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

Abstract

The invention discloses a porcine epidemic diarrhea virus monoclonal antibody and application thereof in preparation of an antigen detection test strip. The detection test strip comprises a polyvinyl chloride bottom plate at 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 method 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 invention relates to the fields of cell biotechnology, immunology technology and animal disease detection technology, and in particular to a porcine epidemic diarrhea virus monoclonal antibody and an application thereof in preparing an antigen detection test strip. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease of pigs characterized by acute diarrhea, vomiting, dehydration, lethargy, and weight loss. It is widespread worldwide and carries a high mortality rate. my country has classified it as a Category II zoonotic disease. To address its causative agent, porcine epidemic diarrhea virus (PEDV), countries are strengthening surveillance efforts to achieve rapid diagnosis and effective prevention and control, thereby curbing the spread of the disease and minimizing losses to the livestock industry.

[0003] In order to meet the needs of large-scale farming and high-standard biosafety control, it is urgent to establish convenient and efficient on-site detection technology to significantly shorten the time of pathogen detection, achieve early identification and precise prevention and control, thereby effectively reducing the risk of epidemic transmission, improving epidemic prevention efficiency, and ensuring the healthy development of the pig industry. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provides a porcine epidemic diarrhea virus monoclonal antibody and its use in preparing an antigen detection test strip. The test strip prepared with the monoclonal antibody of the present invention is suitable for on-site diagnosis. The test strip is simple to use, has rapid detection, high sensitivity and strong specificity, and the test results are clear and easy to judge. The entire detection process does not require any instruments and the operator does not require any professional training.

[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows: The present invention provides a porcine epidemic diarrhea virus monoclonal antibody, wherein the porcine epidemic diarrhea virus monoclonal antibody is monoclonal antibody 3B5 or monoclonal antibody 8G6. The monoclonal antibody 3B5 comprises a light chain variable region and a heavy chain variable region; wherein, The light chain variable region of 3B5 includes three complementarity determining regions, namely 3B5-LCDR-1, 3B5-LCDR-2, and 3B5-LCDR-3, whose amino acid sequences are SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively; 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, and SEQ ID No: 12, respectively; The monoclonal antibody 8G6 comprises a light chain variable region and a heavy chain variable region; wherein, 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 SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21, respectively; The heavy chain variable region of 8G6 includes three complementarity determining regions, namely 8G6-HCDR-1, 8G6-HCDR-2 and 8G6-HCDR-3, and their amino acid sequences are SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30, respectively.

[0006] Furthermore, the light chain variable region of the monoclonal antibody 3B5 contains four light chain framework regions, namely 3B5-LFR-1, 3B5-LFR-2, 3B5-LFR-3 and 3B5-LFR-4, whose 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 3B5 contains four heavy chain framework regions, namely 3B5-HFR-1, 3B5-HFR-2, 3B5-HFR-3, and 3B5-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 8G6 contains four light chain framework regions, namely 8G6-LFR-1, 8G6-LFR-2, 8G6-LFR-3, and 8G6-LFR-4; their amino acid sequences are 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 8G6 contains four heavy chain framework regions, namely 8G6-HFR-1, 8G6-HFR-2, 8G6-HFR-3 and 8G6-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.

[0007] Furthermore, 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 cloned antibody 3B5 are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively.

[0008] Furthermore, 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.

[0009] The above-mentioned porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 are both prepared by using the N protein of porcine epidemic diarrhea virus YN17 as an immunogen.

[0010] The above monoclonal antibody is secreted by a hybridoma cell line immunized with the N protein of porcine epidemic diarrhea virus YN17.

[0011] The present invention also provides a use of the porcine epidemic diarrhea virus monoclonal antibody in preparing a porcine epidemic diarrhea virus antigen detection test strip based on a double antibody sandwich method.

[0012] The present invention also provides a porcine epidemic diarrhea virus red latex microsphere antigen detection test strip, comprising the above-mentioned monoclonal antibody 3B5 and / or the above-mentioned monoclonal antibody 8G6.

[0013] Furthermore, the test strip includes 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 test line (T line) and a quality control line (C line) are provided on the nitrocellulose membrane along the flow direction; wherein, The conjugate pad is sprayed with red latex microsphere-labeled antibodies, the detection line is sprayed with coated antibodies, and the quality control line is sprayed with coated commercial goat anti-mouse IgG.

[0014] Furthermore, the antibody labeled with the red latex microspheres is monoclonal antibody 3B5 or monoclonal antibody 8G6; and the antibody labeled with the red latex microspheres has a labeled amount of 50 μg / mL (the labeled amount means that in the process of preparing the labeled antibody, 50 μg of the antibody is added to each 1 mL of the latex microsphere reaction system for reaction, covalent binding, or adsorption with the microspheres); The coated antibody is monoclonal antibody 3B5 or monoclonal antibody 8G6; 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.

[0015] The purpose of labeling antibodies with red latex microspheres is to allow the antibodies to attach to the latex microspheres, so that they can recognize the target antigen and display color signals in the test strip.

[0016] The present invention also provides a method for preparing the porcine epidemic diarrhea virus red latex microsphere antigen detection test strip, comprising the following steps: 1) Add red latex microspheres to MES buffer, mix with EDC and NHS in sequence, then centrifuge, resuspend with boric acid buffer several times, and finally disperse by ultrasonication to obtain activated red latex microsphere suspension. 2) labeling the activated red latex microsphere suspension with the monoclonal antibody to obtain a red latex microsphere-labeled antibody; 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 above-mentioned monoclonal antibodies and commercial goat anti-mouse IgG were 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.

[0017] Furthermore, 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; the concentration of MES buffer is 0.03 mol / L.

[0018] b. According to the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) to the 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 on a rotary mixer at room temperature. c. Centrifuge, discard the supernatant, add borate buffer and resuspend, repeat centrifugation several times, and after the final resuspension, ultrasonically disperse to obtain an activated red latex microsphere suspension; wherein the borate buffer concentration is 0.02 mmol / L and its pH is 7.0; The red latex microspheres mentioned above contain carboxyl groups (commonly known as carboxyl-modified microspheres), allowing antibodies to be covalently attached to the microspheres via their amino groups via EDC / NHS chemistry. Therefore, during the EDC / NHS activation reaction, EDC and NHS should be added in a 1:1:1 molar ratio based on the molar weight of these carboxyl groups. This ensures that all carboxyl groups are effectively activated, facilitating subsequent antibody conjugation.

[0019] The red latex microsphere-labeled antibody is prepared by the following steps: i. The monoclonal antibody was added to the activated red latex microsphere suspension and mixed at room temperature on a rotary mixer; wherein the labeled amount of the monoclonal antibody was 50 μg / mL; ii. Then, add BSA (bovine serum albumin) 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 (bovine serum albumin), 0.5g PVP (polyvinylpyrrolidone), 0.5g F68, 0.5mL PEG200 (polyethylene glycol 200), 0.5mL TritonX-100 (a nonionic surfactant in Chinese), and 1mL Tween-20 in 100mL of water.

[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 function: Stabilize the structure of antibodies or proteins to 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; Helps maintain the stability and consistent performance of latex markers.

[0021] The blocking solution of the conjugate pad is prepared by adding 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 to 100 mL of water, and the pH value thereof is 7.6.

[0022] The blocking solution of the sample pad uses 0.5 mol / L Tris-HCl buffer as the base solution, and is supplemented with 0.50% PVPK-40 (wt%), 0.25% SDS-L (wt%), 0.02% NaN3 (wt%), and 0.2% casein (wt%).

[0023] The spraying volume of the red latex microsphere-labeled antibody was 5 μL / cm.

[0024] 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.

[0025] The spray volume of monoclonal antibody coating solution and commercial goat anti-mouse IgG coating solution is 1 μL / cm The detection method of the above-mentioned porcine epidemic diarrhea virus red latex microsphere antigen detection test strip is to perform a double antibody sandwich reaction on the porcine epidemic diarrhea virus antigen in the sample to be tested (pig fecal swab, the sample volume is 80μL), so that it aggregates at the T line and develops color.

[0026] Beneficial effects of the present invention: The present invention prepares monoclonal antibodies by prokaryotic expression of proteins, and screens out two specific porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6. This pair of antibodies has a good effect in paired screening of antigen detection test strips. The red latex microsphere antigen detection test strip method established using this antibody pair has high sensitivity, strong specificity, simple operation, and good stability. It is suitable for rapid on-site detection and has good application prospects in the clinical diagnosis and epidemiological monitoring of viral diarrhea.

[0027] This method uses a double-antibody sandwich assay, offering advantages such as rapid detection, high sensitivity, and strong specificity. The test results are intuitive, clear, and easy to interpret. The entire testing process requires no equipment or specialized operator training, making it simple to operate and particularly suitable for rapid on-site diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 2 is a diagram of enzyme digestion identification, protein expression and purification of the prokaryotic expression plasmid pET-28a-PEDV-N in the examples; In the figure, A is the enzyme digestion identification diagram of the prokaryotic expression plasmid pET-28a-PEDV-N; B is a diagram showing the expression and purification of the protein from the prokaryotic expression plasmid pET-28a-PEDV-N; Figure 2 This is an IFA detection image of the monoclonal antibody secreted by the hybridoma cell line PEDV-MAb-3B5 in the embodiment; Figure 3 This is a WB detection image of the monoclonal antibody secreted by the hybridoma cell line PEDV-MAb-8G6 in the example; Figure 4 Schematic diagram of the structure of the red latex microsphere test strip for detecting porcine epidemic diarrhea virus antigen; In the figure, sample pad 1, conjugate pad 2, nitrocellulose membrane 3, test line 4, quality control line 5, absorbent pad 6, polyvinyl chloride base plate 7; Figure 5 This is the sensitivity test result of the red latex microsphere antigen test strip for porcine epidemic diarrhea virus; Figure 6 This is the specific detection result of the red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus.

[0029] Figure 7 This is a schematic diagram of the display of the red latex microsphere test strip after testing for porcine epidemic diarrhea virus antigen in Example 4. Among them, a is a schematic diagram of a positive result, b is a schematic diagram of a negative result, and c is a schematic diagram of an invalid result. DETAILED DESCRIPTION

[0030] The present invention will be described in further detail below in conjunction with specific examples for those skilled in the art to understand. The experimental methods used in the following examples are conventional methods unless otherwise specified. Meanwhile, the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0031] Example 1 Preparation of hybridoma cell lines PEDV-MAb-3B5 and PEDV-MAb-8G6 1. Virus strains, cells, serum, and experimental animals Strain: Porcine epidemic diarrhea virus YN17 (PEDV-YN17, whose genome sequence is published in the NCBI database, Genbank: MZ604716).

[0032] Cells: African green monkey kidney cells (Vero-ccl, also known as Vero cells) were purchased from Shanghai Cell Bank.

[0033] Bacteria: Escherichia coli DH5α used for plasmid construction was purchased from Beyotime Biotechnology.

[0034] Plasmid vector: Prokaryotic expression vector pET-28a (+), purchased from Addgene platform.

[0035] Experimental animals: 5- to 6-week-old BALB / c female experimental mice were purchased from Hubei Provincial Laboratory Animal Research Center.

[0036] 2. Construction, protein expression and purification of recombinant plasmid pET-28a-PEDV-N The nucleotide sequence of the N gene of PEDV-YN17 strain is shown in SEQ ID NO: 39: Its amino acid sequence is shown in SEQ ID NO:40: MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHADLRYRTRTEGVFWVAKEGAKTEPTNL GVRKASEKPIIPNFSQQLPSVVEIVEPNTPPTSRANSRSRSRGNGNNRSRSPSNNRGNNQSRGNSQNRGNNQGRGASQNRGGNNNNNNKSRNQSKNRNQSNDRGGVTSRDD LVAAVKDALKSLGIGENPDKLKQQQKPKQERSDSSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAA LLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKPQRKKEKKNKSETTQQLNEEAIYDDVGVPSDVTHANLEWDTAVDGGDTAVEIINEIFDTGN; Using Oligo 6.0 software, a pair of primers were designed targeting the ORF of the N gene. The primers were synthesized by Qingke Biotechnology Co., Ltd. The sequences are as follows: PEDV-NF: 5'-GCGGATCCATGGCTTCTGTCAGTTTTCAGG-3' (SEQ ID No: 37); PEDV-NR: 5'-TGCTCGAGATTTCCTGTGTCGAAGATCTCG-3' (SEQ ID No: 38).

[0037] Using the N gene of PEDV YN17 strain as a template, the above primers were used to perform RT-PCR amplification to obtain the target fragment of the N gene. 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 target fragment of the N gene were ligated in vitro using T4 DNA Ligase to obtain a recombinant plasmid. The recombinant plasmids that were positive for enzyme digestion were selected and sent to Wuhan Qingke Biological Company for sequencing verification. The correctly identified recombinant plasmid was named pET-28a-PEDV-N. The plasmid electrophoresis results are as follows Figure 1 As shown in A, the results showed that the pET-28a-PEDV-N plasmid was successfully constructed.

[0038] The plasmid pET-28a-PEDV-N was transformed into E. coli Rosetta (DE3) competent cells were used to induce protein expression by IPTG, the supernatant was collected for purification, and the purified product was detected by SDS-PAGE. Figure 1 As shown in B, the detection results showed that the expressed N protein was approximately 60 kDa in size, which was consistent with the expected size.

[0039] 3. Animal immunization Healthy female BALB / c mice aged four to six weeks were selected and immunized by subcutaneous multi-point injection with an immunization volume of 0.5 mL / point. Based on the concentration of purified N protein and the final immunization dose, an appropriate amount of protein was emulsified with an equal volume of Freund's complete adjuvant, followed by subcutaneous injection for the first immunization. An appropriate amount of protein was mixed with an equal volume of Freund's incomplete adjuvant for emulsification, followed by a second immunization. Two weeks later, blood was collected by tail cutting and serum was separated. The serum antibody titer was detected by indirect ELISA, and mice with relatively high serum antibody levels were selected for subsequent hybridoma preparation experiments.

[0040] 4. Establishment of hybridoma cell lines PEDV-MAb-3B5 and PEDV-MAb-8G6 Mouse peritoneal macrophages were prepared as feeder cells using conventional methods. Splenocytes were then fused with myeloma cells (SP2 / 0) at a ratio of 5:1 using the fusion agent PEG4000. Hybridomas positive for antibody secretion were screened by indirect ELISA and cloned by limiting dilution. Ultimately, two hybridoma cell lines, PEDV-MAb-3B5 and PEDV-MAb-8G6, were obtained, stably secreting monoclonal antibodies against the PEDV N protein.

[0041] Example 2 Preparation of porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 from hybridoma cell lines PEDV-MAb-3B5 and PEDV-MAb-8G6 1. A method for preparing porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 from hybridoma cell lines PEDV-MAb-3B5 and PEDV-MAb-8G6, comprising the following steps: 0.5 mL of sterilized paraffin oil was injected into the mouse peritoneal cavity, and then 10 6 The hybridoma cells PEDV-MAb-3B5 and PEDV-MAb-8G6 were cultured. After 7-10 days, the ascites in the mouse abdomen was extremely swollen and the ascites was extracted. The ascites contained a large amount of monoclonal antibodies, which were purified and divided into several groups for use.

[0042] 2. Determination of the above-mentioned porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6: (1) Indirect immunofluorescence (IFA) with monoclonal antibodies Vero cells were seeded in 24-well plates. When the cells grew to 80-90% confluence, they were inoculated with PEDV YN17 for IFA detection.

[0043] The results are as follows Figure 2 As shown, the prepared monoclonal antibodies 2B2, 3B5, 4E6, 5G3 and 8G6 reacted positively with PEDV YN17 and produced specific green fluorescence; the negative control group had no fluorescence, indicating that the prepared MAb had strong specificity.

[0044] (2) Monoclonal antibody Western blotting (WB) Vero cells were seeded into 10 cm cell culture dishes. When the cells grew to 80-90% confluence, they were inoculated with PEDV YN17. Samples were collected for WB detection at 12 h and 24 h.

[0045] The results are as follows Figure 3 As shown, the prepared monoclonal antibodies 2B2, 3B5, 4E6, 5G3 and 8G6 reacted positively with PEDV YN17, producing a specific band at a size of 60 kDa, while the negative control group had no specific band, indicating that the prepared mouse monoclonal antibody MAb has strong specificity.

[0046] a. The sequence of monoclonal antibody 3B5 is as follows: 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; The four 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 four 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; 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: 8 and SEQ ID No: 17 respectively; The nucleic acid sequence encoding the light chain variable region of the above monoclonal antibody 3B5, the nucleic acid sequence of the heavy chain variable region are shown in SEQ ID No: 9 and SEQ ID No: 18 respectively; The specific sequence of the above monoclonal antibody 3B5 is as follows: 3B5-LCDR-1: QNIVDSNGNTY (SEQ ID No. 1); 3B5-LCDR-2: KVSNRFS (SEQ ID No. 2); 3B5-LCDR-3: FQGSHVPPT (SEQ ID No. 3); 3B5-LFR-1: DVLMTQTPLSLPVSLGDQASISC (SEQ ID No. 4); 3B5-LFR-2: WYLQKPGQSPKLLIY (SEQ ID No. 5); 3B5-LFR-3: GVPDRFSGSGSGTDFTLQISRVEAEDLGVYYC (SEQ ID No. 6); 3B5-LFR-4: FGGGTKLEIK (SEQ ID No. 7); The amino acid sequence of the light chain variable region, wherein the underlined part represents the CDR sequence: DVLMTQTPLSLPVSLGDQASISCRSS QNIVDSNGNTY LEWYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLQISRVEAEDLGVYYC FQGSHVPPT FGGGTKLEIK (SEQ ID No. 8); The nucleic acid sequence of the light chain variable region: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTAGATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCCAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAGC (SEQ ID No.9); 3B5-HCDR-1: EYTIH (SEQ ID No. 10); 3B5-HCDR-2:GINPDHGGTRFNHKFKG (SEQ ID No. 11); 3B5-HCDR-3: EVFDY (SEQ ID No. 12); 3B5-HFR-1: EVQLQQSGPELVKPGTSVKISCKTSGYTFT (SEQ ID No. 13); 3B5-HFR-2: WVKQSHGKSLEWIG (SEQ ID No. 14); 3B5-HFR-3:KATLTVDKSSSTVYMDLRSLTSEDSAVYYCTR (SEQ ID No. 15); 3B5-HFR-4: WGQGTSLTVSS (SEQ ID No. 16); The amino acid sequence of the heavy chain variable region, where the CDR sequences are underlined: EVQLQQSGPELVKPGTSVKISCKTSGYTFT EYTIH WVKQSHGKSLEWIG GINPDHGGTRFNHKFKG KATLTVDKSSSTVYMDLRSLTSEDSAVYYCTR EVFDY WGQGTSLTVSS (SEQ ID No. 17); Nucleic acid sequence of the heavy chain variable region: GAGGTCCAGTTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATATACCATACACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTGACATGGTGGTAC TAGGTTCAACCACAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGTCTACATGGACCTCCGCAGCCTGACATCTGAAGATTCTGCAGTCTATTACTGTACAAGAGAGGTCTTTGACTACTGGGGCCAAGGCACCAGTCTCACAGTCTCCTCAG (SEQ ID No.18).

[0047] b. The sequence of monoclonal antibody 8G6 is as follows: The three complementarity determining regions of the light chain variable region are 8G6-LCDR-1, 8G6-LCDR-2, and 8G6-LCDR-3, and their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20, and SEQ ID No: 21, respectively; the three complementarity determining regions of the heavy chain variable region are 8G6-HCDR-1, 8G6-HCDR-2, and 8G6-HCDR-3, and their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29, and SEQ ID No: 30, respectively; The four light chain framework regions of the light chain variable region are 8G6-LFR-1, 8G6-LFR-2, 8G6-LFR-3, and 8G6-LFR-4, and their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, and SEQ ID No: 25, respectively; the amino acid sequences of the heavy chain framework regions 8G6-HFR-1, 8G6-HFR-2, 8G6-HFR-3, and 8G6-HFR-4 of the heavy chain variable region are shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, and SEQ ID No: 34, respectively; 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; The nucleic acid sequences encoding the light chain variable region and heavy chain variable region of the monoclonal antibody 8G6 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively; Among them: The specific sequence of monoclonal antibody 8G6 is as follows: 8G6-LCDR-1: RASQSISDYLH (SEQ ID No. 19); 8G6-LCDR-2: YASQSIS (SEQ ID No. 20); 8G6-LCDR-3: QNGHSFPALT (SEQ ID No. 21); 8G6-LFR-1: DIVMTQSPATLSVTPGDRVFLSC (SEQ ID No. 22); 8G6-LFR-2: WYQQKSHESPRLLIK (SEQ ID No. 23); 8G6-LFR-3: GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID No. 24); 8G6-LFR-4: FGAGTRLELK (SEQ ID No. 25); The amino acid sequence of the light chain variable region, where the CDR sequences are underlined: DIVMTQSPATLSVTPGDRVFLSC RASQSISDYLH WYQQKSHESPRLLIK YASQSIS GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC QNGHSFPALT FGAGTRLELK (SEQ ID No. 26); Nucleic acid sequence of the light chain variable region: GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTTTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACTGTCAAAATGGTCACAGCTTTCCAGCGCTCACATTCGGTGCTGGGACCAGGCTGGAGCTGAAAC (SEQ ID No. 27); 8G6-HCDR-1: SYAMS (SEQ ID No. 28); 8G6-HCDR-2: AISSGGTYTYYPDSVKG (SEQ ID No. 29); 8G6-HCDR-3: QGDFGGDWYFDV (SEQ ID No. 30); 8G6-HFR-1: EVQLVESGGGLVKPPGSLKLSCAASGFTFT (SEQ ID No. 31); 8G6-HFR-2: WVRQTPEKRLEWVA (SEQ ID No. 32); 8G6-HFR-3: RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR (SEQ ID No. 33); 8G6-HFR-4: WGAGTTVTVSS (SEQ ID No. 34); Amino acid sequence of the heavy chain variable region, wherein underlined sequences represent CDR sequences: EVQLVESGGGLVKPPGSLKLSCAASGFTFT SYAMS WVRQTPEKRLEWVA AISSGGTYTYYPDSVKG RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR QGDFGGDWYFDV WGAGTTVTVSS (SEQ ID No. 35); Nucleic acid sequence of the heavy chain variable region: GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTCCAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAGCCATTAGTAGTGGTGGTACTTACACCTACTATCCAG ACAGTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGACAGGGGGGATTTCGGCGGCGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG (SEQ IDNo.36).

[0048] Example 3: Preparation of red latex microsphere antigen detection test strips for porcine epidemic diarrhea virus using porcine epidemic diarrhea virus monoclonal antibodies 3B5 and 8G6 1. Cells, plasmids, serum and main reagents: Plasmid pET-28a-PEDV-N (constructed in Example 1), hybridoma cell lines PEDV-MAb-3B5 and PEDV-MAb-8G6 secreting PEDV-N protein monoclonal antibodies 3B5 and 8G6 (prepared in Example 1). Porcine epidemic diarrhea virus (PEDV-YN17), 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 rotavirus A (PoRVA) were provided by Luo Rui's research group at Huazhong Agricultural University. Fecal swabs from porcine diarrhea were collected from Chaoyang City, Liaoning Province, Chenzhou City, Hunan Province, Jincheng City, Shanxi Province, and Shaoguan City, Guangdong Province.

[0049] 2. Preparation of the Materials for the Red Latex Microsphere Antigen Test Strip for Porcine Epidemic Diarrhea Virus 1. Preparation of monoclonal antibody 3B5-red latex microsphere marker In an EP tube, add 975 μL of 0.03 mol / L MES buffer, then add 25 μL of red latex microspheres with a solid content of 4% and vortex mix; add 5 μL of 10 mg / mL NHS solution and vortex mix, then add 5 μL of EDC of the same concentration and vortex shake, then mix at room temperature for 20 min on a rotary mixer at 20 rpm; centrifuge at 4°C, 13,000 rpm for 10 min, discard the supernatant, and add 1 mL of 0.02 mmol / L Resuspend in borate buffer at pH 7.5, centrifuge twice, and ultrasonically disperse for 2 minutes. Dilute monoclonal antibody 3B5 to 0.2 mg / mL with ultrapure water, add 100 μg to the activated microsphere suspension, and mix on a rotary mixer at 20 rpm for 2 hours at room temperature. Add BSA to a final concentration of 0.5%, and block on a rotary mixer at 20 rpm for 1 hour at room temperature. Centrifuge at 13,000 rpm at 4°C for 10 minutes, discard the supernatant, resuspend in 1 mL of storage solution, and store at 4°C. This yields the porcine epidemic diarrhea virus monoclonal antibody 3B5-red latex microsphere marker of the present invention.

[0050] The above-mentioned preservation solution is prepared by adding 1.21g Tris, 5g sucrose, 0.5g BSA (bovine serum albumin), 0.5g PVP (polyvinyl pyrrolidone), 0.5g F68, 0.5mL PEG200 (polyethylene glycol 200), 0.5mL TritonX-100 (Chinese name: TritonX-100, a non-ionic surfactant), and 1mL Tween-20 to 100mL of water.

[0051] 2. Preparation of conjugate pad (1) Prepare the conjugate pad blocking solution Weigh 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, dissolve them in ddH2O, adjust the pH to 7.6, dilute to 100 mL in a volumetric flask, and filter through a 0.22 µm filter to obtain the conjugate pad blocking solution.

[0052] (2) Preparation of conjugate pad The glass cellulose membrane (model: Fusion4) was immersed in the conjugate pad blocking solution for 30 minutes and dried in a 37°C oven to obtain a blocked conjugate pad.

[0053] 3. Preparation of sample pad (1) Prepare sample pad blocking solution The sample pad blocking solution 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.

[0054] (2) Preparation of sample pad Soak the glass cellulose membrane (model BX-03) in the sample pad blocking solution for 30 minutes and dry it in a 37°C oven to obtain a blocked binding pad.

[0055] 4. Spray the conjugate pad with the monoclonal antibody 3B5 against porcine epidemic diarrhea virus - red latex microsphere marker The monoclonal antibody 3B5-red latex microsphere marker was sprayed onto the treated conjugate pad 2 at a spray rate of 5 μL / cm. After spraying, the pad was dried in a 37° C. oven for 2 hours and cut into 3 mm wide strips for later use.

[0056] 5. Spraying inspection line and quality control line (1) Preparation of antibody coating solution Add 1.0% (w / v) trehalose, 1.0% (w / v) sorbitol, and 0.3% (w / v) Tween-20 to 0.02 mol / L phosphate buffered saline (PBS) with a pH of 7.2, stir well, and filter through a 0.22 μm filter to obtain the antibody coating solution.

[0057] (2) Preparation of test lines and quality control lines The non-spotted surface of the nitrocellulose membrane 3 was adhered to the polyvinyl chloride base plate 7. The specific model 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 antibody coating solution, and the commercial goat anti-mouse IgG was diluted to 0.5 mg / mL with antibody coating solution. They were then sprayed at positions 4 and 5 of the nitrocellulose membrane at a spray volume of 1 μL / cm as the test line and quality control line, respectively.

[0058] The distance between the test line and the quality control line is 5 mm. After spraying, it is dried in a 37°C oven for 2 hours before use.

[0059] 3. Assembly of test strips like Figure 4As 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 porcine epidemic diarrhea virus monoclonal antibody 3B5-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.

[0060] Detection of the sensitivity and specificity of the above test strips 1. Test strip sensitivity test 3.89×10 4 TCID 50 The PEDV strain with a concentration of 1 / mL was diluted 10-fold in a series with the above diluent, and 80 μL was aspirated for spot detection, and the results were observed within 10 minutes.

[0061] The results are as follows Figure 5 As shown, the lowest detection limit of the test strip is 3.89×10 1 TCID 50 / mL.

[0062] 2. Specificity detection of test strips Dilute the inactivated viruses of PEDV, PRV, PRRSV, TGEV, PDCoV, ASFV, and PoRVA 20-fold with the above diluent, then aspirate 80 μL for spot detection and observe the results within 10 minutes.

[0063] The results are as follows Figure 6 As shown, there is no cross reaction of the test strips and the test strips have good specificity.

[0064] Example 4 Application of the above-mentioned red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus 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.

[0065] 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.

[0066] 2. Detection Take 80 μL of the supernatant and drop it on the above test strip. Observe the result within 10 minutes. The test result is determined based on: like Figure 7 As shown: When red appears on both the quality control line and the test line, it is a positive result, that is, the sample contains porcine epidemic diarrhea virus ( Figure 7 a); If the quality control line appears red and the test line does not appear red, it is a negative result, that is, the sample does not contain porcine epidemic diarrhea virus ( Figure 7 b); If the quality control line does not appear red, and the test line appears red, it means that the test paper is invalid ( Figure 7 c).

[0067] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A porcine epidemic diarrhea virus monoclonal antibody, characterized in that: The monoclonal antibody against epidemic diarrhea virus is monoclonal antibody 3B5 or monoclonal antibody 8G6, The monoclonal antibody 3B5 comprises a light chain variable region and a heavy chain variable region; wherein, The light chain variable region of 3B5 includes three complementarity determining regions, namely 3B5-LCDR-1, 3B5-LCDR-2, and 3B5-LCDR-3, whose amino acid sequences are SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively; 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, and SEQ ID No: 12, respectively; The monoclonal antibody 8G6 comprises a light chain variable region and a heavy chain variable region; wherein, 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 SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21, respectively; The heavy chain variable region of 8G6 includes three complementarity determining regions, namely 8G6-HCDR-1, 8G6-HCDR-2 and 8G6-HCDR-3, and their amino acid sequences are SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30, respectively.

2. The porcine epidemic diarrhea virus monoclonal antibody according to claim 1, characterized in that: The light chain variable region of the monoclonal antibody 3B5 contains four light chain framework regions, namely 3B5-LFR-1, 3B5-LFR-2, 3B5-LFR-3 and 3B5-LFR-4, whose 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 3B5 contains four heavy chain framework regions, namely 3B5-HFR-1, 3B5-HFR-2, 3B5-HFR-3, and 3B5-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 8G6 contains four light chain framework regions, namely 8G6-LFR-1, 8G6-LFR-2, 8G6-LFR-3, and 8G6-LFR-4; their amino acid sequences are 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 8G6 contains four heavy chain framework regions, namely 8G6-HFR-1, 8G6-HFR-2, 8G6-HFR-3 and 8G6-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 porcine epidemic diarrhea virus monoclonal antibody according to claim 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 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 cloned antibody 3B5 are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively.

4. The porcine epidemic diarrhea virus monoclonal antibody according to claim 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 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.

5. Use of the porcine epidemic diarrhea virus monoclonal antibody according to claim 1 in preparing a porcine epidemic diarrhea virus antigen detection test strip based on a double antibody sandwich method.

6. A red latex microsphere antigen detection test strip for porcine epidemic diarrhea virus, characterized by: The invention comprises the monoclonal antibody 3B5 according to claim 1 and / or the monoclonal antibody 8G6 according to claim 1.

7. The test strip according to claim 6, characterized in that: 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 4 and a quality control line are arranged on the nitrocellulose membrane along the flow direction; wherein, The conjugate pad is sprayed with red latex microsphere-labeled antibodies, the detection line is sprayed with coated antibodies, and the quality control line is sprayed with coated commercial goat anti-mouse IgG.

8. The test strip according to claim 7, wherein: The antibody labeled with the red latex microspheres is monoclonal antibody 3B5 or monoclonal antibody 8G6; and the antibody labeled with the red latex microspheres has an antibody labeling amount of 50 μg / mL; The coated antibody is monoclonal antibody 3B5 or monoclonal antibody 8G6; 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 porcine epidemic diarrhea 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 with boric acid buffer several times, and finally disperse by ultrasonication to obtain 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 4% solid content red latex microspheres to MES buffer and vortex mix; wherein the concentration of MES buffer is 0.03 mol / L; b. Weigh 10 mg / mL of EDC solution and 10 mg / mL of NHS solution according to the molar ratio of EDC, NHS and red microsphere surface of 1:1:1, then add NHS solution and vortex mix, then add EDC solution and vortex oscillation, and mix in a rotary mixer at room temperature. c. Centrifuge, discard the supernatant, add borate buffer and resuspend, repeat centrifugation several times, and after the final resuspension, ultrasonically disperse to obtain an activated red latex microsphere suspension; wherein the borate buffer concentration is 0.02 mmol / L and its pH is 7.0; 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, BSA was added to a final concentration of 0.5%, and the mixture was blocked and mixed on a rotary mixer at room temperature. The mixture was centrifuged, the supernatant was discarded, and the suspension was resuspended in preservation solution and stored at 4°C. The preservation solution was 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 added to 100mL of water. The blocking solution of the conjugate pad was 2g sucrose, 0.3g PVPK-30, 2g BSA, 0.02g NaN3, 0.29g Na2HPO4·12H2O, and 0.02g KH2PO4 added to 100mL of water, with a pH 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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