Monoclonal antibody of African swine fever virus and application thereof

Abstract

The invention discloses an African swine fever virus monoclonal antibody and application thereof. A detection test strip comprises a polyvinyl chloride bottom plate on the lowest layer, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially laid on the polyvinyl chloride bottom plate in the 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 two sides of the nitrocellulose membrane; a detection line and a quality control line are arranged on the nitrocellulose membrane along the flowing direction; an antibody marked by red latex microspheres is sprayed on the combination pad, a coated antibody is sprayed on the detection line, and coated commercial goat anti-mouse IgG is sprayed on the quality control line. The test strip adopts a double-antibody sandwich method for detection, and has the advantages of high detection speed, high sensitivity, strong specificity and the like. The detection result is visual, clear and easy to interpret; the whole detection process does not need any instrument and equipment, operators do not need professional training, operation is easy and convenient, and the method 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, cyanotic 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 report animal epidemic 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, and the whole 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: The present application provides an African swine fever virus monoclonal antibody, which 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, 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; 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; 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, 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; 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.

[0007] 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; 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; The light chain variable region of the monoclonal antibody 8A5 comprises four light chain framework regions, namely 8A5-LFR-1, 8A5-LFR-2, 8A5-LFR-3 and 8A5-LFR-4, whose 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, 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.

[0008] 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. 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.

[0009] 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.

[0010] The above-mentioned monoclonal antibody is secreted by a hybridoma cell strain immunized by the p30 recombinant protein of the African swine fever virus.

[0011] 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.

[0012] 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.

[0013] Further, the test strip comprises a lowermost polyvinyl chloride base plate, and a sample pad, a conjugate 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 conjugate pad, and the conjugate 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, The conjugate pad is sprayed with a red latex microsphere-labeled antibody, the detection line is sprayed with a coated antibody, and the quality control line is sprayed with a coated commercial sheep anti-mouse IgG.

[0014] Further, the red latex microsphere-labeled antibody is monoclonal antibody 3C5 or monoclonal antibody 8A5; and the antibody labeling amount in the red latex microsphere-labeled antibody is 50 μg / mL (the labeling amount means that 50 μg of the antibody is used in 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 antibody); The coated antibody is monoclonal antibody 3C5 or monoclonal antibody 8A5; and 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.

[0015] The purpose of the red latex microsphere-labeled antibody is to attach the antibody to the latex microspheres, so that it plays a role of recognizing the target antigen and displaying a color signal in the test strip.

[0016] The application further provides a preparation method of the above African swine fever virus red latex microsphere antigen test strip, comprising the following steps: 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 an activated red latex microsphere suspension, 2) labeling the above monoclonal antibody on the activated red latex microsphere suspension to obtain a red latex microsphere-labeled antibody; 3) respectively coating the sample pad and the conjugate pad with blocking solution and storing at 4°C; 4) spraying and fixing the red latex microsphere-labeled antibody on the conjugate pad; 5) coating the above monoclonal antibody and commercial sheep anti-mouse IgG respectively, and then spraying them on the nitrocellulose membrane as a detection line and a quality control line; 6) pasting the water absorption pad, the conjugate pad and the sample pad on a polyvinyl chloride bottom plate on the nitrocellulose membrane to assemble the test strip.

[0017] Further, the activated red latex microsphere suspension is prepared by the following steps: a. adding red latex microspheres with a solid content of 4% into MES buffer solution and vortexing uniformly; wherein the concentration of the MES buffer solution is 0.03 mol / L.

[0018] b. According to the 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 molar ratio of 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 added and vortexed, then the EDC solution is added and vortexed, and then the mixture is mixed in a rotary mixer at room temperature, 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 microspheres suspension; wherein the concentration of boric acid buffer is 0.02 mmol / L and its pH is 8.0; The above red latex microspheres have carboxyl groups (commonly carboxyl modified microspheres), which can be covalently connected to the microspheres through the amino groups of the antibodies by EDC / NHS chemical coupling. Therefore, when doing EDC / NHS activation reaction, EDC and NHS should be added according to the molar ratio of 1:1:1 based on the molar amount of these carboxyl groups; in this way, all carboxyl groups can be effectively activated to facilitate subsequent antibody coupling.

[0019] The red latex microsphere labeled antibody is prepared by the following steps: i. The above monoclonal antibody 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 is 50 μg / mL; ii. Then add BSA (bovine serum albumin) with a final concentration of 0.5%, mix in a rotary mixer at room temperature, centrifuge, discard the supernatant, resuspend with a preservative, and store at 4°C; wherein the preservative is 100 mL of water containing 1.21 g of Tris, 5 g of sucrose, 0.5 g of BSA (bovine serum albumin), 0.5 g of PVP (polyvinylpyrrolidone, English name: Polyvinylpyrrolidone), 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), 1 mL of Tween-20.

[0020] The above F68: trade name: Pluronic F68 Alias: polyoxypropylene-polyoxyethylene block copolymer F68 Chemical name: polyblock copolymer PEG-PPG-PEG (PEO-PPO-PEO) Chinese name: F68, Pluronic F68, block copolymer F68 F68 action: Stabilize antibody or protein structure, prevent non-specific adsorption; Improve the dispersibility of latex microspheres or colloidal particles; Prevent aggregation or precipitation during freeze-thaw process; Enhance the wettability of the storage solution on the test strip; Help maintain the stability and performance consistency of the latex marker.

[0021] The blocking solution of the conjugate pad is 100 mL of water added with 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, and the pH value is 7.6.

[0022] 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%).

[0023] The spraying amount of the red latex microsphere marker antibody is 5 μL / cm.

[0024] In the step 5), the coating method of the monoclonal antibody and the commercialized sheep anti-mouse IgG is as follows: The monoclonal antibody and the commercialized sheep anti-mouse IgG are respectively diluted to 0.3 mg / mL and 0.5 mg / mL with the antibody coating solution to obtain the monoclonal antibody coating solution and the commercialized sheep anti-mouse IgG coating solution.

[0025] The detection method of the above African swine fever virus red latex microsphere antigen test strip is to carry out double antibody sandwich reaction on the African swine fever virus antigen in the sample (pig fecal swab, the sample amount is 80 μL) to be detected, so that the antigen is gathered and colored at the T line.

[0026] The beneficial effects of the present application are: The present application prepares monoclonal antibodies by prokaryotic expression of proteins, and screens two specific African swine fever virus monoclonal antibodies 3C5 and 8A5, which have good effect in antigen test strip pairing screening. The antibody has high sensitivity, strong specificity, simple operation, good stability, and is suitable for on-site rapid detection, and has good application prospect in clinical diagnosis and epidemiological monitoring of viral diarrhea.

[0027] The African swine fever virus red latex microsphere antigen detection test paper strip of the application 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 easy to judge. The entire detection process does not require any instrument equipment, and the operator also does not need professional training, and the operation is simple, and is particularly suitable for on-site rapid diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a restriction enzyme identification, protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-ASFV-p30 in the embodiment of the application; Figure 2 It is a serum titer detection result diagram of mice immunized with the recombinant p30 protein. A diagram is the antibody titer of mouse serum two weeks after the second immunization, and B diagram is the antibody titer of mouse serum two weeks after the third immunization.

[0029] Figure 3 It is an IFA detection diagram of monoclonal antibodies secreted by the hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5 in the embodiment of the application; Figure 4 It is a WB detection diagram of monoclonal antibodies secreted by the hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5 in the embodiment of the application; Figure 5 It is a structural schematic diagram of a red latex microsphere test paper strip for detecting African swine fever virus antigens in the embodiment of the application; In the figure, the sample pad 1, the binding pad 2, the nitrocellulose membrane 3, the detection line 4, the quality control line 5, the water absorption pad 6, and the polyvinyl chloride bottom plate 7 are shown. Figure 6 It is a sensitivity detection result diagram of the African swine fever virus red latex microsphere antigen detection test paper strip in the embodiment of the application; Figure 7 It is a specificity detection result diagram of the African swine fever virus red latex microsphere antigen detection test paper strip in the embodiment of the application.

[0030] Figure 8 It is a display schematic diagram of the red latex microsphere test paper strip for detecting African swine fever virus antigens after detection in the embodiment of the application, Wherein, a is a positive result schematic diagram, b is a negative result schematic diagram, and c is an invalid result schematic diagram. In the figure, C represents the quality control area, and T represents the detection area. DETAILED DESCRIPTION

[0031] The application will be further described in detail below with reference to specific embodiments so as to be understood by those skilled in the art.

[0032] Example 1 Preparation of hybridoma cell strains ASFV-MAb-3C5 and ASFV-MAb-8A5 1. Strains, cells, serum and experimental animals Strains: Inactivated African swine fever virus provided by Professor Luo Rui's research group of Huazhong Agricultural University.

[0033] Cells: Human embryonic kidney cells 293 (HEK293) were purchased from Shanghai Cell Bank.

[0034] Strains: Escherichia coli DH5a used for plasmid construction was purchased from Biyun Tian Biological.

[0035] Plasmid vectors: Prokaryotic expression vector pET-28a(+) and eukaryotic expression vector pcDNA3.1(+) were purchased from Addgene platform.

[0036] Experimental animals: 5-6 week old BALB / c female experimental mice were purchased from Hubei Experimental Animal Research Center.

[0037] 2. Construction of recombinant plasmid pET-28a-ASFV-p30, protein expression and purification According to the p30 gene sequence of ASFV as shown in SEQ ID No: 39: ATGGATTTTATTTTAAATATATCCATGAAAATGGAGGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCTTGTATAATTGGTTTTCTGTTGAGATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTCAGTACTGTTAAGTATGATATTGTGAAATCTGCTCATATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAGGAGACAGAATCCTCAGCATCATCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATAAATGCGCATCCTCCTTTGAAACATTGTTTGAGCAAGAGCCCTCATCAGAGGAACCTAAAGACTCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAACTTTATTCAAACCATTCATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTTATTAAACTTTTAAAAAAAAAATAA The encoded amino acid sequence is shown as SEQ ID No: 40: MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSAHIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNKCASSFETLFEQEPSSEEPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKK.

[0038] A pair of primers were designed by Oligo 6.0 software, with the ORF of ASFV gene as the target region, and the primers were synthesized by Genescript. The sequences are as follows: ASFV-p30-F: 5'-CAGCCATATGGATTTTATTTTAAATATATCCATGAAAATG-3' (SEQ ID No: 37); ASFV-p30-R: 5'-TGCTCGAGAATGTAGGTGAGATAAAAGCTTA-3' (SEQ ID No: 38).

[0039] The p30 gene of ASFV was used as a template for RT-PCR amplification of the p30 gene target fragment using the above primers. The prokaryotic expression vector pET-28a(+) was digested with NdeI and XhoI, and the gel was recovered. 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 that was positive in the enzyme digestion was selected and sent to Wuhan Qikexi Biological Company for sequencing verification. The recombinant plasmid that was correctly identified 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.

[0040] 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 recombinant p30 protein was about 35 kDa in size, which was consistent with the expected size.

[0041] 3. Animal immunization Healthy female BALB / c mice aged four to six weeks were selected for immunization by subcutaneous multi-point injection, with an immunization 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 injected subcutaneously for the first immunization. An appropriate amount of protein was mixed with an equal volume of Freund's incomplete adjuvant for emulsification, and then used for the second immunization. Two weeks later, the tail blood was collected and the 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.

[0042] 4. Establishment of hybridoma cell lines ASFV-MAb-3C5 and ASFV-MAb-8A5 Mouse peritoneal macrophages were prepared as feeder cells by routine method, and spleen cells were fused with myeloma cells (SP2 / 0) at a ratio of 5:1 in the presence of PEG4000, and the hybridoma cells secreting positive antibodies were screened by indirect ELISA, and cloned by limiting dilution method. Finally, two hybridoma cell lines ASFV-MAb-3C5 and ASFV-MAb-8A5 capable of stably secreting ASFV p30 protein monoclonal antibodies were obtained.

[0043] Example 2 Preparation of African swine fever virus monoclonal antibodies 3C5 and 8A5 from hybridoma cell lines ASFV-MAb-3C5 and ASFV-MAb-8A5 1. A method for preparing African swine fever virus monoclonal antibodies 3C5 and 8A5 from hybridoma cell lines ASFV-MAb-3C5 and ASFV-MAb-8A5, comprising the following steps: 0.5 mL of sterilized paraffin oil was injected into the abdominal cavity of the mouse, and 10 6 hybridoma cells ASFV-MAb-3C5 and ASFV-MAb-8A5 were injected into the abdominal cavity of the mouse one week later, respectively. 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.

[0044] 2. Determination of the above African swine fever virus monoclonal antibodies 3C5 and 8A5: (1) Indirect immunofluorescence (IFA) of monoclonal antibodies 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.

[0045] The results are shown in Figure 3 Table 1, and 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.

[0046] (2) Western Blot of monoclonal antibody protein HEK293 cells were inoculated in a 10 cm cell culture dish, and when the cells grew to 80-90%, pcDNA3.1(+) empty vector and pcDNA3.1-ASFV-p30 plasmid were transfected, respectively, and the sample was collected 12 h later for Western Blot detection.

[0047] The results are shown in Figure 4As shown, the prepared monoclonal antibodies 3C5 and 8A5 do not specifically bind to the target protein, and no specific band is detected, indicating that the antigen epitopes recognized by the two prepared MAbs are spatial epitopes, not linear epitopes.

[0048] a. The sequence of monoclonal antibody 3C5 is as follows: The three complementarity determining regions of the light chain variable region are 3C5-LCDR-1, 3C5-LCDR-2 and 3C5-LCDR-3, respectively, and their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2, 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, respectively, and their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, respectively; 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, respectively, and their amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, 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, respectively, and their amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, respectively; 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; 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; The specific sequence of the above-mentioned monoclonal antibody 3C5 is as follows: 3C5-LCDR-1: RSSQTIVHSNGITYLE (SEQ ID No. 1); 3C5-LCDR-2: KVSNRFS (SEQ ID No. 2); 3C5-LCDR-3: FQGSHVPWT (SEQ ID No. 3); 3C5-LFR-1: DVLMTQTPFSLPVSLGDQASISC (SEQ ID No. 4); 3C5-LFR-2: WYMQKPGQSPKLLIY (SEQ ID No. 5); 3C5-LFR-3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID No. 6); 3C5-LFR-4: FGGGTKLEIK (SEQ ID No. 7); Amino acid sequence of the light chain variable region, with the CDR sequences underlined: DVLMTQTPFSLPVSLGDQASISC RSSQTIVHSNGITYLE WYMQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPWT FGGGTKLEIK (SEQ ID No. 8); Nucleic acid sequence of the light chain variable region: GATGTTTTGATGACCCAAACTCCATTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAATCACCTATTTAGAGTGGTACATGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 9); 3C5-HCDR-1: SYAMS (SEQ ID No. 10); 3C5-HCDR-2: TISRFSTYTYYPDSVKG (SEQ ID No. 11); 3C5-HCDR-3: QGYYSGEGDYFDY (SEQ ID No. 12); 3C5-HFR-1: EVMLVESGGGLVKPGGSLKVSCAASGFTFS (SEQ ID No. 13); 3C5-HFR-2: WVRQTPEKRLEWVA (SEQ ID No. 14); 3C5-HFR-3: RFTISRDNAKNTLSLQMSSLRSEDTAIYYCIR (SEQ ID No. 15); 3C5-HFR-4: WGQGTTLTVSS (SEQ ID No. 16); Amino acid sequence of the heavy chain variable region, wherein underlined sequences represent CDR sequences: EVMLVESGGGLVKPGGSLKVSCAASGFTFS SYAMS WVRQTPEKRLEWVA TISRFSTYTYYPDSVKG RFTISRDNAKNTLSLQMSSLRSEDTAIYYCIR QGYYSGEGDYFDY WGQGTTLTVSS (SEQ ID No. 17); Nucleic acid sequence of the heavy chain variable region: GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAAGTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGTCAGACTCCGGAGAAGAGACTGGAGTGGGTCGCAACCATTAGTCGTTTTAGTACTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTCCCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATTTATTACTGTATAAGACAGGGTTATTACTCCGGGGAGGGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No. 18).

[0049] b. The sequence of monoclonal antibody 8A5 is as follows: The 3 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 3 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; 4 light chain framework regions of the light chain variable region, 8A5-LFR-1, 8A5-LFR-2, 8A5-LFR-3, 8A5-LFR-4, respectively, the amino acid sequences of which are shown in SEQ ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, respectively; 4 heavy chain framework regions of the heavy chain variable region, 8A5-HFR-1, 8A5-HFR-2, 8A5-HFR-3, 8A5-HFR-4, respectively, the amino acid sequences of which are shown in SEQ ID No:31, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, respectively; the amino acid sequence of the light chain variable region, the amino acid sequence of the heavy chain variable region are shown in SEQ ID No:26 and SEQ ID No:35, respectively; the nucleic acid sequence encoding the light chain variable region of the monoclonal antibody 8A5, the nucleic acid sequence of the heavy chain variable region are shown in SEQ ID No:27 and SEQ ID No:36, respectively; The specific sequences of the above-mentioned monoclonal antibody 8A5 are as follows: 8A5-LCDR-1: RASESVDTYGNSFMH (SEQ ID No. 19); 8A5-LCDR-2: LASNLES (SEQ ID No. 20); 8A5-LCDR-3: QQNNEDPYT (SEQ ID No. 21); 8A5-LFR-1: NIVLTQSPASLAVSLGQRATISC (SEQ ID No. 22); 8A5-LFR-2: WYQQKPGQPPKLLIY (SEQ ID No. 23); 8A5-LFR-3: GVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID No. 24); 8A5-LFR-4: FGGGTKLEIK (SEQ ID No. 25); The amino acid sequence of the light chain variable region, wherein the underlined part represents the CDR sequence: NIVLTQSPASLAVSLGQRATISC RASESVDTYGNSFMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSRTDFTLTIDPVEADDAATYYC QQNNEDPYT FGGGTKLEIK (SEQ ID No. 26); Nucleic acid sequence of the heavy chain variable region: AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGCGTTGATACTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC (SEQ ID No. 27); 8A5-HCDR-1: SYWIH (SEQ ID No. 28); 8A5-HCDR-2: RIYPGTGISFYNEKFKG (SEQ ID No. 29); 8A5-HCDR-3: RIFDDYPNWYFDV (SEQ ID No. 30); 8A5-HFR-1: QVQLKQSGAELVRPGASVKLSCQTSGYIFT (SEQ ID No. 31); 8A5-HFR-2: WVKQRSGQGLEWIA (SEQ ID No. 32); 8A5-HFR-3: KATLTADKSSSTAYMQLSSLKSEDSAVYFCAR (SEQ ID No. 33); 8A5-HFR-4: WGAGTTVTVSS (SEQ ID No. 34); Amino acid sequence of the heavy chain variable region, wherein underlined sequences represent CDR sequences: QVQLKQSGAELVRPGASVKLSCQTSGYIFT SYWIH WVKQRSGQGLEWIA RIYPGTGISFYNEKFKG KATLTADKSSSTAYMQLSSLKSEDSAVYFCAR RIFDDYPNWYFDV WGAGTTVTVSS (SEQ ID No. 35); Nucleic acid sequence of the heavy chain variable region: CAGGTCCAGCTGAAGCAGTCTGGAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCCAGACTTCTGGATACATCTTCACCAGCTACTGGATTCACTGGGTTAAACAGAGGTCTGGACAGGGCCTTGAGTGGATTGCAAGGATTTATCCTGGAACTGGTATTAGTTTCTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGAAATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGACGGATCTTTGATGATTACCCTAACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG (SEQ ID No. 36).

[0050] Example 3 Preparation of ASFV red latex microsphere antigen test strip using ASFV monoclonal antibodies 3C5 and 8A5 I. Cells, plasmids, serum and main reagents: 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.

[0051] II. Preparation of ASFV red latex microsphere antigen test strip 1. Preparation of monoclonal antibody 3C5-red latex microsphere marker In the 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; after vortexing with 5 μL of 10 mg / mL NHS solution, 5 μL of EDC with the same concentration was vortexed, and then vortexed on a rotary mixer at 20 r / min for 20 min at room temperature; centrifuged 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 vortexed on a rotary mixer at 20 r / min for 2 h at room temperature; 0.5% BSA was added, and closed at room temperature on a rotary mixer at 20 r / min for 1 h; centrifuged 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 stored at 4°C. Thus, the monoclonal antibody 3C5-red latex microsphere marker of African swine fever virus is obtained.

[0052] 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.

[0053] 2. Preparation of the binding pad (1) Preparation of the binding pad blocking solution 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, dissolved with ddH2O, the pH was adjusted to 7.6, the volume bottle was made up to 100 mL, and then filtered with a 0.22 μm filter membrane to obtain the binding pad blocking solution.

[0054] (2) Preparation of the binding pad The glass cellulose membrane (model Fusion4) was soaked in the binding pad blocking solution for 30 min and dried in a 37°C oven to obtain the binding pad.

[0055] 3. Preparation of the sample pad (1) Preparation of the sample pad blocking solution The sample pad sealing liquid uses 0.5 mol / L Tris-HCl buffer as a base liquid, and additionally contains 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, and 0.2% casein.

[0056] (2) Preparation of the sample pad The glass cellulose membrane (type GL-B04) is soaked in the sample pad sealing liquid for 30 min and dried in a 37°C oven, thereby obtaining the sealed binding pad.

[0057] 4. Spraying of monoclonal antibody 3C5-red latex microsphere marker of African swine fever virus on the binding pad The monoclonal antibody 3C5-red latex microsphere marker is sprayed on the treated binding 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 pad is dried in a 37°C oven for 2 hours, and is cut into a 3 mm wide strip for standby use.

[0058] 5. Spraying of the detection line and the quality control line (1) Preparation of the antibody coating liquid The antibody coating liquid is prepared by adding 1.0% (w / v) trehalose, 1.0% (w / v) sorbitol, and 0.3% (w / v) Tween-20 to 0.02 mol / L phosphate buffer solution (PBS) with a pH value of 7.2, uniformly stirring, and filtering through a 0.22 μm filter membrane.

[0059] (2) Preparation of the detection line and the quality control line The nitrocellulose membrane 3 is non-point spotted and pasted on the polyvinyl chloride bottom plate 7, and the type of the nitrocellulose membrane 3 is Millipore Pall 90s; the monoclonal antibody 8A5 of African swine fever virus is diluted to 0.3 mg / mL with the antibody coating liquid, and the commercial sheep anti-mouse IgG is diluted to 0.5 mg / mL with the antibody coating liquid, and then is sprayed on the positions of the nitrocellulose membranes 4 and 5 at a spraying amount of 1 μL / cm, respectively, as the detection line and the quality control line.

[0060] The distance between the detection line and the quality control line is 5 mm, and after the spraying is completed, the sample pad is dried in a 37°C oven for 2 hours for standby use.

[0061] III. Assembly of the test strip As Figure 5As shown, the nitrocellulose membrane 3 prepared in the above step 4 is pasted on the polyvinyl chloride base plate 7; the conjugate pad 2 sprayed with the African swine fever virus monoclonal antibody 3C5-red latex microsphere marker prepared in step 3 is pasted on the top of the nitrocellulose membrane 3, covering 1-2 mm on the nitrocellulose membrane 3; the conjugate pad 2 is adjacent to the test line 4 on the nitrocellulose membrane 3; the absorbent pad 6 is pasted on the top of the nitrocellulose membrane 3, covering 1-2 mm on the nitrocellulose membrane 3; the absorbent pad 6 is adjacent to the quality control line 5 on the nitrocellulose membrane 3; the sample pad 1 is pasted on the top of the conjugate pad 2, covering 1-2 mm on the conjugate pad 2; the colloidal gold test strip is cut into 3 mm wide test strips using a strip cutter, and then placed in a test cartridge and a desiccant in an aluminum foil bag for sealed storage.

[0062] Detection of the sensitivity and specificity of the above test strips 1. Test strip sensitivity test Put 10 6.5 TCID 50 After the inactivated ASFV strain with a concentration of 10 / mL was diluted 10-fold in a series with the above diluent, 80 μL was aspirated for spot detection and the results were observed within 10 minutes.

[0063] The results are as follows Figure 6 As shown, the minimum detection limit of the test strip is 10 3.5 TCID 50 / mL.

[0064] 2. Specificity detection of test strips Dilute the inactivated ASFV, PEDV, and PCV-2 viruses 20 times with the above diluent, then aspirate 80 μL for spot detection and observe the results within 10 minutes.

[0065] The results are as follows Figure 7 As shown, there is no cross reaction of the test strips and the test strips have good specificity.

[0066] Example 4 Application of the above-mentioned African swine fever virus red latex microsphere antigen detection test strip 1. Pretreatment of the sample to be tested Take a small amount of pig feces and add it to 1 mL of diluent to make a suspension. Mix thoroughly and let it stand for 5 minutes. Keep the supernatant or centrifuge and keep the supernatant.

[0067] The above dilution solution is as follows: weigh 0.3092 g of boric acid, dissolve it completely in 400 mL of ddH2O, adjust the pH to 8.0 with NaOH, make the volume to 500 mL, and then add 2.5 mL of NP-40.

[0068] 2. Detection Drop 80 μL of the supernatant on the test strip as described above, and observe the result within 10 min; the determination of the detection result is based on: As shown in Figure 8 : when red color appears on both the quality control line 4 and the detection line 5, it is a positive result, i.e. the sample contains African swine fever virus (a); Figure 8 when red color appears on the quality control line 5 but not on the detection line 4, it is a negative result, i.e. the sample does not contain African swine fever virus (b), Figure 8 when no red color appears on the quality control line 5, the test strip is invalid (c). Figure 8

[0069] The other parts not described in detail are prior art. Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, which all belong to the protection scope of the present application.​​​

Claims

1. An African swine fever virus monoclonal antibody, characterized by: 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 includes three complementarity determining regions, 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 includes 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; The monoclonal antibody 8A5 comprises a light chain variable region and a heavy chain variable region; wherein, The light chain variable region of 8A5 includes three complementarity determining regions, namely 8A5-LCDR-1, 8A5-LCDR-2, and 8A5-LCDR-3; their amino acid sequences are SEQ ID No: 19, SEQ ID No: 20, and SEQ ID No: 21, respectively; The heavy chain variable region of 8A5 includes three complementarity determining regions, namely 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 and 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 contains four light chain framework regions, namely 3C5-LFR-1, 3C5-LFR-2, 3C5-LFR-3 and 3C5-LFR-4; their amino acid sequences are 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 contains four heavy chain framework regions, namely 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 contains four light chain framework regions, namely 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 and 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 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. The nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region encoding the African swine fever virus monoclonal antibody 3C5 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 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; the nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region encoding the African swine fever virus monoclonal antibody 8A5 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

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

6. A red latex microsphere antigen detection test strip for African swine fever virus, characterized by: Including the African swine fever virus monoclonal antibody according to claim 1.

7. The test strip according to claim 6, wherein: The test strip comprises a bottom polyvinyl chloride base plate, on which a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad are sequentially laid along the flow direction; the sample pad is partially overlapped on the conjugation pad, and the conjugation pad and the water-absorbing 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 along the flow direction; wherein, The conjugate pad is sprayed with antibodies labeled with red latex microspheres, the detection line 4 is sprayed with coated antibodies, and the quality control line is sprayed with coated commercial goat anti-mouse IgG.

8. The test strip according to claim 7, wherein: The antibody labeled with the red latex microspheres is monoclonal antibody 3C5 or monoclonal antibody 8A5; and the antibody labeled with the red latex microspheres has an antibody labeling amount of 50 μg / mL; The coated antibody is monoclonal antibody 3C5 or monoclonal antibody 8A5; and the concentration of the coated antibody is 0.3 mg / mL; the concentration of the coated commercial goat anti-mouse IgG is 0.5 mg / mL.

9. A method for preparing the African swine fever virus red latex microsphere antigen detection test strip according to claim 7, characterized in that: The following steps are involved: 1) Add red latex microspheres to MES buffer, mix with EDC and NHS in sequence, then centrifuge, resuspend in boric acid buffer several times, and finally disperse by ultrasonication to obtain an activated red latex microsphere suspension; 2) labeling the activated red latex microsphere suspension with the monoclonal antibody according to claim 1 to obtain an antibody labeled with red latex microspheres; 3) Separately block the sample pad and conjugate pad with blocking solution and store at 4°C. 4) Spray the red latex microsphere-labeled antibody onto the conjugate pad; 5) The monoclonal antibody of claim 1 and commercial goat anti-mouse IgG are coated separately and then sprayed onto nitrocellulose membranes as test lines and quality control lines respectively; 6) Attach the absorbent pad, conjugate pad, and sample pad to the PVC base on the nitrocellulose membrane to assemble the test strip.

10. The preparation method according to claim 9, characterized in that: The activated red latex microsphere suspension is prepared by the following steps: a. Add red latex microspheres with a solid content of 4% to MES buffer and vortex mix; wherein the concentration of MES buffer is 0.03 mol / L, b. According to the molar ratio of EDC, NHS and red microsphere surface of 1:1:1, weigh 10 mg / mL EDC solution and 10 mg / mL NHS solution, then add NHS solution and vortex mix, then add EDC solution and vortex shake, and then mix in a rotary mixer at room temperature. c. Centrifuge, discard the supernatant, resuspend in boric acid buffer, repeat centrifugation several times, and after the final resuspension, ultrasonically disperse to obtain an activated red latex microsphere suspension; wherein the boric acid buffer concentration 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 according to 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 the monoclonal antibody is 50 μg / mL; ii. Then, add BSA to a final concentration of 0.5%, block and mix on a rotary mixer at room temperature, centrifuge, discard the supernatant, resuspend in preservation solution, and store at 4°C; the preservation solution consists of 1.21g Tris, 5g sucrose, 0.5g BSA, 0.5g PVP, 0.5g F68, 0.5mL PEG200, 0.5mL TritonX-100, and 1mL Tween-20 in 100mL of water; The blocking solution of the conjugate pad is 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 added to 100 mL of water, with a pH value of 7.6; The blocking solution of the sample pad used 0.5 mol / L Tris-HCl buffer as the base solution, plus 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, and 0.2% casein; The spraying volume of the red latex microsphere-labeled antibody was 5 μL / cm; In step 5), the method for coating with monoclonal antibodies and commercial goat anti-mouse IgG is as follows: The monoclonal antibody and commercial goat anti-mouse IgG were diluted to 0.3 mg / mL and 0.5 mg / mL respectively with antibody coating solution to obtain monoclonal antibody coating solution and commercial goat anti-mouse IgG coating solution.

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