establishment of a non-diagnostic method for detecting oxacin class compounds.

9. An immunoassay kit or immunoassay device for an oxacin-like compound, characterized in that, Contains the composition of claim 6 or 7, or is prepared using the composition of claim 6 or 7.

10. A method for detecting non-diagnostic oxacin compounds, characterized in that, The composition according to claim 6 or 7 is used.

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