Rapid identification of latex double immunochromatographic test strip for detecting tgev and pedv and preparation method thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a latex double immunochromatographic test strip for rapidly identifying porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) and a preparation method thereof. The soluble TGEV N protein and PEDV N protein with immunogenicity and reactivity are obtained by using a prokaryotic expression system, mice are immunized by using the immunogenicity, and the specific TGEV N protein and PEDV N protein monoclonal antibodies are obtained by using cell fusion and subcloning screening technology. The antibody variable region gene sequence is obtained by using nested PCR amplification technology and can be obtained by using genetic engineering or protein engineering method subsequently. Then, the latex double immunochromatographic test strip for detecting TGEV and PEDV and the preparation method thereof are developed based on the specific TGEV N protein and PEDV N protein monoclonal antibodies.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a latex double immunochromatographic test strip for rapidly identifying porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) and a preparation method thereof. BACKGROUND

[0002] Clinically, diarrhea viruses often exist in the form of mixed infection of two or more viruses, which potentially increases the difficulty of prevention and control. Under the current condition that there is no effective vaccine, it is crucial to establish an antigen diagnosis method capable of rapidly and conveniently identifying and detecting multiple viruses at the same time for preventing and controlling the spread of epidemic diseases.

[0003] Porcine transmissible gastroenteritis coronavirus (TGEV) and porcine epidemic diarrhea virus (PEDV) are two enteric coronaviruses causing diarrhea in piglets, and their clinical symptoms and epidemiology are very similar. Both of them show symptoms of watery diarrhea, vomiting and dehydration, especially high mortality rate in newborn piglets. The commonly used pathogen detection methods for both of the pathogens include virus isolation and identification, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR. However, these methods have long detection time, complicated operation, require specific or expensive large instruments and professional technicians, and are difficult to meet the requirements of rapid, inexpensive and efficient monitoring of primary breeding farms.

[0004] Lateral flow immunochromatography technology is a technology for realizing macroscopic visualization or microscopic antigen-antibody reaction through signal amplification by coupling different signal markers on the surface of antigens or antibodies. This technology is simple to operate and can be completed without professional operation, and does not require specific instruments, and the naked eye can obtain the detection result, and the time consumption is short, which can be completed within 15 minutes. The detection time is greatly shortened. The commonly used signal markers are mainly colloidal gold particles and latex microspheres. Although there are various markers on the market, only colloidal gold particles and latex microspheres can be used for large-scale production.

[0005] TGEV N protein is an important nucleocapsid structural protein, which can stimulate the body to produce a large amount of antibodies in the early stage of viral infection. Similar to TGEV N protein, PEDV N protein, as a major nucleocapsid protein, is expressed abundantly and has good genetic sequence conservation, and can stimulate specific antibodies in the early stage of viral infection. Therefore, both of the N proteins have important application value in immunological diagnosis and development of new vaccines. SUMMARY

[0006] The present application uses a prokaryotic expression system to obtain soluble TGEV N protein and PEDV N protein with immunogenicity and reactivity, respectively, to immunize mice with the immunogenicity, and uses cell fusion and subcloning screening technology to obtain specific TGEV N protein and PEDV N protein monoclonal antibodies. The antibody variable region gene sequence is obtained by nested PCR amplification technology, and can be obtained by genetic engineering or protein engineering method subsequently. Then, based on the TGEV N protein and PEDV N protein specific monoclonal antibody, a convenient, rapid and high sensitivity latex double immunochromatography test strip for detecting TGEV and PEDV and a preparation method are developed.

[0007] The present application specifically includes the following contents:

[0008] The present application provides a latex double immunochromatography test strip for rapidly detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The test strip comprises a PVC bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad; the nitrocellulose membrane is provided with a quality control line C and detection lines T1 and T2. The binding pad is sprayed with latex microsphere labeled TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10 as capture antibodies, the detection line T1 is coated with PEDV N protein monoclonal antibody 1E1 as a detection antibody, the detection line T2 is coated with TGEV N protein monoclonal antibody 2A9 as a detection antibody, and the quality control line C is coated with goat anti-mouse IgG.

[0009] The heavy chain variable region of the monoclonal antibody 1B7 of the present application comprises CDR1 with an amino acid sequence of GFNIKDTY, CDR2 with an amino acid sequence of IDPADGYT and CDR3 with an amino acid sequence of ARPGTLDY, and the heavy chain variable region of the monoclonal antibody 2A9 of the present application comprises CDR1 with an amino acid sequence of GYTFTDFN, CDR2 with an amino acid sequence of INPNNGRS and CDR3 with an amino acid sequence of ARRHWDWYFDV.

[0010] The light chain variable region of the monoclonal antibody 1B7 of the present application comprises CDR1 with an amino acid sequence of KSVSTSGYSY, CDR2 with an amino acid sequence of LVS and CDR3 with an amino acid sequence of QHIRELTR, and the light chain variable region of the monoclonal antibody 2A9 of the present application comprises CDR1 with an amino acid sequence of KSLLHSN, CDR2 with an amino acid sequence of QMS and CDR3 with an amino acid sequence of AQNLEFPWT.

[0011] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B7 of the present application is shown as SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A9 of the present application is shown as SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 4.

[0012] The DNA sequence encoding the heavy chain variable region of the monoclonal antibody 1B7 is shown as SEQ ID NO: 5, and the DNA sequence encoding the light chain variable region of the monoclonal antibody 1B7 is shown as SEQ ID NO: 6; the DNA sequence encoding the heavy chain variable region of the monoclonal antibody 2A9 is shown as SEQ ID NO: 7, and the DNA sequence encoding the light chain variable region of the monoclonal antibody 2A9 is shown as SEQ ID NO: 8.

[0013] The heavy chain constant region of the porcine transmissible gastroenteritis coronavirus N protein monoclonal antibody of the present application is IgG1 or IgG2b type. The light chain constant region of the porcine transmissible gastroenteritis coronavirus N protein monoclonal antibody of the present application is Kappa type.

[0014] The heavy chain variable region of the monoclonal antibody 1F10 of the present application comprises CDR1 with the amino acid sequence GYTFTTYY, CDR2 with the amino acid sequence IYPGNINT, and CDR3 with the amino acid sequence ARISSALPY, and the heavy chain variable region of the monoclonal antibody 1E1 of the present application comprises CDR1 with the amino acid sequence GYTFTAYV, CDR2 with the amino acid sequence INPYNDRT, and CDR3 with the amino acid sequence ARDDDDYEEGFAY.

[0015] The light chain variable region of the monoclonal antibody 1F10 of the present application comprises CDR1 with the amino acid sequence RSLLNTSSQKSY, CDR2 with the amino acid sequence FAS, and CDR3 with the amino acid sequence QQHFSTPPT, and the light chain variable region of the monoclonal antibody 1E1 of the present application comprises CDR1 with the amino acid sequence QKCQYIWLSL, CDR2 with the amino acid sequence SCI, and CDR3 with the amino acid sequence SAHSGAYT.

[0016] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F10 of the present application is shown as SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 10; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E1 of the present application is shown as SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 12.

[0017] The DNA sequence encoding the heavy chain variable region of the monoclonal antibody 1F10 is shown as SEQ ID NO: 13, and the DNA sequence encoding the light chain variable region of the monoclonal antibody 1F10 is shown as SEQ ID NO: 14; the DNA sequence encoding the heavy chain variable region of the monoclonal antibody 1E1 is shown as SEQ ID NO: 15, and the DNA sequence encoding the light chain variable region of the monoclonal antibody 1E1 is shown as SEQ ID NO: 16.

[0018] The heavy chain constant region of the PEDV N protein monoclonal antibody of the present application is of IgG1 type. The light chain constant region of the PEDV N protein monoclonal antibody of the present application is of Kappa type.

[0019] Preferably, the preparation method of the binding pad is as follows:

[0020] The binding pad is blocked with a binding pad blocking solution;

[0021] After the latex microspheres are activated with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended with MES buffer, and ultrasonically dispersed to fully disperse the fluorescent microspheres, TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10 are added, incubated at room temperature, then a latex microsphere marker blocking solution is added for blocking, incubated at room temperature, and centrifuged; the precipitate is resuspended with a latex microsphere marker protective agent to obtain a latex microsphere marker mixed solution of TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10;

[0022] The prepared latex microsphere marker mixed solution is uniformly sprayed on the blocked binding pad using an instrument;

[0023] The binding pad blocking solution is an ultrapure water solution containing 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, and 1.5% m / v PVP-40.

[0024] The latex microsphere marker blocking solution is an ultrapure water solution containing 10% m / v BSA;

[0025] The latex microsphere marker protective agent is a Tris-HCl solution containing 3% m / v sucrose, 1% m / v PVP-40, and 1% v / v Tween-20.

[0026] The latex microsphere marker mixed solution of TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10 consists of 40 μg TGEV N protein monoclonal antibody 1B7 and 60 μg PEDV N protein monoclonal antibody 1F10 per 1 mL of latex microsphere solution.

[0027] Preferably, the detection line T1 is coated with 1 mg / mL PEDV N protein monoclonal antibody 1E1.

[0028] Preferably, the detection line T2 is coated with 1 mg / mL TGEV N protein monoclonal antibody 2A9.

[0029] Preferably, the quality control line C is coated with 1 mg / mL goat anti-mouse IgG.

[0030] Preferably, the sample pad is soaked with a sample pad blocking solution; the sample pad blocking solution is a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate.

[0031] In a sixth aspect, the present application provides a preparation method of the rapid detection of swine infectious gastroenteritis and porcine epidemic diarrhea virus latex double immunochromatography test strip. The method comprises the following steps:

[0032] (1) preparing a conjugate pad coated with latex microsphere labeled TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10;

[0033] (2) spraying PEDV N protein monoclonal antibody 1E1 (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) on the nitrocellulose membrane with an interval of 3 mm, respectively as the detection line T1 and the quality control line C; spraying TGEV N protein monoclonal antibody 2A9 (1 mg / mL) 5 mm away from the PEDV N protein monoclonal antibody 1E1 (1 mg / mL) as the detection line T2.

[0034] (3) sequentially lapping the sample pad, the conjugate pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate to obtain the latex double immunochromatography test strip for identifying and detecting swine infectious gastroenteritis and porcine epidemic diarrhea virus.

[0035] In a seventh aspect, the present application provides a detection method of the test strip, which comprises the following steps:

[0036] (1) processing of TGEV negative and positive control samples

[0037] PK15 cells were inoculated with TGEV to cover the monolayer, and after 60 hours of inoculation, they were repeatedly frozen and thawed 3 times in a-80℃ refrigerator, centrifuged at 12000 rpm and 4℃ for 10 min, the cell debris was removed, and the supernatant was taken as the detected antigen. The uninfected PK15 cells were treated in the same way as the negative control.

[0038] (2) processing of PEDV negative and positive control samples

[0039] VeroE6 cells were inoculated with PEDV and incubated for 60 hours, then frozen and thawed 3 times in a -80℃ refrigerator, centrifuged at 12000 rpm for 10 minutes at 4℃, and the supernatant was taken as the antigen to be detected. Uninoculated VeroE6 cells were treated in the same way as the negative control.

[0040] (3) Result determination

[0041] After the reaction, the positive and negative results were determined by visual inspection.

[0042] The present application has the following advantages:

[0043] The latex double immunochromatography test strip for identifying and detecting porcine epidemic gastroenteritis virus and porcine epidemic diarrhea virus provided by the present application has no non-specific reaction when detecting TGEV or PEDV alone, and does not have cross-reactions with other pig-derived enteric coronaviruses SADS-CoV and PDCoV that have similar clinical symptoms. At the same time, the test strip can detect the minimum limit of viruses in the TGEV and PEDV positive controls, which is 10 4.69 TCID 50 / 0.1mL and 10 3.09 TCID 50 / 0.1mL, and the prepared test strip has good specificity, sensitivity, simple operation, short time, etc. It provides a rapid detection method for the rapid identification and detection of two kinds of diarrhea viruses in the field or laboratory. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 It is the PCR amplification result of TGEV N gene. M: DL2000 relative molecular mass standard; 1: control water; 2: N gene, about 1000bp in size.

[0045] Figure 2 It is the expression and purification result of TGEV pET32a-N recombinant protein. (A) M: protein Marker; 1: pET32a-N before induction; 2: pET32a-N after induction; 3: pET32a-N lysis supernatant; 4: pET32a-N after lysis. (B) M: protein Marker; 1: purified pET32a-N protein.

[0046] Figure 3 It is the ELISA detection of mouse antibody titer after immunization of TGEV N protein. 1-3: 1, 2 and 3 immunized mice, NC: negative control.

[0047] Figure 4SDS-PAGE identification results of TGEV N protein antibody purification. M: protein Marker, 1: 1B7 monoclonal antibody; 2: 2A9 monoclonal antibody.

[0048] Figure 5 IFA identification results of TGEV N protein monoclonal antibody reactivity. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) SP2 / 0 cell culture solution.

[0049] Figure 6 Western blot identification results of TGEV N protein monoclonal antibody reactivity. M: protein Marker, Mock: PK-15 cell control; TGEV: TGEV infected PK-15 cell.

[0050] Figure 7 TGEV N protein monoclonal antibody subclass identification results.

[0051] Figure 8 TGEV N protein monoclonal antibody variable region PCR amplification results. (A) Heavy chain variable region PCR amplification results. M: DL2000 Marker; 1: water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody; (B) Light chain K variable region PCR amplification results. M: DL2000 Marker; 1: water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody.

[0052] Figure 9 PEDV N gene PCR amplification and pET32a-N recombinant plasmid enzyme digestion identification results. (A) M: DL2000 relative molecular mass standard; 1: water control; 2: N gene, about 1300 bp in size; (B) M: DL5000 relative molecular mass standard; 1: pET32a-N recombinant plasmid double enzyme digestion with BamHI and XhoI.

[0053] Figure 10 pET32a-N recombinant protein expression and purification results. (A) M: protein Marker; 1: pET32a-N before induction; 2: pET32a-N after induction; 3: pET32a-N lysis supernatant; 4: pET32a-N after lysis precipitation; (B) M: protein Marker; 1: supernatant; 2: flow-through; 3-4: 25 μM imidazole elution; 5-6: 50 μM imidazole elution; 7-8: 75 μM imidazole elution; 9-10: 100 μM imidazole elution; 11: 150 μM imidazole elution; 12: 200 μM imidazole elution; 13: 300 μM imidazole elution; 14: 500 μM imidazole elution.

[0054] Figure 11The figure shows the results of ELISA detection of the titer of polyclonal antibody against mouse PEDV N protein.

[0055] Figure 12 SDS-PAGE identification of purified PEDV N protein monoclonal antibody. M: Protein Marker; 1: 1F10 monoclonal antibody purification; 2: 1E1 monoclonal antibody purification.

[0056] Figure 13 To verify the reactivity of the PEDV N protein monoclonal antibody for IFA. (A) Supernatant of 1F10 hybridoma cells; (B) Supernatant of 1E1 hybridoma cells; (C) Supernatant of SP2 / 0 cells.

[0057] Figure 14 To identify the reactivity of the PEDV N protein monoclonal antibody by Western blot; M: protein marker, MockVeroE6 cell control; PEDV: PEDV-infected VeroE6 cells.

[0058] Figure 15 The results of the identification of PEDV N protein monoclonal antibody subclasses.

[0059] Figure 16 The results show the PCR amplification of the variable region of the PEDV N protein monoclonal antibody. (A) PCR amplification results of the heavy chain variable region. M: DL2000 Marker; 1F10: PCR amplification of the heavy chain variable region; 1E1: PCR amplification of the heavy chain variable region; (B) PCR amplification results of the light chain K variable region. M: DL2000 Marker; 1F10: PCR amplification of the light chain variable region; 1E1: PCR amplification of the light chain variable region.

[0060] Figure 17 Schematic diagram of TGEV-PEDV dual-strand latex test strip.

[0061] Figure 18 Schematic diagram for determining the results of TGEV-PEDV dual-strand latex test strips.

[0062] Figure 19 Sensitivity test results of TGEV-PEDV dual-use latex test strips. 1-9: First, both PEDV and TGEV positive controls were started from 2... 1 Serial dilution to 2 9 Then, each of the above samples was diluted 1:5 as an independent sample, and finally tested one by one using test strips. 10:NC

[0063] Figure 20Specific detection results of TGEV-PEDV double test strip; 1: positive control for detecting PEDV and TGEV at the same time; 2: positive control for detecting PEDV only; 3: positive control for detecting TGEV only; 4: detecting SADS positive antigen; 5: detecting PDCoV positive antigen; 6: NC. DETAILED DESCRIPTION

[0064] The application will be described in more detail below with specific embodiments, so as to facilitate the understanding of the technical solutions of the application, but not for limiting the protection scope of the application.

[0065] Example 1 Preparation of TGEV N protein monoclonal antibody

[0066] 1. Construction, expression and purification of TGEV N protein recombinant plasmid

[0067] 1.1 Construction of pET32a-N recombinant plasmid

[0068] The TGEV strain is HLJ-17 strain (Genbank accession number: MT522161.1), and the N protein of the strain is used as a reference to design specific primers. The upstream P1 is 5'-CGC GGATCC ATGGCCAACCAGGGACAACG-3', and the downstream P2 is 5'-CCG CTCGAG TTAGTTCGTTACCTCATCAATTATC-3', and the 5' end of the upstream and downstream primers is introduced with a BamHI and XhoI enzyme cutting site (underlined part). After synthesis of the primers, the TGEV cell supernatant RNA of the infected PK15 cells is extracted, reverse transcribed into cDNA, and then used as a template for PCR amplification. The PCR reaction system is: PrimeSTAR Max Premix (2x) 25 μL, P1 and P2 each 1 μL, cDNA 1 μL, ddH2O supplemented to 50 μL. The reaction program is: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 10 min. The PCR product is detected by 1% agarose gel electrophoresis, and the result is shown in Figure 1

[0069] The N gene PCR gel recovery product of TGEV is double-enzyme cut with BamHⅠand XhoI, and then linked, and the DH5α competent cells are transformed. The constructed recombinant plasmid is sequenced and identified, and the positive recombinant plasmid is named as pET32a-N.

[0070] ​1. Induction expression and purification of recombinant TGEV N protein

[0071] The recombinant plasmid pET32a-N was transformed into E. coli BL21(DE3) and a single colony was picked into 5 mL LB medium containing ampicillin. When the OD 600 was about 0.8, the expression was induced by adding IPTG to a final concentration of 1 mmol / L. Five hours after induction, the precipitate was collected by centrifugation at 12000 rpm for 2 min. The precipitate was resuspended in PBS and treated by ultrasonication for 5 min (ultrasonic for 3 s and stop for 3 s). The supernatant and the precipitate were collected by centrifugation at 12000 rpm for 10 min at 4°C. The supernatant and the precipitate were boiled in 5x loading buffer for 10 min and subjected to SDS-PAGE. The results showed that pET32a-N was expressed in both the supernatant and the precipitate (Fig. 2A), indicating that the TGEV N protein was partially soluble. Figure 2

[0072] The expression of pET32a-N was scaled up according to the above method. The lysed supernatant after ultrasonication was subjected to nickel column purification. The specific operation steps of purification were as follows:

[0073] (1) Resin loading: an empty column was taken and 2 mL nickel column NTA resin was added. When the storage solution descended to the surface of the resin, the column was washed once with 5 times the column volume of distilled water, and then the column was equilibrated with 5 times the column volume of equilibration buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.4);

[0074] (2) Sample loading: when the equilibration buffer descended to the surface of the resin, 3 mL of the lysed supernatant containing the recombinant protein was added. The sample loading was repeated 2-3 times, each time for 2 min, and the sample flow-through was collected. The column was washed once with 5 times the column volume of equilibration buffer;

[0075] (3) Protein elution: the protein sample was eluted with equilibration buffer containing different concentrations of imidazole (25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM and 300 mM), 1 mL each time, and each concentration was repeated twice. The purpose was to determine the optimal concentration of imidazole for elution and protein elution;

[0076] (4) Column washing and preservation: the column was washed once with 5 times the volume of 0.5 M NaOH, and then washed once with distilled water. Subsequently, an appropriate amount of 70% anhydrous ethanol was added, and the column was stored in a 4°C refrigerator. Each collected sample was subjected to SDS-PAGE identification. As shown in Fig. 2B, a TGEV N protein with high purity was obtained. Figure 2

[0077] 2. Animal immunization ​​

[0078] Select 6W (week) age female BALB / c mice 3, the purified N recombinant protein according to 30 μg / only immunization mice. First immunization, N protein and Freund's complete adjuvant mixed with equal volume after emulsification, using multiple point subcutaneous injection of mice (back one point, two points in the abdomen). Every 2W for booster immunization, a total of four times, booster immunization is N recombinant protein and Freund's incomplete adjuvant mixed with equal volume emulsification, immunization method with the first immunization. Four 7d (days) after the tail blood to determine the antibody titer.

[0079] 3. ELISA method for detecting polyclonal antibody titer

[0080] TGEV virus liquid and coating solution 1:1 dilution coated ELISA plate, 50 μL / well, 4℃ overnight coating, PBST wash four times, 2% fucose 4℃ blocking 10h. Positive serum and negative serum with PBST dilution, dilution gradient from 1:100 to 1:12800, dilution of 8 gradient, 37℃ for 1h, PBST wash four times, HRP labeled goat anti-mouse IgG (1:20000 dilution), PBST wash four times, TMB color development, placed on a microplate reader to determine OD 450 .

[0081] The results are shown in Figure 3 , with non-immune mouse serum as negative control (NC), serum 1:102400 dilution, 1, 2 and 3 immunized mice OD 450 still greater than the NC group, indicating that the antibody titer can reach 1:102400 or more.

[0082] 4. Preparation of monoclonal antibody

[0083] The specific operation steps are as follows:

[0084] (1) Preparation of feeder cells: HAT culture fluid injection female BALB / c mouse abdominal cavity, slowly repeated aspiration, liquid suction, spread in 96-well plates.

[0085] (2) Cell fusion: the spleen cells and appropriate amount of SP20 cells in the presence of fusion agent PEG cell fusion. The cells after fusion were plated in 96-well plates containing feeder cells.

[0086] (3) Screening of positive clones:

[0087] a) indirect ELISA detection method, purified TGEV N protein coated ELISA plate, detection of antibody secretion by fusion cells.

[0088] b) Indirect immunofluorescence (IFA) detection method, the supernatant in ELISA antibody positive cell hole, add TGEV infected PK15 cells, then add FITC labeled goat anti-mouse IgG, after the reaction is completed, observe the results under fluorescence microscope.

[0089] (4) Subcloning of positive hybridoma cells: ELISA and IFA positive wells were selected, and the selected positive hybridoma cells were subcloned by limiting dilution method. Under an inverted microscope, mark the wells with only single clone growth, take the supernatant, and detect the antibody by the above-mentioned ELISA and IFA methods. The positive cells enter the next round of subcloning, and a total of three times.

[0090] (5) Preparation of ascites: 10-12W Balb / c mice were selected, and each mouse was injected with Freund's incomplete adjuvant 0.5mL in the abdominal cavity. After 1W, each mouse was injected with 5x10 5 hybridoma cells (0.2mL) in the abdominal cavity, and 7-10 days later, the abdominal cavity of the mouse was obviously swollen. The ascites was collected, the titer was detected by ELISA, and it was stored at -80℃.

[0091] (6) Purification of ascites: affinity chromatography purification was performed according to PIERCE company NAbTM Protein G Spin Purification Kit, and the purity was identified by SDS-PAGE electrophoresis. The heavy chain and light chain of 1B7 monoclonal antibody and 2A9 monoclonal antibody obtained after purification were about 55kDa and about 25kDa Figure 4 respectively, indicating that the purification method after purification was correct, and the purity was high.

[0092] 5. Identification of monoclonal antibody

[0093] (1) Specificity identification: including IFA and western blot verification.

[0094] a) IFA verification: TGEV infected PK15 cells, 36h after virus infection, cells were fixed with polyethylene glycol, after cell fixation, ascites was diluted with PBS (500 times) and added to virus infected cells, then incubated with FITC-anti mouse secondary antibody (100 times dilution), and then observed under fluorescence microscope after reaction. The results are shown in Figure 5 A and 5B, monoclonal antibodies 1B7 and 2A9 can detect specific green fluorescent signal in TGEV infected PK15 cells, and no fluorescent signal is observed in TGEV infected cells with SP2 / 0 cell supernatant Figure 5

[0095] ​b) Western blot verification: The purified TGEV N protein or the virus infected and uninfected PK15 cells were collected and subjected to SDS-PAGE, then the protein gel was transferred to NC membrane for western blot verification. The primary antibody was monoclonal antibody 1B7 or 2A9, and the secondary antibody was HRP-anti-mouse IgG. The results are shown in Figure 2, and both monoclonal antibodies 1B7 and 2A9 can recognize the TGEV N protein in virus infected cells. Figure 6

[0096] (2) Subclass identification: The obtained monoclonal antibodies were subjected to antibody subclass identification according to the operation instruction of SBA Clonotyping™ System / HRP Antibody Subclass Identification Kit of Southern Biotech Company. The results are shown in Figure 3, and the heavy chain constant region of 1B7 monoclonal antibody is IgG1 type, and that of 2A9 is IgG2b type, and the light chain constant region of them is Kappa type. Figure 7

[0097] 6. PCR amplification and sequence determination of variable region genes of monoclonal antibodies

[0098] Firstly, the RNA of monoclonal antibody hybridoma cells was extracted, and the cDNA was synthesized by reverse transcription using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0099] The variable region genes of antibodies were amplified by nested PCR. Firstly, the variable region genes of antibodies were amplified by using the first round of mouse antibody IgG1 and kappa light chain primers as templates, and then the variable region genes of antibodies were amplified by using the first round of products as templates and the second round of mouse antibody IgG1 and kappa light chain primers. The PCR reaction system was: PrimeSTAR Max Premix (2x) 25 μL, P1 and P2 each 1 μL, cDNA 1 μL, ddH2O supplemented to 50 μL. The reaction program was: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min. The primers for amplifying the variable region genes of antibodies were referred to the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, A. D., Wang, Q., Gazumyan, A., Nussenzweig, M. C., 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.).​​

[0100] After amplification, 1% agarose gel electrophoresis was performed, and the heavy chain and kappa light chain variable region genes of the monoclonal antibodies 1B7 and 2A9 were about 300 bp in size (Figs. 8A and 8B), and the target fragments were recovered by cutting the gel. The recovered target fragments were inserted into the pMD-19T vector, and sequence determination was performed. Figure 8

[0101] The DNA sequence of the heavy chain variable region of the monoclonal antibody 1B7 is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; the DNA sequence of the heavy chain variable region of the monoclonal antibody 2A9 is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0102] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B7 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A9 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0103] The heavy chain variable region of the monoclonal antibody 1B7 includes CDR1 with an amino acid sequence of GFNIKDTY, CDR2 with an amino acid sequence of IDPADGYT, and CDR3 with an amino acid sequence of ARPGTLDY; the heavy chain variable region of the monoclonal antibody 2A9 includes CDR1 with an amino acid sequence of GYTFTDFN, CDR2 with an amino acid sequence of INPNNGRS, and CDR3 with an amino acid sequence of ARRHWDWYFDV.

[0104] The light chain variable region of the monoclonal antibody 1B7 includes CDR1 with an amino acid sequence of KSVSTSGYSY, CDR2 with an amino acid sequence of LVS, and CDR3 with an amino acid sequence of QHIRELTR; the light chain variable region of the monoclonal antibody 2A9 includes CDR1 with an amino acid sequence of KSLLHSN, CDR2 with an amino acid sequence of QMS, and CDR3 with an amino acid sequence of AQNLEFPWT.

[0105] Example 2 Preparation of a PEDV N protein monoclonal antibody

[0106] 1. Construction, expression, and purification of a PEDV N protein recombinant plasmid

[0107] 1.1 Construction of a pET32a-N recombinant plasmid

[0108] ​PEDV CV777 strain N protein (Genbank accession number: AF353511.1) as a reference, specific primers were designed, upstream P1: 5'-CGC GGATCC ATGGCTTCTGTCAGCTTTCA-3', downstream P2: 5'-CCG CTCGAG TTAATTTCCTGTATCGAAGATC-3', introducing BamHI and XhoI enzyme cutting sites (underlined part) at 5' end of upstream and downstream primers. Amplification, enzyme cutting and ligation were performed according to 1.1 in Reference Example 1.

[0109] PEDV N gene PCR amplification results are shown in Figure 9 A, and double enzyme cutting identification results are shown in Figure 9 B.

[0110] 1.2 Induction expression and purification of PEDV N recombinant protein

[0111] Induction, expression and purification of PEDV N recombinant protein were performed according to 1.2 in Reference Example 1. As shown in Figure 10 A and 10B, PEDV N protein with high purity was obtained.

[0112] 2. Animal immunization

[0113] The method was the same as 2 in Example 1.

[0114] 3. ELISA method for detecting polyclonal antibody titer

[0115] The detection method was the same as 3 in Example 1.

[0116] The results showed that the OD values of serum 1, 2 and 3 were 0. 1, 0. 2 and 0. 3 respectively when the serum was diluted 12800 times, indicating that the antibody titer could reach more than 1:12800. 450 / NC>2.0. Figure 11

[0117] 4. Preparation of monoclonal antibody

[0118] Preparation of monoclonal antibody and ascites purification method were the same as 4 in Example 1. After ascites purification, SDS-PAGE identification was performed, and the results are shown in Figure 12 .

[0119] 5. Identification of monoclonal antibody

[0120] After PEDV infected VeroE6 cells, PEDV N protein monoclonal antibody was verified for its reactivity by IFA and western blot. IFA verification results are shown in Figure 13 ​As shown in A and 13B, the obtained monoclonal antibodies 1F10 and 1E1 can detect specific green fluorescent signals in PEDV infected VeroE6 cells, while no fluorescent signals are observed in PEDV infected cells treated with supernatant of SP2 / 0 cells Figure 13 C) Western blot results are shown in Figure 14 As shown in A and 13B, the obtained monoclonal antibodies 1F10 and 1E1 can detect specific green fluorescent signals in PEDV infected VeroE6 cells, while no fluorescent signals are observed in PEDV infected cells treated with supernatant of SP2 / 0 cells

[0121] The obtained monoclonal antibodies were subjected to antibody subclass identification according to the operation instruction of antibody subclass identification kit. The results are shown in Figure 15 As shown in A and 13B, the obtained monoclonal antibodies 1F10 and 1E1 can detect specific green fluorescent signals in PEDV infected VeroE6 cells, while no fluorescent signals are observed in PEDV infected cells treated with supernatant of SP2 / 0 cells

[0122] 6. PCR amplification and sequence determination of variable region genes of monoclonal antibodies

[0123] The variable region sequences of monoclonal antibodies 1F10 and 1E1 against PEDV N protein were amplified according to the method in 6 of Example 1. The amplification results are shown in Figure 16 A and 16B. The recovered target fragments were inserted into pMD-19T vector for sequence determination.

[0124] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10; the amino acid sequence of the heavy chain variable region of monoclonal antibody 1E1 of the present application is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12.

[0125] The DNA sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 13, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 14; the DNA sequence of the heavy chain variable region of monoclonal antibody 1E1 of the present application is shown in SEQ ID NO: 15, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0126] The heavy chain variable region of monoclonal antibody 1F10 comprises CDR1 with the amino acid sequence of GYTFTTYY, CDR2 with the amino acid sequence of IYPGNINT, and CDR3 with the amino acid sequence of ARISSALPY; the heavy chain variable region of monoclonal antibody 1E1 of the present application comprises CDR1 with the amino acid sequence of GYTFTAYV, CDR2 with the amino acid sequence of INPYNDRT, and CDR3 with the amino acid sequence of ARDDDDYEEGFAY.

[0127] The light chain variable region of the monoclonal antibody 1F10 comprises a CDR1 with an amino acid sequence of RSLLNTSSQKSY, a CDR2 with an amino acid sequence of FAS, and a CDR3 with an amino acid sequence of QQHFSTPPT, and the light chain variable region of the monoclonal antibody 1E1 of the present application comprises a CDR1 with an amino acid sequence of QKCQYIWLSL, a CDR2 with an amino acid sequence of SCI, and a CDR3 with an amino acid sequence of SAHSGAYT.

[0128] Example 3 Preparation of a latex double immunochromatographic test strip for detecting porcine transmissible gastroenteritis coronavirus

[0129] 1. Preparation of the conjugate pad

[0130] 1.1 Preparation of a latex microsphere labeling mixed solution of the TGEV N protein monoclonal antibody 1B7 and the PEDV N protein monoclonal antibody 1F10

[0131] Take 975 μL of 0.1M pH 5.6 MES buffer into a 2mL centrifuge tube, then add 25 μL of latex microspheres with a solid content of 4% (Suzhou Fudoo Biotechnology Co., Ltd.) and mix gently; then add 10 μL of 20mg / mL EDC (purchased from sigma, catalog number: 03449) and 5 μL of 20mg / mL NHS (purchased from sigma, catalog number: 8045180025), mix thoroughly, and activate at room temperature for 20min, centrifuge at 17000r for 20min; remove the supernatant, resuspend with 1mL of MES buffer, and centrifuge again at 17000r for 20min; at the same time, add 40 μg of TGEV N protein monoclonal antibody 1B7 and 60 μg of PEDV N protein monoclonal antibody 1F10, and incubate at room temperature for 2h; add 200 μL of 10w / v% BSA solution for blocking, incubate at room temperature for 1h, centrifuge at 17000r for 10min, and discard the supernatant; finally, resuspend with 2mL of latex microsphere marker protector; if there is obvious sedimentation that cannot be mixed, ultrasonic at 100W for 3s, stop for 3s, and ultrasonic for a total of 1min, and store at 4°C for standby.

[0132] 1.2 Preparation of a latex microsphere conjugate pad

[0133] The binding pad was completely soaked in the binding pad blocking solution (5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, 1.5% m / v PVP-40 and 0.05% Krovin 300 in ultrapure water) at 37-38°C for 2 h, and then taken out to be dried in an oven for 2-4 h. The mixed solution of the latex microspheres labeled with the prepared TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10 was uniformly sprayed on the binding pad by a film sprayer, and then dried in an oven at 37°C for 2-3 h, and stored in a sealed manner.

[0134] 2. Preparation of sample pad

[0135] The sample pad was soaked in the sample pad blocking solution (2% BSA, 0.5% S17, 2% PEG200000, 0.5% sodium caseinate and 0.05% Krovin 300 in Tris-HCl solution) at 37-38°C for 2 h, and then taken out to be dried in an oven for 2-4 h, and then dried in an aluminum foil bag for standby.

[0136] 3. Preparation of nitrocellulose membrane

[0137] The PEDV N protein monoclonal antibody 1E1 (1 mg / mL) and the goat anti-mouse IgG antibody (1 mg / mL) were sprayed on the nitrocellulose membrane with an interval of 3 mm, as the detection line T1 and the quality control line C, respectively; and the TGEV protein monoclonal antibody 2A9 (1 mg / mL) was sprayed 5 mm away from the PEDV N protein monoclonal antibody 1E1 (1 mg / mL), as the detection line T2.

[0138] 4. Assembly of test strip

[0139] The sample pad, the binding pad, the nitrocellulose membrane and the water absorption pad were sequentially connected on the PVC base plate to obtain a test strip, the test strip was cut according to the size of the test strip shell, and then the cut test strip was loaded into the test strip shell. The structure diagram of the test strip is shown in Figure 17 .

[0140] Example 4 Application of a latex double immunochromatographic test strip for detecting porcine transmissible gastroenteritis and porcine epidemic diarrhea coronavirus

[0141] 1. Method for using the test strip

[0142] (1) Detection: the test strip was taken out and balanced to room temperature, the sample to be detected was added to the sample addition area, and the result was determined after standing at room temperature.

[0143] (2) Result determination: The sample to be detected is diluted 1:5 with sample diluent (PBS, pH 7.4), 100 μL of the sample is dropped on the sample pad, and the result is observed within 15 min at room temperature; if the quality control line C and the detection line T1 develop color, and the detection line T2 does not develop color, it indicates that PEDV is positive and TGEV is negative; if the quality control line C and the detection line T2 develop color, and the detection line T1 does not develop color, it indicates that TGEV is positive and PEDV is negative; if the quality control line C, the detection line T1 and T2 all develop color, it indicates that both PEDV and TGEV are positive; if the quality control line C develops color, and the detection lines T1 and T2 do not develop color, it is negative; if the quality control line C does not develop color, it is determined that the test strip is invalid. As shown in Figure 18 .

[0144] 2. Sensitivity of the test strip

[0145] TGEV (TCID 50 10 7.8 / 0.1 mL) and PEDV (TCID 50 10 6.2 / 0.1 mL) virus solution are diluted 1:5, and then 2-fold dilution is performed to detect the sensitivity of the established test strip. The results show that when the virus is diluted 1:2 8 , the detection value is still positive, and when it is diluted 1:2 9 , the detection value is negative. Therefore, the sensitivity of the test strip prepared in the present application can reach 1:2 8 , that is, the minimum detection amount of TGEV and PEDV virus is 10 4.69 TCID 50 / 0.1 mL and 10 3.09 TCID 50 / 0.1 mL, respectively, as shown in Figure 19 .

[0146] 3. Specificity of the test strip

[0147] TGEV, PEDV, SADS-CoV and PDCoV are detected by the test strip, it is found that when only PEDV is detected, T1 develops color and is positive, and T2 does not develop color and is negative; when only TGEV is detected, T2 develops color and is positive, and T1 does not develop color and is negative; the above indicates that there is no cross between the two. SADS-CoV and PDCoV do not react, which indicates that the method established in the present application can specifically recognize TGEV and PEDV, and there is no non-specific cross reaction with the rest of the easily confused porcine diarrhea coronavirus, and the results are shown in Figure 20 .

[0148] 4. Stability test

[0149] Stability is an important factor for field application. The test strips were stored at room temperature (18-25℃) and 4℃, respectively. Stability test was performed at 0, 1, 3, 5, 7, 9, 12 and 15 months. As shown in Table 1, the test strips can be stored at room temperature for 7 months and at 4℃ for 12 months.

[0150] Table 1 Stability test results of test strips

[0151]

[0152] Note: - : negative; +, ++, +++ : positive degree in turn enhanced.

[0153] The above-described embodiments are only the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Any equivalent changes or modifications made according to the structure, features and principles described in the patent scope of the present application should be included in the patent scope of the present application.

Claims

1. A colloidal gold double immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus, characterized in that, The test strip comprises a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad; the nitrocellulose membrane is provided with a quality control line C and detection lines T1 and T2; the conjugate pad is sprayed with latex microsphere-labeled pig infectious gastroenteritis virus N protein monoclonal antibody A and porcine epidemic diarrhea virus N protein monoclonal antibody A as capture antibodies; the detection line T1 is coated with epidemic diarrhea virus N protein monoclonal antibody B as a detection antibody; the detection line T2 is coated with pig infectious gastroenteritis virus N protein monoclonal antibody B as a detection antibody; and the quality control line C is coated with goat anti-mouse IgG; The pig epidemic diarrhea virus N protein monoclonal antibody A has a heavy chain variable region comprising CDR1 with an amino acid sequence of GYTFTTYY, CDR2 with an amino acid sequence of IYPGNINT and CDR3 with an amino acid sequence of ARISSALPY, and a light chain variable region comprising CDR1 with an amino acid sequence of RSLLNTSSQKSY, CDR2 with an amino acid sequence of FAS and CDR3 with an amino acid sequence of QQHFSTPPT; The pig epidemic diarrhea virus N protein monoclonal antibody B has a heavy chain variable region comprising CDR1 with an amino acid sequence of GYTFTAYV, CDR2 with an amino acid sequence of INPYNDRT and CDR3 with an amino acid sequence of ARDDDDYEEGFAY, and a light chain variable region comprising CDR1 with an amino acid sequence of QKCQYIWLSL, CDR2 with an amino acid sequence of SCI and CDR3 with an amino acid sequence of SAHSGAYT.

2. The test strip according to claim 1, wherein The pig epidemic diarrhea virus N protein monoclonal antibody A has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10; The pig epidemic diarrhea virus N protein monoclonal antibody B has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 11 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

12.

3. The test strip according to claim 2, wherein The pig epidemic diarrhea virus N protein monoclonal antibody A has a heavy chain variable region with a gene sequence as shown in SEQ ID NO: 13 and a light chain variable region with a gene sequence as shown in SEQ ID NO: 14; The pig epidemic diarrhea virus N protein monoclonal antibody B has a heavy chain variable region with a gene sequence as shown in SEQ ID NO: 15 and a light chain variable region with a gene sequence as shown in SEQ ID NO:

16.

4. The test strip of claim 1, wherein, The pig infectious gastroenteritis virus N protein monoclonal antibody is as follows: The pig infectious gastroenteritis virus N protein monoclonal antibody A has a heavy chain variable region comprising CDR1 with an amino acid sequence of GFNIKDTY, CDR2 with an amino acid sequence of IDPADGYT and CDR3 with an amino acid sequence of ARPGTLDY, and a light chain variable region comprising CDR1 with an amino acid sequence of KSVSTSGYSY, CDR2 with an amino acid sequence of LVS and CDR3 with an amino acid sequence of QHIRELTR; The heavy chain variable region of the porcine transmissible gastroenteritis virus N protein monoclonal antibody B comprises CDR1 with the amino acid sequence of GYTFTDFN, CDR2 with the amino acid sequence of INPNNGRS, and CDR3 with the amino acid sequence of ARRHWDWYFDV; the light chain variable region comprises CDR1 with the amino acid sequence of KSLLHSN, CDR2 with the amino acid sequence of QMS, and CDR3 with the amino acid sequence of AQNLEFPWT.

5. The test strip according to claim 4, characterized in that, The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the porcine transmissible gastroenteritis virus N protein monoclonal antibody A are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the porcine transmissible gastroenteritis virus N protein monoclonal antibody B are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

6. The test strip according to claim 5, characterized in that, The DNA sequences encoding the heavy chain variable region and the light chain variable region of the porcine transmissible gastroenteritis virus N protein monoclonal antibody A are shown in SEQ ID NO: 5 and SEQ ID NO: 6; The DNA sequences encoding the heavy chain variable region and the light chain variable region of the porcine transmissible gastroenteritis virus N protein monoclonal antibody B are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

7. A method of preparing the test strip of claim 1, characterized in that, It comprises: (1) a binding pad coated with latex microsphere-labeled porcine transmissible gastroenteritis virus N protein monoclonal antibody A and porcine epidemic diarrhea virus N protein monoclonal antibody A; (2) spraying porcine epidemic diarrhea virus N protein monoclonal antibody B and goat anti-mouse IgG antibody on the nitrocellulose membrane as a detection line T1 and a quality control line C, respectively; spraying porcine transmissible gastroenteritis virus N protein monoclonal antibody B 0.5 mm beside the porcine epidemic diarrhea virus N protein monoclonal antibody B as a detection line T2; (3) sequentially laminating the sample pad, the binding pad, the nitrocellulose membrane, and the water-absorbing pad on a PVC base plate to obtain a latex double immunochromatography test strip for differentiating and detecting porcine transmissible gastroenteritis and porcine epidemic diarrhea virus.

8. The method of claim 7, wherein, The preparation method of the binding pad is as follows: The binding pad is closed and treated with a binding pad blocking solution; After the latex microspheres are activated with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, centrifugation is performed, the latex microspheres are resuspended with MES buffer, and ultrasonic dispersion is performed to fully disperse the fluorescent microspheres, followed by adding porcine transmissible gastroenteritis coronavirus N protein monoclonal antibody A and porcine epidemic diarrhea virus N protein monoclonal antibody A, incubation at room temperature, adding a latex microsphere marker blocking solution for blocking, incubation at room temperature, centrifugation, and resuspending the precipitate with a latex microsphere marker protective agent to obtain a latex microsphere-labeled mixed solution of porcine transmissible gastroenteritis virus N protein monoclonal antibody A and porcine epidemic diarrhea virus N protein monoclonal antibody A; The prepared latex microsphere-labeled mixed solution is uniformly sprayed on the closed binding pad.

9. The method of claim 8, wherein, The binding pad blocking solution is an ultrapure water solution containing 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40; The latex microsphere marker blocking solution is an ultrapure water solution containing 10% m / v BSA; The latex microsphere marker protective agent is a Tris-HCl solution containing 3% m / v sucrose, 1% m / v PVP-40 and 1% v / v Tween-20.

10. The method of claim 8, wherein, The sample pad is soaked with a sample pad blocking solution; the sample pad blocking solution is a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate.

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