A florfenicol and tilmicosin dual detection test paper and its preparation method and application

Abstract

The present invention discloses a florfenicol and tilmicosin double detection test paper and a preparation method and application, belonging to the field of veterinary drug residue analysis and immunology technology. The test paper includes a monoclonal antibody against florfenicol and a monoclonal antibody against tilmicosin, and can detect florfenicol and tilmicosin simultaneously. When the monoclonal antibody is prepared, florfenicol and tilmicosin are respectively used as haptens, and are coupled with the amino group of the carrier protein BSA to prepare florfenicol and tilmicosin artificial complete antigens. The prepared artificial complete antigens FF-BSA and TIL-BSA have good immunogenicity, and the two monoclonal monomers obtained by immunizing mice have the advantages of high specificity, high affinity and high sensitivity, which lays the foundation for the preparation of florfenicol and tilmicosin double detection test paper. The test paper detection has the advantages of simplicity, rapidity, sensitivity, accuracy, high throughput, etc., and can realize on-site real-time detection.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary drug residue analysis and immunology, and in particular to a florfenicol and tilmicosin double detection test paper and a preparation method and application thereof. Background Art

[0002] Florfenicol (FFrfenicol, FF), also known as fluorosulfamic acid, is a new generation of chloramphenicol antibiotics for animals. It has the characteristics of a broad antibacterial spectrum and low toxicity and side effects. It is an animal-specific antibacterial drug approved by the Ministry of Agriculture and is widely used in animal breeding. Although florfenicol is less toxic than chloramphenicol, incorrect or excessive use will inevitably cause florfenicol residues in livestock and poultry aquatic products. In particular, strains resistant to florfenicol have appeared, and the use of florfenicol is on the rise, which poses a potential threat to human health. Therefore, the study of its residue monitoring technology is of great significance to protecting people's health and the safety of aquatic products.

[0003] As a broad-spectrum antibiotic, macrolide antibiotics can be divided into 12- to 16-membered macrolide antibacterial drugs according to the carbon lactone ring in the molecular structure. Tilmicosin (TILmicosin, TIL) belongs to the 16-membered macrolide antibiotics. It is a new antibiotic for livestock and poultry synthesized from tylosin in recent years. It is mainly used to treat infections caused by Pasteurella and hemolytic Mannheimia in cattle. Improper use of Tilmicosin can cause adverse reactions such as shortness of breath and vomiting in livestock and poultry. Excessive use can cause death. In addition, improper use of drugs can cause veterinary drug residues in livestock products, which in turn cause harm to the human body.

[0004] At present, the main method for detecting the residues of florfenicol and tilmicosin is chromatographic analysis, such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Although chromatographic analysis is sensitive, accurate, has high separation and can perform qualitative and quantitative research on multi-residue detection, it requires expensive instruments, cumbersome pretreatment, and skilled professional operators. If instrumental analysis is used to detect large quantities of samples, the cost will be very high, and most of the current national testing institutions are only equipped with sophisticated analytical instruments at the provincial level, so it is necessary to establish a detection system from screening to confirmation. The methods for confirming the residues of florfenicol and tilmicosin are relatively complete, but there are few studies on detection methods that can be used for the initial screening of large quantities of samples. Immunological detection methods, especially colloidal gold test strips, have the advantages of rapidity, high sensitivity, simple operation, strong adaptability, and high throughput, and are suitable for high-throughput sample screening. Therefore, colloidal gold test strips have more advantages for rapid detection of florfenicol and tilmicosin residues in animal feed, soil or edible animal tissues. Summary of the invention

[0005] The purpose of the present invention is to provide a florfenicol and tilmicosin dual detection test paper and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The test paper is a colloidal gold immunochromatography dual test paper capable of simultaneously detecting florfenicol and tilmicosin, has the advantages of being simple, rapid, sensitive and accurate, and can realize on-site real-time detection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a method for preparing an anti-florfenicol monoclonal antibody. The method comprises the following steps: coupling an anti-florfenicol hapten with a carrier protein BSA by a glutaraldehyde method to synthesize FF-BSA; and immunizing mice with the FF-BSA to obtain the anti-florfenicol monoclonal antibody.

[0008] The present invention also provides an anti-florfenicol monoclonal antibody prepared by the preparation method, wherein the heavy chain variable region gene sequence of the anti-florfenicol monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the light chain variable region gene sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0009] The present invention also provides a method for preparing an anti-tilmicosin monoclonal antibody, comprising the steps of coupling the tilmicosin hapten with a carrier protein BSA by a succinic anhydride method to synthesize TIL-BSA; and then immunizing mice with the TIL-BSA to obtain the anti-tilmicosin monoclonal antibody.

[0010] The present invention also provides an anti-tilmicosin monoclonal antibody prepared by the preparation method, wherein the heavy chain variable region gene sequence of the anti-tilmicosin monoclonal antibody is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; the light chain variable region gene sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.

[0011] The present invention also provides a florfenicol and tilmicosin double detection test paper, comprising the anti-florfenicol monoclonal antibody and the anti-tilmicosin monoclonal antibody.

[0012] Preferably, it further comprises: a detection membrane, a conjugation pad, a sample pad, a water absorbent pad and a support plate, wherein the detection membrane is pasted to the center of the support plate, the conjugation pad and the sample pad are pasted to the sample end of the detection membrane in sequence, and the water absorbent pad is pasted to the other end of the detection membrane.

[0013] Preferably, the conjugate pad is loaded with the anti-florfenicol monoclonal antibody and the anti-tilmicosin monoclonal antibody.

[0014] The present invention also provides a method for preparing the florfenicol and tilmicosin dual detection test paper, comprising the following steps:

[0015] Step 1: Spray the FF-BSA, the TIL-BSA and the rabbit anti-mouse IgG on the center of the nitrocellulose detection membrane respectively to form detection line T1, detection line T2 and quality control line C blots to prepare the detection membrane;

[0016] Step 2: spraying two gold-labeled antibodies, anti-florfenicol monoclonal antibody and anti-tilmicosin monoclonal antibody, onto glass wool to prepare a conjugate pad;

[0017] Step 3: Paste the test membrane to the center of the support plate, and then paste the binding pad and sample pad to the sample end of the test membrane in sequence, with each layer overlapping by 1-2 mm; then paste the absorbent pad to the other end of the test membrane, overlapping with the test membrane by 1-2 mm, to obtain the florfenicol and tilmicosin dual detection test paper.

[0018] Preferably, the anti-florfenicol monoclonal antibody titer is 1:2.048×10 6 ,IC 50 The titer of anti-tilmicosin monoclonal antibody was 1:2.048×10 6 ,IC 50 It is 2.31 ng / mL.

[0019] The present invention also provides an application of the florfenicol and tilmicosin dual detection test paper in detecting florfenicol and tilmicosin residues.

[0020] The present invention discloses the following technical effects:

[0021] (1) In the preparation of monoclonal antibodies, the present invention uses florfenicol (FF) and tilmicosin (TIL) as haptens, and couples FF and TIL with carrier proteins by glutaraldehyde method and succinic anhydride method, respectively, to prepare FF-BSA artificial complete antigen and TIL-BSA artificial complete antigen. The antibodies obtained after immunizing mice with the immunogens FF-BSA and TIL-BSA have good titers and half-inhibitory concentrations, indicating that they have better immunogenicity. In addition, the two monoclonal antibodies obtained have the advantages of high specificity, high affinity and high sensitivity, laying a foundation for the preparation of florfenicol and tilmicosin dual detection test strips.

[0022] (2) The colloidal gold immunochromatographic double test paper established by the present invention can simultaneously detect florfenicol and tilmicosin. The content of florfenicol and tilmicosin in animal feed or the residues in edible animal tissues can be detected in one measurement, which can save the number of sample analyses and has better economic value.

[0023] (3) Compared with the existing high performance liquid chromatography detection technology, the colloidal gold immunochromatography double test paper of the present invention has the advantages of rapidity, specificity, sensitivity, accuracy, high throughput, simplicity and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of the synthesis principle of the florfenicol artificial complete antigen (FF-BSA) of the present invention.

[0026] Figure 2 Schematic diagram of the synthesis principle of the tilmicosin artificial complete antigen (TIL-BSA) of the present invention.

[0027] Figure 3 These are the SDS-PAGE identification results of florfenicol artificial complete antigen and tilmicosin artificial complete antigen; line M is the standard protein marker; line 1 is BSA; line 2 is FF-BSA; line 3 is BSA; and line 4 is TIL-BSA.

[0028] Figure 4 The present invention is a schematic diagram of the structure of the florfenicol and tilmicosin colloidal gold immunochromatographic double detection test paper; 1-detection membrane, 2-binding pad, 3-sample pad, 4-water absorption pad, 5-support plate.

[0029] Figure 5 It is a schematic diagram of the determination results of different sample combinations detected by the florfenicol and tilmicosin colloidal gold immunochromatographic double detection test strip of the present invention;

[0030] Figure 6 It is the test result of the florfenicol and tilmicosin colloidal gold immunochromatography double test paper of the present invention; wherein: 1- T1, T2 test lines and C quality control line are all colored, which is a negative result; 2- T1 and C quality control lines are colored, and T2 test line is not colored, which is tilmicosin positive and florfenicol negative; 3- T2 and C quality control lines are colored, and T1 test line is not colored, which is florfenicol positive and tilmicosin negative; 4- T1, T2 test lines are not colored, and C quality control line is colored, which is a double positive result of florfenicol and tilmicosin;

[0031] Figure 7Comparison of FF and TIL in samples by double test strips and HPLC; A: comparison results of FF; B: comparison results of TIL. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Preparation of Florfenicol and Tilmicosin Monoclonal Antibodies

[0038] 1.1 Preparation of artificial complete antigens of florfenicol and tilmicosin

[0039] (1) Florfenicol FF was coupled to the carrier protein BSA by the glutaraldehyde method to prepare FF-BSA artificial complete antigen. The specific method is as follows:

[0040] Weigh 72.5 mg of florfenicol and 31.2 mg of anhydrous sodium carbonate respectively, dissolve in 3 mL of double distilled water, place on a magnetic stirrer, heat to 50-60°C, stir until the solution is clear, and obtain completely hydrolyzed florfenicol, i.e., florfenicol amide; add 10 mg of BSA to the above solution, stir evenly, add 100 µL of glutaraldehyde, stir at room temperature overnight, and finally dialyze the obtained mixture with PBS at 4°C for 3 days, changing the solution 4 times a day, and obtain FF-BSA artificial complete antigen. The experimental principle is as follows: Figure 1 shown.

[0041] (2) Tilmicosin TIL was coupled to the carrier protein BSA by the succinic anhydride method to prepare TIL-BSA artificial complete antigen. The specific method is as follows:

[0042] Dissolve 0.87 g of tilmicosin in 5 mL of anhydrous acetone, add 26 mg of 4-dimethylaminopyridine (DMAP), place on a magnetic stirrer, heat to 50-60°C, slowly add 0.24 g of succinic anhydride, react for 4 h to obtain tilmicosin succinate, then add 10 mg of EDC, stir at room temperature for 30 min, finally add 10 mg of BSA, stir at room temperature for 4 h, dialyze the mixture with PBS at 4°C for 3 days, change the solution 4 times a day, and obtain TIL-BSA artificial complete antigen. The experimental principle is as follows: Figure 2 shown.

[0043] 1.2 Animal immunization

[0044] The prepared immunogen FF-BSA ( Figure 3 ) were added with Freund's complete adjuvant and Freund's incomplete adjuvant respectively to make Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen (the volume ratio of FF-BSA to Freund's complete adjuvant and Freund's incomplete adjuvant was 1:1). Three female BALB / c mice aged 6 to 8 weeks (purchased from the Animal Experiment Center of the School of Medicine of Zhengzhou University) were immunized with FF-BSA Freund's complete adjuvant immunogen by multiple subcutaneous injections on the back, 10 μg / mouse; 21 days and 42 days after the first immunization, BALB / c mice were boosted with FF-BSA Freund's incomplete adjuvant immunogen in the same way and dose; 3 to 4 days before cell fusion, BALB / c mice were super-immunized with FF-BSA without adjuvant by tail vein injection, and the immunization dose was 20 μg / mouse. One week after the last booster immunization, the tails of the three mice were cut and blood was collected, and then the titer and sensitivity of the polysera of the three mice were measured by indirect ELISA and indirect competitive ELISA. Among them, the immune effect of mouse No. 1 was the best, with a titer of 1:51200 and a half inhibitory concentration IC 50was 15.6 ng / mL (Table 1, Table 2), therefore, mouse No. 1 was selected for fusion in the next step.

[0045] The same method was used to prepare the immunogen TIL-BSA ( Figure 3 ) were used for animal immunization. After 4 immunizations, the titer and sensitivity of the polyclonal antiserum of the three mice were measured by indirect ELISA and indirect competitive ELISA. Among them, the immunization effect of mouse No. 3 was the best, with a titer of 1:51200 and a half inhibitory concentration IC 50 was 7.81 ng / mL (Table 1, Table 2), therefore, mouse No. 3 was selected for fusion in the next step.

[0046] Table 1 Titer of mouse polyclonal antibody

[0047]

[0048] Table 2 Sensitivity of mouse polyclonal antibody

[0049]

[0050] 1.3 Cell fusion and subcloning

[0051] Establishment of anti-FF hybridoma cell line: On the third day after super-strong immunization of mouse No. 1, the spleen cells of the immunized mouse and mouse myeloma cells SP2 / 0 were fused at a ratio of 10:1 by polyethylene glycol method. The fused cells were screened with HAT selection medium, distributed in 96-well cell culture plates with feeder cells, and cultured in a 37°C, 5% CO2 incubator. After 10 days, FF-OVA (synthesis method is the same as FF-BSA) was used as a coating source, and the wells with hybridoma cells were positively screened by indirect ELISA method, and then the positive hybridoma cells were subcloned by limiting dilution method until a hybridoma cell line that can stably secrete anti-FF monoclonal antibodies was obtained (named 5F7). Establishment of anti-TIL hybridoma cell line: In the same way, mouse No. 3 was selected and cell fusion and subcloning were performed according to the above method, and finally a hybridoma cell line that can stably secrete anti-TIL monoclonal antibodies (named 1D9) was obtained.

[0052] 1.4 Preparation and identification of monoclonal antibody ascites

[0053] Two multiparous female BALB / c mice were selected and intraperitoneally injected with 500 μL of sterilized paraffin. One week later, 2×10 5F7 and 1D9 hybridoma cell lines were injected intraperitoneally into the two mice, respectively. 5 One week later, the abdomen of the mouse was swollen and the ascites was extracted. The supernatant was taken after centrifugation and the two monoclonal antibodies in the ascites were purified by the ammonium sulfate method.

[0054] The titer and sensitivity of the purified two monoclonal antibodies in ascites were measured by indirect ELISA and indirect competitive ELISA, respectively. The results showed that the titer of 5F7 monoclonal antibody could reach 1:2.048×10 6 ,IC 50 The heavy chain nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the light chain nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; the titer of 1D9 monoclonal antibody can reach 1: 2.048×10 6 ,IC 50 The concentration of 5F7 monoclonal antibody in the samples was 2.31 ng / mL (Table 3, Table 4), and the heavy chain nucleotide sequence was shown in SEQ ID NO: 5, and the amino acid sequence was shown in SEQ ID NO: 6; the light chain nucleotide sequence was shown in SEQ ID NO: 7, and the amino acid sequence was shown in SEQ ID NO: 8. The subtype identification results showed that 5F7 monoclonal antibody belonged to IgG1, k type; 1D9 monoclonal antibody belonged to IgG2b, k type.

[0055] The 5F7 heavy chain variable region gene sequence (SEQ ID NO: 1) is:

[0056] TCTGGGGGAGGCTTAGTGAAGCCTGGCGGGTCCCTGAAACTCTCCTGTGCAGCCTCTTTCGCCACTTTCAGTAACTATGCCATGTCTTGGATTCGCCAGACTCCAGAGAAGAGGGCGGAGTGGGTCGGATCCCATAGTAGTGCTGGTACAACCTACTATCCAGACAG TGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCGGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTCAGAGACAGGTACGACGAAGCTTTGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA.

[0057] The amino acid sequence of the 5F7 heavy chain variable region (SEQ ID NO: 2) is:

[0058] SGGGLVKPGGSLKLSCAASFATFSNYAMSWIRQTPEKRAEWVGSHSSAGTTYYPDSVKGRFTISRDNARNILYLQMSGLRSEDTAMYYCVRDRYDEALDYWGQGTTVTVSS.

[0059] The 5F7 light chain variable region gene sequence (SEQ ID NO: 3) is:

[0060] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGCAGAAAGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAATTACATGGAATGGTACCAGCAGAAGCTAGGATCCTCCCCAAAGTCTGGATTTATGACACATCCAAACTGG CTCCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCATCAGTGGAGTAGTTACCCACTCACGTTCGGTGCAGGGACCAAGCTGGAGCTGAAAC.

[0061] The amino acid sequence of the 5F7 light chain variable region (SEQ ID NO: 4) is:

[0062] QIVLTQSPAIMSASPGQKVTITCSASSSVNYMEWYQQKLGSSPKVWIYDTSKLAPGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPLTFGAGTKLELK.

[0063] The 1D9 heavy chain variable region gene sequence (SEQ ID NO: 5) is:

[0064] GAGTCTGGGGGAGGCTTAGTGAAGCCTGGCGGGTCCCTGAAACTCTCCTGTGCAGCCTCTACTTTCACTTTCAGTAACTATCCAATGTCTTGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTCACAGTTGTGGTACAACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCGGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGACCGAGGTACGACGATGAGTTGGAGGACTACTGGGGTCAAGGAACCTCAGTCACCG。

[0065] The amino acid sequence of the 1D9 heavy chain variable region (SEQ ID NO: 6) is as follows:

[0066] ESGGGLVKPGGSLKLSCAASTFTFSNYPMSWIRQTPEKRLEWVASIHSCGTTYYPDSVKGRFTISRDNARNILYLQMSGLRSEDTAMYYCVRPRYDDELEDYWGQGTSVT。

[0067] The gene sequence of the 1D9 light chain variable region (SEQ ID NO: 7) is as follows:

[0068] CTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCCTGAGCTACAGGGCCAGCAAATCTGTCAGTACATCCGGCTATACCTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCACCAGATTCGTGAATCTACAAGGTCGGAGGGGGGACCAAGCTGGAAATCAAAC。

[0069] The amino acid sequence of the 1D9 light chain variable region (SEQ ID NO: 8) is as follows:

[0070] LTQSPASLAVSLGQRATLSYRASKSVSTSGYTYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCHQIRESTRSEGGPSWKSN.

[0071] Table 3 mAb potency determination

[0072]

[0073] Table 4 Inhibition curve determination of mAbs

[0074]

[0075] Example 2 Preparation of rabbit anti-mouse IgG

[0076] Healthy New Zealand rabbits weighing about 2.0 kg were immunized with purified mouse IgG. The first immunization was with antigen emulsified in Freund's complete adjuvant, and 50 μg / rabbit was injected subcutaneously at multiple points. Each booster immunization was 3 weeks apart, and antigen emulsified in Freund's incomplete adjuvant was injected intramuscularly. Two weeks after the last booster immunization, when the immune serum antibody titer was higher than 1:40 as determined by ELISA, whole blood of the highly immune rabbits was collected, the serum was separated, and the rabbit anti-mouse IgG was purified by the caprylic acid-ammonium sulfate method.

[0077] Example 3 Preparation of gold-labeled antibodies

[0078] 1.1 Preparation of colloidal gold

[0079] Take 100mL of ultrapure water and place it in a 500mL clean conical flask, add 1mL of 1% (w / v) chloroauric acid solution and boil; quickly add 1mL of freshly prepared 1% (w / v) sodium citrate solution while stirring, boil for about 3min until the color of the solution changes from yellow to purple-red, and continue boiling for 2min; wait for the solution to cool to room temperature, add ultrapure water to 100mL, adjust the pH to 9.0 with 0.2mol / L K2CO3, and store at 4℃ away from light for later use.

[0080] 1.2 Determination of optimal marker protein concentration

[0081] Take the anti-FF and TIL monoclonal antibody IgG to be labeled and dialyze them with 20mmol / L sodium borate solution (pH8.0) at 4℃ overnight. Dilute the FF and TIL monoclonal antibodies to be labeled in a microplate with 25μL ultrapure water at 1:2, 1:4, 1:8, 1:16, 1:32, etc.; add 125μL colloidal gold solution to each well and let it stand at room temperature for 5min; add 1250μL 1mol / L NaCl solution; the color of each well changes from red to blue as the protein concentration decreases. The protein concentration at the highest dilution of the monoclonal antibody that does not turn blue is the optimal colloidal gold labeling concentration. When labeled with colloidal gold, the protein concentration increases by 20%.

[0082] 1.3 Colloidal gold labeling of monoclonal antibodies

[0083] Take 2mL of the monoclonal antibody IgG to be labeled with the optimal protein concentration, add 10mL of colloidal gold solution (pH 9.0), mix quickly, and let it act at room temperature for 10min~15min; add 10% of the volume of the mixed solution containing 10% (w / v) bovine serum albumin (BSA) in 20mmol / L sodium borate solution, mix quickly, and let it act at room temperature for 10min~15min; centrifuge at 4℃ and 15000g for 30min, and carefully remove the supernatant; resuspend the precipitate with 20mmol / L sodium borate solution containing 1% (w / v) BSA, centrifuge as above, and discard the supernatant; repeat washing once, resuspend the precipitate with 1mL of 20mmol / L sodium borate solution containing 1% (w / v) BSA, and store at 4℃ for later use.

[0084] Example 4 Preparation of test paper

[0085] 1.1 Preparation of detection membrane

[0086] The nitrocellulose test membrane was placed on the XYZ 3000 spraying instrument platform and fixed with a pressure strip; TIL-BSA, FF-BSA artificial antigens and rabbit anti-mouse IgG antibody were diluted to 1 mg / mL with PBS buffer, filtered through a 0.22 mL filter membrane, and then filtered through a 1 membrane. Figure 3 ) and rabbit anti-mouse IgG antibody solution were sprayed on the center of the nitrocellulose test membrane to form test lines (T1 line, T2 line) and quality control line (C line) blots; the test lines and the test lines, as well as the test lines and the quality control lines were 0.5 cm apart; the test membrane was placed in a 42°C drying oven for 30 min or dried naturally at room temperature, and then stored in a dry and sealed place at 4°C.

[0087] 1.2 Preparation of conjugate pad

[0088] Place the glass wool on the XYZ 3000 sprayer platform and fix it with a pressure strip; take 1 mL of gold-labeled antibody and add 2 mL of 20 mmol / L sodium borate solution (pH 8.0) containing 2% (w / v) BSA, 3% (w / v) sucrose, 0.6 mol / L NaCl, 0.2% Tween 20 (v / v) and 0.1% (w / v) sodium azide; spray the gold-labeled antibody solution on the glass wool at 15 L / cm; place it in a 50°C drying oven for 30 min to dry; place the conjugate pad in a plastic bag, add desiccant and seal it at 4°C for storage until use.

[0089] 1.3 Preparation of sample pad

[0090] Soak the glass wool strips in a PBS (pH 7.2) solution containing 0.1 mol / L NaCl, 0.2% Tween 20 (v / v) and 0.1% (w / v) sodium azide; dry them in a 50°C drying oven for 30 min; place the sample pad in a plastic bag, add desiccant and seal it at room temperature for later use.

[0091] 1.4 Preparation of absorbent pad

[0092] Place the absorbent pad in a plastic bag, add desiccant and store it in a sealed container at room temperature for later use.

[0093] 1.5 Preparation of support plate

[0094] A double-sided tape is attached to a PVC support plate to prepare a support plate.

[0095] 1.6 Assembly of test strips

[0096] Use the LM5000 test strip assembly instrument or manually to assemble the above materials into a test strip. First, paste the test film 1 to the center of the support plate 5, and then paste the two binding pads 2 and sample pads 3 containing gold-labeled antibodies (anti-FF gold-labeled antibodies and anti-TIL gold-labeled antibodies) to the sample end of the test film 1 in sequence, with each layer overlapping by 1 mm to 2 mm, and then paste the absorbent pad 4 to the other end of the test film, overlapping with the test film 1 by 1 mm to 2 mm (such as Figure 4 ).

[0097] Florfenicol and Tilmicosin dual test strips detection principle:

[0098] The florfenicol and tilmicosin dual test strip uses two monoclonal antibodies, florfenicol and tilmicosin, and is designed and assembled based on modern immunological technology and chromatography technology. During the chromatography process of the sample to be tested, the florfenicol and tilmicosin in the sample compete with the florfenicol-protein conjugate and tilmicosin-protein conjugate on the two detection lines (T1 line and T2 line) on the nitrocellulose detection membrane for their respective gold-labeled specific monoclonal antibodies. The color changes of the T1 line and T2 line on the test strip are compared with the quality control line (C line) to determine the florfenicol and tilmicosin content in the sample (e.g. Figure 5 ).

[0099] Example 5 Detection method

[0100] 1.1 Sample pretreatment

[0101] (1) Pretreatment of eggs

[0102] Take 5g of egg white to be tested and add 10mL of ethyl acetate, vortex on a vortexer for 10min, centrifuge at 3500rpm for 10min, take the supernatant, blow dry with nitrogen, and finally dissolve the residue with 5mL of PBS buffer (0.01M pH7.4).

[0103] (2) Pretreatment of chicken and pork samples

[0104] After homogenizing the tissue sample to be tested, take 5 g of the homogenized tissue and add 10 mL of ethyl acetate, vortex on a vortexer for 10 min, centrifuge at 3500 rpm for 10 min, take the supernatant, blow dry with nitrogen, and finally dissolve the residue with 5 mL of PBS buffer (0.01 M pH 7.4).

[0105] (3) Pretreatment of chicken and pig feed samples

[0106] After grinding the feed sample to be tested into powder, take 5g of feed powder and add 10mL of ethyl acetate, vortex on a vortexer for 10min, centrifuge at 3500rpm for 10min, take the supernatant, blow dry with nitrogen, and finally dissolve the residue with 5mL of PBS buffer (0.01MpH7.4).

[0107] (4) Pretreatment of soil samples

[0108] Soil samples were collected from farms, dried naturally, crushed, and stones and animal and plant residues were picked out. After grinding, the soil was sieved with a 100-mesh filter. 5 g of the sieved soil was weighed and added to 10 mL of ethyl acetate. The soil was vortexed on a vortex machine for 10 min, centrifuged at 3500 rpm for 10 min, and the supernatant was taken and dried with nitrogen. Finally, 5 mL of PBS buffer (0.01 M pH 7.4) was used to dissolve the residue.

[0109] 1.2 Detection

[0110] Take 100µL of the sample to be tested and add it to the microplate. Immerse the sample end of the colloidal gold immunochromatography double test paper in the sample solution for 10s to 20s. Take out the test paper and place it horizontally for 5min to 10min to observe the results.

[0111] 1.3 Result determination

[0112] like Figure 2 As shown in the figure, the test paper shows three reddish-brown bands (T1, T2 test lines and C quality control line) which is negative, indicating that the FF in the tissue to be tested is negative, and TIL is negative (FF - / TIL - );

[0113] If only one reddish brown band (C quality control line) is present, it is positive, indicating that the sample to be tested contains both FF and TIL residues (FF + / TIL + );

[0114] The test paper shows two reddish-brown bands (T2 detection line and C quality control line), indicating that the sample to be tested is FF positive and TIL negative (FF + / TIL - );

[0115] The test paper shows two reddish-brown bands (T1 detection line and C quality control line), indicating that the sample to be tested is FF negative and TIL positive (FF - / TIL + );

[0116] If the test paper does not show any stripes, it indicates that the test operation is improper or the test paper is invalid, and another test paper must be taken for retesting.

[0117] From the above experiments and results, it can be seen that the test paper is suitable for testing samples including animal feed, animal muscle tissue, soil samples, etc. The sample processing method involved in the present invention is simple and easy to operate. The main organic reagent used for sample processing is ethyl acetate, which is relatively less harmful to the health of the operator. In addition, the semi-quantitative detection of FF and TIL can be achieved by naked eye observation using the test paper, and quantitative detection can be achieved by scanning the grayscale of the T line of the test paper with the BioDot-TSR3000 reader.

[0118] Example 6 Testing of the performance of colloidal gold immunochromatographic dual test paper

[0119] 1.1 Identification of the specificity of the dual test strip

[0120] The organic arsenic additives florfenicol, tilmicosin, chloramphenicol, doxycycline, tylosin, trimethoprim, and tylosin were selected as standard substances, and these seven standards were diluted into 10 ng / mL solutions with PBS, and specific identification was performed according to the above detection method. The identification results showed that the double test paper only reacted specifically to florfenicol and tilmicosin.

[0121] 1.2 Identification of the sensitivity of the double test paper

[0122] Use PBS to dilute FF and TIL standards to concentrations of 0, 1, 2, 3, 4, and 5 ng / mL, respectively. Prepare solutions containing FF / TIL 0 / 0, 0 / 1, 1 / 2, 2 / 3, 3 / 4, and 4 / 5 ng / mL. Then use double test strips for detection. Repeat the measurement for 3 times for each sample and read the results within 10 minutes. The minimum concentration of FF that can make the T1 line disappear, and the minimum concentration of TIL that can make the T2 line disappear are the detection limits of the test strips, that is, the sensitivity.

[0123] The results showed that the detection limit of the dual test strip for FF was 2ng / mL, and the detection limit for TIL was 3ng / mL.

[0124] 1.3 Double test paper stability test

[0125] Since the test strips have good stability and can be stored for more than 1 year under normal circumstances, in order to shorten the test strip shelf life determination time, this test uses an accelerated stability test to evaluate the stability of the two-piece test strips. The test strips are stored at 45°C. The test is based on the Arrhenius formula to deduce the relationship between different temperatures and the number of days of the accelerated test. Storage at 45°C for 37.5 days is equivalent to storage at room temperature of 25°C for 1 year. Therefore, in this test, the same batch of test strips were stored at 45°C in the dark, and the test strips were taken out at 0, 10, 20, 30, 40, and 50 days, respectively. The test strips were used to detect 50 negative samples and 50 positive samples containing FF 2ng / mL and TIL 3ng / mL, respectively. The color development of the T line of the negative sample of the test strip and the false negative rate and false positive rate of the test strip were observed, so as to determine the shelf life of the test strip.

[0126] like Figure 6 As shown in the results, it can be seen that no false positives or false negatives were observed within 40 days at 45°C, and the T line color was normal. However, the color of the test paper became weaker after being stored at 45°C for 50 days, indicating that the stability of the test paper has deteriorated. Therefore, the shelf life of the test paper at room temperature (about 25°C) should be set at 1 year, but it should be noted that the storage conditions need to be dry and dark.

[0127] 1.4 Double test strips for natural samples

[0128] Take 10 egg white samples, 10 chicken tissue samples, 10 pork tissue samples, 10 chicken feed samples, 10 pig feed samples, and 10 soil samples that have been tested by HPLC (the detection targets are FF and TIL, and the HPLC test results are attached). Use FF and TIL colloidal gold immunochromatography double test strips to detect the samples at the same time, and use the test strip scanner to scan the grayscale, calculate the content of FF and TIL in the samples, and repeat the measurement 3 times for each sample to get the average value. Use the concentration detected by HPLC in each sample as the horizontal axis and the concentration detected by the test strip as the vertical axis to draw a scatter plot, generate a regression curve, and use the R of the regression curve as the 2 To evaluate the correlation between the test results obtained by the two detection methods, and thus to evaluate whether the test paper can be used to detect the residues of FF and TIL in the samples.

[0129] The results showed that among the 60 samples tested by the test strip, 20 samples were FF positive (Table 5) and 29 samples were TIL positive (Table 6). A scatter plot was drawn based on the HPLC results ( Figure 7 ). The R of the results of HPLC and test paper method for detecting FF and TIL content in samples 2 The values ​​of FF and TIL residues in animal feed, edible animal tissue or soil samples were 0.9742 and 0.9554, respectively, both greater than 0.9000, indicating that the results of the two methods are well correlated, and the corresponding regression line slopes are 0.9303 and 0.9312, respectively, both less than 1, indicating that the results obtained by the test paper detection method are slightly lower than those of HPLC. In summary, the double test paper can be used for the rapid detection of FF and TIL residues in animal feed, edible animal tissue or soil samples, and the results are accurate and reliable.

[0130] Table 5 Compliance rate of FF detected by test paper and HPLC

[0131]

[0132] Table 6 Compliance rate of test strips and HPLC in detecting TIL

[0133]

[0134] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention. Sequence Listing <110> Zhengzhou University, Henan Biobio Bioengineering Co., Ltd. <120> A florfenicol and tilmicosin dual detection test paper and its preparation method and application <160> 8 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 333 <212> DNA <213> Artificial Sequence <400> 1 tctgggggag gcttagtgaa gcctggcggg tccctgaaac tctcctgtgc agcctctttc 60 gccactttca gtaactatgc catgtcttgg attcgccaga ctccagagaa gagggcggag 120 tgggtcggat cccatagtag tgctggtaca acctactatc cagacagtgt gaagggccga 180 ttcaccatct ccagagataa tgccaggaac atcctgtacc tgcaaatgag cggtctgagg 240 tctgaggaca cggccatgta ttactgtgtc agagacaggt acgacgaagc tttggactac 300 tggggccaag ggaccacggt caccgtctcc tca 333 <210> 2 <211> 111 <212> PRT <213> Artificial Sequence <400> 2 Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys 1 5 10 15 Ala Ala Ser Phe Ala Thr Phe Ser Asn Tyr Ala Met Ser Trp Ile Arg 20 25 30 Gln Thr Pro Glu Lys Arg Ala Glu Trp Val Gly Ser His Ser Ser Ala 35 40 45 Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 50 55 60 Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg Asp Arg Tyr Asp Glu 85 90 95 Ala Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 110 <210> 3 <211> 319 <212> DNA <213> Artificial Sequence <400> 3 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggca gaaagtcacc 60 ataacctgca gtgccagctc aagtgtaaat tacatggaat ggtaccagca gaagctagga 120 tcctccccca aagtctggat ttatgacaca tccaaactgg ctcctggagt ccctgctcgc 180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240 gatgctgcct cttatttctg ccatcagtgg agtagttacc cactcacgtt cggtgcaggg 300 accaagctgg agctgaaac 319 <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <400> 4 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Glu Trp Tyr Gln Gln Lys Leu Gly Ser Ser Pro Lys Val Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 5 <211> 331 <212> DNA <213> Artificial Sequence <400> 5 gagtctgggg gaggcttagt gaagcctggc gggtccctga aactctcctg tgcagcctct 60 gagtctgggg gaggcttagt gaagcctggc gggtccctga aactctcctg tgcagcctct 60 actttcactt tcagtaacta tccaatgtct tggattcgcc agactccaga gaagaggctg 120 actttcactt tcagtaacta tccaatgtct tggattcgcc agactccaga gaagaggctg 120 gagtgggtcg catccattca cagttgtggt acaacctact atccagacag tgtgaagggc 180 gagtgggtcg catccattca cagttgtggt acaacctact atccagacag tgtgaagggc 180 cgattcacca tctccagaga taatgccagg aacatcctgt acctgcaaat gagcggtctg 240 cgattcacca tctccagaga taatgccagg aacatcctgt acctgcaaat gagcggtctg 240 aggtctgagg acacggccat gtattactgt gtaagaccga ggtacgacga tgagttggag 300 aggtctgagg acacggccat gtattactgt gtaagaccga ggtacgacga tgagttggag 300 gactactggg gtcaaggaac ctcagtcacc g 331 gactactggg gtcaaggaac ctcagtcacc g 331 <210> 6 <211> 110 <212> PRT <213> Artificial Sequence <400> 6 Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser 1 5 10 15 Cys Ala Ala Ser Thr Phe Thr Phe Ser Asn Tyr Pro Met Ser Trp Ile Cys Ala Ala Ser Thr Phe Thr Phe Ser Asn Tyr Pro Met Ser Trp Ile 20 25 30 Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser Ile His Ser Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser Ile His Ser 35 40 45 Cys Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Cys Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg Pro Arg Tyr Asp 85 90 95 Asp Glu Leu Glu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 <210> 7 <211> 321 <212> DNA <213> Artificial Sequence <400> 7 ctgacacagt ctcctgcttc cttagctgta tctctggggc agagggccac cctgagctac 60 agggccagca aatctgtcag tacatccggc tatacctata tgcactggaa ccaacagaaa 120 ccaggacagc cacccagact cctcatctat cttgtatcca acctagaatc tggggtccct 180 gccaggttca gtggcagtgg gtctgggaca gacttcaccc tcaacatcca tcctgtggag 240 gaggaggatg ctgcaaccta ttactgtcac cagattcgtg aatctacaag gtcggagggg 300 ggaccaagct ggaaatcaaa c 321 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <400> 8 Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala 1 5 10 15 Thr Leu Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Thr 20 25 30 Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu 35 40 45 Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu 65 70 75 80 Glu Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ile Arg Glu Ser Thr 85 90 95 Arg Ser Glu Gly Gly Pro Ser Trp Lys Ser Asn 100 105

Claims

1. An anti-florfenicol monoclonal antibody, characterized in that: The heavy chain variable region gene sequence of the anti-florfenicol monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the light chain variable region gene sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO:

4.

2. An anti-tilmicosin monoclonal antibody, characterized in that: The heavy chain variable region gene sequence of the anti-tilmicosin monoclonal antibody is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; the light chain variable region gene sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO:

8.

3. A florfenicol and tilmicosin dual detection test paper, characterized in that: The invention comprises the anti-florfenicol monoclonal antibody according to claim 1 and the anti-tilmicosin monoclonal antibody according to claim 2.

4. The florfenicol and tilmicosin dual detection test paper according to claim 3, characterized in that: Also includes: A detection membrane, a binding pad, a sample pad, a water absorbent pad and a support plate, wherein the detection membrane is pasted on the center of the support plate, the binding pad and the sample pad are pasted on the sample end of the detection membrane in sequence, and the water absorbent pad is pasted on the other end of the detection membrane.

5. The florfenicol and tilmicosin dual detection test paper according to claim 4, characterized in that: The anti-florfenicol monoclonal antibody and the anti-tilmicosin monoclonal antibody are loaded on the conjugate pad.

6. A method for preparing a florfenicol and tilmicosin dual detection test paper according to any one of claims 3 to 5, characterized in that: The following steps are involved: Step 1: Using the glutaraldehyde method, the florfenicol hapten is coupled with the carrier protein BSA to synthesize FF-BSA; The tilmicosin hapten is coupled with the carrier protein BSA by the succinic anhydride method to synthesize TIL-BSA; the FF-BSA, the TIL-BSA and the rabbit anti-mouse IgG are sprayed on the center of the nitrocellulose detection membrane respectively to form detection line T1, detection line T2 and quality control line C blots to prepare the detection membrane; Step 2: spraying the two gold-labeled antibodies, the anti-florfenicol monoclonal antibody of claim 1 and the anti-tilmicosin monoclonal antibody of claim 2, onto glass wool to prepare a conjugate pad; Step 3: Paste the detection membrane to the center of the support plate, and then paste the binding pad and the sample pad to the sample end of the detection membrane in sequence, with each layer overlapping by 1-2 mm; then paste the absorbent pad to the other end of the detection membrane, overlapping with the detection membrane by 1-2 mm, to obtain the florfenicol and tilmicosin dual detection test paper.

7. The method for preparing the florfenicol and tilmicosin dual detection test paper according to claim 6, characterized in that: The titer of the anti-florfenicol monoclonal antibody is 1:2.048×10 6 ,IC 50 The titer of anti-tilmicosin monoclonal antibody was 1:2.048×10 6 ,IC 50 It is 2.31 ng / mL.

8. Use of the florfenicol and tilmicosin dual detection test paper as claimed in any one of claims 3 to 5 in detecting florfenicol and tilmicosin residues.

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