A test strip and method for detecting flunixin meglumine and metabolites thereof

Abstract

The application discloses a test strip and a method for detecting flunixin meglumine and metabolites thereof. The test strip comprises a test paper and a microporous reagent, wherein the test paper comprises a reaction film, a sample absorption pad, a water absorption pad and a bottom plate, the reaction film is provided with a detection line coated with a flunixin meglumine hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody, and the microporous reagent is freeze-dried with a flunixin meglumine monoclonal antibody-colloidal gold marker. The method for detecting flunixin meglumine and metabolites thereof by using the test strip is simple, rapid, intuitive, accurate, convenient to carry, widely applicable, low in cost and easy to popularize and use.

Description

TECHNICAL FIELD

[0001] The present application relates to a test strip and method for detecting flunixin meglumine and its metabolites, in particular to a colloidal gold test strip for detecting flunixin meglumine and its metabolites, which is particularly suitable for detecting flunixin meglumine and its metabolite residues in liquid milk. BACKGROUND

[0002] Flunixin meglumine (FM) is the only non-steroidal anti-inflammatory drug for animals, which is combined with solubilizer meglumine and flunixin in a 1:1 form. FM is a potent cyclooxygenase inhibitor that prevents the production of inflammatory substances such as thromboxane, prostaglandin, etc. by selectively inhibiting cyclooxygenase, thereby playing a role in anti-inflammatory, antipyretic, analgesic. It is often used in clinical to relieve inflammation and pain caused by internal organs, muscles, bones, breast, uterus disorders, etc. of pigs, horses, and cows, as well as to control various acute inflammations caused by infectious diseases, treat canine endotoxemia, etc. Due to its small dosage, rapid absorption, long duration, significant efficacy, and light adverse reactions, it is favored by veterinarians and is the largest non-steroidal anti-inflammatory drug consumed in veterinary clinics at home and abroad. FM is easily metabolized and eliminated in the body, about 90% of the original drug is excreted out of the body through urine and feces 24 hours after administration, and its main metabolites are 4-hydroxyflunixin, 5-hydroxyflunixin and 2-methylhydroxy, among which 5-hydroxyflunixin is identified as a marker residue in cow's milk.

[0003] In the past two decades, many studies have shown that FM and other non-steroidal anti-inflammatory drug residues in animal-derived foods may pose potential risks to human health, such as gastrointestinal damage, hematopoietic and renal system toxicity, hepatotoxicity, and aseptic meningitis, etc. In order to reduce these toxic effects, the European Union, the United States, Canada, Japan and China have determined the maximum residue limits (MRLs) of flunixin meglumine and its metabolite 5-hydroxyflunixin in animal tissues and milk to be 6-60 μg / kg. Therefore, it is essential to establish a rapid and effective detection method for FM and its metabolites in animal-derived foods.

[0004] The currently reported methods for detecting flunixin meglumine mainly include instrument methods such as liquid chromatography and liquid chromatography-mass spectrometry. These methods all need to be operated under laboratory conditions, with complicated and time-consuming sample pretreatment, and require expensive instrument equipment, resulting in high detection cost, long time consumption, and complex operation, which has great limitations in practical application and is difficult to meet the needs of rapid detection of a large number of samples and on-site samples. Therefore, the development of a colloidal gold test strip for detecting flunixin meglumine and its metabolites in liquid milk, which is simple, rapid and suitable, can meet the needs of on-site screening and monitoring of a large number of samples, and can better meet the detection work of food supervision departments in China. SUMMARY

[0005] The present application aims to provide a colloidal gold test strip capable of detecting flunixin meglumine and its metabolite residues in liquid milk, and to provide a detection method which is efficient, accurate, simple, suitable for on-site monitoring and large sample screening.

[0006] The test strip for detecting flunixin meglumine and its metabolite provided by the present application comprises a test paper and a micropore reagent; the test paper comprises a bottom plate, a sample absorption pad, a reaction membrane and a water absorption pad, which are sequentially connected, the reaction membrane has a detection line coated with flunixin meglumine hapten-carrier protein conjugate and a quality control line coated with sheep anti-mouse antibody; the micropore reagent has flunixin meglumine monoclonal antibody-colloidal gold label freeze-dried thereon, and has a micropore plug.

[0007] The flunixin meglumine monoclonal antibody is prepared by taking flunixin meglumine hapten-carrier protein conjugate as an immunogen.

[0008] The flunixin meglumine hapten-carrier protein conjugate is obtained by coupling flunixin meglumine hapten and carrier protein, and the carrier protein is bovine serum albumin, egg white albumin, hemocyanin, thyroid protein or human serum albumin; the synthesis method of the flunixin meglumine hapten is as follows: 2.33 g of 2-amino-6-(trifluoromethyl)benzene propionic acid is dissolved in 100 mL of N,N-dimethylformamide, 1.88 g of 2-chloronicotinic acid is added, and the mixture is stirred thoroughly, 0.78 g of anhydrous KOH is added, a reflux condenser is installed, and the mixture is heated at 70℃ for 6 h, then the reaction is stopped, the mixture is cooled to room temperature, 200 mL of water is added, 6 mol / L hydrochloric acid is added to adjust the pH value to 6, 100 mL of ethyl acetate is added for extraction, the water phase is separated, the organic phase is evaporated to dryness, and the residue is separated by silica gel column elution with a mixed solution of dichloromethane and methanol in a volume ratio of 10:1 to obtain flunixin meglumine hapten, and the molecular structural formula of the flunixin meglumine hapten is as follows:

[0009]

[0010] The bottom plate is a PVC bottom plate or other hard non-water-absorbing material; the sample absorption pad is made of polyester fiber or glass fiber material; the water absorption pad is water absorption filter paper; and the reaction membrane is nitrocellulose membrane or cellulose acetate membrane.

[0011] Another object of the present application is to provide a method for preparing the above-mentioned test strip, which comprises the following steps:

[0012] 1) preparing a micropore reagent freeze-dried with flunixin meglumine monoclonal antibody-colloidal gold label;

[0013] 2) preparing a reaction membrane having a detection line coated with flunixin meglumine hapten-carrier protein conjugate and a quality control line coated with sheep anti-mouse antibody.

[0014] 3) Assembling the prepared reaction membrane of 2) with sample absorption pad, water absorption pad and bottom plate into a test paper;

[0015] 4) Assembling the prepared microporous reagent with freeze-dried flunixin meglumine monoclonal antibody-gold label and test paper of 1) and 3) into a test strip.

[0016] Specifically, the steps include:

[0017] 1) Preparing flunixin meglumine hapten by reacting 2-amino-6-(trifluoromethyl)benzene propionic acid with 2-chloronicotinic acid;

[0018] 2) Preparing flunixin meglumine hapten-carrier protein conjugate by coupling flunixin meglumine hapten with carrier protein;

[0019] 3) Immunizing mice with flunixin meglumine hapten-carrier protein conjugate, and obtaining hybridoma cell strain secreting flunixin meglumine monoclonal antibody by fusing mouse spleen cells and mouse myeloma cells and screening;

[0020] 4) Immunizing healthy goats with mouse IgG to obtain goat anti-mouse antibody;

[0021] 5) Coating flunixin meglumine hapten-carrier protein conjugate and goat anti-mouse antibody on the test line (T) and the control line (C) of the reaction membrane, respectively;

[0022] 6) Preparing colloidal gold by reacting trisodium citrate with chloroauric acid;

[0023] 7) Obtaining flunixin meglumine monoclonal antibody-gold label by adding prepared flunixin meglumine monoclonal antibody to prepared colloidal gold;

[0024] 8) Freezing flunixin meglumine monoclonal antibody-gold label in microporous reagent, and adding microporous plug to the microporous reagent;

[0025] 9) Soaking sample absorption pad in 0.2 mol / L phosphate buffer solution containing 1% bovine serum albumin and having pH of 7.2 for 2 h, and drying at 37℃ for 2 h for standby;

[0026] 10) Sticking sample absorption pad, reaction membrane and water absorption pad in sequence on the bottom plate, and finally cutting into small strips with width of 3.95 mm;

[0027] 11) Assembling prepared microporous reagent and test paper into a test strip, and storing under the condition of 2-8℃ for 12 months.

[0028] Another object of the present application is to provide a method for detecting flunixin meglumine and its metabolite residues in liquid milk by using the above test strip, which comprises the following steps:

[0029] (1) sample pre-treatment;

[0030] (2) detection with test strips;

[0031] (3) analysis of detection results.

[0032] wherein the flunixin meglumine metabolite is 5-hydroxyflunixin.

[0033] The flunixin meglumine and metabolite rapid detection test strip of the present application uses highly specific antigen-antibody reactions and immunochromatographic analysis techniques, and flunixin meglumine monoclonal antibody-colloidal gold label is freeze-dried in the micropore reagent. Flunixin meglumine and metabolites in the sample are first fully reacted with the same, forming drug-antibody-colloidal gold label. Then the test strip is inserted, and the drug in the sample competes with flunixin meglumine hapten-carrier protein conjugate on the reaction membrane detection line to bind flunixin meglumine monoclonal antibody-colloidal gold label during the flow process. Whether the sample liquid contains flunixin meglumine and metabolite residues is determined according to the depth of the red band on the detection line.

[0034] During detection, the sample is dropped into the micropore reagent after treatment, and the test strip is inserted into the micropore after full reaction. When the concentration of flunixin meglumine and metabolites in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold label will bind to flunixin meglumine hapten-carrier protein conjugate fixed on the reaction membrane during chromatography, and each of the detection line (T) and the quality control line (C) will appear a red band, and the color of the T line is deeper than that of the C line or consistent with that of the C line. If the concentration of flunixin meglumine and metabolites in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold label will bind to flunixin meglumine and metabolites, so that no red band will appear at the T line due to the competition reaction, or the color is shallower than that of the C line. As shown in the following table. Figure 5

[0035] Negative: When the quality control line (C) shows a red band, the detection line (T) also shows a red band, and the color of the (T) line is close to or deeper than that of the (C) line, it is determined to be negative.

[0036] Positive: When the quality control line (C) shows a red band, and the detection line (T) does not show color or the color of the (T) line is shallower than that of the (C) line, it is determined to be positive.

[0037] Invalid: When the quality control line (C) does not show a red band, whether the detection line (T) shows a red band or not, the test strip is determined to be invalid.

[0038] ​The test strip of the present application has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for use by various units, simple storage, and long shelf life. The high-specificity fengnisin meglumine monoclonal antibody is used to ensure the reliability of the detection result; the gold-labeled antibody is freeze-dried in the micropore reagent, and in the detection process, the gold-labeled antibody can fully contact and react with the sample liquid to be detected, thereby reducing errors and increasing the reaction sensitivity of the whole system. The method for detecting fengnisin meglumine and its metabolites by using the test strip of the present application is simple, rapid, intuitive, accurate, widely applicable, low in cost, and easy to popularize and use. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Synthesis diagram of fengnisin meglumine hapten.

[0040] Figure 2 Nuclear magnetic resonance hydrogen spectrum diagram of fengnisin meglumine hapten.

[0041] Figure 3 Schematic diagram of test paper section structure.

[0042] Figure 4 Micropore reagent diagram.

[0043] Figure 5 Test strip detection result determination diagram. DETAILED DESCRIPTION

[0044] The present application will be further described below in combination with specific examples. It should be understood that these examples are only used to illustrate the present application, and are not used to limit the scope of the present application.

[0045] Example 1: Preparation of test strip for detecting fengnisin meglumine and its metabolites

[0046] The preparation method of the test strip mainly includes the following steps:

[0047] 1) Preparation of micropore reagent freeze-dried with fengnisin meglumine monoclonal antibody-gold label;

[0048] 2) Preparation of reaction membrane with detection line coated with fengnisin meglumine hapten-carrier protein conjugate and quality control line coated with sheep anti-mouse antibody;

[0049] 3) Assembly of the reaction membrane prepared in 2) with a sample absorption pad, a water absorption pad, and a bottom plate into a test paper;

[0050] 4) Assembly of the micropore reagent freeze-dried with fengnisin meglumine monoclonal antibody-gold label prepared in 1) and the test paper prepared in 3) into a test strip.

[0051] The following is described in detail in steps:

[0052] 1. Synthesis of flunixin meglumine hapten (synthetic route is attached) Figure 1 )

[0053] 2.33 g of 2-amino-6-(trifluoromethyl)phenylpropionic acid (CAS No.: 1806406-46-1) was dissolved in 100 mL of N,N-dimethylformamide (DMF), and 1.88 g of 2-chloronicotinic acid (CAS No.: 2942-59-8) was added. The mixture was stirred thoroughly, and 0.78 g of anhydrous KOH was added. A reflux condenser was attached, and the mixture was heated to 70 °C for 6 h. The reaction was stopped, cooled to room temperature, and 200 mL of water was added. The pH was adjusted to 6 with 6 mol / L hydrochloric acid. The mixture was extracted with 100 mL of ethyl acetate, and the aqueous phase was separated. The organic phase was evaporated to dryness by rotary evaporation, loaded onto a silica gel column, and eluted with a 10:1 dichloromethane-methanol mixture to obtain 1.72 g of flunixin meglumine hapten, with a yield of 48.58%.

[0054] Hydrogen nuclear magnetic resonance (HNMR) of the hapten 1 H-NMR) measurements, results as follows Figure 2 As shown: 1 ¹H NMR (500MHz, Chloroform-d): δ (ppm) 10.11 (s, 2H), 9.77 (dd, J = 5.0, 2.3Hz, 2H), 9.69 (s, 2H), 9.15 (dd, J = 8.8, 2.2Hz, 2H), 7.75 (dd, J = 8.7, 5.0Hz, 2H), 7.68 (dd, J = 10.4, 1.6Hz, 2H), 7.64 (dd, J = 10.4, 7.5Hz, 2H), 7.26 (dd, J = 7.5, 1.6Hz, 2H), 3.50–3.33 (m, 4H), 2.93 (d, J = 16.8Hz, 2H), confirming the correct hapten structure.

[0055] 2. Preparation of immunogens

[0056] Take 13.2 mg of flunixin meglumine hapten, dissolve and clarify it in 1 mL of DMF, add 12.87 mg of N-hydroxysuccinimide (NHS) and 21.44 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dissolve and mix thoroughly, and react at room temperature for 2 h to obtain hapten activation solution A; take 50 mg of bovine serum albumin (BSA), dissolve it in 4 mL of 0.1 mol / L pH 9.5 CB buffer to obtain solution B; add solution A dropwise to solution B, react at room temperature for 6 h, stop the reaction, and purify by dialyzing with 0.02 mol / L PBS buffer for 3 days, changing the buffer 3 times a day, centrifuge and aliquot to obtain flunixin meglumine hapten-BSA conjugate, which is the immunogen.

[0057] 3. Preparation of the coating agent

[0058] Take flunixin meglumine hapten 15.73 mg, dissolve in 1 mL DMF, add NHS 12.87 mg, EDC 21.44 mg, dissolve thoroughly, react at room temperature for 2 h to obtain hapten activation solution A; take ovalbumin (OVA) 100 mg, dissolve in 0.1 mol / L CB buffer solution pH 9.5 4 mL to obtain B solution; add A solution dropwise to B solution, react at room temperature for 6 h, stop the reaction, purify by dialysis with 0.02 mol / L PBS buffer solution for 3 days, change the solution 3 times a day, centrifuge and dispense to obtain flunixin meglumine hapten-OVA conjugate, which is the coating original.

[0059] 4. Preparation of flunixin meglumine monoclonal antibody

[0060] (1) Animal immunization

[0061] The immunogen obtained in step 2 is injected into Balb / c mice, and the immunization dose is 150 μg per mouse to make the mice produce antisera.

[0062] (2) Cell fusion and cloning

[0063] Take the spleen cells of the immunized Balb / c mice, and fuse them with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio). The cell supernatant is determined by indirect competitive ELISA, and positive wells are screened. The positive wells are cloned by limited dilution method until a hybridoma cell strain stably secreting monoclonal antibody is obtained.

[0064] (3) Cell cryopreservation and recovery

[0065] The hybridoma cells are prepared into a cell suspension of 1×10 6 / mL with a cryopreservation solution, and stored in liquid nitrogen for a long time. When recovered, the cryopreservation tube is taken out and immediately placed in a 37℃ water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, it is transferred to a culture bottle for culture.

[0066] (4) Preparation and purification of monoclonal antibody

[0067] Incremental culture method: the hybridoma cells are cultured in a cell culture medium at 37℃, and the obtained culture solution is purified by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, which is stored at -20℃.

[0068] The cell culture medium is RPMI1640 culture medium added with calf serum and sodium bicarbonate, so that the final concentration of calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium is 7.4.

[0069] 5. Preparation of goat anti-mouse antibody

[0070] Sheep are used as immunized animals, and mouse-derived antibodies are used as immunogens to immunize the pathogen-free sheep, and sheep anti-mouse antibodies are obtained.

[0071] 6. Preparation of flunixin meglumine monoclonal antibody-colloidal gold marker

[0072] (1) Preparation of colloidal gold

[0073] Dilute 1% chloroauric acid with double-distilled deionized water to 0.01% (mass fraction), take 100 mL and place in a conical flask, heat to boiling with a constant-temperature electromagnetic stirrer, add 1.5 mL of 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir at a uniform speed and heat until the solution is transparent red, then stop, cool to room temperature, restore to the original volume with deionized water, and store at 4°C. The prepared colloidal gold is pure, transparent, and free of precipitates and floating materials, and the color is wine red under sunlight.

[0074] (2) Preparation of flunixin meglumine monoclonal antibody-colloidal gold marker

[0075] Under magnetic stirring, adjust the pH value of the colloidal gold to 7.2 with 0.2 mol / L potassium carbonate solution, add the above flunixin meglumine monoclonal antibody to the colloidal gold solution at 5-50 μg of antibody per mL of colloidal gold solution, continue to stir and mix for 30 min; after standing for 10 min, add 10% BSA to make the final concentration of BSA in the colloidal gold solution 1%, and stand for 10 min. Centrifuge at 12,000 r / min at 4°C for 40 min, discard the supernatant, wash the precipitate with resuspension buffer twice, resuspend the precipitate with resuspension buffer with a volume of 1 / 10 of the initial colloidal gold volume, and store at 4°C.

[0076] Resuspension buffer: 0.02 mol / L phosphate buffer containing 0.1%-0.5% BSA and 2%-4% sucrose, pH 7.2.

[0077] 7. Preparation of microwell reagent

[0078] Add 100 μL of flunixin meglumine monoclonal antibody-colloidal gold marker to the microwell reagent microwell plate, place in a freeze-drying machine, pre-freeze at -50°C for 3 h, then vacuum dry for 15 h, take out, and obtain the microwell reagent with flunixin meglumine monoclonal antibody-colloidal gold marker, and seal and store.

[0079] 8. Preparation of sample absorption pad

[0080] Soak the sample absorption pad in 0.2 mol / L phosphate buffer containing 1% bovine serum albumin and having a pH of 7.2 for 2 h, and dry at 37°C for 2 h for standby use.

[0081] 9. Preparation of reaction membrane

[0082] The flunixin meglumine hapten-OVA conjugate is coated on the reaction membrane to form the detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form the quality control line.

[0083] Coating process: the flunixin meglumine hapten-OVA conjugate is diluted to 1 mg / mL with 0.01 mol / L, pH 7.2 phosphate buffer, and coated on the detection line (T line) of the nitrocellulose membrane with a Bio dot membrane marker, with a coating amount of 1.0 μL / cm; the goat anti-mouse antibody is diluted to 200 μg / mL with 0.01 mol / L, pH 7.2 phosphate buffer, and coated on the quality control line (C line) of the nitrocellulose membrane with a Bio dot membrane marker, with a coating amount of 1.0 μL / cm. The coated reaction membrane is dried at 37°C for 16 h and stored for use.

[0084] 10. Assembly of test strip

[0085] According to the test paper profile structure shown in Figure 3 , the sample absorption pad (1), the reaction membrane (2), and the water absorption pad (3) are sequentially adhered to the PVC base plate (6) in order; the end of the sample absorption pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the water absorption pad, the beginning of the sample absorption pad is aligned with the beginning of the PVC base plate, and the end of the water absorption pad is aligned with the end of the PVC base plate; the reaction membrane has a detection line (4) and a quality control line (5), both the detection line (T line) and the quality control line (C line) are strip-shaped bands perpendicular to the length of the test paper; the detection line is located on the side close to the end of the sample absorption pad; the quality control line is located on the side away from the end of the sample absorption pad; and the test paper is cut into small strips of 3.95 mm in width by a machine.

[0086] According to the microporous reagent diagram shown in Figure 4 , the microporous reagent 7 has a microporous plug 8.

[0087] The above test paper and microporous reagent are assembled into a test strip, which is stored in an environment of 2-8°C, and has a shelf life of 12 months.

[0088] Example 2: Detection of flunixin meglumine and its metabolites in liquid milk

[0089] 1. Sample pretreatment

[0090] After the sample is restored to room temperature, it is directly detected; the sample to be detected must be a uniform liquid and cannot have lumps, sourness, or precipitates.

[0091] 2. Detection with test strip

[0092] Take 200 μL of the sample to be tested with a micropipette, slowly suck and mix well with the reagent in the microwell; after incubation at 35℃ for 5 min, insert the test strip into the microwell; start timing when the liquid begins to flow, react for 5 min, and determine the result.

[0093] 3. Analyze the test results

[0094] Negative (-): The color of the T line is deeper than that of the C line or consistent with that of the C line, indicating that the concentration of flunixin meglumine and its metabolites in the sample is lower than the detection limit, such as Figure 5 a, 5b.

[0095] Positive (+): The color of the T line is lighter than that of the C line or the T line does not develop color, indicating that the concentration of flunixin meglumine and its metabolites in the sample is equal to or higher than the detection limit, such as Figure 5 c, 5d.

[0096] Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated, such as Figure 5 e, 5f.

[0097] Example 3: Sample detection example

[0098] 1. Detection limit test

[0099] Take blank raw milk, pasteurized milk, UHT sterilized milk, and goat milk samples, and add flunixin meglumine to a final concentration of 0.5 μg / L, 1 μg / L, and 2 μg / L, and add 5-hydroxyflunixin to a final concentration of 0.75 μg / L, 1.5 μg / L, and 3 μg / L, respectively, and take the test strip for detection. Each sample was measured three times.

[0100] When detecting raw milk, pasteurized milk, UHT sterilized milk, and goat milk samples with the test strip, when there is no flunixin meglumine and its added concentration is 0.5 μg / L, the test strip shows that the color of the T line is deeper than that of the C line or consistent with that of the C line, which is negative; when the added concentration of flunixin meglumine is 1 μg / L and 2 μg / L, the test strip shows that the color of the T line is lighter than that of the C line or the T line does not develop color, which is positive, indicating that the detection limit of the test strip for flunixin meglumine in liquid milk is 1 μg / L.

[0101] When detecting raw milk, pasteurized milk, UHT sterilized milk, and goat milk samples with the test strip, when there is no 5-hydroxyflunixin and its added concentration is 0.75 μg / L, the test strip shows that the color of the T line is deeper than that of the C line or consistent with that of the C line, which is negative; when the added concentration of 5-hydroxyflunixin is 1.5 μg / L and 3 μg / L, the test strip shows that the color of the T line is lighter than that of the C line or the T line does not develop color, which is positive, indicating that the detection limit of the test strip for 5-hydroxyflunixin in liquid milk is 1.5 μg / L.

[0102] 2. False positive rate, false negative rate test

[0103] Take 20 portions of blank raw milk, pasteurized milk, UHT sterilized milk, goat milk samples and positive raw milk, pasteurized milk, UHT sterilized milk, goat milk samples added with flunixin meglumine to a final concentration of 1 μg / L and 5-hydroxyflunixin to a final concentration of 1.5 μg / L, respectively, and use three batches of test strips produced to detect, and calculate the positive and negative rates.

[0104] The results show that when the positive raw milk, pasteurized milk, UHT sterilized milk, goat milk samples are detected by the test strips produced in three batches, the results are all positive, and it can be known that the positive coincidence rate is 100%, and the false negative rate is 0; when the blank raw milk, pasteurized milk, UHT sterilized milk, goat milk samples are detected, the results are all negative, and it can be known that the negative coincidence rate is 100%, and the false positive rate is 0. It is shown that the test strip of the application can rapidly detect flunixin meglumine and its metabolite 5-hydroxyflunixin residues in liquid milk.

[0105] 3. Specificity test

[0106] When 500 μg / L aspirin, indomethacin, naproxen, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib and other non-steroidal anti-inflammatory drugs are detected by the test strip, the T line on the test strip shows a color that is deeper than the color of the C line, and is negative, which shows that the test strip has small cross-reaction to these structural analogs, and has good specificity.

Claims

1. A test strip for detecting flunixin meglumine and its metabolites, comprising a test paper and a microporous reagent, the test paper comprising a reaction membrane, a sample absorption pad, a water absorption pad and a base plate, the reaction membrane having a detection line coated with flunixin meglumine hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse antibody, and the microporous reagent having flunixin meglumine monoclonal antibody-colloidal gold label lyophilized thereon; the flunixin meglumine monoclonal antibody is prepared by using flunixin meglumine hapten-carrier protein conjugate as immunogen; the flunixin meglumine hapten-carrier protein conjugate is obtained by coupling flunixin meglumine hapten with carrier protein, and the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin; characterized in that The synthesis method of the flunixin meglumine hapten is as follows: 2-amino-6-(trifluoromethyl)benzene propionic acid 2.33 g is dissolved in N,N-dimethylformamide 100 mL, 2-chloronicotinic acid 1.88 g is added, and stirred thoroughly, anhydrous KOH 0.78 g is added, a reflux condenser is installed, and heated at 70℃ for 6 h, the reaction is stopped, cooled to room temperature, water 200 mL is added, 6 mol / L hydrochloric acid is added to adjust the pH value to 6, ethyl acetate 100 mL is added for extraction, the water phase is separated, the organic phase is evaporated to dryness, and silica gel column is used for separation, and elution is performed with a dichloromethane-methanol mixed solution with a volume ratio of 10:1 to obtain the flunixin meglumine hapten, and the molecular structural formula is as follows: ； The flunixin meglumine metabolite is 5-hydroxyflunixin.

2. The test strip of claim 1, wherein The test paper is composed of a sample absorption pad, a reaction film, and a water absorption pad which are sequentially pasted on a bottom plate, and the microporous reagent has a microporous plug.

3. A method for preparing the test paper strip according to any one of claims 1-2, comprising the steps of: 1) preparing a microporous reagent with flunixin meglumine monoclonal antibody-gold label freeze-dried; 2) preparing a reaction film with a detection line coated with flunixin meglumine hapten-carrier protein conjugate and a quality control line coated with sheep anti-mouse antibody; 3) assembling the reaction film prepared in 2) with a sample absorption pad, a water absorption pad, and a bottom plate into a test paper; 4) assembling the microporous reagent with flunixin meglumine monoclonal antibody-gold label freeze-dried prepared in 1) and the test paper prepared in 3) into a test paper strip.

4. A method for detecting flunixin meglumine and its metabolite residues in liquid milk, comprising the steps of: 1) sample pretreatment; 2) detection by the test paper strip according to any one of claims 1-2; 3) analysis of the detection results.

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