African swine fever virus monoclonal antibody and application thereof

Abstract

The application discloses an African swine fever virus monoclonal antibody and application thereof, and a test paper strip which comprises a polyvinyl chloride bottom plate at the bottom, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially arranged on the polyvinyl chloride bottom plate in a flowing direction; the sample pad is partially overlapped on the combination pad, and the combination pad and the water absorption pad are respectively overlapped on both sides of the nitrocellulose membrane; a detection line and a quality control line are arranged on the nitrocellulose membrane in the flowing direction; the combination pad is sprayed with red latex microsphere marked antibodies, the detection line is sprayed with coated antibodies, and the quality control line is sprayed with coated commercialized sheep anti-mouse IgG. The test paper strip adopts a double-antibody sandwich method for detection, and has the advantages of fast detection speed, high sensitivity, strong specificity and the like. The detection result is intuitive and clear, and is easy to judge; the whole detection process does not need any instrument and equipment, and an operator also does not need professional training, and the operation is simple and convenient, and is particularly suitable for on-site rapid diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of cell biology technology, immunology technology and animal epidemic detection technology, and particularly relates to an African swine fever virus monoclonal antibody and application thereof. BACKGROUND

[0002] African swine fever (ASF) is an acute, severe, highly contagious disease caused by African swine fever virus (ASFV), mainly infecting domestic pigs and wild boars. The clinical manifestations are high fever, anorexia, cyanosis of the skin, vomiting, hemorrhagic diarrhea and high mortality, and the mortality rate can be as high as 100%. The disease does not infect humans, but it is extremely harmful to the pig industry, and has been listed as a legal reportable animal disease by the World Organization for Animal Health (WOAH) and as a class A animal infectious disease in China.

[0003] In recent years, African swine fever has continued to spread globally, causing serious economic losses and great prevention and control pressure to the pig industry. Due to the strong resistance of ASFV in the environment, complex transmission routes, lack of safe and effective commercial vaccines, and great challenges in epidemic control. Therefore, strengthening the monitoring and early warning of African swine fever virus has become the key to prevention and control work.

[0004] In order to adapt to the modern large-scale breeding system and the requirements of biological safety and prevention and control, it is urgent to establish a sensitive, rapid and convenient on-site detection technology to realize the instant identification and accurate disposal of suspected cases, effectively block the virus transmission chain, reduce the risk of epidemic spread, improve the prevention and control efficiency, and effectively guarantee the sustainable and healthy development of the pig breeding industry. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide an African swine fever virus monoclonal antibody and application thereof. The African swine fever virus monoclonal antibody of the present application is monoclonal antibody 3C5 or monoclonal antibody 8A5. The antigen detection test strip prepared by using the monoclonal antibody is suitable for on-site diagnosis. The use of the test strip is simple, rapid, sensitive and specific. The detection result is clear and easy to judge. The entire detection process does not require any instrument and the operator does not need any professional training.

[0006] To achieve the above purpose, the technical scheme designed by the present application is as follows:

[0007] The present application provides an African swine fever virus monoclonal antibody, which is monoclonal antibody 3C5 or monoclonal antibody 8A5,

[0008] The monoclonal antibody 3C5 comprises a light chain variable region and a heavy chain variable region; wherein,

[0009] The light chain variable region of 3C5 comprises three complementarity determining regions, namely 3C5-LCDR-1, 3C5-LCDR-2 and 3C5-LCDR-3, whose amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, respectively;

[0010] The heavy chain variable region of 3C5 comprises three complementarity determining regions, namely 3C5-HCDR-1, 3C5-HCDR-2 and 3C5-HCDR-3, whose amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, respectively;

[0011] The monoclonal antibody 8A5 comprises a light chain variable region and a heavy chain variable region; wherein,

[0012] The light chain variable region of 8A5 comprises three complementarity determining regions, namely 8A5-LCDR-1, 8A5-LCDR-2 and 8A5-LCDR-3, whose amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21, respectively;

[0013] The heavy chain variable region of 8A5 comprises three complementarity determining regions, namely 8A5-HCDR-1, 8A5-HCDR-2 and 8A5-HCDR-3, whose amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30, respectively.

[0014] Further, the light chain variable region of the monoclonal antibody 3C5 comprises four light chain framework regions, namely 3C5-LFR-1, 3C5-LFR-2, 3C5-LFR-3 and 3C5-LFR-4, whose amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7, respectively;

[0015] The heavy chain variable region of the monoclonal antibody 3C5 comprises four heavy chain framework regions, namely 3C5-HFR-1, 3C5-HFR-2, 3C5-HFR-3 and 3C5-HFR-4, whose amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15 and SEQ ID No: 16, respectively;

[0016] The light chain variable region of the monoclonal antibody 8A5 contains four light chain framework regions, namely 8A5-LFR-1, 8A5-LFR-2, 8A5-LFR-3 and 8A5-LFR-4, and their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24 and SEQ ID No: 25, respectively.

[0017] The heavy chain variable region of the monoclonal antibody 8A5 contains four heavy chain framework regions, namely 8A5-HFR-1, 8A5-HFR-2, 8A5-HFR-3 and 8A5-HFR-4, and their amino acid sequences are shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33 and SEQ ID No: 34, respectively.

[0018] Further, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C5 are shown in SEQ ID No: 8 and SEQ ID No: 17, respectively, and the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the African swine fever virus monoclonal antibody 3C5 are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively.

[0019] Further, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8A5 are shown in SEQ ID No: 26 and SEQ ID No: 35, respectively, and the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the African swine fever virus monoclonal antibody 8A5 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

[0020] The above-mentioned African swine fever virus monoclonal antibody 3C5 and monoclonal antibody 8A5 are prepared by using the p30 recombinant protein of the African swine fever virus as an immunogen.

[0021] The above-mentioned monoclonal antibody is secreted by a hybridoma cell strain immunized by the p30 recombinant protein of the African swine fever virus.

[0022] The application also provides a use of the above-mentioned African swine fever virus monoclonal antibody in the preparation of an African swine fever virus antigen detection test strip based on a double antibody sandwich method.

[0023] The application also provides an African swine fever virus red latex microsphere antigen detection test strip, which comprises the above-mentioned African swine fever virus monoclonal antibody.

[0024] Further, the detection test strip comprises a lowermost polyvinyl chloride base plate, a sample pad, a binding pad, a nitrocellulose membrane 3 and a water absorption pad are sequentially arranged on the polyvinyl chloride base plate in the flow direction; the sample pad is partially overlapped on the binding pad, and the binding pad and the water absorption pad are respectively overlapped on both sides of the nitrocellulose membrane; a detection line (T line) and a quality control line (C line) are arranged on the nitrocellulose membrane in the flow direction; wherein,

[0025] The binding pad is sprayed with red latex microsphere labeled antibodies, the detection line is sprayed with coated antibodies, and the quality control line is sprayed with coated commercial sheep anti-mouse IgG.

[0026] Further, the red latex microsphere labeled antibodies are monoclonal antibody 3C5 or monoclonal antibody 8A5; and the antibody labeling amount in the red latex microsphere labeled antibodies is 50 μg / mL (the labeling amount means that 50 μg of antibodies are added to each 1 mL of the latex microsphere reaction system for reaction, covalent binding or adsorption with the microspheres in the process of preparing the labeled antibodies);

[0027] The coated antibodies are monoclonal antibody 3C5 or monoclonal antibody 8A5; and the concentration of the coated antibodies is 0.3 mg / mL; and the concentration of the coated commercial sheep anti-mouse IgG is 0.5 mg / mL.

[0028] The purpose of the red latex microsphere labeled antibodies is to attach the antibodies to the latex microspheres, so that the antibodies can recognize the target antigens and display color signals in the detection test strip.

[0029] The application further provides a preparation method of the above African swine fever virus red latex microsphere antigen detection test strip, comprising the following steps:

[0030] 1) adding red latex microspheres into MES buffer solution, sequentially mixing with EDC and NHS, then centrifuging, resuspending with boric acid buffer solution for multiple times, and finally ultrasonic dispersion to obtain activated red latex microsphere suspension,

[0031] 2) labeling the activated red latex microsphere suspension with the above monoclonal antibodies to obtain red latex microsphere labeled antibodies;

[0032] 3) respectively blocking the sample pad and the binding pad, and storing at 4°C;

[0033] 4) spraying and fixing the red latex microsphere labeled antibodies on the binding pad;

[0034] 5) coating the monoclonal antibodies and the commercial sheep anti-mouse IgG respectively, and then spraying them onto the nitrocellulose membrane as the detection line and the quality control line;

[0035] 6) Paste the water absorption pad, the combination pad, the sample pad on the polyvinyl chloride base plate on the nitrocellulose membrane, assemble the test paper strip.

[0036] Further, the activated red latex microsphere suspension is prepared by the following steps:

[0037] a. Red latex microspheres with a solid content of 4% are added to MES buffer and vortexed; wherein the concentration of the MES buffer is 0.03 mol / L.

[0038] b. According to the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (English full name: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, abbreviated as EDC), N-hydroxysuccinimide (English full name: N-hydroxysuccinimide, abbreviated as NHS) and the surface of the red microspheres is 1:1:1, 10 mg / mL of EDC solution and 10 mg / mL of NHS solution are weighed, then the NHS solution is vortexed, then the EDC solution is vortexed, and then the rotation mixer is mixed at room temperature,

[0039] c. Centrifugation, discard the supernatant, add boric acid buffer for resuspension, repeat centrifugation for several times, after the last resuspension, ultrasonic dispersion, get activated red latex microsphere suspension; wherein the concentration of boric acid buffer is 0.02 mmol / L and its pH is 8.0;

[0040] The above-mentioned red latex microspheres have carboxyl groups (commonly carboxyl-modified microspheres), which can be covalently connected to the amino groups of the antibodies through EDC / NHS chemical coupling. Therefore, when doing EDC / NHS activation reaction, according to the molar amount of these carboxyl groups, EDC and NHS are added according to the molar ratio of 1:1:1; in this way, all carboxyl groups can be effectively activated, which is conducive to the subsequent antibody coupling.

[0041] The red latex microsphere-labeled antibody is prepared by the following steps:

[0042] i. The above-mentioned monoclonal antibody is added to the activated red latex microsphere suspension, and the rotation mixer is mixed at room temperature; wherein the labeling amount of the monoclonal antibody is 50 μg / mL;

[0043] ii. Then add a final concentration of 0.5% BSA (bovine serum albumin), block mixing at room temperature in a rotating mixer, centrifuge, discard the supernatant, resuspend with a preservation solution, and store at 4°C; wherein the preservation solution is 100 mL of water with 1.21 g Tris, 5 g sucrose, 0.5 g BSA (bovine serum albumin), 0.5 g PVP (polyvinylpyrrolidone, English name: Polyvinylpyrrolidone), 0.5 g F68, 0.5 mL PEG200 (polyethylene glycol 200), 0.5 mL Triton X-100 (Chinese name Triton X-100, a non-ionic surfactant), 1 mL Tween-20.

[0044] The above F68: trade name: Pluronic F68

[0045] Alias: polyoxypropylene-polyoxyethylene block copolymer F68

[0046] Chemical name: polyblock copolymer PEG-PPG-PEG (PEO-PPO-PEO)

[0047] Chinese name: F68, Pluronic F68, block copolymer F68

[0048] F68 function:

[0049] Stabilize antibody or protein structure, prevent non-specific adsorption;

[0050] Improve the dispersibility of latex microspheres or colloidal particles;

[0051] Prevent aggregation or precipitation during freeze-thawing;

[0052] Enhance the wettability of the storage solution on the test strip;

[0053] Help maintain the stability and performance consistency of the latex marker.

[0054] The blocking solution of the conjugate pad is 100 mL of water with 2 g sucrose, 0.3 g PVPK-30, 2 g BSA, 0.02 g NaN3, 0.29 g Na2HPO4·12H2O, and 0.02 g KH2PO4, with a pH of 7.6.

[0055] The blocking solution of the sample pad uses 0.5 mol / L Tris-HCl buffer as the base solution, plus 0.50% PVPK-40 (wt%), 0.25% SDS-L (wt%), 0.02% NaN3 (wt%), and 0.2% casein (wt%).

[0056] The spraying amount of the red latex microsphere labeled antibody is 5 μL / cm.

[0057] In the step 5), the monoclonal antibody and the commercialized goat anti-mouse IgG are coated by the following method:

[0058] The monoclonal antibody and the commercialized goat anti-mouse IgG are diluted to 0.3 mg / mL and 0.5 mg / mL respectively by using the antibody coating solution to obtain the monoclonal antibody coating solution and the commercialized goat anti-mouse IgG coating solution.

[0059] The detection method of the African swine fever virus red latex microsphere antigen detection test strip is a double antibody sandwich reaction of the African swine fever virus antigen in the sample (pig fecal swab, the sample amount is 80 muL), so that the antigen is gathered and colored at the T line.

[0060] The beneficial effects of the application are:

[0061] The application uses the prokaryotic expression protein to prepare the monoclonal antibody, and two specific African swine fever virus monoclonal antibodies 3C5 and 8A5 are screened out, which have good effects in the antigen detection test strip pairing screening.

[0062] The African swine fever virus red latex microsphere antigen detection test strip of the application uses the double antibody sandwich method for detection, and has the advantages of fast detection speed, high sensitivity, strong specificity and the like. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 It is the enzyme digestion identification of the prokaryotic expression plasmid pET-28a-ASFV-p30 in the embodiment of the application, the expression and purification diagram of the protein;

[0064] Figure 2 It is the serum titer detection result diagram of the mouse immunized by the recombinant p30 protein, A is the antibody titer of the mouse serum after two weeks of the second immunization, and B is the antibody titer of the mouse serum after two weeks of the third immunization.

[0065] Figure 3 It is the IFA detection diagram of the monoclonal antibody secreted by the hybridoma cell strain ASFV-MAb-3C5 and ASFV-MAb-8A5 in the embodiment of the application;

[0066] Figure 4 It is the WB detection diagram of the monoclonal antibody secreted by the hybridoma cell strain ASFV-MAb-3C5 and ASFV-MAb-8A5 in the embodiment of the application;

[0067] Figure 5 Structure diagram of a red latex microsphere test strip for detecting African swine fever virus antigen in an embodiment of the present application;

[0068] In the figure, sample pad 1, binding pad 2, nitrocellulose membrane 3, detection line 4, quality control line 5, water absorption pad 6, and polyvinyl chloride bottom plate 7.

[0069] Figure 6 Figure of sensitivity detection result of the red latex microsphere antigen test strip for African swine fever virus in an embodiment of the present application;

[0070] Figure 7 Figure of specificity detection result of the red latex microsphere antigen test strip for African swine fever virus in an embodiment of the present application.

[0071] Figure 8 Display diagram of the red latex microsphere test strip for detecting African swine fever virus antigen in an embodiment of the present application after detection,

[0072] Wherein, a is a positive result diagram, b is a negative result diagram, and c is an invalid result diagram.

[0073] In the figure, C represents the quality control area, and T represents the detection area. DETAILED DESCRIPTION

[0074] The present application will be further described in detail below with reference to specific embodiments so as to be understood by those skilled in the art.

[0075] Example 1 Preparation of hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5

[0076] 1. Strains, cells, serum, and experimental animals

[0077] Strains: Inactivated African swine fever virus provided by Professor Luo Rui of Huazhong Agricultural University.

[0078] Cells: Human embryonic kidney cells 293 (HEK293) were purchased from Shanghai Cell Bank.

[0079] Strains: Escherichia coli DH5α used for plasmid construction was purchased from Biyun Tian Biological.

[0080] Plasmid vectors: Prokaryotic expression vector pET-28a(+) and eukaryotic expression vector pcDNA3.1(+) were purchased from Addgene platform.

[0081] Experimental animals: 5-6-week-old BALB / c female experimental mice were purchased from Hubei Experimental Animal Research Center.

[0082] 2. Construction, protein expression and purification of recombinant plasmid pET-28a-ASFV-p30

[0083] According to the p30 gene sequence of ASFV, as shown in SEQ ID No: 39:

[0084] ATGGATTTTATTTTAAATATATCCATGAAAATGGAGGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCTTGTATAATTGGTTTTCTGTTGAGATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTCAGTACTGTTAAGTATGATATTGTGAAATCTGCTCATATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAGGAGACAGAATCCTCAGCATCATCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATAAATGCGCATCCTCCTTTGAAACATTGTTTGAGCAAGAGCCCTCATCAGAGGAACCTAAAGACTCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAACTTTATTCAAACCATTCATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTTATTAAACTTTAAAAAAAAAATAA

[0085] The encoded amino acid sequence is shown in SEQ ID No: 40:

[0086] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSAHIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNKCASSFETLFEQEPSSEEPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKK.

[0087] A pair of primers were designed by Oligo 6.0 software, and the primers were synthesized by Genescript. The sequences are as follows:

[0088] ASFV-p30-F: 5'-CAGCCATATGGATTTTATTTTAAATATATCCATGAAAATG-3' (SEQ ID No: 37);

[0089] ASFV-p30-R: 5'-TGCTCGAGAATGTAGGTGAGATAAAAGCTTA-3' (SEQ ID No: 38).

[0090] The p30 gene of ASFV was used as a template, and the target fragment of the p30 gene was obtained by RT-PCR amplification using the above primers. The prokaryotic expression vector pET-28a(+) was digested with NdeI and XhoI, and then gel recovery was performed. The double-digested prokaryotic expression vector pET-28a(+) and the p30 gene target fragment were ligated in vitro using T4 DNA Ligase to obtain a recombinant plasmid. The recombinant plasmid with positive enzyme digestion was selected and sent to Wuhan Genescript for sequencing verification. The recombinant plasmid with correct identification was named pET-28a-ASFV-p30. The results of plasmid electrophoresis are shown in Figure 1 A, and the results showed that the pET-28a-ASFV-p30 plasmid was successfully constructed. The pcDNA3.1-ASFV-p30 plasmid was constructed in the same way.

[0091] The pET-28a-ASFV-p30 plasmid was transformed into E. coli Rosetta (DE3) competent cells, and protein expression was induced by IPTG. The inclusion bodies were collected for purification, and the purified product was detected by SDS-PAGE. As shown in Figure 1 B, the detection results showed that the size of the expressed recombinant p30 protein was about 35 kDa, which was consistent with the expected size.

[0092] 3. Animal immunization

[0093] Healthy female BALB / c mice of four to six weeks of age were selected, and immunization was performed by subcutaneous multi-point injection with a volume of 0.5 mL per point. According to the concentration of the purified p30 protein and the final immunization dose, an appropriate amount of protein was emulsified with an equal volume of Freund's complete adjuvant, and then the first immunization was performed by subcutaneous injection. An appropriate amount of protein was mixed with an equal volume of Freund's incomplete adjuvant for emulsification, and then the second immunization was performed. Two weeks later, tail blood was collected, and serum was separated. The serum antibody titer was detected by indirect ELISA, as shown in Figure 2 The mice with relatively high serum antibody levels were selected for subsequent hybridoma preparation experiments.

[0094] 4. Establishment of hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5

[0095] Mouse peritoneal macrophages were prepared as feeder cells according to a conventional method, and spleen cells were fused with myeloma cells (SP2 / 0) at a ratio of 5:1 under the action of fusion agent PEG4000. Antibody-secreting positive hybridoma cells were screened by indirect ELISA, and cloning was performed by limited dilution. Finally, two hybridoma cell strains, ASFV-MAb-3C5 and ASFV-MAb-8A5, which can stably secrete ASFV p30 protein monoclonal antibodies, were obtained.

[0096] Example 2 Preparation of African swine fever virus monoclonal antibodies 3C5 and 8A5 from hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5

[0097] 1. A method for preparing African swine fever virus monoclonal antibodies 3C5 and 8A5 from hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5, comprising the following steps:

[0098] 0.5 mL of sterilized paraffin oil was injected into the peritoneal cavity of a mouse, and 10 6 days later, 0.5 mL of sterilized paraffin oil was injected into the peritoneal cavity of the mouse again. After 7-10 days, when the ascites in the abdominal cavity of the mouse was extremely swollen, the ascites was extracted, and a large amount of monoclonal antibodies were contained in the ascites. After purification, the ascites was ready for use.

[0099] 2. Determination of the above African swine fever virus monoclonal antibodies 3C5 and 8A5:

[0100] (1) Indirect immunofluorescence (IFA) of monoclonal antibodies

[0101] HEK293 cells were inoculated in a 24-well plate, and when the cells grew to 80-90%, pcDNA3.1-ASFV-p30 plasmid was transfected, and IFA detection was performed 24 h later.

[0102] The results are shown in Figure 3 Fig. 2. The prepared monoclonal antibodies 3C5 and 8A5 specifically bind to HEK293 cells transfected with pcDNA3.1-ASFV-p30 plasmid, producing green fluorescence; the negative control group has no fluorescence, indicating that the prepared MAb has strong specificity.

[0103] (2) Western Blot of monoclonal antibody protein

[0104] HEK293 cells were inoculated in 10 cm cell culture dishes, and when the cells grew to 80-90%, pcDNA3.1(+) empty plasmid and pcDNA3.1-ASFV-p30 plasmid were transfected respectively, and samples were collected 12 h later for Western Blot detection.

[0105] The results are shown in Figure 4 As shown in the results, the prepared monoclonal antibodies 3C5 and 8A5 do not specifically bind to the target protein, and no specific bands are detected, indicating that the antigen epitopes recognized by the two prepared MAb are spatial epitopes, not linear epitopes.

[0106] The sequence of the monoclonal antibody 3C5 is as follows:

[0107] The three complementarity determining regions of the light chain variable region are 3C5-LCDR-1, 3C5-LCDR-2 and 3C5-LCDR-3, and their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, respectively; the three complementarity determining regions of the heavy chain variable region are 3C5-HCDR-1, 3C5-HCDR-2 and 3C5-HCDR-3, and their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, respectively;

[0108] The four light chain framework regions of the light chain variable region are 3C5-LFR-1, 3C5-LFR-2, 3C5-LFR-3 and 3C5-LFR-4, and their amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7, respectively; the four heavy chain framework regions of the heavy chain variable region are 3C5-HFR-1, 3C5-HFR-2, 3C5-HFR-3 and 3C5-HFR-4, and their amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15 and SEQ ID No: 16, respectively;

[0109] The amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region are shown in SEQ ID No: 8 and SEQ ID No: 17, respectively;

[0110] The nucleic acid sequence encoding the light chain variable region of the above-mentioned monoclonal antibody 3C5 and the nucleic acid sequence encoding the heavy chain variable region are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively;

[0111] The specific sequence of the above-mentioned monoclonal antibody 3C5 is as follows:

[0112] 3C5-HCDR-1 : RSSQTIVHSNGNTYADSVKG (SEQ ID No. 1);

[0113] 3C5-HCDR-2: KVSNRFS (SEQ ID No. 2);

[0114] 3C5-HCDR-3: FQGSHVPWT (SEQ ID No. 3);

[0115] 3C5-HFR-1 : EIVLTQSPATLSLSPGERATLSCRASKG (SEQ ID No. 4);

[0116] 3C5-HFR-2: WYQQKPGKAPKLLIY (SEQ ID No. 5);

[0117] 3C5-HFR-3: RFTISRDNAADYYTL (SEQ ID No. 6);

[0118] 3C5-HFR-4: FGQGTKLEIK (SEQ ID No. 7);

[0119] Amino acid sequence of the heavy chain variable region, with the CDR sequences underlined:

[0120] EIVLTQSPATLSLSPGERATLSCRASKG RSSQTIVHSNGITYLE WYQQKPGKAPKLLIY KVSNRFS RFTISRDNAADYYTL FQGSHVPWT FGQGTKLEIK (SEQ ID No. 8);

[0121] Nucleic acid sequence of the heavy chain variable region:

[0122] GATGTTTTGATGACCCAAACTCCATTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAATCACCTATTTAGAGTGGTACATGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 9);

[0123] 3C5-HCDR-1: SYAMS (SEQ ID No. 10);

[0124] 3C5-HCDR-2: TISRFSTYTYYPDSVKG (SEQ ID No. 11);

[0125] 3C5-HCDR-3: QGYYSGEGDYFDY (SEQ ID No. 12);

[0126] 3C5-HFR-1: EVMLVESGGGLVKPGGSLKVSCAASGFTFS (SEQ ID No. 13);

[0127] 3C5-HFR-2: WVRQTPEKRLEWVA (SEQ ID No. 14);

[0128] 3C5-HFR-3: RFTISRDNAKNTLSLQMSSLRSEDTAIYYCIR (SEQ ID No. 15);

[0129] 3C5-HFR-4: WGQGTTLTVSS (SEQ ID No. 16);

[0130] Amino acid sequence of the heavy chain variable region, wherein underlined sequences represent CDR sequences:

[0131] EVMLVESGGGLVKPGGSLKVSCAASGFTFS SYAMS WVRQTPEKRLEWVA TISRFSTYTYYPDSVKGRFTISRDNAKNTLSLQMSSLRSEDTAIYYCIR QGYYSGEGDYFDY WGQGTTLTVSS (SEQ ID No. 17);

[0132] Nucleic acid sequence of the heavy chain variable region:

[0133] GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAAGTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGTCAGACTCCGGAGAAGAGACTGGAGTGGGTCGCAACCATTAGTCGTTTTAGTACTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTCCCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATTTATTACTGTATAAGACAGGGTTATTACTCCGGGGAGGGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No. 18).

[0134] b. The sequence of monoclonal antibody 8A5 is as follows:

[0135] The three complementarity determining regions of the light chain variable region are 8A5-LCDR-1, 8A5-LCDR-2, and 8A5-LCDR-3, respectively, and their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20, and SEQ ID No: 21, respectively; the three complementarity determining regions of the heavy chain variable region are 8A5-HCDR-1, 8A5-HCDR-2, and 8A5-HCDR-3, respectively, and their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29, and SEQ ID No: 30, respectively;

[0136] The amino acid sequences of the four light chain framework regions of the light chain variable region are 8A5-LFR-1, 8A5-LFR-2, 8A5-LFR-3, and 8A5-LFR-4, respectively, and the amino acid sequences of the four heavy chain framework regions of the heavy chain variable region are 8A5-HFR-1, 8A5-HFR-2, 8A5-HFR-3, and 8A5-HFR-4, respectively, as shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, and SEQ ID No: 34, respectively.

[0137] The amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID No: 26 and SEQ ID No: 35, respectively.

[0138] The nucleic acid sequences encoding the light chain variable region and the heavy chain variable region of the monoclonal antibody 8A5 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

[0139] The specific sequences of the monoclonal antibody 8A5 are as follows:

[0140] 8A5-LCDR-1: RASESVDTYGNSFMH (SEQ ID No. 19);

[0141] 8A5-LCDR-2: LASNLES (SEQ ID No. 20);

[0142] 8A5-LCDR-3: QQNNEDPYT (SEQ ID No. 21);

[0143] 8A5-LFR-1: NIVLTQSPASLAVSLGQRATISC (SEQ ID No. 22);

[0144] 8A5-LFR-2: WYQQKPGQPPKLLIY (SEQ ID No. 23);

[0145] 8A5-LFR-3: GVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID No. 24);

[0146] 8A5-LFR-4: FGGGTKLEIK (SEQ ID No. 25);

[0147] The amino acid sequence of the light chain variable region, in which the CDR sequences are indicated by underlines:

[0148] NIVLTQSPASLAVSLGQRATISC RASESVDTYGNSFMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSRTDFTLTIDPVEADDAATYYC QQNNEDPYT FGGGTKLEIK (SEQ ID No. 26);

[0149] Nucleic acid sequence of the heavy chain variable region:

[0150] AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGCGTTGATACTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC (SEQ ID No. 27);

[0151] 8A5-HCDR-1: SYWIH (SEQ ID No. 28);

[0152] 8A5-HCDR-2: RIYPGTGISFYNEKFKG (SEQ ID No. 29);

[0153] 8A5-HCDR-3: RIFDDYPNWYFDV (SEQ ID No. 30);

[0154] 8A5-HFR-1: QVQLKQSGAELVRPGASVKLSCQTSGYIFT (SEQ ID No. 31);

[0155] 8A5-HFR-2: WVKQRSGQGLEWIA (SEQ ID No. 32);

[0156] 8A5-HFR-3: KATLTADKSSSTAYMQLSSLKSEDSAVYFCAR (SEQ ID No. 33);

[0157] 8A5-HFR-4: WGAGTTVTVSS (SEQ ID No. 34);

[0158] Amino acid sequence of the heavy chain variable region, with the CDR sequences underlined:

[0159] QVQLKQSGAELVRPGASVKLSCQTSGYIFT SYWIH WVKQRSGQGLEWIA RIYPGTGISFYNEKFKG KATLTADKSSSTAYMQLSSLKSEDSAVYFCAR RIFDDYPNWYFDV WGAGTTVTVSS (SEQ ID No. 35);

[0160] Nucleic acid sequence of the heavy chain variable region:

[0161] CAGGTCCAGCTGAAGCAGTCTGGAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCCAGACTTCTGGATACATCTTCACCAGCTACTGGATTCACTGGGTTAAACAGAGGTCTGGACAGGGCCTTGAGTGGATTGCAAGGATTTATCCTGGAACTGGTATTAGTTTCTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGAAATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGACGGATCTTTGATGATTACCCTAACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG (SEQ ID No. 36).

[0162] Example 3 Preparation of ASFV red latex microsphere antigen test strip using ASFV monoclonal antibodies 3C5 and 8A5

[0163] I. Cells, plasmids, serum and main reagents:

[0164] Plasmid pET-28a-ASFV-p30 (constructed in Example 1), hybridoma cell strains secreting ASFV-p30 protein monoclonal antibodies 3C5 and 8A5, ASFV-MAb-3C5, ASFV-MAb-8A5 (prepared in Example 1). Inactivated African swine fever virus (ASFV), porcine circovirus type 2 (PCV-2), porcine epidemic diarrhea virus (PEDV) were provided by Professor Luo Rui's research group of Huazhong Agricultural University.

[0165] II. Preparation of African swine fever virus red latex microsphere antigen detection test strip

[0166] 1. Preparation of monoclonal antibody 3C5-red latex microsphere marker

[0167] In an EP tube, 975 μL of 0.03 mol / L MES buffer was added, and then 25 μL of red latex microspheres with a solid content of 4% was added and vortexed for mixing; 5 μL of 10 mg / mL NHS solution was added and vortexed for mixing, followed by the addition of 5 μL of EDC with the same concentration and vortexing, and then the mixture was mixed at 20 r / min on a rotary mixer at room temperature for 20 min; centrifugation was performed at 4°C and 13000 r / min for 10 min, the supernatant was discarded, 1 mL of 0.02 mmol / L pH8.0 boric acid buffer was added for resuspension, and the centrifugation was repeated twice, and ultrasonic dispersion was performed for 2 min; the monoclonal antibody 3C5 was diluted to 0.2 mg / mL with ultrapure water, and 50 μg was added to the activated microsphere suspension, and mixed at 20 r / min on a rotary mixer at room temperature for 2 h; 0.5% BSA was added, and the mixture was blocked at 20 r / min on a rotary mixer at room temperature for 1 h; centrifugation was performed at 4°C and 13000 r / min for 10 min, the supernatant was discarded, 1 mL of the storage solution was added for resuspension, and the mixture was stored at 4°C. Thus, the monoclonal antibody 3C5-red latex microsphere marker of the African swine fever virus was obtained.

[0168] The above-mentioned storage solution is 100 mL of water to which 1.21 g of Tris, 5 g of sucrose, 0.5 g of BSA (bovine serum albumin), 0.5 g of PVP (polyvinyl pyrrolidone, English name: Polyvinyl pyrrolidone), 0.5 g of F68, 0.5 mL of PEG200 (polyethylene glycol 200), 0.5 mL of TritonX-100 (Chinese name: TritonX-100, a non-ionic surfactant), and 1 mL of Tween-20 are added.

[0169] 2. Preparation of the binding pad

[0170] (1) Preparation of the binding pad blocking solution

[0171] 2 g of sucrose, 0.3 g of PVPK-30, 2 g of BSA, 0.02 g of NaN3, 0.29 g of Na2HPO4·12H2O, and 0.02 g of KH2PO4 were weighed and dissolved in ddH2O, the pH was adjusted to 7.6, the volume was adjusted to 100 mL in a volumetric flask, and then filtered through a 0.22 μm filter membrane to obtain the binding pad blocking solution.

[0172] (2) Preparation of the binding pad

[0173] The glass cellulose membrane (type Fusion4) is soaked in the blocking solution of the conjugate pad for 30 min and dried in a 37°C oven to obtain the conjugate pad.

[0174] 3. Preparation of the sample pad

[0175] (1) Preparation of the blocking solution of the sample pad

[0176] The blocking solution of the sample pad is prepared by using 0.5 mol / L Tris-HCl buffer as the base solution and adding 0.50% PVP K-40, 0.25% SDS-L, 0.02% NaN3 and 0.2% casein.

[0177] (2) Preparation of the sample pad

[0178] The glass cellulose membrane (type GL-B04) is soaked in the blocking solution of the sample pad for 30 min and dried in a 37°C oven to obtain the blocked conjugate pad.

[0179] 4. Spraying of the monoclonal antibody 3C5-red latex microsphere marker of the African swine fever virus on the conjugate pad

[0180] The monoclonal antibody 3C5-red latex microsphere marker is sprayed on the treated conjugate pad 2, and the spraying amount of the monoclonal antibody 3C5-red latex microsphere marker is 5 μL / cm; after the spraying is completed, the sample is dried in a 37°C oven for 2 hours and cut into a 3 mm wide strip for standby use.

[0181] 5. Spraying of the detection line and the quality control line

[0182] (1) Preparation of the antibody coating solution

[0183] The antibody coating solution is prepared by adding 1.0% (w / v) trehalose, 1.0% (w / v) sorbitol and 0.3% (w / v) Tween-20 into 0.02 mol / L phosphate buffer solution (PBS) with a pH value of 7.2, stirring uniformly and filtering through a 0.22 μm filter membrane.

[0184] (2) Preparation of the detection line and the quality control line

[0185] The nitrocellulose membrane 3 is non-pointed and pasted on the polyvinyl chloride base plate 7, and the type of the nitrocellulose membrane 3 is Millipore Pall 90s; the monoclonal antibody 8A5 of the African swine fever virus is diluted to 0.3 mg / mL with the antibody coating solution, and the commercialized sheep anti-mouse IgG is diluted to 0.5 mg / mL with the antibody coating solution, and then sprayed on the positions of the nitrocellulose membranes 4 and 5 as the detection line and the quality control line respectively with a spraying amount of 1 μL / cm.

[0186] The distance between the detection line and the quality control line is 5 mm, and after the spraying is completed, the sample is dried in a 37°C oven for 2 hours for standby use.

[0187] III. Assembly of the test strips

[0188] like Figure 5 As shown, the nitrocellulose membrane 3 prepared in step 4 is pasted onto the polyvinyl chloride base plate 7; the conjugate pad 2, which is coated with the monoclonal antibody 3C5-red latex microsphere marker for African swine fever virus prepared in step 3, is pasted on top of the nitrocellulose membrane 3, covering the nitrocellulose membrane 3 by 1-2 mm; the conjugate pad 2 is adjacent to the detection line 4 on the nitrocellulose membrane 3; the absorbent pad 6 is pasted on top of the nitrocellulose membrane 3, covering the nitrocellulose membrane 3 by 1-2 mm; the absorbent pad 6 is adjacent to the quality control line 5 on the nitrocellulose membrane 3; the sample pad 1 is pasted on top of the conjugate pad 2, covering the conjugate pad 2 by 1-2 mm; the colloidal gold test strip is cut into 3 mm wide test strips using a strip cutter, and then placed into the test card shell and desiccant together in an aluminum foil bag, which is then sealed and stored.

[0189] The above test strips were used to detect the sensitivity and specificity.

[0190] 1. Test strip sensitivity test

[0191] 10 6.5 TCID 50 The ASFV inactivated strain at a concentration of / mL was serially diluted 10-fold with the above diluent, and 80μL of the solution was taken for spot testing. The results were observed within 10 minutes.

[0192] The results are as follows Figure 6 As shown, the lower limit of detection for the test strip is 10. 3.5 TCID 50 / mL.

[0193] 2. Specificity testing of test strips

[0194] Dilute the inactivated ASFV, PEDV, and PCV-2 viruses 20 times with the above diluent, then take 80 μL of the sample for testing, and observe the results within 10 minutes.

[0195] The results are as follows Figure 7 As shown, the test strips exhibit no cross-reactivity and demonstrate good specificity.

[0196] Example 4: Application of the above-mentioned African swine fever virus red latex microsphere antigen test strip

[0197] 1. Pretreatment of the sample to be tested

[0198] Add a small amount of pig manure to 1 mL of diluent to make a suspension. Mix thoroughly and let stand for 5 minutes. Retain the supernatant or centrifuge and retain the supernatant.

[0199] The above diluent is prepared by: weighing 0.3092 g of boric acid, dissolving it completely in 400 mL of ddH2O, adjusting the pH to 8.0 with NaOH, bringing the volume to 500 mL, and then adding 2.5 mL of NP-40.

[0200] 2. Detection

[0201] Take 80 μL of the supernatant and drop it onto the test strip described above. Observe the results within 10 minutes. The determination of the test results is based on:

[0202] like Figure 8 As shown: A positive result is indicated when both control line 4 and test line 5 appear red, meaning the sample contains African swine fever virus (ASFV). Figure 8 a)

[0203] A negative result is indicated when control line 5 appears red and test line 4 does not appear red, meaning the sample does not contain African swine fever virus. Figure 8 b)

[0204] If no red line appears at control line 5, the test strip is invalid. Figure 8 c).

[0205] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An African swine fever virus monoclonal antibody, characterized in that: the monoclonal antibody is monoclonal antibody 3C5 or monoclonal antibody 8A5, the monoclonal antibody 3C5 comprises a light chain variable region and a heavy chain variable region; wherein, the light chain variable region of 3C5 comprises three complementarity determining regions, respectively 3C5-LCDR-1, 3C5-LCDR-2 and 3C5-LCDR-3; their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, respectively; the heavy chain variable region of 3C5 comprises three complementarity determining regions, respectively 3C5-HCDR-1, 3C5-HCDR-2 and 3C5-HCDR-3; their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, respectively; the monoclonal antibody 8A5 comprises a light chain variable region and a heavy chain variable region; wherein, the light chain variable region of 8A5 comprises three complementarity determining regions, respectively 8A5-LCDR-1, 8A5-LCDR-2, 8A5-LCDR-3; their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21, respectively; the heavy chain variable region of 8A5 comprises three complementarity determining regions, respectively 8A5-HCDR-1, 8A5-HCDR-2 and 8A5-HCDR-3; their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, respectively.

2. The African swine fever virus monoclonal antibody according to claim 1, characterized in that: the light chain variable region of the monoclonal antibody 3C5 comprises four light chain framework regions, respectively 3C5-LFR-1, 3C5-LFR-2, 3C5-LFR-3 and 3C5-LFR-4; their amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7, respectively; the heavy chain variable region of the monoclonal antibody 3C5 comprises four heavy chain framework regions, respectively 3C5-HFR-1, 3C5-HFR-2, 3C5-HFR-3 and 3C5-HFR-4; their amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15 and SEQ ID No: 16, respectively; the light chain variable region of the monoclonal antibody 8A5 comprises four light chain framework regions, respectively 8A5-LFR-1, 8A5-LFR-2, 8A5-LFR-3 and 8A5-LFR-4; their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24 and SEQ ID No: 25, respectively; The heavy chain variable region of the monoclonal antibody 8A5 contains four heavy chain framework regions, namely 8A5-HFR-1, 8A5-HFR-2, 8A5-HFR-3 and 8A5-HFR-4; their amino acid sequences are shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, respectively.

3. The African swine fever virus monoclonal antibody according to claim 1 or 2, characterized in that: The amino acid sequence of the light chain variable region of the monoclonal antibody 3C5, the amino acid sequence of the heavy chain variable region are shown in SEQ ID No: 8 and SEQ ID No: 17, respectively; the nucleotide sequence encoding the light chain variable region of the African swine fever virus monoclonal antibody 3C5, the nucleotide sequence of the heavy chain variable region are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively.

4. The African swine fever virus monoclonal antibody according to claim 1 or 2, characterized in that: The amino acid sequence of the light chain variable region of the monoclonal antibody 8A5, the amino acid sequence of the heavy chain variable region are shown in SEQ ID No: 26 and SEQ ID No: 35, respectively; the nucleotide sequence encoding the light chain variable region of the African swine fever virus monoclonal antibody 8A5, the nucleotide sequence of the heavy chain variable region are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

5. Use of the African swine fever virus monoclonal antibody of claim 1 in the preparation of an African swine fever virus antigen detection test strip based on a double antibody sandwich method.

6. An African swine fever virus red latex microsphere antigen detection test strip, characterized in that: The test strip comprises a polyvinyl chloride bottom plate at the bottom, and a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the polyvinyl chloride bottom plate in the flow direction; the sample pad is partially overlapped on the binding pad, and the binding pad and the water absorption pad are respectively overlapped on both sides of the nitrocellulose membrane; a detection line and a quality control line are arranged on the nitrocellulose membrane in the flow direction; wherein, The binding pad is sprayed with red latex microsphere labeled antibody, the detection line 4 is sprayed with coated antibody, and the quality control line is sprayed with coated commercial sheep anti-mouse IgG; The red latex microsphere labeled antibody is the monoclonal antibody 3C5 of claim 1; and the coated antibody is the monoclonal antibody 8A5 of claim 1.

7. The test strip of claim 6, wherein: In the red latex microsphere labeled antibody, the antibody labeling amount is 50 μg / mL; The concentration of the coated antibody is 0.3 mg / mL; and the concentration of the coated commercial sheep anti-mouse IgG is 0.5 mg / mL.

8. A method for preparing the African swine fever virus red latex microsphere antigen detection test strip according to claim 7, characterized in that: The method comprises the following steps: 1) Add red latex microspheres to MES buffer, sequentially mix with EDC and NHS, then centrifuge, resuspend with boric acid buffer for multiple times, and finally ultrasonic dispersion to obtain activated red latex microsphere suspension; 2) Label the monoclonal antibody 3C5 of claim 1 with the activated red latex microsphere suspension to obtain red latex microsphere labeled antibody; 3) Seal the sample pad and the binding pad with blocking solution respectively and store at 4°C; 4) Spray and fix the red latex microsphere labeled antibody on the binding pad; 5) Coat the monoclonal antibody 8A5 of claim 1 and the commercial sheep anti-mouse IgG respectively, and then spray them on the nitrocellulose membrane as the detection line and the quality control line respectively; 6) Paste the water absorption pad, the binding pad, the sample pad on the polyvinyl chloride base plate of the nitrocellulose membrane, and assemble the test strip.

9. The preparation method of claim 8, wherein: The activated red latex microsphere suspension is prepared by the following steps: a. Red latex microspheres with a solid content of 4% are added to MES buffer and vortexed to mix; wherein the concentration of the MES buffer is 0.03 mol / L, b. 10 mg / mL EDC solution and 10 mg / mL NHS solution are weighed according to the molar ratio of EDC, NHS and the surface of the red microspheres 1:1:1, then the NHS solution is vortexed to mix, and then the EDC solution is vortexed and shaken, and then mixed in a rotary mixer at room temperature, c. Centrifugation, discard the supernatant, resuspend with boric acid buffer, repeat centrifugation several times, after the last resuspension, ultrasonic dispersion, obtain the activated red latex microsphere suspension; wherein the concentration of the boric acid buffer is 0.02 mmol / L and its pH is 8.0; The red latex microsphere labeled antibody is prepared by the following steps: i. The monoclonal antibody 3C5 of claim 1 is added to the activated red latex microsphere suspension and mixed in a rotary mixer at room temperature; wherein the labeled amount of monoclonal antibody 3C5 is 50 μg / mL; ii. Then add BSA with a final concentration of 0.5%, mix in a rotary mixer at room temperature, centrifuge, discard the supernatant, resuspend with a preservation solution, and store at 4°C; wherein the preservation solution is 100 mL of water added with 1.21 g Tris, 5 g sucrose, 0.5 g BSA, 0.5 g PVP, 0.5 g F68, 0.5 mL PEG200, 0.5 mL TritonX-100, 1 mL Tween-20; The blocking solution of the binding pad is 100 mL of water added with 2 g sucrose, 0.3 g PVPK-30, 2 g BSA, 0.02 g NaN3, 0.29 g Na2HPO4·12H2O, 0.02 g KH2PO4, and its pH value is 7.6; The blocking solution of the sample pad uses 0.5 mol / L Tris-HCl buffer as the base solution, plus 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, 0.2% casein; The spraying amount of the red latex microsphere labeled antibody is 5 μL / cm; In step 5), the coating methods of the monoclonal antibody and the commercialized sheep anti-mouse IgG are as follows: The monoclonal antibody 8A5 and the commercialized sheep anti-mouse IgG are diluted to 0.3 mg / mL and 0.5 mg / mL respectively with the antibody coating solution to obtain the monoclonal antibody coating solution and the commercialized sheep anti-mouse IgG coating solution.

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