Porcine delta coronavirus monoclonal antibody and application thereof in preparation of antigen detection test strip

Abstract

The invention discloses a porcine delta coronavirus monoclonal antibody and application thereof in preparation of an antigen detection test strip. The detection test strip comprises a polyvinyl chloride bottom plate on the lowest layer, and a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad are sequentially laid on the polyvinyl chloride bottom plate in the flowing direction; the sample pad is partially overlapped on the combination pad, and the combination pad and the water absorption pad are respectively overlapped on two sides of the nitrocellulose membrane; a detection line and a quality control line are arranged on the nitrocellulose membrane along the flowing direction; an antibody marked by red latex microspheres is sprayed on the combination pad, a coated antibody is sprayed on the detection line, and coated commercial goat anti-mouse IgG is sprayed on the quality control line. The test strip adopts a double-antibody sandwich method for detection, and has the advantages of high detection speed, high sensitivity, strong specificity and the like. The detection result is visual, clear and easy to interpret; the detection process does not need any instrument and equipment, operators do not need professional training, operation is easy and convenient, and the method is particularly suitable for on-site rapid diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of cell biology technology, immunology technology and animal epidemic detection technology, and particularly relates to a porcine deltacoronavirus monoclonal antibody and application thereof in preparation of antigen detection test strips. BACKGROUND

[0002] Porcine deltacoronavirus (PDCoV) is a newly emerging enteropathogenic coronavirus, mainly infecting piglets and growing pigs, and the clinical manifestations are vomiting, diarrhea, dehydration and weight loss, which can cause severe mortality in suckling piglets and pose a serious threat to the pig industry.

[0003] PDCoV is mainly transmitted through the fecal-oral route, has strong environmental resistance, and can be transmitted through various media such as contaminated feed, drinking water, transportation tools, personnel and wild animals, making it difficult to control the epidemic. Although PDCoV is similar to porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (PoRV) in clinical symptoms, it has unique genetic characteristics and immunogenicity, and there is no commercial vaccine available, so the prevention and control measures still mainly rely on biological safety measures and early diagnosis.

[0004] In recent years, with the development of large-scale breeding and the increase of pig farm mobility, the risk of PDCoV transmission is rising, which brings continuous challenges to the breeding industry. Therefore, it is of great significance to establish a sensitive, rapid and specific on-site detection technology to identify infected individuals in a timely manner, control PDCoV epidemic and ensure the safety of pig production. This has important practical value for improving prevention and control efficiency, reducing economic losses, promoting pig farm health management and biological safety system construction. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provides a porcine deltacoronavirus monoclonal antibody and application thereof in preparation of antigen detection test strips. The test strip prepared from the monoclonal antibody 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 entire detection process does not require any instrument or professional training of the operator.

[0006] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows: The present application provides a porcine deltacoronavirus monoclonal antibody, which comprises a monoclonal antibody 18E2 or a monoclonal antibody 15D1, The monoclonal antibody 18E2 comprises a light chain variable region and a heavy chain variable region; wherein, 18E2's light chain variable region includes three complementarity determining regions, namely 18E2-LCDR-1, 18E2-LCDR-2 and 18E2-LCDR-3; their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, respectively; 18E2's heavy chain variable region includes three complementarity determining regions, namely 18E2-HCDR-1, 18E2-HCDR-2 and 18E2-HCDR-3; their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, respectively; The monoclonal antibody 15D1 includes a light chain variable region and a heavy chain variable region; wherein, 15D1's light chain variable region includes three complementarity determining regions, namely 15D1-LCDR-1, 15D1-LCDR-2 and 15D1-LCDR-3; their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21, respectively; 15D1's heavy chain variable region includes three complementarity determining regions, namely 15D1-HCDR-1, 15D1-HCDR-2 and 15D1-HCDR-3; their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30, respectively.

[0007] Further, the light chain variable region of the monoclonal antibody 18E2 contains four light chain framework regions, namely LFR-1, LFR-2, LFR-3 and LFR-4; their amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7, respectively; The heavy chain variable region of the monoclonal antibody 18E2 contains four heavy chain framework regions, namely HFR-1, HFR-2, HFR-3 and HFR-4; their amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, respectively; The light chain variable region of the monoclonal antibody 15D1 contains four light chain framework regions, namely LFR-1, LFR-2, LFR-3 and LFR-4; their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24 and SEQ ID No: 25, respectively; The heavy chain variable region of the monoclonal antibody 15D1 contains four heavy chain framework regions HFR-1, HFR-2, HFR-3 and HFR-4, and their amino acid sequences are shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33 and SEQ ID No: 34, respectively.

[0008] Further, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 18E2 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 porcine delta coronavirus monoclonal antibody 18E2 are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively.

[0009] Further, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 15D1 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 porcine delta coronavirus monoclonal antibody 15D1 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

[0010] The above-mentioned porcine epidemic diarrhea virus monoclonal antibodies 18E2 and 15D1 are prepared by using the recombinant N protein of PDCoV CHN-HG-2017 (GenBank number: MF095123.1) as an immunogen.

[0011] The above-mentioned monoclonal antibodies are secreted by hybridoma cell strains immunized by the recombinant N protein of PDCoV CHN-HG-2017.

[0012] The application also provides a use of the above-mentioned porcine delta coronavirus monoclonal antibody in the preparation of a porcine epidemic diarrhea virus antigen detection test strip based on a double antibody sandwich method.

[0013] The application also provides a porcine delta coronavirus red latex microsphere antigen detection test strip, which comprises the above-mentioned monoclonal antibody.

[0014] Further, the test strip comprises a lowermost polyvinyl chloride bottom plate 7, and a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 3 and a water absorption pad 6 are sequentially arranged on the polyvinyl chloride bottom plate 7 in the flow direction; the sample pad is partially overlapped on the conjugate pad 2, and the conjugate pad 2 and the water absorption pad 6 are respectively overlapped on both sides of the nitrocellulose membrane 3; a detection line 4 and a quality control line 5 (C line) are arranged on the nitrocellulose membrane 3 in the flow direction; wherein, The red latex microsphere marked antibody is sprayed on the binding pad 2, the coated antibody is sprayed on the detection line 4, and the coated commercial sheep anti-mouse IgG is sprayed on the quality control line 5.

[0015] Further, the red latex microsphere marked antibody is monoclonal antibody 18E2 or monoclonal antibody 15D1, and the antibody marking amount in the red latex microsphere marked antibody is 50 mu g / mL (the marking amount means that 50 mu g of the antibody is used in each 1 mL of the latex microsphere reaction system for reaction, covalent combination or adsorption with the microsphere in the process of preparing the marked antibody). The coated antibody is monoclonal antibody 18E2 or monoclonal antibody 15D1, 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.

[0016] 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.

[0017] The application further provides a preparation method of the pig delta coronavirus red latex microsphere antigen test strip. 1) The red latex microspheres are added into the MES buffer solution, mixed with EDC and NHS in sequence, then centrifuged, resuspended with the boric acid buffer solution for multiple times, and finally ultrasonically dispersed to obtain an activated red latex microsphere suspension, 2) The activated red latex microsphere suspension is marked with the above-mentioned monoclonal antibody to obtain a red latex microsphere marked antibody; 3) The sample pad and the binding pad are respectively sealed with a blocking solution and stored at 4 DEG C; 4) The red latex microsphere marked antibody is sprayed and fixed on the binding pad; 5) The above-mentioned monoclonal antibody and the commercial sheep anti-mouse IgG are respectively coated, and then sprayed on the nitrocellulose membrane as the detection line and the quality control line; 6) The water absorption pad, the binding pad and the sample pad are pasted on the polyvinyl chloride bottom plate of the nitrocellulose membrane to assemble the test strip.

[0018] Further, the activated red latex microsphere suspension is prepared by the following steps: a. The red latex microspheres with a solid content of 4% are added into the MES buffer solution and vortexed uniformly; wherein the concentration of the MES buffer solution is 0.03 mol / L.

[0019] 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 (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC for short), N-hydroxysuccinimide (N-hydroxysuccinimide, NHS for short) and the surface of the red microspheres, then the NHS solution is added and vortexed, then the EDC solution is added and vortexed, and then the mixture is mixed at room temperature in a rotary mixer, c. Centrifugation, discard the supernatant, add boric acid buffer for resuspension, repeat centrifugation for several times, after the last resuspension, ultrasonic dispersion, obtain activated red latex microspheres suspension; wherein the concentration of boric acid buffer is 0.02 mmol / L and its pH is 7.0; The above-mentioned red latex microspheres have carboxyl groups (commonly carboxyl modified microspheres), so that the antibody can be covalently connected to the microspheres through its amino group by EDC / NHS chemical coupling. Therefore, when doing EDC / NHS activation reaction, EDC and NHS are 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.

[0020] Further, the red latex microsphere labeled antibody is prepared by the following steps: i. The above-mentioned monoclonal antibody is added to the activated red latex microsphere suspension, and mixed at room temperature in a rotary mixer; wherein the labeled amount of monoclonal antibody is 50 μg / mL; ii. Then add BSA (bovine serum albumin) with a final concentration of 0.5%, mix and block at room temperature in a rotary mixer, centrifuge, discard the supernatant, add the preservation solution for resuspension, and store at 4°C; wherein the preservation solution is 100 mL of water added with 1.21 g of Tris, 5 g of sucrose, 0.5 g of BSA (bovine serum albumin), 0.5 g of PVP (Polyvinylpyrrolidone, English name: Polyvinylpyrrolidone), 0.5 g of F68, 0.5 mL of PEG200 (Polyethylene glycol 200), 0.5 mL of TritonX-100 (Chinese name: TritonX-100, a non-ionic surfactant), 1 mL of Tween-20.

[0021] The above-mentioned F68: trade name: Pluronic F68 Alias: polyoxypropylene-polyoxyethylene block copolymer F68 Chemical name: polyblock copolymer PEG-PPG-PEG (PEO-PPO-PEO) Chinese name: F68, Pluronic F68, block copolymer F68 F68 action: Stabilize antibody or protein structure, prevent non-specific adsorption; Improve the dispersibility of latex microspheres or colloidal particles; Prevent aggregation or precipitation during freeze-thaw process; Enhance the wettability of the storage solution on the test strip; Help maintain the stability and performance consistency of the latex marker.

[0022] The blocking solution of the conjugate pad is 100 mL of water added with 2 g of sucrose, 0.3 g of PVPK-30, 2 g of BSA, 0.02 g of NaN3, 0.29 g of Na2HPO4·12H2O, and 0.02 g of KH2PO4, and the pH value is 7.6.

[0023] 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.

[0024] The spraying amount of the red latex microsphere labeled antibody is 5 muL / cm.

[0025] In the step 5), the coating method of the monoclonal antibody and the commercialized sheep anti-mouse IgG is as follows: The monoclonal antibody and the commercialized sheep anti-mouse IgG are respectively diluted to 0.3 mg / mL and 0.5 mg / mL with the antibody coating solution to obtain a monoclonal antibody coating solution and a commercialized sheep anti-mouse IgG coating solution.

[0026] The spraying amount of the monoclonal antibody coating solution and the commercialized sheep anti-mouse IgG coating solution is 1 muL / cm. The detection method of the above-mentioned red latex microsphere antigen detection test strip for porcine delta coronavirus is a double-antibody sandwich reaction of the porcine delta coronavirus antigen in the sample (porcine fecal swab, the sample amount is 80 muL) to be detected, so that the antigen is gathered and colored at the T line.

[0027] The beneficial effects of the present application are: The present application prepares monoclonal antibodies by prokaryotic expression of proteins, and screens two specific porcine delta coronavirus monoclonal antibodies 18E2 and 15D1, which have good effects in antigen detection test strip pairing screening. The use of the antibodies has high sensitivity, strong specificity, simple operation, good stability, and is suitable for on-site rapid detection, and has good application prospect in clinical diagnosis and epidemiological monitoring of viral diarrhea.

[0028] The detection test strip of the application adopts a double-antibody sandwich method for detection, and has the advantages of fast detection speed, high sensitivity, strong specificity and the like. The detection result is intuitive and clear, and easy to judge; the entire detection process does not require any instrument equipment, and the operator also does not need professional training, and the operation is simple, and is particularly suitable for on-site rapid diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-PDCoV-N in the embodiment of the application; A is an enzyme digestion identification diagram of the prokaryotic expression plasmid pET-28a-PDCoV-N, B is a protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-PDCoV-N; Figure 2 It is a serum titer detection result diagram after immunizing mice with the recombinant N protein, In the figure, A is the antibody titer of the mouse serum two weeks after the second immunization, and B is the antibody titer of the mouse serum two weeks after the third immunization.

[0030] Figure 3 It is an IFA detection diagram of the monoclonal antibodies secreted by the hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 in the embodiment of the application; Figure 4 It is a WB detection diagram of the monoclonal antibodies secreted by the hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 in the embodiment of the application; Figure 5 It is a structural schematic diagram of a red latex microsphere test strip for detecting porcine delta coronavirus antigens in the embodiment of the application; 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; Figure 6 It is a sensitivity detection result diagram of a red latex microsphere antigen test strip for porcine delta coronavirus in the embodiment of the application; Figure 7 It is a specificity detection result diagram of a red latex microsphere antigen test strip for porcine delta coronavirus in the embodiment of the application.

[0031] Figure 8 It is a display schematic diagram after detection of a red latex microsphere test strip for detecting porcine delta coronavirus antigens in the embodiment of the application, Wherein a is a positive result schematic diagram, b is a negative result schematic diagram, and c is an invalid result schematic diagram. DETAILED DESCRIPTION

[0032] The application will be further described in detail below with reference to specific examples so as to be understood by those skilled in the art. The experimental methods used in the following examples are conventional methods unless otherwise specified, and meanwhile, the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0033] Example 1 Preparation of hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 1. Strains, cells, serum and experimental animals Strain: Porcine deltacoronavirus PDCoV CHN-HG-2017 (GenBank number: MF095123.1).

[0034] Cells: Porcine kidney epithelial cells (LLCPK1) were purchased from Shanghai Cell Bank.

[0035] Strain: Escherichia coli DH5α used for plasmid construction was purchased from Biyun Tian Biological.

[0036] Plasmid vector: Prokaryotic expression vector pET-28a(+) was purchased from Addgene platform Experimental animals: 5-6-week-old BALB / c female experimental mice were purchased from Hubei Experimental Animal Research Center.

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

[0038] A pair of primers were designed by Oligo 6.0 software, and the primers were synthesized by Genescript. The sequences are as follows: PDCoV-N-F: 5'-GCCATATGATGGCTGCACCAGTGGTCCCTA-3' (SEQ ID No: 37); PDCoV-N-R: 5'-TGCTCGAGCGCTGCTGATTCCTGCTTTATC-3' (SEQ ID No: 38).

[0039] The N gene of PDCoV was used as a template, and the N gene target fragment was obtained by RT-PCR amplification with the above primers. The prokaryotic expression vector pET-28a(+) was double-digested with NdeI and XhoI, and then gel recovery was performed. 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-PDCoV-N. The plasmid electrophoresis result is shown in Figure 1 A, and the results showed that the pET-28a-PDCoV-N plasmid was successfully constructed.

[0040] The pET-28a-PDCoV-N plasmid was transformed into E. coliRosetta (DE3) competent cells, and induce protein expression by IPTG, collect supernatant for purification, and detect the purified product by SDS-PAGE. As shown in Figure 1 B, the detection results show that the size of the expressed recombinant N protein is about 50 kDa, which is consistent with the expected size.

[0041] 3. Animal immunization Select healthy female BALB / c mice of four to six weeks old, and immunize by subcutaneous multi-point injection with a volume of 0.5 mL per point. According to the N protein concentration after purification and the final immunization dose, emulsify an appropriate amount of protein with an equal volume of Freund's complete adjuvant, and then perform the first immunization by subcutaneous injection. Emulsify an appropriate amount of protein with an equal volume of Freund's incomplete adjuvant, and then perform the second immunization. Two weeks later, collect blood from the tail, and separate the serum. Detect the serum antibody titer by indirect ELISA, as shown in Figure 2 Select mice with relatively high serum antibody levels for subsequent hybridoma preparation experiments.

[0042] 4. Establishment of hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 Prepare mouse peritoneal macrophages as feeder cells according to the conventional method, and fuse the spleen cells with myeloma cells (SP2 / 0) at a ratio of 5:1 under the action of fusion agent PEG4000. Screen the hybridoma cells that secrete antibodies by indirect ELISA, and clone them by limited dilution method. Finally, two hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 that can stably secrete PDCoV N protein monoclonal antibodies are obtained.

[0043] Example 2 Preparation of porcine delta coronavirus monoclonal antibodies 18E2 and 15D1 from hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 1. The method for preparing porcine delta coronavirus monoclonal antibodies 18E2 and 15D1 from hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1, comprising the following steps: Inject 0.5 mL of sterilized paraffin oil into the peritoneal cavity of the mouse, and then inject 10 6 hybridoma cells PDCoV-MAb-18E2 and PDCoV-MAb-15D1 into the peritoneal cavity of the mouse, respectively, one week later. When the ascites in the abdominal cavity of the mouse is extremely swollen, extract the ascites, which contains a large amount of monoclonal antibodies. After purification, the product is ready for use.

[0044] 2. Determination of the above porcine delta coronavirus monoclonal antibodies 18E2 and 15D1: (1) Indirect immunofluorescence (IFA) LLCPK1 cells were inoculated in 24-well plates, and when the cells grew to 80-90%, they were infected with PDCoV CHN-HG-2017 for IFA detection.

[0045] The results are shown in Figure 3 As shown in the table, the prepared monoclonal antibodies 15D1, 16B12, 18E2 and 20H12 all showed positive reaction with PDCoV CHN-HG-2017, producing specific green fluorescence; the negative control group showed no fluorescence, indicating that the prepared MAb has strong specificity.

[0046] (2) Western Blot of Monoclonal Antibody Protein LLCPK1 cells were inoculated in 10 cm cell culture dishes, and when the cells grew to 80-90%, they were infected with PDCoV CHN-HG-2017, and samples were collected at 12 h and 24 h for WB detection.

[0047] The results are shown in Figure 4 As shown in the table, the prepared monoclonal antibodies 15D1, 16B12, 18E2 and 20H12 all showed positive reaction with PDCoV CHN-HG-2017, producing specific green fluorescence; the negative control group showed no fluorescence, indicating that the prepared MAb has strong specificity.

[0048] a. The sequence of monoclonal antibody 18E2 is as follows: The three complementarity determining regions of the light chain variable region are 18E2-LCDR-1, 18E2-LCDR-2 and 18E2-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 HCDR-1, HCDR-2 and 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 4 light chain framework regions of the light chain variable region 18E2-LFR-1, 18E2-LFR-2, 18E2-LFR-3, 18E2-LFR-4, the amino acid sequences of which are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, respectively; the 4 heavy chain framework regions of the heavy chain variable region are 18E2-HFR-1, 18E2-HFR-2, 18E2-HFR-3, 18E2-HFR-4, the amino acid sequences of which are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, respectively; The amino acid sequence of the light chain variable region, the amino acid sequence of the heavy chain variable region are shown in SEQ ID No: 8 and SEQ ID No: 17, respectively; The nucleic acid sequence encoding the light chain variable region of the above-mentioned monoclonal antibody 18E2, 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 sequences of the above-mentioned monoclonal antibody 18E2 are as follows: The sequence of the monoclonal antibody 18E2 is as follows: 18E2-LCDR-1: RSSQSLVHSNGNTYLH (SEQ ID No. 1); 18E2-LCDR-2: KVSNRFS (SEQ ID No. 2); 18E2-LCDR-3: SQSTHVPRT (SEQ ID No. 3); 18E2-LFR-1: DVVMTQTPLSLPVSLGDQASISC (SEQ ID No. 4); 18E2-LFR-2: WYLQKPGQSPKLLIY (SEQ ID No. 5); 18E2-LFR-3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID No. 6); 18E2-LFR-4: FGGGTKLEIK (SEQ ID No. 7); The amino acid sequence of the light chain variable region, in which the CDR sequence is indicated by underlining: DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPRTFGGGTKLEIK (SEQ ID No. 8); Nucleic acid sequence of the heavy chain variable region: GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGAGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 9); 18E2-HCDR-1: SYDIS (SEQ ID No. 10); 18E2-HCDR-2: VIWTGGGTNYKSAFMS (SEQ ID No. 11); 18E2-HCDR-3: DTGFYFDY (SEQ ID No. 12); 18E2-HFR-1: QVQLKESGPGLVAPSQSLSITCTVSGFLLI (SEQ ID No. 13); 18E2-HFR-2: WIRQSPGKGLEWLG (SEQ ID No. 14); 18E2-HFR-3: RLTISKDNSKSQVFLKMNSLQTDDTAIYYCVR (SEQ ID No. 15); 18E2-HFR-4: WGQGTTLTVSS (SEQ ID No. 16); Amino acid sequence of the heavy chain variable region, with the CDR sequences underlined: QVQLKESGPGLVAPSQSLSITCTVSGFLLI SYDIS WIRQSPGKGLEWLG VIWTGGGTNYKSAFMS RLTISKDNSKSQVFLKMNSLQTDDTAIYYCVR DTGFYFDY WGQGTTLTVSS (SEQ ID No. 17); Nucleic acid sequence of the heavy chain variable region: CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATTACCTGCACTGTCTCTGGGTTCTTATTAATCAGTTACGATATAAGTTGGATTCGCCAGTCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGGTGGAGGCACAAATTATAAATCAGCTTTCATGTCCAGACTGACCATCAGTAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATATATTACTGTGTAAGAGACACTGGGTTTTATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No. 18).

[0049] b. The sequence of the monoclonal antibody 15D1 is as follows: The 3 complementarity determining regions of the light chain variable region are LCDR-1, LCDR-2, and 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 3 complementarity determining regions of the heavy chain variable region are HCDR-1, HCDR-2, and 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 4 light chain framework regions of the light chain variable region are 15D1-LFR-1, 15D1-LFR-2, 15D1-LFR-3, and 15D1-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 4 heavy chain framework regions of the heavy chain variable region are 15D1-HFR-1, 15D1-HFR-2, 15D1-HFR-3, and 15D1-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; 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: 26 and SEQ ID No: 35, respectively; The nucleic acid sequence encoding the light chain variable region of the above-mentioned monoclonal antibody 15D1, the nucleic acid sequence of the heavy chain variable region are respectively shown as SEQ ID No: 27 and SEQ ID No: 36; The specific sequence of the above-mentioned monoclonal antibody 15D1 is as follows: 15D1-LCDR-1: RSSQSLVHSNGNTYLH (SEQ ID No. 19); 15D1-LCDR-2: KVSNRFS (SEQ ID No. 20); 15D1-LCDR-3: SQSTHVPWT (SEQ ID No. 21); 15D1-LFR-1: DVVMTQTPLSLPVSLGDQASISC (SEQ ID No. 22); 15D1-LFR-2: WYLQKPGQSPKLLIY (SEQ ID No. 23); 15D1-LFR-3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID No. 24); 15D1-LFR-4: FGGGTKLEIK (SEQ ID No. 25); The amino acid sequence of the light chain variable region, wherein the underlined part represents the CDR sequence: DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK (SEQ ID No. 26); The nucleic acid sequence of the light chain variable region: GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 27); 15D1-HCDR-1: SYDIS (SEQ ID No. 28); 15D1-HCDR-2: VIWTGGGTNYNSAFMS (SEQ ID No. 29); 15D1-HCDR-3: DTGFYFDY (SEQ ID No. 30); 15D1-HFR-1: QVQLKESGPGLVAPSQSLSITCTVSGFSLI (SEQ ID No. 31); 15D1-HFR-2: WIRQPPGKGLEWLG (SEQ ID No. 32); 15D1-HFR-3: RLNISKDNSKSQVFLKMNSLQTDDTAIYYCVR (SEQ ID No. 33); 15D1-HFR-4: WGQGTTLTVSS (SEQ ID No. 34); Amino acid sequence of the heavy chain variable region, wherein underlined sequences represent CDR sequences: QVQLKESGPGLVAPSQSLSITCTVSGFSLI SYDIS WIRQPPGKGLEWLG VIWTGGGTNYNSAFMS RLNISKDNSKSQVFLKMNSLQTDDTAIYYCVR DTGFYFDY WGQGTTLTVSS (SEQ ID No. 35); Nucleic acid sequence of the heavy chain variable region: CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATTACCTGCACTGTCTCTGGGTTCTCATTAATCAGCTATGATATAAGCTGGATTCGCCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGGTGGAGGCACAAATTATAATTCAGCTTTCATGTCCAGACTGAACATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATATATTACTGTGTAAGAGACACTGGGTTTTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCGG (SEQ ID No. 36).

[0050] Example 3 Preparation of red latex microsphere antigen test strip for porcine delta coronavirus using porcine delta coronavirus monoclonal antibodies 18E2 and 15D1 I. Cells, plasmids, serum and main reagents: Plasmid pET-28a-PDCoV-N (constructed in Example 1), hybridoma cell strains secreting PDCoV-N protein monoclonal antibodies 18E2 and 15D1, PDCoV-MAb-18E2, PDCoV-MAb-15D1 (prepared in Example 1). Inactivated porcine delta coronavirus (PDCoV), porcine group A rotavirus (PoRVA), porcine reproductive and respiratory syndrome virus (PRRSV), porcine transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV) were provided by Professor Luo Rui's research group of Huazhong Agricultural University. Porcine diarrhea fecal swabs were collected from Chaoyang City, Liaoning Province, Chenzhou City, Hunan Province, Jincheng City, Shanxi Province, Shaoguan City, Guangdong Province, etc.

[0051] II. Preparation of each material of the red latex microsphere antigen test strip for porcine delta coronavirus 1. Preparation of monoclonal antibody 18E2-red latex microsphere marker In the 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%, vortex to mix; after vortexing to mix after adding 5 μL of 10 mg / mL NHS solution, vortexing to mix after adding 5 μL of EDC of the same concentration, vortexing to mix at 20 r / min on a rotary mixer at room temperature for 20 min; centrifuge at 4°C, 13000 r / min for 10 min, discard the supernatant, resuspend with 1 mL of 0.02 mmol / L borate buffer at pH 8.5, repeat centrifugation twice, and ultrasonic dispersion for 2 min; dilute the monoclonal antibody 18E2 with ultrapure water to 0.2 mg / mL, take 50 μg and add to the activated microsphere suspension, mix at 20 r / min on a rotary mixer at room temperature for 2 h; add BSA to a final concentration of 0.5%, block at room temperature on a rotary mixer at 20 r / min for 1 h; centrifuge at 4°C, 13000 r / min for 10 min, discard the supernatant, resuspend with 1 mL of storage solution (0.01 mol / L Tris buffer at pH 9.0), and store at 4°C. Thus, the monoclonal antibody 18E2-red latex microsphere marker of porcine delta coronavirus is obtained.

[0052] The above storage solution is 100 mL of water to which 1.21 g of Tris, 5 g of sucrose, 0.5 g of BSA (bovine serum albumin), 0.5 g of PVP (polyvinyl pyrrolidone, English name: Polyvinyl pyrrolidone), 0.5 g of F68, 0.5 mL of PEG200 (polyethylene glycol 200), 0.5 mL of TritonX-100 (Chinese name TritonX-100, a non-ionic surfactant), and 1 mL of Tween-20 are added.

[0053] 2. Preparation of the binding pad (1) Preparation of the binding pad blocking solution Weigh 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, dissolve with ddH2O, adjust the pH to 7.6, and dilute to 100 mL with a volumetric flask. Filter with a 0.22 μm filter membrane to obtain the binding pad coating solution.

[0054] (2) Preparation of the binding pad Soak the glass cellulose membrane (model BX-01) in the binding pad coating solution for 30 min, and dry it in a 37°C oven to obtain the binding pad.

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

[0056] (2) Preparation of the sample pad The glass cellulose membrane (model BX-03) is soaked in the sample pad sealing liquid for 30 min and dried in a 37°C oven to obtain the sealed binding pad.

[0057] 4. Spraying of the monoclonal antibody 18E2-red latex microsphere marker of porcine delta coronavirus on the binding pad The monoclonal antibody 18E2-red latex microsphere marker is sprayed on the treated binding pad 2, and the spraying amount of the monoclonal antibody 18E2-red latex microsphere marker is 5 μL / cm; after the spraying is completed, the sample pad is dried in a 37°C oven for 2 hours, and then cut into a 3 mm wide strip for standby.

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

[0059] (2) Preparation of the detection line and the quality control line The non-spotting surface of the nitrocellulose membrane 3 is pasted on the polyvinyl chloride bottom plate 7, and the model of the nitrocellulose membrane 3 is Millipore Pall 90s; the monoclonal antibody 15D1 of porcine delta coronavirus is diluted to 0.3 mg / mL with the antibody coating liquid, and the commercialized sheep anti-mouse IgG is diluted to 0.5 mg / mL with the antibody coating liquid, and then sprayed on the positions of the nitrocellulose membranes 4 and 5 at a spraying amount of 1 μL / cm to serve as the detection line and the quality control line, respectively.

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

[0061] III. Assembly of the test strip As Figure 5As shown, the nitrocellulose membrane 3 prepared in the above step 4 is pasted on the polyvinyl chloride base plate 7; the conjugate pad 2 sprayed with the monoclonal antibody 18E2-red latex microsphere marker against swine delta coronavirus prepared in step 3 is pasted on the top of the nitrocellulose membrane 3, covering 1-2 mm on the nitrocellulose membrane 3; the conjugate pad 2 is adjacent to the test line 4 on the nitrocellulose membrane 3; the absorbent pad 6 is pasted on the top of the nitrocellulose membrane 3, covering 1-2 mm on the nitrocellulose membrane 3; the absorbent pad 6 is adjacent to the quality control line 5 on the nitrocellulose membrane 3; the sample pad 1 is pasted on the top of the conjugate pad 2, covering 1-2 mm on the conjugate pad 2; the colloidal gold test strip is cut into 3 mm wide test strips using a strip cutter, and then placed in a test cartridge and a desiccant in an aluminum foil bag for sealed storage.

[0062] Detection of the sensitivity and specificity of the above test strips 1. Test strip sensitivity test Put 10 7 TCID 50 After the inactivated PDCoV strain with a concentration of 10 / mL was diluted 10-fold in series with the above diluent, 80 μL was aspirated for spot detection and the results were observed within 10 minutes.

[0063] The results are as follows Figure 6 As shown: The minimum detection limit of the test strip is 10 3 TCID 50 / mL.

[0064] 2. Specificity detection of test strips Dilute the inactivated PDCoV, PoRVA, PRRSV, TGEV, PRV, and PEDV viruses 20-fold with the above diluent, then aspirate 80 μL for spot detection and observe the results within 10 minutes.

[0065] The results are as follows Figure 7 As shown: There is no cross reaction of the test strip and the test strip has good specificity.

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

[0067] The above dilution solution is: 0.78 g NaH2PO4, 1.42 g Na2HPO4, 2.92 g NaCl, dissolved in ddH2O, adjusted to pH 7.4, and the volumetric flask is made up to 500 mL, and then 2.5 mL NP-40 is added.

[0068] 2. Detection Drop 80 μL of the supernatant on the test strip described in the application, and observe the results within 10 min; the determination basis of the detection results is as follows: As shown in Figure 8 : when red color appears on the quality control line and the detection line, it is a positive result, i.e. the sample contains porcine delta coronavirus (a); Figure 8 when red color appears on the quality control line and no red color appears on the detection line, it is a negative result, i.e. the sample does not contain porcine delta coronavirus (b), Figure 8 if no red line appears on the quality control line, the test strip is invalid (c). Figure 8

[0069] Other parts not described in detail are prior art. Although the above embodiment describes the application in detail, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which belong to the protection scope of the application.​​​

Claims

1. A porcine delta coronavirus monoclonal antibody, characterized in that: The porcine delta coronavirus monoclonal antibody includes monoclonal antibody 18E2 or monoclonal antibody 15D1, The monoclonal antibody 18E2 comprises a light chain variable region and a heavy chain variable region; wherein, The light chain variable region of 18E2 includes three complementarity determining regions, 18E2-LCDR-1, 18E2-LCDR-2, and 18E2-LCDR-3; their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively; The heavy chain variable region of 18E2 includes three complementarity determining regions, namely 18E2-HCDR-1, 18E2-HCDR-2, and 18E2-HCDR-3; their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11, and SEQ ID No: 12, respectively; The monoclonal antibody 15D1 comprises a light chain variable region and a heavy chain variable region; wherein, The light chain variable region of 15D1 includes three complementarity determining regions, 15D1-LCDR-1, 15D1-LCDR-2, and 15D1-LCDR-3; their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20, and SEQ ID No: 21, respectively; The heavy chain variable region of 15D1 includes three complementarity determining regions, namely 15D1-HCDR-1, 15D1-HCDR-2 and 15D1-HCDR-3; their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30, respectively.

2. The porcine delta coronavirus monoclonal antibody according to claim 1, characterized in that: The light chain variable region of the monoclonal antibody 18E2 contains four light chain framework regions, namely LFR-1, LFR-2, LFR-3 and LFR-4; their amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7, respectively; The heavy chain variable region of the monoclonal antibody 18E2 contains four heavy chain framework regions, namely HFR-1, HFR-2, HFR-3 and 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 15D1 contains four light chain framework regions: LFR-1, LFR-2, LFR-3, and LFR-4; their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, and SEQ ID No: 25, respectively; The heavy chain variable region of the monoclonal antibody 15D1 contains four heavy chain framework regions HFR-1, HFR-2, HFR-3 and 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 delta coronavirus 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 porcine delta coronavirus monoclonal antibody 18E2 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 of the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 18E2 are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively.

4. The porcine deltacoronavirus 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 porcine delta coronavirus monoclonal antibody 15D1 are shown in SEQ ID No: 26 and SEQ ID No: 35, respectively; the nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region encoding the porcine delta coronavirus monoclonal antibody 15D1 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively.

5. Use of the porcine deltacoronavirus 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 test strip for porcine delta coronavirus, characterized by: The monoclonal antibody 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 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 18E2 or monoclonal antibody 15D1; and the antibody labeled with the red latex microspheres has an antibody labeling amount of 50 μg / mL; The coated antibody is monoclonal antibody 18E2 or monoclonal antibody 15D1; and the concentration of the coated antibody is 0.3 mg / mL; the concentration of the coated commercial goat anti-mouse IgG is 0.5 mg / mL; 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.

9. A method for preparing the porcine delta coronavirus 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 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.

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 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and red microsphere surface of 1:1:1, then add NHS solution and vortex mix. Then add EDC solution and vortex shake, 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 microsphere-labeled antibody is prepared by the following steps: i. The monoclonal antibody according to claim 1 is added to the activated red latex microsphere suspension and mixed in a rotary mixer at room temperature; wherein the labeled amount of the monoclonal antibody is 50 μg / mL; ii. Then, add BSA to a final concentration of 0.5%, block and mix on a rotary mixer at room temperature, centrifuge, discard the supernatant, resuspend in preservation solution, and store at 4°C; the preservation solution consists of 1.21g Tris, 5g sucrose, 0.5g BSA, 0.5g PVP, 0.5g F68, 0.5mL PEG200, 0.5mL TritonX-100, and 1mL Tween-20 in 100mL of water; The blocking solution of the conjugate pad is 2 g sucrose, 0.3 g PVPK-30, 2 g BSA, 0.02 g NaN3, 0.29 g Na2HPO4·12H2O, and 0.02 g KH2PO4 added to 100 mL of water, with a pH value of 7.6; The blocking solution of the sample pad used 0.5 mol / L Tris-HCl buffer as the base solution, plus 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, and 0.2% casein; The spraying volume of the red latex microsphere-labeled antibody was 5 μL / cm; In step 5), the method for coating with monoclonal antibodies and commercial goat anti-mouse IgG is as follows: The monoclonal antibody and commercial goat anti-mouse IgG were diluted to 0.3 mg / mL and 0.5 mg / mL respectively with antibody coating solution to obtain monoclonal antibody coating solution and commercial goat anti-mouse IgG coating solution; The spraying volume of the monoclonal antibody coating solution and the commercial goat anti-mouse IgG coating solution was 1 μL / cm.

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