Olaquindox metabolite hapten, artificial antigen and application thereof

Abstract

The present invention discloses a hapten of olaquindox metabolite, an artificial antigen and its application. The artificial antigen and monoclonal antibody prepared from the hapten of olaquindox metabolite provided by the present invention are used for ELISA detection with strong specificity, IC 50 The present invention also establishes a colloidal gold immunochromatographic technique for detecting the quinolone metabolite MQCA, which quickly and conveniently realizes the qualitative detection of MQCA. The sensitivity in standard solution reaches 1 μg / kg, and the detection sensitivity in actual samples reaches 0.5 μg / kg. It has good stability and meets the storage, transportation and use requirements in market applications.

Description

Technical Field

[0001] The present invention relates to the field of biochemical engineering, and in particular to a quinolone metabolite hapten, an artificial antigen and applications thereof. Background Art

[0002] Olaquindox, also known as quinamide, and marketed under the trade names Beiyunuo and Kuaiyuling, is widely used in veterinary clinics due to its broad-spectrum antimicrobial activity. However, olaquindox exhibits moderate to significant cumulative toxicity and is a significant teratogen in most animals. It also has potential triple toxicity in humans: teratogenicity, mutagenicity, and carcinogenicity. Therefore, olaquindox is prohibited as a feed additive.

[0003] Olaquindox itself is unstable and rapidly metabolizes in animals. It produces more than a dozen metabolites, most of which are not detectable. 3-Methoxyquinoxaline-2-carboxylic acid (MQCA) is the primary metabolite, which is relatively stable in the body and a designated residue by the Codex Alimentarius Commission. Therefore, MQCA is often used as a target for olaquindox residue analysis and monitoring. To ensure the safety of animal-derived foods, developing methods for detecting olaquindox metabolites in animal-derived foods is of great significance.

[0004] Among the relevant detection technologies, the detection method of quinolone metabolites is mainly based on instrumental methods. However, due to the expensive equipment and instruments required, the detection time is long, and professional personnel are required to operate, it is impossible to truly achieve on-site detection and rapid clinical inspection, which brings great inconvenience to daily detection work.

[0005] The key to immunoassay detection technology lies in the performance of the antigen and antibody, and the key to these two is the hapten. Therefore, to obtain antigens and antibodies with excellent performance, the structural design of the hapten is particularly important. In the existing technology, the structural modification of the MQCA hapten is based on the derivation of a linear active arm from the carboxyl terminus, which fails to retain the characteristic carboxyl group directly connected to the quinoxaline ring, resulting in the preparation of artificial antigens with insufficient sensitivity. Therefore, the development of highly specific MQCA haptens or artificial antigens is of vital importance for rapid, highly sensitive, and low-cost detection methods for olaquinoxalate. Summary of the Invention

[0006] In order to solve the problem of lack of rapid, sensitive and simple MQCA immunoassay method for olaquindox metabolite in the prior art, the present invention provides an olaquindox metabolite hapten, an artificial antigen and applications thereof.

[0007] The first object of the present invention is to provide a hapten of olaquindox metabolite.

[0008] The second object of the present invention is to provide the use of a compound having a structural formula as shown in formula (I) in the preparation of an artificial antigen of an olaquindox metabolite.

[0009]

[0010] The third object of the present invention is to provide an artificial antigen of olaquindox metabolite.

[0011] The fourth object of the present invention is to provide the use of the artificial antigen of olaquindox metabolite in the preparation of antibodies for detecting olaquindox metabolites.

[0012] The fifth object of the present invention is to provide an artificial antigen combination of olaquindox metabolites.

[0013] The sixth object of the present invention is to provide the use of the artificial antigen combination of olaquindox metabolites in the preparation of products for detecting olaquindox metabolites.

[0014] A seventh object of the present invention is to provide a kit for detecting olaquinoxaline metabolites.

[0015] In order to achieve the above object, the present invention is implemented through the following scheme:

[0016] The hapten prepared by the present invention completely retains the characteristic group of the quinoxaline ring in the olaquinoxaline metabolite, and also retains the carboxyl group connected to the quinoxaline ring. The designed coupling arm is a straight chain with an active group, so that the small molecule structure on the prepared artificial antigen retains the original characteristics intact, is more conducive to presenting the structural characteristics of the olaquinoxaline metabolite, and improves the immunogenicity of the antigen.

[0017] A hapten of a quinolone metabolite, the structural formula of which is shown in formula (I),

[0018]

[0019] The preparation method of the compound represented by the structural formula (I) comprises the following steps: compound a reacts under acidic conditions to obtain compound b; compound b and compound c react fully to obtain;

[0020]

[0021] The compound a is 3-methylquinoxaline-2-carboxylic acid, and its structural formula is The structural formula of the compound b is

[0022] The structural formula of the compound c is

[0023] Preferably, the compound a is reacted under acidic conditions to obtain compound b, comprising the following steps: fully reacting the compound a with hydrobromic acid, adjusting the pH of the obtained reactant to 4-5, then extracting with n-butanol, collecting the obtained organic phase, performing solid-liquid separation, and collecting the precipitate to obtain a crude compound b.

[0024] Preferably, the compound b and compound c are fully reacted, comprising the following steps: fully reacting the compound b, potassium carbonate and the compound c, extracting the resulting reactant with ethyl acetate, collecting the resulting aqueous phase, adjusting the pH to 4-5, collecting the precipitated solid, and then washing it with a methanol-ethyl acetate solution to obtain the product.

[0025] More preferably, the molar ratio of the compound b to the compound c is 1:(1.5-3).

[0026] More preferably, the molar ratio of the compound b to the compound c is 1:2.

[0027] More preferably, in the methanol-ethyl acetate solution, the volume ratio of methanol to ethyl acetate is 1:(2-6).

[0028] Further preferably, in the methanol-ethyl acetate solution, the volume ratio of methanol to ethyl acetate is 1:4.

[0029] Specifically, the preparation method of the compound represented by the structural formula (I) comprises the following steps:

[0030] 200 mg of compound a (MQCA, CAS No. 55495-69-7, 0.98 mmol) was placed in a 50 ml round-bottom flask and 5 ml of 48% aqueous hydrobromic acid was added. The resulting reaction mixture was reacted at 60°C for 3-5 hours. After completion of the reaction, 40 ml of purified water was added, and the pH was adjusted to 4-5 with solid sodium bicarbonate. The mixture was extracted twice with n-butanol, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain 184 mg of crude compound b.

[0031] Dissolve 184 mg of crude compound b (0.97 mmol) in 5 ml of dimethylformamide (DMF), then add 268 mg of potassium carbonate (1.94 mmol) and 237 mg of compound c (1.94 mmol) in sequence. After thorough stirring, the reaction mixture is allowed to react at room temperature for 8-12 hours. After completion of the reaction, add 30 ml of pure water and extract twice with ethyl acetate. The aqueous phase is then adjusted to a pH of 4-5 with 4 M hydrochloric acid, and the precipitated solid is collected. The resulting solid is washed three times with a 20% methanol / ethyl acetate (v:v) mixture and then dried.

[0032] The use of the compound represented by the structural formula (I) in the preparation of artificial antigens of olaquindox metabolites should also be within the scope of protection of the present invention.

[0033]

[0034] An artificial antigen of olaquindox metabolite, whose structural formula is shown in formula (II),

[0035]

[0036] Among them, Protein is a carrier protein.

[0037] Preferably, the carrier protein is bovine serum albumin or lactoferrin.

[0038] The method for preparing the artificial antigen of olaquindox metabolite comprises the following steps: coupling the olaquindox metabolite hapten with a carrier protein under acidic conditions to obtain the artificial antigen.

[0039] Preferably, the method comprises the following steps: fully reacting the olaquindox metabolite hapten with an activator and the carrier protein, fully reacting the obtained reactant with sodium borohydride, and dialyzing to obtain the product.

[0040] More preferably, the solvent for the reaction is dimethylformamide.

[0041] Specifically, the carrier protein is bovine serum albumin, and the method for preparing the artificial antigen of olaquindox metabolite comprises the following steps:

[0042] 50 mg of bovine serum albumin was fully dissolved in 5 mL of MES buffer at pH 6.0 to obtain a carrier protein solution; 10 mg of the olaquindox metabolite hapten was fully dissolved in 0.5 mL of DMF and added dropwise to the carrier protein solution under stirring, and stirred at room temperature in the dark for 16 to 24 hours to obtain a carrier protein activation solution; 50 mg of sodium borohydride was dissolved in 0.5 mL of ice water and added dropwise to the carrier protein activation solution under stirring, and stirred at room temperature in the dark for 1 to 2 hours, and then the resulting solution was dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day. The dialyzed product obtained was the olaquindox metabolite artificial antigen.

[0043] Specifically, the carrier protein is lactoferrin, and the method for preparing the artificial antigen of olaquindox metabolite comprises the following steps:

[0044] 30 mg of lactoferrin was fully dissolved in 3 mL of MES buffer at pH 6.0 to obtain a carrier protein solution; 4 mg of the olaquindox metabolite hapten was fully dissolved in 0.2 mL of DMF and added dropwise to the carrier protein solution under stirring, and stirred at room temperature in the dark for 16 to 24 hours to obtain a carrier protein activation solution; 30 mg of sodium borohydride was dissolved in 0.8 mL of ice water and added dropwise to the carrier protein activation solution under stirring, and stirred at room temperature in the dark for 1 to 2 hours; the resulting solution was then dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day. The dialyzed product obtained was the olaquindox metabolite artificial antigen.

[0045] The use of any artificial antigen of olaquindox metabolite in the preparation of antibodies for detecting olaquindox metabolites should also be within the scope of protection of the present invention.

[0046] An antibody to olaquindox metabolite is prepared by immunizing an animal with the artificial olaquindox metabolite antigen whose carrier protein is hemocyanin as an immunogen.

[0047] Preferably, the antibody to the olaquindox metabolite is a monoclonal antibody, which is obtained by immunizing an animal with the artificial antigen of the olaquindox metabolite whose carrier protein is hemocyanin as an immunogen to obtain hybridoma cells, culturing the obtained hybridoma cells and collecting the cells for animal immunization to obtain ascites, and then identifying and purifying the antibody.

[0048] The invention discloses an artificial antigen combination of olaquindox metabolite, comprising a coating agent and an immunogen, wherein the coating agent is obtained by coupling the olaquindox metabolite hapten with bovine serum albumin; and the immunogen is obtained by coupling the olaquindox metabolite hapten with lactoferrin.

[0049] The use of the artificial antigen combination of olaquindox metabolites in the preparation of products for detecting olaquindox metabolites should also be within the scope of protection of the present invention.

[0050] Preferably, the olaquinoxaline metabolite is 3-methylquinoxaline-2-carboxylic acid.

[0051] A kit for detecting olaquindox metabolites comprises the olaquindox metabolite artificial antigen combination.

[0052] Preferably, it comprises an immunochromatographic test strip and a micropore; the immunochromatographic test strip comprises a bottom plate, on which a sample pad, a coated reaction membrane and a water-absorbing pad are sequentially overlapped, the reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area, the detection area is coated with the coating antigen in the artificial antigen combination of olaquindox metabolites, and the quality control area is coated with IgG; the micropore is filled with an antibody for detecting olaquindox metabolites, and the antibody is prepared from the immunogen in the artificial antigen combination of olaquindox metabolites.

[0053] Preferably, the sample pad is soaked in a sample treatment solution, which is a 0.1 M PB buffer solution containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide.

[0054] Preferably, the IgG is murine IgG.

[0055] Specifically, the preparation method of the coated reaction membrane includes the following steps: using a nitrocellulose membrane as a reaction membrane, adjusting the concentration of the coating source to 0.05-0.2 mg / mL with a coating buffer to obtain a detection coating source solution; and adjusting the concentration of mouse IgG to 0.1-0.5 mg / mL with a coating buffer to obtain a quality control coating source solution; according to a membrane liquid volume of 0.8-1.2 μL / cm, the detection coating source solution and the quality control coating source solution are respectively sprayed onto the detection area and control area corresponding to the reaction membrane, with a spacing of 2.5 mm between the detection area and the control area, and placed in a 45°C oven for 12-16 hours to obtain the obtained membrane; the coating buffer is a 0.1M PB buffer containing 1wt% sucrose and 0.05wt% sodium azide, with a pH of 7.4.

[0056] Preferably, the antibody to the olaquindox metabolite is a monoclonal antibody, which is obtained by immunizing an animal with the artificial antigen of the olaquindox metabolite whose carrier protein is hemocyanin as an immunogen to obtain hybridoma cells, culturing the obtained hybridoma cells and collecting the cells for animal immunization to obtain ascites, and then identifying and purifying the antibody.

[0057] More preferably, the monoclonal antibody is labeled with colloidal gold.

[0058] Further preferably, the method for labeling the monoclonal antibody with colloidal gold comprises the following steps: taking 1 g of chloroauric acid, dissolving it with pure water and ultrasonically dissolving it to 100 ml, thereby obtaining a chloroauric acid solution; taking 1 ml of the chloroauric acid solution and adding it to 100 ml of pure water, heating it to boiling, adding 0.5 ml of a 0.06 wt% sodium citrate solution, continuing to heat it for 10 minutes, cooling it to room temperature, and then adding pure water to a volume of 100 ml, thereby obtaining a nanogold solution;

[0059] The pH of the colloidal gold solution was adjusted with 0.1 mol / L KCO and 0.1 mol / L HCl, respectively (the amount added was the minimum amount required to prevent discoloration during the subsequent labeling process). 5 μg of the monoclonal antibody was added to the nanogold solutions at different pH values ​​and allowed to react at room temperature for 5 minutes. The color change of the solution was observed, and the pH value at which the solution remained red was recorded. 10 μl of 10 wt% BSA was added for blocking, and the solution was centrifuged at 12,000 rpm. The supernatant was discarded, and the resulting precipitate was collected to obtain the product.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The artificial antigen and monoclonal antibody prepared from the hapten of olaquindox metabolite provided by the present invention are used for ELISA detection with high specificity and IC 50 The present invention also establishes a colloidal gold immunochromatographic technique for detecting the quinolone metabolite MQCA, which quickly and conveniently realizes the qualitative detection of MQCA. The sensitivity in standard solution reaches 1 μg / kg, and the detection sensitivity in actual samples reaches 0.5 μg / kg. It has good stability and meets the storage, transportation and use requirements in market applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a synthetic route for the hapten of olaquindox metabolite.

[0063] Figure 2 The mass spectrometry results of olaquindox metabolite hapten.

[0064] Figure 3 This is the synthetic route of artificial antigen of olaquindox metabolite.

[0065] Figure 4 is the standard curve of MQCA standard solution. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0067] Example 1 Preparation and Identification of Olaquindox Metabolite Haptens

[0068] 1. Preparation of olaquindox metabolite hapten

[0069] According to Figure 1 The synthetic route shown is used to prepare the quinolone metabolite hapten, and the specific steps are as follows:

[0070] (1) 200 mg of compound a (MQCA, CAS No. 55495-69-7, 0.98 mmol) was placed in a 50 ml round-bottom flask, and 5 ml of aqueous hydrobromic acid (48% by mass) was added. The resulting reaction mixture was reacted at 60°C for 3-5 h. After the reaction was complete, 40 ml of purified water was added, and the pH was adjusted to 4-5 with solid sodium bicarbonate. The mixture was extracted twice with n-butanol, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain 184 mg of crude compound b.

[0071] (2) Dissolve 184 mg of crude compound b (0.97 mmol) in 5 ml of dimethylformamide (DMF), then add 268 mg of potassium carbonate (1.94 mmol) and 237 mg of compound c (1.94 mmol) in sequence. After thorough stirring, the reaction mixture is reacted at room temperature for 8 to 12 hours. After the reaction is complete, add 30 ml of pure water and extract twice with ethyl acetate. The pH value of the aqueous phase is adjusted to 4 to 5 with 4 M hydrochloric acid, and the precipitated solid is collected. The resulting solid is washed three times with a 20% methanol-ethyl acetate mixture (i.e., the volume ratio of methanol to ethyl acetate is 20:80) and dried to obtain 85 mg of quinolone metabolite hapten.

[0072] 2. Identification of haptens of olaquindox metabolites

[0073] The structural formula of olaquindox metabolite hapten is shown in formula (I):

[0074]

[0075] The mass spectrometry results of quinolone metabolite hapten are as follows Figure 2 As shown, specifically: 231(M-1).

[0076] Example 2 Preparation and Identification of Artificial Antigens of Olaquindox Metabolites

[0077] The structural formula of the artificial antigen of olaquindox metabolite provided by the present invention is shown in formula (II):

[0078]

[0079] The protein is a carrier protein, and the carrier protein is bovine serum albumin (BSA) or lactoferrin (LF).

[0080] According to Figure 3 The synthetic route shown is used to prepare the artificial antigen of olaquindox metabolite.

[0081] 1. The specific steps for preparing artificial antigens of quinolone metabolites with BSA as carrier protein are as follows:

[0082] (1) Weigh 50 mg of BSA and dissolve it in 5 mL of MES buffer (pH 6.0) to obtain a carrier protein solution;

[0083] (2) 10 mg of the quinolone metabolite hapten prepared in Example 1 was fully dissolved in 0.5 mL of DMF and added dropwise to the carrier protein solution prepared in step (1) under stirring, and stirred in the dark at room temperature for 16 to 24 hours to obtain a carrier protein activation solution;

[0084] (3) Dissolve 50 mg of sodium borohydride in 0.5 mL of ice water and add dropwise to the carrier protein activation solution prepared in step (2) under stirring, and stir at room temperature in the dark for 1 to 2 hours;

[0085] (4) dialyzing the solution obtained in step (3) with 0.01 mol / L PBS at room temperature for 3 days, changing the dialysate 3 times a day to remove unreacted small molecules. The resulting dialyzate is the artificial antigen of quinolone metabolite;

[0086] (5) Aliquot and store at 4°C until use.

[0087] 2. The specific steps for preparing artificial antigens of quinolone metabolites with LF as carrier protein are as follows:

[0088] (1) Weigh 30 mg of LF and dissolve it in 3 mL of MES buffer (pH 6.0) to obtain a carrier protein solution;

[0089] (2) 4 mg of the quinolone metabolite hapten prepared in Example 1 was fully dissolved in 0.2 mL of DMF and added dropwise to the carrier protein solution prepared in step (1) under stirring, and stirred in the dark at room temperature for 16 to 24 h to obtain a carrier protein activation solution;

[0090] (3) Dissolve 30 mg of sodium borohydride in 0.8 mL of ice water and add dropwise to the carrier protein activation solution prepared in step (2) under stirring, and stir at room temperature in the dark for 1 to 2 hours;

[0091] (4) dialyzing the solution obtained in step (3) with 0.01 mol / L PBS at room temperature for 3 days, changing the dialysate 3 times a day to remove unreacted small molecules. The resulting dialyzate is the artificial antigen of quinolone metabolite;

[0092] (5) Aliquot and store at 4°C until use.

[0093] Example 3 Preparation of monoclonal antibodies to olaquindox metabolites

[0094] 1. Animal immunization

[0095] The artificial antigen, a metabolite of olaquindox, prepared in Example 2, was used as the immunogen, emulsified with an equal volume of Freund's adjuvant, and immunized into BALB / C mice at a dose of 50 μg to 100 μg per mouse. Immunizations were repeated 2 weeks apart, and after three immunizations, tail vein blood was collected to determine serum titers. If the antibody titer did not meet the required level, booster immunizations were performed.

[0096] After the antibody titer stopped increasing, subcutaneous booster immunization was performed with 100 μg of whole antigen.

[0097] 2. Hybridoma Cell Preparation

[0098] Five days after the last booster immunization, mouse spleen cells were harvested and fused with human osteosarcoma SP20 cells. The fused cells were selected in HAT medium and cultured five days later with complete medium replaced with HAT medium.

[0099] The cell supernatant was tested by ELISA, and cells in wells with strongly positive results were cloned and cultured using the limiting dilution method. After three clone culture tests, cells in wells that were positive were identified as hybridoma cells secreting monoclonal antibodies.

[0100] 3. Monoclonal Antibody Preparation

[0101] After the hybridoma cells are expanded and cultured, they are inoculated into the peritoneal cavity of mice to produce ascites containing the antibody. The ascites is purified using the octanoic acid-ammonium sulfate precipitation method to obtain highly pure and specific monoclonal antibodies to the olaquindox metabolite.

[0102] Example 4 ELISA performance evaluation

[0103] 1. Experimental methods

[0104] The performance of the artificial antigen and monoclonal antibody prepared by the present invention was evaluated by ELISA method, which includes the following steps:

[0105] (1) Antigen coating

[0106] The olaquindox metabolite artificial antigen prepared in Example 2 with BSA as the carrier protein was diluted to 0.08 μg / mL using carbonate buffer at pH 9.6 as the coating diluent. 100 μL / well was added to a polystyrene microplate, coated overnight at 4°C, dried, and washed three times with PBST.

[0107] (2) Closed

[0108] Add phosphate buffer containing 1% wt BSA at 280 μL / well, block at 37°C for 1 hour, spin dry, wash three times with PBST, dry and vacuum pack for storage.

[0109] (3) Primary antibody dilution

[0110] The monoclonal antibody to the olaquindox metabolite prepared in Example 3 was diluted to 0.1 μg / mL using phosphate buffer (pH 7.4) containing 0.05% wt sodium azide and stored at 4° C. for later use.

[0111] (4) Preparation of standard solution

[0112] The MQCA standard was dissolved in 0.01 M PBS to obtain MQCA standard solutions with concentrations of 0 μg / L, 0.5 μg / L, 1.5 μg / L, 4.5 μg / L, 13.5 μg / L and 40.5 μg / L, respectively.

[0113] (5) Sample addition and primary antibody incubation

[0114] 100 μL / well of MQCA standard solution of various concentrations was added to the microwells of the ELISA plate coated with artificial antigen of olaquindox metabolite, and then 20 μL / well of olaquindox metabolite monoclonal antibody solution was added accordingly, reacted at 37°C for 0.5h, and dried.

[0115] (6) Washing the primary antibody

[0116] Add 280 μL / well PBST, wash three times, and pat dry.

[0117] (7) Secondary antibody incubation

[0118] Add 100 μL / well HRP enzyme-labeled goat anti-mouse IgG enzyme-labeled secondary antibody and react at 37°C for 0.5 h.

[0119] (8) Washing the secondary antibody

[0120] Add 280 μL / well PBST, wash again three times, and pat dry.

[0121] (9) Color development

[0122] Add 100 μL / well TMB color development solution and react at 37°C for 15 min; add 50 μL / well 1 M sulfuric acid to stop color development.

[0123] (10) Absorbance determination

[0124] Place the microporous microplate obtained in the previous step into a microplate reader and set the microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0125] 2. Experimental results

[0126] Table 1 OD values ​​of MQCA standard solutions at different concentrations determined by ELISA

[0127]

[0128] The OD value measurement results of MQCA standard solutions with different concentrations are shown in Table 1. Based on the data shown in Table 1, ELISA Calc software was used to perform four-parameter logistic curve fitting, and the following was obtained: Figure 4 The linear equation of the standard curve shown is: y = (AD) / [1 + (x / C) ^ B] + D, r 2=0.99928; where A = 1.12999, B = 0.89404, C = 0.13580, D = 0.14166, x represents the concentration of the analyte, and y represents the OD value. IC 50 The value was 0.188 μg / L, and it was linear in the range of 0.04 μg / L to 3.24 μg / L.

[0129] Example 5 A colloidal gold qualitative immunochromatographic kit for detecting olaquindox metabolites

[0130] 1. Composition of the kit

[0131] (1) Colloidal gold qualitative immunochromatographic test paper card

[0132] The colloidal gold qualitative immunochromatographic test paper card contained in this kit is prepared by the following method:

[0133] Preparation of coating reaction membrane: Using nitrocellulose membrane (NC membrane) as the reaction membrane, the carrier protein prepared in Example 2 was adjusted to 0.05-0.2 mg / mL with a coating buffer (0.1 M PB buffer containing 1 wt% sucrose and 0.05 wt% sodium azide, pH = 7.4) to obtain a detection coating original solution; and the concentration of mouse IgG was also adjusted to 0.1-0.5 mg / mL with a coating buffer to obtain a quality control coating original solution; according to the membrane liquid volume of 0.8-1.2 μL / cm, the detection coating original solution and the quality control coating original solution were sprayed onto the detection area (T line) and control area (C line) corresponding to the reaction membrane, respectively. The interval between the detection area and the control area was 2.5 mm, and the reaction membrane was placed in a 45 ° C oven for 12-16 h to obtain a coating reaction membrane, which was placed in a constant temperature and humidity storage box for standby.

[0134] Preparation of sample pad: Soak a 30×30 cm blank sample pad in the sample treatment solution (0.01 M PBT buffer containing 0.05 wt% Tween 20, 1 wt% sucrose, 0.1 wt% BSA, and 0.05 wt% sodium azide) for 5 minutes. Then, remove it and dry it at 37°C for 16 hours to obtain the sample pad. Place it in a constant temperature and humidity storage box until used.

[0135] Assembly: Attach the sample pad, coated reaction membrane, and absorbent pad to the center of a PVC board, overlapping them in sequence, with the absorbent pad adjacent to the control area of ​​the coated reaction membrane and the sample pad adjacent to the detection area. This creates a colloidal gold qualitative immunochromatographic test card. Cut the test card into 3mm wide strips and load them into the test card, with the sample pad adjacent to the sample well.

[0136] (2) Gold-labeled micropores

[0137] The gold-labeled microwells included in this kit were prepared by the following method:

[0138] Preparation of nanogold solution: Dissolve 1g of chloroauric acid with pure water and ultrasonically, then dilute to 100ml to obtain chloroauric acid solution. Store at 4°C in the dark until ready for use. Add 1ml of chloroauric acid solution to 100ml of pure water, heat to boiling, then add 0.5ml of 0.06wt% sodium citrate solution. Continue heating for 10 minutes. After cooling to room temperature, add pure water to a volume of 100ml to obtain the nanogold solution. Store in the dark at room temperature until ready for use. All glassware used should be soaked in a mixture of potassium permanganate and sulfuric acid overnight, cleaned, and dried before use.

[0139] Labeling of olaquindox metabolite monoclonal antibodies: The nanogold solution was dispensed into 1 ml bottles and the pH of the colloidal gold solution was adjusted with 0.1 mol / L KCO and 0.1 mol / L HCl, respectively (the amount added was the minimum amount required to prevent discoloration during the subsequent labeling process). 5 μg of the olaquindox metabolite monoclonal antibody prepared in Example 3 was added to the nanogold solutions at different pH values. The reaction was allowed to proceed at room temperature for 5 minutes. The color change of the solution was observed, and the pH value at which the solution remained red was recorded. 10 μl of 10 wt% BSA was added for blocking, and the solution was centrifuged at 12,000 rpm. The supernatant was discarded, and the resulting precipitate was the nanogold-labeled olaquindox metabolite monoclonal antibody.

[0140] Gold-labeled microwells were packaged as follows: 1 ml of gold diluent (water containing 2 wt% Tris, 5 wt% BSA, 0.05 wt% thimerosal, and 5 wt% sucrose) was used to reconstitute the gold-labeled monoclonal antibody to the quinolone metabolite, and 20 μl / well was dispensed into the microwells. The microwells were dried at 37°C for 16 hours to obtain gold-labeled microwells, which were then stored for future use.

[0141] 2. How to use the kit

[0142] (1) Detection

[0143] Add 100 μl of the test solution to the gold-labeled microwells at a rate of 100 μl / well. Repeatedly pipette and swirl to reconstitute the solution. After 3 minutes, transfer the solution from the gold-labeled microwells to the sample wells of the colloidal gold qualitative immunochromatographic test strip. Start the timer after sample addition and observe the results after 5-8 minutes. After 8 minutes, the test is considered invalid. Three replicates are performed.

[0144] (2) Visual interpretation

[0145] Observe the color of the T line and C line of the colloidal gold qualitative immunochromatographic test paper card. When the color of the T line is stronger than that of the C line or there is no obvious difference in color with the C line, it indicates that the test result is negative (-); when the color of the T line is significantly weaker than that of the C line or the T line does not show color, it indicates that the test result is positive (+); when the C line does not show color, it indicates that the test result is invalid due to incorrect operation process or invalid test strip.

[0146] Example 6 Performance Evaluation of Colloidal Gold Qualitative Immunochromatography Kit

[0147] 1. Sensitivity

[0148] The MQCA standard was diluted with 0.01 M PBS buffer to obtain MQCA standard solutions of different concentrations, which were used as test solutions and detected using the colloidal gold qualitative immunochromatographic kit of Example 5. The experiment was repeated three times.

[0149] The results are shown in Table 2. It can be seen that the detection limit of the kit for quinolone metabolite MQCA is as low as 1 μg / L, with high sensitivity.

[0150] Table 2 Determination results of quinolone metabolite standard solutions at different concentrations

[0151]

[0152] Note: “+” represents positive, “-” represents negative.

[0153] 2. Stability

[0154] The colloidal gold qualitative immunochromatographic test paper card is stored at room temperature. In order to evaluate its stability, an accelerated destructive test is performed and the card is placed at 45°C for 60 consecutive days.

[0155] On day 0, day 5, day 10, day 20, day 30, day 40, day 50, and day 60, the MQCA standard was diluted with 0.01 M PBS buffer to obtain MQCA standard solutions of different concentrations, which were used as test solutions and detected using the colloidal gold qualitative immunochromatography kit of Example 5. The experiment was repeated for three groups.

[0156] As shown in Table 3, it can be seen that after 60 days of sealed storage at room temperature and 45°C, the T / C color depth interpretation results of the colloidal gold qualitative immunochromatographic test strips did not change significantly, indicating that the colloidal gold qualitative immunochromatographic test strips can be stably stored for at least 60 days at 45°C in the accelerated experiment. Therefore, the colloidal gold qualitative immunochromatographic test strips for olaquindox metabolites prepared by the present invention can be stably stored at room temperature for more than one year, meeting the market requirements during storage and transportation.

[0157] Table 3 Accelerated destructive test results

[0158]

[0159]

[0160] 3. Spiked testing of actual tissue samples

[0161] 2 g of fat-free and homogenized tissue sample (muscle tissue) was placed in a 15 mL centrifuge tube, 3 mL of acetonitrile and 2 g of solid sodium sulfate were added, and the mixture was thoroughly shaken or shaken for 1 minute. After mixing, the mixture was centrifuged at 4000 rpm at room temperature for 5 minutes. 2.5 mL of the supernatant was blown dry at 75°C under nitrogen or air. 0.5 mL of n-hexane and 0.3 mL of 0.01 M PBS buffer were added to the residue, mixed thoroughly, and allowed to stand to separate (if emulsification occurs, the mixture can be placed in an 80°C water bath for 5 minutes or centrifuged at 4000 rpm at room temperature for 2 minutes). The upper layer of liquid was discarded, and the lower layer was collected as the test solution. Detection was performed using the colloidal gold qualitative immunochromatography kit of Example 5, and the experiment was repeated three times.

[0162] Minimum detection limit: 10 blank samples were spiked with olaquindox metabolite standard solution at gradient addition levels of 0 μg / kg, 0.15 μg / kg, 0.5 μg / kg, 1 μg / kg and 2 μg / kg.

[0163] As shown in Table 4, the test results for the 10 samples were highly reproducible. When the olaquindox metabolite content in the samples was below 0.5 μg / kg, all samples were negative; when it was above 0.5 μg / kg, all samples were positive. This kit can detect MQCA in real tissue samples with a detection limit as low as 0.5 μg / kg.

[0164] Table 4 Results of spiked test of actual tissue samples

[0165]

[0166] Note: 1 and 2 are from different markets.

[0167] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A hapten of olaquindox metabolite, characterized in that Its structural formula is shown in formula (I), 2. Use of a compound having a structural formula (I) in the preparation of artificial antigens of olaquindox metabolites, 3. An artificial antigen of olaquindox metabolite, characterized in that Its structural formula is shown in formula (II), Among them, Protein is a carrier protein.

4. The artificial antigen of olaquindox metabolite according to claim 3, characterized in that The carrier protein is bovine serum albumin or lactoferrin.

5. Use of the artificial antigen of olaquindox metabolite according to claim 3 or 4 in the preparation of antibodies for detecting olaquindox metabolites.

6. An artificial antigen combination of olaquindox metabolites, characterized in that: The invention comprises a coating agent and an immunogen, wherein the coating agent is obtained by coupling the hapten of the olaquindox metabolite according to claim 1 with bovine serum albumin; and the immunogen is obtained by coupling the hapten of the olaquindox metabolite according to claim 1 with lactoferrin.

7. Use of the artificial antigen combination of olaquindox metabolites according to claim 6 in the preparation of a product for detecting olaquindox metabolites.

8. The use according to claim 7, characterized in that The olaquinoxaline metabolite is 3-methylquinoxaline-2-carboxylic acid.

9. A kit for detecting olaquinoxaline metabolites, characterized in that: The invention comprises the artificial antigen combination of olaquindox metabolites according to claim 6.

10. The kit according to claim 9, characterized in that Contains immunochromatographic test strips and microwells; The immunochromatographic test strip comprises a bottom plate, on which a sample pad, a coated reaction membrane and a water-absorbing pad are sequentially overlapped, the reaction membrane being a nitrocellulose membrane having a detection area and a quality control area, the detection area being coated with the coating agent according to claim 6, and the quality control area being coated with IgG; The microwells are filled with antibodies for detecting olaquinoxaline metabolites, and the antibodies are prepared from the immunogen described in claim 6.

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