Porcine epidemic diarrhea virus monoclonal antibody and application thereof in preparation of antigen detection test strip

Abstract

The application discloses a porcine epidemic diarrhea virus monoclonal antibody and application thereof in preparation of an antigen detection test strip, and the detection test strip comprises a polyvinyl chloride bottom plate at the bottom, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad 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 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 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 disease detection technology, and particularly relates to a porcine epidemic diarrhea virus monoclonal antibody and application thereof in preparation of an antigen detection test strip. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease, mainly manifested as acute diarrhea, vomiting, dehydration, listlessness and weight loss in pigs, and is widely prevalent in the world with a high mortality rate. China has listed it as a class II animal infectious disease. In view of the pathogen of PEDV, countries have strengthened monitoring efforts in order to achieve rapid diagnosis and effective prevention and control, so as to curb the spread of the epidemic and reduce the losses of the breeding industry.

[0003] In order to meet the needs of large-scale breeding and high-standard biological safety prevention and control, it is urgent to establish a convenient and efficient on-site detection technology to significantly shorten the pathogen detection time, realize early identification and accurate prevention and control, so as to effectively reduce the risk of epidemic spread, improve the efficiency of epidemic prevention, and ensure the healthy development of the pig industry. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provides a porcine epidemic diarrhea virus monoclonal antibody and application thereof in preparation of an antigen detection test strip. The monoclonal antibody prepared by the present application is suitable for on-site diagnosis, and the test strip is simple to operate, rapid to detect, high in sensitivity and strong in specificity. The detection result is clear and easy to judge, and the whole detection process does not require any instrument or professional training of the operator.

[0005] To achieve the above-mentioned purpose, the technical scheme designed by the present application is as follows:

[0006] The present application provides a porcine epidemic diarrhea virus monoclonal antibody, which is monoclonal antibody 3B5 or monoclonal antibody 8G6,

[0007] The monoclonal antibody 3B5 comprises a light chain variable region and a heavy chain variable region; wherein,

[0008] The light chain variable region of 3B5 comprises three complementarity determining regions, namely 3B5-LCDR-1, 3B5-LCDR-2 and 3B5-LCDR-3, and their amino acid sequences are SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3 in turn;

[0009] The heavy chain variable region of 3B5 includes three complementarity determining regions, namely 3B5-HCDR-1, 3B5-HCDR-2 and 3B5-HCDR-3, whose amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, respectively;

[0010] The monoclonal antibody 8G6 includes a light chain variable region and a heavy chain variable region; wherein,

[0011] The light chain variable region of 8G6 includes three complementarity determining regions, namely 8G6-LCDR-1, 8G6-LCDR-2 and 8G6-LCDR-3, whose amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, respectively;

[0012] The heavy chain variable region of 8G6 includes three complementarity determining regions, namely 8G6-HCDR-1, 8G6-HCDR-2 and 8G6-HCDR-3, whose amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, respectively.

[0013] Further, the light chain variable region of the monoclonal antibody 3B5 includes four light chain framework regions, namely 3B5-LFR-1, 3B5-LFR-2, 3B5-LFR-3 and 3B5-LFR-4, whose amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, respectively;

[0014] The heavy chain variable region of the monoclonal antibody 3B5 includes four heavy chain framework regions, namely 3B5-HFR-1, 3B5-HFR-2, 3B5-HFR-3 and 3B5-HFR-4, whose amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, respectively;

[0015] The light chain variable region of the monoclonal antibody 8G6 includes four light chain framework regions, namely 8G6-LFR-1, 8G6-LFR-2, 8G6-LFR-3 and 8G6-LFR-4, whose amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, respectively;

[0016] The heavy chain variable region of the monoclonal antibody 8G6 contains four heavy chain framework regions, namely 8G6-HFR-1, 8G6-HFR-2, 8G6-HFR-3 and 8G6-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.

[0017] 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 3B5 are shown in SEQ ID No: 8 and SEQ ID No: 17, respectively; the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3B5 are shown in SEQ ID No: 9 and SEQ ID No: 18, 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 8G6 are shown in SEQ ID No: 26 and SEQ ID No: 35, respectively; the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8G6 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

[0019] The above-mentioned porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 are prepared by taking the N protein of the porcine epidemic diarrhea virus YN17 as an immunogen.

[0020] The above-mentioned monoclonal antibodies are secreted by hybridoma cell strains immunized by the N protein of the porcine epidemic diarrhea virus YN17.

[0021] The application also provides a use of the above-mentioned porcine epidemic diarrhea virus monoclonal antibody in the preparation of a porcine epidemic diarrhea virus antigen detection test strip based on a double antibody sandwich method.

[0022] The application also provides a porcine epidemic diarrhea virus red latex microsphere antigen detection test strip, which comprises the above-mentioned monoclonal antibody 3B5 or / and the above-mentioned monoclonal antibody 8G6.

[0023] Further, the test strip comprises a lowermost polyvinyl chloride bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the polyvinyl chloride bottom plate in a 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 test line (T line) and a quality control line (C line) are arranged on the nitrocellulose membrane in the flow direction; wherein,

[0024] The conjugated pad is sprayed with red latex microsphere marked antibody, the detection line is sprayed with coated antibody, and the quality control line is sprayed with coated commercial sheep anti-mouse IgG.

[0025] Further, the red latex microsphere marked antibody is monoclonal antibody 3B5 or monoclonal antibody 8G6, and the antibody marking amount in the red latex microsphere marked antibody is 50 μg / mL (the marking amount means that 50 μg of antibody is used in each 1 mL of latex microsphere reaction system in the process of preparing the marked antibody, for reaction, covalent binding or adsorption with the microsphere);

[0026] The coated antibody is monoclonal antibody 3B5 or monoclonal antibody 8G6, 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.

[0027] The purpose of the red latex microsphere marked antibody is to attach the antibody to the latex microsphere, so that it plays a role of recognizing the target antigen and displaying a color signal in the test strip.

[0028] The application also provides a preparation method of the above-mentioned porcine epidemic diarrhea virus red latex microsphere antigen test strip, comprising the following steps:

[0029] 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,

[0030] 2) marking the activated red latex microsphere suspension with the above-mentioned monoclonal antibody to obtain red latex microsphere marked antibody;

[0031] 3) respectively sealing the sample pad and the conjugated pad with blocking solution and storing at 4°C;

[0032] 4) spraying and fixing the red latex microsphere marked antibody on the conjugated pad;

[0033] 5) coating the above-mentioned monoclonal antibody and commercial sheep anti-mouse IgG respectively, and then spraying them on the nitrocellulose membrane as the detection line and the quality control line;

[0034] 6) pasting the water absorption pad, the conjugated pad and the sample pad on the polyvinyl chloride bottom plate of the nitrocellulose membrane to assemble the test strip.

[0035] Further, the activated red latex microsphere suspension is prepared by the following steps:

[0036] 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.

[0037] b. 10 mg / mL of EDC solution and 10 mg / mL of NHS solution are weighed according to the molar ratio of 1:1:1 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, then add the NHS solution and vortex uniformly, then add the EDC solution and vortex oscillation, and then mix in the rotary mixer at room temperature,

[0038] 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 7.0;

[0039] The above 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, EDC and NHS should be added according to the molar ratio of 1:1:1 according to the molar amount of these carboxyl groups; in this way, all carboxyl groups can be effectively activated to facilitate subsequent antibody coupling.

[0040] The red latex microsphere labeled antibody is prepared by the following steps:

[0041] i. The above monoclonal antibody is added to the activated red latex microsphere suspension, and mixed in the rotary mixer at room temperature; wherein the labeled amount of monoclonal antibody is 50 μg / mL;

[0042] ii. Then add BSA (bovine serum albumin) with a final concentration of 0.5%, block and mix in the rotary mixer at room temperature, centrifuge, discard the supernatant, add the preservative solution for resuspension, and store at 4°C; wherein the preservative solution is 100 mL of water added 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 TritonX-100 (Chinese name: TritonX-100, a non-ionic surfactant), 1 mL Tween-20.

[0043] The above F68: trade name: Pluronic F68

[0044] Alias: polyoxypropylene-polyoxyethylene block copolymer F68

[0045] Chemical name: Polyblock copolymer PEG-PPG-PEG (PEO-PPO-PEO)

[0046] Chinese name: F68, Pluronic F68, block copolymer F68

[0047] F68 action:

[0048] Stabilize antibody or protein structure, prevent non-specific adsorption;

[0049] Improve the dispersibility of latex microspheres or colloidal particles;

[0050] Prevent aggregation or precipitation during freeze-thaw process;

[0051] Enhance the wettability of the storage solution on the test strip;

[0052] Help maintain the stability and performance consistency of latex markers.

[0053] 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, with a pH value of 7.6.

[0054] 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%).

[0055] The spraying amount of the red latex microsphere labeled antibody is 5 μL / cm.

[0056] In step 5), the coating method of the monoclonal antibody and the commercialized sheep anti-mouse IgG is as follows:

[0057] The monoclonal antibody 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.

[0058] The spraying amount of the monoclonal antibody coating solution and the commercialized sheep anti-mouse IgG coating solution is 1 μL / cm.

[0059] The detection method of the above-mentioned red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus is to perform a double antibody sandwich reaction on the porcine epidemic diarrhea virus antigen in the sample (porcine fecal swab, with a sample amount of 80 μL) to be detected, so that it gathers and develops color at the T line.

[0060] Advantages of the present application:

[0061] The present application prepares monoclonal antibodies by prokaryotic expression of proteins, and screens two specific porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6, which have good effects in antigen detection test strip pairing screening, and the antibodies have high sensitivity, strong specificity, simple operation and good stability in the established red latex microsphere antigen detection test strip method, are suitable for on-site rapid detection, and have good application prospects in clinical diagnosis and epidemiological monitoring of viral diarrhea.

[0062] The present application adopts a double-antibody sandwich method for detection, has the advantages of fast detection speed, high sensitivity and strong specificity. The detection result is intuitive and clear, easy to judge; the whole 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

[0063] Figure 1 It is a protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-PEDV-N in the examples;

[0064] In the figure, A is the enzyme digestion identification diagram of the prokaryotic expression plasmid pET-28a-PEDV-N;

[0065] B is a protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-PEDV-N;

[0066] Figure 2 It is a single antibody IFA detection diagram of the hybridoma cell strain PEDV-MAb-3B5 secreted in the examples;

[0067] Figure 3 It is a WB detection diagram of the single antibody secreted by the hybridoma cell strain PEDV-MAb-8G6 in the examples;

[0068] Figure 4 It is a structural schematic diagram of the red latex microsphere test strip for detecting porcine epidemic diarrhea virus antigens;

[0069] 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;

[0070] Figure 5 It is a sensitivity detection result diagram of the red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus;

[0071] Figure 6 It is a specificity detection result diagram of the red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus.

[0072] Figure 7A schematic diagram of the display after detection of the red latex microsphere test strip for detecting porcine epidemic diarrhea virus antigen in Example 4,

[0073] wherein a is a positive result schematic diagram, b is a negative result schematic diagram, and c is an invalid result schematic diagram. DETAILED DESCRIPTION

[0074] The application will be further described in conjunction with specific examples so as to be understood by those skilled in the art. In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0075] Example 1 Preparation of hybridoma cell strains PEDV-MAb-3B5 and PEDV-MAb-8G6

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

[0077] Strain: Porcine epidemic diarrhea virus YN17 (PEDV-YN17, the genomic sequence of which is disclosed in the NCBI database, Genbank: MZ604716).

[0078] Cells: African green monkey kidney cells (Vero-ccl, i.e. Vero cells) were purchased from Shanghai Cell Bank.

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

[0080] Plasmid vector: prokaryotic expression vector pET-28a(+) was 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 of recombinant plasmid pET-28a-PEDV-N, protein expression and purification

[0083] According to the nucleotide sequence of the N gene sequence of the PEDV-YN17 strain as shown in SEQ ID NO: 39:

[0084]

[0085] The amino acid sequence of which is shown as SEQ ID NO: 40:

[0086] MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHADLRYRTRTEGVFWVAKEGAKTEPTNLGVRKASEKPIIPNFSQQLPSVVEIVEPNTPPTSRANSRSRSRGNGNNRSRSPSNNRGNNQSRGNSQNRGNNQGRGASQNRGGNNNNNNKSRNQSKNRNQSNDRGGVTSRDDLVAAVKDALKSLGIGENPDKLKQQQKPKQERSDSSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKPQRKKEKKNKSETTQQLNEEAIYDDVGVPSDVTHANLEWDTAVDGGDTAVEIINEIFDTGN;

[0087] A pair of primers were designed by taking the ORF of N gene as the target region using Oligo 6.0 software, and the primers were synthesized by Genescript. The sequences are as follows:

[0088] PEDV-N-F: 5'-GCGGATCCATGGCTTCTGTCAGTTTTCAGG-3' (SEQ ID No: 37);

[0089] PEDV-N-R: 5'-TGCTCGAGATTTCCTGTGTCGAAGATCTCG-3' (SEQ ID No: 38).

[0090] The N gene target fragment was obtained by RT-PCR amplification using the PEDV YN17 strain N gene as the template and the above primers. The prokaryotic expression vector pET-28a(+) was double-digested with BamHI and XhoI, and then gel-recovered. The double-digested prokaryotic expression vector pET-28a(+) and the N gene target fragment were connected 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-PEDV-N. The plasmid electrophoresis result is as follows:Figure 1 As shown in A, the results show that the pET-28a-PEDV-N plasmid is successfully constructed.

[0091] The plasmid pET-28a-PEDV-N is transformed into E. coli Rosetta (DE3) competent cells, and the protein expression is induced by IPTG, the supernatant is collected for purification, and the purified product is detected by SDS-PAGE. As shown in B, the detection results show that the size of the expressed N protein is about 60 kDa, which is consistent with the expected size. Figure 1

[0092] 3、Animal immunization

[0093] Healthy female BALB / c mice of four to six weeks old are selected, and are immunized by subcutaneous multi-point injection, with an immunization volume of 0.5 mL per point. According to the N protein concentration after purification and the final immunization dose, an appropriate amount of protein is emulsified with an equal volume of Freund's complete adjuvant, and then is injected subcutaneously for the first immunization. An appropriate amount of protein is mixed with an equal volume of Freund's incomplete adjuvant for emulsification, and then is used for the second immunization. Two weeks later, the tail is cut off for blood collection, and the serum is separated. The serum antibody titer is detected by indirect ELISA, and the mice with relatively high serum antibody levels are selected for subsequent hybridoma preparation experiments.

[0094] 4、Establishment of hybridoma cell strains PEDV-MAb-3B5 and PEDV-MAb-8G6

[0095] Mouse peritoneal macrophages are prepared as feeder cells according to a conventional method, and the spleen cells and myeloma cells (SP2 / 0) are fused at a ratio of 5:1 under the action of fusion agent PEG4000. The hybridoma cells secreting antibodies are screened by indirect ELISA, and are cloned by limited dilution method. Finally, two hybridoma cell strains PEDV-MAb-3B5 and PEDV-MAb-8G6 capable of stably secreting PEDV N protein monoclonal antibodies are obtained.

[0096] Example 2 Preparation of porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 from hybridoma cell strains PEDV-MAb-3B5 and PEDV-MAb-8G6

[0097] 1. A method for preparing porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 from hybridoma cell strains PEDV-MAb-3B5 and PEDV-MAb-8G6, comprising the following steps:

[0098] 0.5 mL of sterilized paraffin oil is injected into the abdominal cavity of the mouse, and 10 6 ​The hybridoma cells PEDV-MAb-3B5 and PEDV-MAb-8G6 were extracted from the abdominal fluid of the mouse when the abdominal fluid was extremely swollen after 7-10 days, and the abdominal fluid contained a large amount of monoclonal antibodies. After purification, the monoclonal antibodies were ready for use.

[0099] 2. Determination of the above-mentioned porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6:

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

[0101] Vero cells were inoculated in a 24-well plate, and when the cells grew to 80-90%, they were infected with PEDV YN17 for IFA detection.

[0102] The results are shown in Table 1. Figure 2 As shown in Table 1, the prepared monoclonal antibodies 2B2, 3B5, 4E6, 5G3 and 8G6 showed positive reactions with PEDV YN17, producing specific green fluorescence; the negative control group showed no fluorescence, indicating that the prepared MAb had strong specificity.

[0103] (2) Western blot (WB) of monoclonal antibodies

[0104] Vero cells were inoculated in a 10-cm cell culture dish, and when the cells grew to 80-90%, they were infected with PEDV YN17, and samples were collected at 12 h and 24 h for WB detection.

[0105] The results are shown in Table 2. Figure 3 As shown in Table 2, the prepared monoclonal antibodies 2B2, 3B5, 4E6, 5G3 and 8G6 showed positive reactions with PEDV YN17, producing specific bands at 60 kDa, and the negative control group showed no specific bands, indicating that the prepared murine monoclonal antibodies MAb had strong specificity.

[0106] a. The sequence of the monoclonal antibody 3B5 is as follows:

[0107] The three complementarity determining regions of the light chain variable region are 3B5-LCDR-1, 3B5-LCDR-2 and 3B5-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 3B5-HCDR-1, 3B5-HCDR-2 and 3B5-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 4 light chain framework regions of the light chain variable region are 3B5-LFR-1, 3B5-LFR-2, 3B5-LFR-3, and 3B5-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 4 heavy chain framework regions of the heavy chain variable region are 3B5-HFR-1, 3B5-HFR-2, 3B5-HFR-3, and 3B5-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 monoclonal antibody 3B5 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 sequences of the monoclonal antibody 3B5 are as follows:

[0112] 3B5-LCDR-1: QNIVDSNGNTY (SEQ ID No. 1);

[0113] 3B5-LCDR-2: KVSNRFS (SEQ ID No. 2);

[0114] 3B5-LCDR-3: FQGSHVPPT (SEQ ID No. 3);

[0115] 3B5-LFR-1: DVLMTQTPLSLPVSLGDQASISC (SEQ ID No. 4);

[0116] 3B5-LFR-2: WYLQKPGQSPKLLIY (SEQ ID No. 5);

[0117] 3B5-LFR-3: GVPDRFSGSGSGTDFTLQISRVEAEDLGVYYC (SEQ ID No. 6);

[0118] 3B5-LFR-4: FGGGTKLEIK (SEQ ID No. 7);

[0119] The amino acid sequence of the light chain variable region, wherein the underlined part represents the CDR sequence:

[0120] DVLMTQTPLSLPVSLGDQASISCRSS QNIVDSNGNTY LEWYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLQISRVEAEDLGVYYC FQGSHVPPT FGGGTKLEIK (SEQ ID No. 8);

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

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

[0123] 3B5-HCDR-1 : EYTIH (SEQ ID No. 10);

[0124] 3B5-HCDR-2: GINPDHGGTRFNHKFKG (SEQ ID No. 11);

[0125] 3B5-HCDR-3: EVFDY (SEQ ID No. 12);

[0126] 3B5-HFR-1 : EVQLQQSGPELVKPGTSVKISCKTSGYTFT (SEQ ID No. 13);

[0127] 3B5-HFR-2: WVKQSHGKSLEWIG (SEQ ID No. 14);

[0128] 3B5-HFR-3: KATLTVDKSSSTVYMDLRSLTSEDSAVYYCTR (SEQ ID No. 15);

[0129] 3B5-HFR-4: WGQGTSLTVSS (SEQ ID No. 16);

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

[0131] EVQLQQSGPELVKPGTSVKISCKTSGYTFT EYTIH WVKQSHGKSLEWIG GINPDHGGTRFNHKFKG KATLTVDKSSSTVYMDLRSLTSEDSAVYYCTR EVFDY WGQGTSLTVSS (SEQ ID No. 17);

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

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

[0134] b. The sequence of monoclonal antibody 8G6 is as follows:

[0135] The 3 complementarity determining regions of the light chain variable region are 8G6-LCDR-1, 8G6-LCDR-2, and 8G6-LCDR-3, whose 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 8G6-HCDR-1, 8G6-HCDR-2, and 8G6-HCDR-3, whose 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, 8G6-LFR-1, 8G6-LFR-2, 8G6-LFR-3, and 8G6-LFR-4, are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, and SEQ ID No: 25, respectively; the amino acid sequences of the heavy chain framework regions of the heavy chain variable region, 8G6-HFR-1, 8G6-HFR-2, 8G6-HFR-3, and 8G6-HFR-4, are 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 8G6 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively;

[0139] The specific sequences of the monoclonal antibody 8G6 are as follows:

[0140] 8G6-LCDR-1: RASQSISDYLH (SEQ ID No. 19);

[0141] 8G6-LCDR-2: YASQSIS (SEQ ID No. 20);

[0142] 8G6-LCDR-3: QNGHSFPALT (SEQ ID No. 21);

[0143] 8G6-LFR-1: DIVMTQSPATLSVTPGDRVFLSC (SEQ ID No. 22);

[0144] 8G6-LFR-2: WYQQKSHESPRLLIK (SEQ ID No. 23);

[0145] 8G6-LFR-3: GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID No. 24);

[0146] 8G6-LFR-4: FGAGTRLELK (SEQ ID No. 25);

[0147] The amino acid sequence of the light chain variable region, with the CDR sequences underlined:

[0148] DIVMTQSPATLSVTPGDRVFLSC RASQSISDYLH WYQQKSHESPRLLIK YASQSIS GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC QNGHSFPALT FGAGTRLELK (SEQ ID No. 26);

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

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

[0151] 8G6-HCDR-1: SYAMS (SEQ ID No. 28);

[0152] 8G6-HCDR-2: AISSGGTYTYYPDSVKG (SEQ ID No. 29);

[0153] 8G6-HCDR-3: QGDFGGDWYFDV (SEQ ID No. 30);

[0154] 8G6-HFR-1: EVQLVESGGGLVKPPGSLKLSCAASGFTFT (SEQ ID No. 31);

[0155] 8G6-HFR-2: WVRQTPEKRLEWVA (SEQ ID No. 32);

[0156] 8G6-HFR-3: RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR (SEQ ID No. 33);

[0157] 8G6-HFR-4: WGAGTTVTVSS (SEQ ID No. 34);

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

[0159] EVQLVESGGGLVKPPGSLKLSCAASGFTFT SYAMS WVRQTPEKRLEWVA AISSGGTYTYYPDSVKG RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR QGDFGGDWYFDV WGAGTTVTVSS (SEQ ID No. 35);

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

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

[0162] Example 3: Preparation of a red latex microsphere antigen test strip for porcine epidemic diarrhea virus using monoclonal antibodies 3B5 and 8G6 against porcine epidemic diarrhea virus

[0163] I. Cells, plasmids, sera, and major reagents:

[0164] Plasmid pET-28a-PEDV-N (constructed in Example 1), hybridoma cell strains secreting PEDV-N protein monoclonal antibodies 3B5 and 8G6 PEDV-MAb-3B5, PEDV-MAb-8G6 (prepared in Example 1). Porcine epidemic diarrhea virus (PEDV-YN17), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), African swine fever virus (ASFV), and porcine group A rotavirus (PoRVA) were provided by the Luo Rui research group of Huazhong Agricultural University. Pig diarrhea fecal swabs were collected from samples in Chaoyang City, Liaoning Province, Chenzhou City, Hunan Province, Jincheng City, Shanxi Province, and Shaoguan City, Guangdong Province.

[0165] II. Preparation of each material of the red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus

[0166] 1. Preparation of monoclonal antibody 3B5-red latex microsphere marker

[0167] In the EP tube, 975 μL of 0.03 mol / L MES buffer was added, and 25 μL of red latex microspheres with a solid content of 4% were added and vortexed. After vortexing, 5 μL of 10 mg / mL NHS solution was added and vortexed, and then 5 μL of the same concentration of EDC was added and vortexed. The mixture was vortexed at 20 r / min on a rotary mixer for 20 min at room temperature. Centrifugation was performed at 4°C and 13000 r / min for 10 min, the supernatant was discarded, and 1 mL of 0.02 mmol / L pH 7.5 borate buffer was added for resuspension. The resuspension was centrifuged twice and ultrasonically dispersed for 2 min. The monoclonal antibody 3B5 was diluted with ultrapure water to 0.2 mg / mL, and 100 μg was added to the activated microsphere suspension. The mixture was vortexed at 20 r / min on a rotary mixer for 2 h at room temperature. A final concentration of 0.5% BSA was added, and the mixture was blocked at 20 r / min on a rotary mixer for 1 h at room temperature. Centrifugation was performed at 4°C and 13000 r / min for 10 min, the supernatant was discarded, and 1 mL of the storage solution was added for resuspension. The mixture was stored at 4°C. Thus, the monoclonal antibody 3B5-red latex microsphere marker for porcine epidemic diarrhea virus was obtained.

[0168] The above-mentioned storage solution 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 (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.

[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, and the volume was made up to 100 mL with 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 (model Fusion4) was soaked in the binding pad blocking solution for 30 min and dried in a 37°C oven to obtain the blocked binding pad.

[0174] 3. Preparation of the sample pad

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

[0176] The sample pad blocking solution was prepared using 0.5 mol / L Tris-HCl buffer as the base solution, with the addition of 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, and 0.2% casein.

[0177] (2) Preparation of the sample pad

[0178] The glass cellulose membrane (model BX-03) was soaked in the sample pad blocking solution for 30 min and dried in a 37°C oven to obtain the blocked binding pad.

[0179] 4. Spraying of the monoclonal antibody 3B5-red latex microsphere marker of porcine epidemic diarrhea virus on the binding pad

[0180] The monoclonal antibody 3B5-red latex microsphere marker was sprayed on the treated binding pad 2, and the spraying amount of the monoclonal antibody 3B5-red latex microsphere marker was 5 μL / cm; after the spraying was completed, the sample was dried in a 37°C oven for 2 hours, and then 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] 1.0% (w / v) trehalose, 1.0% (w / v) sorbitol, and 0.3% (w / v) Tween-20 were added to 0.02 mol / L phosphate buffer solution (PBS) with a pH value of 7.2, and then stirred uniformly and filtered through a 0.22 μm filter membrane to obtain the antibody coating solution.

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

[0185] The non-spotting surface of the nitrocellulose membrane 3 was pasted on the polyvinyl chloride base plate 7, and the type of the nitrocellulose membrane 3 was Millipore Pall 90s; the monoclonal antibody 8G6 against porcine epidemic diarrhea virus was diluted to 0.3 mg / mL with the antibody coating solution, and the commercialized goat anti-mouse IgG was diluted to 0.5 mg / mL with the antibody coating solution, and then 1 μL / cm of the spraying amount was sprayed on the positions of the nitrocellulose membranes 4 and 5 as the detection line and the quality control line, respectively.

[0186] The distance between the detection line and the quality control line was 5 mm, and after the spraying was completed, the nitrocellulose membranes were dried in an oven at 37°C for 2 hours for standby.

[0187] III. Assembly of test strip

[0188] As shown in Figure 4 , the nitrocellulose membrane 3 prepared in step 4 above was pasted on the polyvinyl chloride base plate 7; the conjugated pad 2 on which the monoclonal antibody 3B5 against porcine epidemic diarrhea virus was sprayed and labeled with red latex microspheres prepared in step 3 was pasted above the nitrocellulose membrane 3, covering 1-2 mm of the nitrocellulose membrane 3; the conjugated pad 2 was adjacent to the detection line 4 on the nitrocellulose membrane 3; the water absorption pad 6 was pasted above the nitrocellulose membrane 3, covering 1-2 mm of the nitrocellulose membrane 3; the water absorption pad 6 was adjacent to the quality control line 5 on the nitrocellulose membrane 3; the sample pad 1 was pasted above the conjugated pad 2, covering 1-2 mm of the conjugated pad 2; after the colloidal gold test strip was cut into a 3 mm wide test strip with a strip cutter, it was put into a detection card shell together with a desiccant and was put into an aluminum foil bag, and was sealed and stored.

[0189] Sensitivity and specificity detection of the test strip

[0190] 1. Sensitivity detection of the test strip

[0191] The PEDV strain of 3.89 x 10 4 TCID 50 / mL was diluted by 10 times successively with the diluent, and 80 μL of the diluted solution was spotted for detection, and the results were observed within 10 min.

[0192] The results are shown in Figure 5 , and the lowest detection limit of the test strip was 3.89 x 10 1 TCID 50 / mL.

[0193] 2. Specificity detection of the test strip

[0194] The PEDV, PRV, PRRSV, TGEV, PDCoV, ASFV, PoRVA inactivated virus was diluted 20 times with the above diluent, 80 μL was taken for sample detection, and the results were observed within 10 min.

[0195] The results are shown in Figure 6 Table 1, the test strip has no cross-reaction, and the test strip has good specificity.

[0196] Example 4 Application of the above porcine epidemic diarrhea virus red latex microsphere antigen detection test strip

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

[0198] A small amount of pig feces was added to 1 mL of diluent to prepare a suspension, and after being mixed thoroughly, it was left to stand for 5 min, and the supernatant was reserved or centrifuged to reserve the supernatant.

[0199] The above diluent is: 0.3092 g of boric acid is weighed, dissolved in 400 mL of ddH2O, and then the pH value is adjusted to 8.0 with NaOH, and then the volume is made up to 500 mL, and then 2.5 mL of NP-40 is added.

[0200] 2. Detection

[0201] 80 μL of the supernatant was dropped on the above test strip, and the results were observed within 10 min; the determination of the detection results is based on:

[0202] As shown in Figure 7 : When red appears on the quality control line and the detection line, it is a positive result, i.e. the sample contains porcine epidemic diarrhea virus (a); when red appears on the quality control line and no red appears on the detection line, it is a negative result, i.e. the sample does not contain porcine epidemic diarrhea virus (b); Figure 7 Figure 7

[0203] If the quality control line does not appear red, and the detection line appears red, it means that the test strip is invalid (c). Figure 7

[0204] The other parts not specifically described are prior art. Although the above examples have 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 other embodiments can be obtained by people according to the present embodiments without creativity, which all belong to the protection scope of the present application.​​​

Claims

1. A porcine epidemic diarrhea virus monoclonal antibody, characterized in that: The epidemic diarrhea virus monoclonal antibody is monoclonal antibody 3B5 or monoclonal antibody 8G6, 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 3B5 are respectively shown as SEQ ID No: 8 and SEQ ID No: 17; the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3B5 are respectively shown as SEQ ID No: 9 and SEQ ID No: 18; 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 8G6 are respectively shown as SEQ ID No: 26 and SEQ ID No: 35; the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8G6 are respectively shown as SEQ ID No: 27 and SEQ ID No:

36.

2. Use of the porcine epidemic diarrhea virus monoclonal antibody of claim 1 in the preparation of a porcine epidemic diarrhea virus antigen detection test strip based on a double antibody sandwich method.

3. A porcine epidemic diarrhea virus red latex microsphere antigen detection test strip, characterized in that: The test strip comprises a lowermost polyvinyl chloride base plate, a sample pad, a binding pad, a nitrocellulose membrane 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 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 is sprayed with coated antibody, and the quality control line is sprayed with coated commercial sheep anti-mouse IgG; wherein the red latex microsphere-labeled antibody is the monoclonal antibody 3B5 of claim 1, and the coated antibody is the monoclonal antibody 8G6 of claim 1.

4. The test strip of claim 3, wherein: In the red latex microsphere-labeled antibody, the monoclonal antibody 3B5 is labeled at a concentration of 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.

5. A method for preparing the PRRSV red latex microsphere antigen detection test strip according to claim 3, 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 multiple times, and finally ultrasonic dispersion to obtain activated red latex microsphere suspension, 2) Label the activated red latex microsphere suspension with monoclonal antibody 3B5; obtain red latex microsphere-labeled antibody; 3) Seal the sample pad and the binding pad with blocking solution and store at 4°C; 4) Spray and fix the red latex microsphere-labeled antibody on the binding pad; 5) Coat the monoclonal antibody 8G6 and the commercial sheep anti-mouse IgG, 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, and the sample pad on the polyvinyl chloride base plate on the nitrocellulose membrane to assemble the test strip.

6. The preparation method of claim 5, wherein: The activated red latex microsphere suspension is prepared by the following steps: a. Add red latex microspheres with solid content of 4% into MES buffer and mix well by vortex; wherein the concentration of the MES buffer is 0.03 mol / L; b. According to the molar ratio of EDC, NHS and the surface of the red microspheres of 1:1:1, weigh 10 mg / mL of EDC solution and 10 mg / mL of NHS solution, then add the NHS solution and mix well by vortex, then add the EDC solution and mix well by vortex, and then mix well in a rotary mixer at room temperature, c. Centrifuge, discard the supernatant, resuspend with boric acid buffer, repeat the centrifugation for several times, after the last resuspension, ultrasonic dispersion, to obtain the activated red latex microsphere suspension; wherein the concentration of the boric acid buffer is 0.02 mmol / L and its pH is 7.0; The red latex microsphere labeled antibody is prepared by the following steps: i. Add monoclonal antibody 3B5 into the activated red latex microsphere suspension and mix well in a rotary mixer at room temperature; wherein the labeling amount of the monoclonal antibody is 50 μg / mL; ii. Then add BSA with a final concentration of 0.5% and mix well by blocking in a rotary mixer at room temperature, centrifuge, discard the supernatant, resuspend with a storage solution and store at 4°C; wherein the storage solution is 100 mL of water added with 1.21 g of Tris, 5 g of sucrose, 0.5 g of BSA, 0.5 g of PVP, 0.5 g of F68, 0.5 mL of PEG200, 0.5 mL of TritonX-100 and 1 mL of Tween-20; the blocking solution of the binding 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; 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 and 0.2% casein; The spraying amount of the red latex microsphere labeled antibody is 5 μL / cm; In the step 5), the coating methods of the monoclonal antibody and the commercialized sheep anti-mouse IgG are as follows: Dilute the monoclonal antibody 8G6 and the commercialized sheep anti-mouse IgG 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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