Anti-canine coronavirus n protein antibody and preparation method thereof, nucleic acid molecule, cell strain and application

Abstract

The application belongs to the technical field of biomedical detection, and discloses an antibody pair against canine coronavirus N protein, a preparation method thereof, a nucleic acid molecule, a cell strain and application. The antibody pair or antigen binding fragment thereof comprises a first antibody or antigen binding fragment thereof, wherein the first antibody or antigen binding fragment thereof comprises amino acid sequences of heavy chain variable region complementarity determining regions 1-3 as shown in SEQ ID NO: 1-3, and amino acid sequences of light chain variable region complementarity determining regions 1-3 as shown in SEQ ID NO: 4-6; and a second antibody or antigen binding fragment thereof, wherein the second antibody or antigen binding fragment thereof comprises amino acid sequences of heavy chain variable region complementarity determining regions 1-3 as shown in SEQ ID NO: 7-9, and amino acid sequences of light chain variable region complementarity determining regions 1-3 as shown in SEQ ID NO: 10-12. The antibody pair provided by the application has a high titer, and has strong binding capacity with canine coronavirus N protein, and can realize high specificity and high sensitivity detection of canine coronavirus without interference of other viruses and drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical detection, and particularly relates to an anti-canine coronavirus N protein antibody pair, its preparation method, nucleic acid molecule, cell line, and application. Background Technology

[0002] Canine coronavirus (CCoV) belongs to the order Nidovirales, family Coronaviridae, and genus Alphacoronavirus. The main sources of infection are infected dogs and their feces and contaminated water. It enters susceptible dogs via the fecal-oral route. Canine coronavirus infection is not species-specific; dogs of different breeds and ages can be infected. After infection, dogs will exhibit clinical symptoms such as lethargy, vomiting, diarrhea, and bloody stools. As the disease progresses, it can lead to severe dehydration or acidosis, ultimately causing death, posing a serious threat to the dog breeding industry.

[0003] Canine coronaviruses are a class of enveloped single-stranded RNA viruses that are spherical or elliptical in shape and have radial protrusions that are characteristic of coronaviruses evenly distributed on their surface. Their structural proteins include the S protein located on the surface of the envelope, the M protein existing on the membrane surface as a transmembrane protein, the E protein located below the M protein, and the N protein located inside the envelope that constitutes the nucleocapsid of the viral particle.

[0004] Currently, canine coronavirus can be detected using methods such as electron microscopy, PCR amplification, recombinase polymerase amplification, virus neutralization assay, indirect ELISA, and indirect immunofluorescence assay. However, most of these methods require specialized equipment, reagents, and professional personnel, and suffer from problems such as long detection cycles, cumbersome operations, high costs, low sensitivity, and poor specificity. Summary of the Invention

[0005] To achieve rapid, highly sensitive, and highly specific detection of canine coronavirus, this invention provides an anti-canine coronavirus N protein antibody pair, its preparation method, nucleic acid molecules, cell lines, and applications.

[0006] In a first aspect, the present invention provides an antibody pair or its antigen-binding fragment using the following technical solution:

[0007] The antibody pair or its antigen-binding fragment comprises: a first antibody or its antigen-binding fragment with amino acid sequences of complementarity-determining regions 1-3 of the heavy chain variable region as shown in SEQ ID NO:1-3, and amino acid sequences of complementarity-determining regions 1-3 of the light chain variable region as shown in SEQ ID NO:4-6; and a second antibody or its antigen-binding fragment with amino acid sequences of complementarity-determining regions 1-3 of the heavy chain variable region as shown in SEQ ID NO:7-9, and amino acid sequences of complementarity-determining regions 1-3 of the light chain variable region as shown in SEQ ID NO:10-12.

[0008] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the first antibody or its antigen-binding fragment is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; the amino acid sequence of the heavy chain variable region of the second antibody or its antigen-binding fragment is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

[0009] In some specific embodiments, the first antibody or its antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, multispecific antibody, monoclonal antibody and polyclonal antibody.

[0010] In some specific embodiments, the first antibody or its antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, multispecific antibody, monoclonal antibody and polyclonal antibody.

[0011] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned antibody pair or its antigen-binding fragment.

[0012] Thirdly, the present invention provides a cell line that secretes the aforementioned antibody pair or its antigen-binding fragment.

[0013] In some specific embodiments, the cell line is a hybridoma cell line and / or an Escherichia coli cell line containing a gene library of antibody pairs or their antigen-binding fragments.

[0014] Fourthly, the present invention provides a kit comprising one or more of the above-mentioned antibody pairs or their antigen-binding fragments, nucleic acid molecules, and cell lines.

[0015] In some specific embodiments, the first antibody or its antigen-binding fragment further includes a detectable marker selected from one or more of a radioactive isotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.

[0016] In some specific embodiments, the second antibody or its antigen-binding fragment further includes a detectable marker selected from one or more of a radioactive isotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.

[0017] In some specific embodiments, the first antibody or its antigen-binding fragment is a capture antibody, and the second antibody or its antigen-binding fragment is a labeled antibody.

[0018] Fifthly, the present invention provides the application of the above-mentioned antibody pairs or their antigen-binding fragments, nucleic acid molecules, cell lines or kits in the detection of canine coronaviruses for non-diagnostic purposes.

[0019] Beneficial effects:

[0020] The first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment provided in this invention both have high affinity for canine coronavirus N protein. When the first antibody or its antigen-binding fragment is used as the capture antibody and the second antibody or its antigen-binding fragment is used as the labeling antibody, the binding of the second antibody to the canine coronavirus N protein can enhance the binding ability of the N protein to the first antibody. When the two are used together, they can be free from cross-interference from other disease virus strains and drugs, achieving high specificity and high sensitivity detection of canine coronavirus N protein, thereby establishing a rapid, simple, low-cost, highly sensitive and specific method for detecting canine coronavirus. Attached Figure Description

[0021] Figure 1 The image shows the detection results of colloidal gold chromatography strips provided in Example 6 of the present invention for inactivated canine coronavirus solution at different dilution ratios;

[0022] Figure 2 The image shows the detection results of colloidal gold chromatography strips provided in Example 7 of the present invention for inactivated canine coronavirus solution at different dilution ratios;

[0023] Figure 3 This is a graph showing the detection results of colloidal gold chromatography strips provided in the comparative examples of the present invention for inactivated canine coronavirus solutions at different dilution ratios. Detailed Implementation

[0024] The N protein in canine coronavirus is highly conserved and binds tightly to the positive-sense RNA of the virus to form a ribonucleoprotein structure, constituting the core of the virus. It plays a crucial role in the replication and translation of the viral genome RNA and is the most abundant protein throughout the entire canine coronavirus infection process. Therefore, this invention provides an antibody pair and its antigen-binding fragment, which can specifically bind to the canine coronavirus N protein, serving as a detection substance to detect the presence of canine coronavirus or its N protein in a sample.

[0025] The antibody pair and its antigen-binding fragment provided by the present invention include a first antibody and its antigen-binding fragment and a second antibody and its antigen-binding fragment.

[0026] In the first antibody and its antigen-binding fragment, adjacent amino acid sequences, such as the heavy chain variable region shown in SEQ ID NO:13 and the light chain variable region shown in SEQ ID NO:14, fold to form a canine coronavirus N protein binding site with a specific three-dimensional spatial structure. The site specifically recognizes and targets the antigenic determinants on the canine coronavirus N protein through complementary determinants 1-3 of the heavy chain variable region shown in SEQ ID NO:1-3 and complementary determinants 1-3 of the light chain variable region shown in SEQ ID NO:4-6.

[0027] In this invention, the amino acid sequence of the heavy chain constant region of the first antibody and its antigen-binding fragment is shown in SEQ ID NO:17, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:18.

[0028] In the second antibody and its antigen-binding fragment, adjacent amino acid sequences, such as the heavy chain variable region shown in SEQ ID NO:15 and the light chain variable region shown in SEQ ID NO:16, fold to form a canine coronavirus N protein binding site with a specific three-dimensional spatial structure. It specifically recognizes and targets another antigenic determinant on the canine coronavirus N protein through complementary determinant regions 1-3 of the heavy chain variable region shown in SEQ ID NO:7-9 and complementary determinant regions 1-3 of the light chain variable region shown in SEQ ID NO:10-12.

[0029] In this invention, the amino acid sequence of the heavy chain constant region of the second antibody and its antigen-binding fragment is shown in SEQ ID NO:19, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:20.

[0030] In this invention, the antibody pair or its antigen-binding fragment can be a polypeptide containing a full-length antibody fragment; or a polypeptide retaining a fragment that has a strong specific binding ability to canine coronavirus N protein, and the appropriate type of polypeptide can be selected as needed; wherein, the polypeptide containing a full-length antibody fragment can be, but is not limited to, one or more of polyclonal antibodies, monoclonal antibodies, and genetically engineered antibodies; the polypeptide retaining a fragment that has a strong specific binding ability to canine coronavirus N protein can be, but is not limited to, one or more of Fab, Fab', F(ab')2, Fd, Fv, or dAb.

[0031] In some specific embodiments, the first antibody or its antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, multispecific antibody, monoclonal antibody, and polyclonal antibody.

[0032] In some specific embodiments, the second antibody or its antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, multispecific antibody, monoclonal antibody, and polyclonal antibody.

[0033] The present invention also provides a nucleic acid molecule that encodes the above-mentioned monoclonal antibody pair or its antigen-binding fragment.

[0034] In some specific embodiments, the nucleic acid molecule includes a fragment encoding the heavy chain variable region of the first antibody and its antigen-binding fragment, the nucleotide sequence of which is shown in SEQ ID NO:21; and also includes a fragment encoding the light chain variable region of the first antibody and its antigen-binding fragment, the nucleotide sequence of which is shown in SEQ ID NO:22.

[0035] In some specific embodiments, the nucleic acid molecule includes a fragment encoding the heavy chain variable region of the second antibody and its antigen-binding fragment, the nucleotide sequence of which is shown in SEQ ID NO:23; and also includes a fragment encoding the light chain variable region of the second antibody and its antigen-binding fragment, the nucleotide sequence of which is shown in SEQ ID NO:24.

[0036] In this invention, the method of preparing the nucleic acid molecule is not limited. Specifically, it can be obtained by extracting from hybridoma cells using DNA extraction technology, or synthesized using engineering recombination technology and chemical synthesis methods.

[0037] The present invention also provides a cell line that can synthesize and secrete a first antibody or its antigen-binding fragment, or synthesize and secrete a second antibody or its antigen-binding fragment, or simultaneously secrete the above substances.

[0038] In some specific implementations, the cell line is a hybridoma cell line, and the specific preparation method is as follows: immune spleen cells are obtained by immunizing mice with canine coronavirus N protein or recombinant canine coronavirus N protein, and the immune spleen cells are fused with mouse myeloma cells to obtain a hybridoma cell line, which can secrete a first antibody or a second antibody.

[0039] In some specific implementations, the cell line is an Escherichia coli cell line containing a gene library of antibody pairs or their antigen-binding fragments. The specific preparation method is as follows: the above-mentioned nucleic acid molecules are assembled into an expression vector to obtain a gene library of antibody pairs or their antigen-binding fragments, and then the obtained gene library is packaged in vitro and used to infect Escherichia coli.

[0040] In this invention, E. coli cell lines containing gene libraries of antibody pairs or their antigen-binding fragments are used to prepare antibody pairs or their antigen-binding fragments, skipping the animal immunization step and simplifying the cell manipulation steps in traditional hybridoma technology, expanding the screening capacity and improving the efficiency of antibody pair or antigen-binding fragment preparation.

[0041] The present invention also provides a method for preparing the above-mentioned antibody pair or its antigen-binding fragment, the method comprising: culturing the above-mentioned cell line under suitable conditions, and recovering the monoclonal antibody pair or its antigen-binding fragment from the cell culture.

[0042] In some specific embodiments, the preparation of monoclonal antibody pairs or their antigen-binding fragments may specifically involve using recombinant DNA technology to obtain recombinant canine coronavirus N protein with an amino acid sequence such as SEQ ID NO:25, using the recombinant canine coronavirus N protein to immunize mice to obtain immune spleen cells, using hybridoma technology to prepare hybridoma cells, and the hybridoma cells secreting and expressing a first antigen or a second antigen.

[0043] In some specific embodiments, the preparation of monoclonal antibody pairs or their antigen-binding fragments may specifically involve using recombinant DNA technology to obtain recombinant canine coronavirus N protein with an amino acid sequence such as SEQ ID NO:25, using the recombinant canine coronavirus N protein to immunize mice to obtain immune spleen cells, using RT-PCR technology to clone the antibody gene from the immune spleen cells, constructing the genome into an expression vector to obtain a gene library, and then transfecting recipient cells, which secrete a first antigen or its antigen-binding fragment, or a second antigen or its antigen-binding fragment.

[0044] The kit provided by this invention includes the aforementioned antibody pairs or their antigen-binding fragments. In this invention, using the aforementioned antibody pairs or their antigen-binding fragments as detection substances, one or more of the following methods can be employed, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, and competitive assays, to qualitatively and / or quantitatively detect canine coronavirus and / or canine coronavirus N protein in a sample.

[0045] In some specific embodiments, depending on the method used for detection, the first antibody or its antigen-binding fragment may also include a detectable label, which may be, but is not limited to, one or more of a radioactive isotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.

[0046] In some specific embodiments, depending on the method used for detection, the second antibody or its antigen-binding fragment may also include a detectable label, which may be, but is not limited to, one or more of a radioactive isotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.

[0047] In this invention, the fluorescent dye may be, but is not limited to, one or more of the following: fluorescein-type fluorescent dyes, rhodamine-type fluorescent dyes, anthocyanin-type fluorescent dyes, or coumarin-type fluorescent dyes.

[0048] In this invention, the enzyme can be, but is not limited to, horseradish peroxidase and / or alkaline phosphatase. The enzyme has a specific catalytic effect on the substrate, generating a colored insoluble product or particles with a certain electron density. In actual detection, it can be observed or measured by the naked eye, microscope or spectrophotometer.

[0049] In some specific embodiments, a first antibody or its antigen-binding fragment is used as a capture antibody, and a second antibody or its antigen-binding fragment is used as a labeling antibody. A double antibody clip method is used to detect canine coronavirus or its N protein or RDB of the N protein. Specifically, the second antibody or its antigen-binding fragment first binds to canine coronavirus or its N protein or RDB of the N protein to form a complex. The complex then binds to the first antibody or its antigen-binding fragment. The qualitative and / or quantitative detection of canine coronavirus or its N protein or RDB of the N protein is achieved by detecting the detectable label.

[0050] This invention provides the application of the above-mentioned antibody pairs or their antigen-binding fragments, nucleic acid molecules, cell lines or kits in the detection of canine coronavirus for non-diagnostic purposes, which can achieve rapid, simple, low-cost, highly sensitive and specific detection of canine coronavirus.

[0051] The amino acid and nucleotide sequences involved in this invention are shown in Table 1:

[0052] Table 1.

[0053]

[0054]

[0055]

[0056] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0057] The cells, reagents, and their sources involved in the embodiments of this invention are specifically shown below:

[0058] Freund's complete adjuvant (Sigma, catalog number F5881);

[0059] Freund's incomplete adjuvant (Sigma, catalog number F5506);

[0060] Myeloma cells (ATCC, catalog number BNCC100908);

[0061] 1640 culture medium (VivaCell, catalog number C3010-0500);

[0062] PEG1500 (Roche, catalog number 10783641001);

[0063] Fetal bovine serum FBS (Sijiqing, product number 11011-8611);

[0064] Penicillin-streptomycin bispecific antibody for cell culture (Shanghai Sangon Biotech, catalog number E607011-0100);

[0065] HAT culture medium additive (Sigma, product number H0262);

[0066] HRP-labeled goat anti-mouse secondary antibody (Xiamen Taijing, product number TJ-211229CN);

[0067] HT culture medium additive (Sigma, catalog number H0137);

[0068] Ascites-specific adjuvant (Beijing Bio-Long, product number KX0210048);

[0069] Protein A column (BorgLon Biotech, catalog number AA301307);

[0070] Horseradish peroxidase (Rise Reagent, catalog number RS20220118);

[0071] DNP antibody (Merck, catalog number D9781-2ML);

[0072] Canine coronavirus monoclonal antibody 5B1 (Zhuhai Bomei Biotechnology Co., Ltd., catalog number M0495-2546);

[0073] Canine coronavirus monoclonal antibody 13H5 (Zhuhai Bomei Biotechnology Co., Ltd., catalog number M0494-1937).

[0074] Example 1.

[0075] This embodiment provides a method for preparing a cell line capable of secreting antibodies against canine coronavirus N protein. The method includes steps such as animal immunization, cellular immunization, screening of positive hybridoma cells, and cloning of positive hybridoma cells, as detailed below:

[0076] 1. Animal immunization

[0077] In animal immunization, 8-week-old BALB / C female mice were used as the immunization subjects, and recombinant canine coronavirus N protein, including the amino acid sequence shown in SEQ ID NO:25, was used as the antigen.

[0078] (1) Preparation of immunogen:

[0079] The first immunogen was prepared by mixing the recombinant SARS-CoV-2 N protein expressed in E. coli with Freund's complete adjuvant in equal volume; the second immunogen was prepared by mixing the recombinant SARS-CoV-2 N protein expressed in E. coli with an equal volume of saline; and the third immunogen was prepared by mixing the recombinant SARS-CoV-2 N protein expressed in E. coli with physiological saline in equal volume.

[0080] (2) Immunization process: First immunization: 50 μg of the first immunogen was injected subcutaneously at 3-4 points on the back of BALB / C female mice; the second immunization was performed two weeks later, with 50 μg of the second immunogen injected subcutaneously at 3-4 points on the back of BALB / C female mice; the third immunization was performed two weeks later, with 50 μg of the second immunogen injected subcutaneously at 3-4 points on the back of BALB / C female mice; the fourth immunization - shock immunization was performed two weeks later, with 50 μg of the third immunogen injected intraperitoneally into BALB / C female mice; the fifth immunization was performed 24 hours later, with 50 μg of the third immunogen injected into the tail vein of BALB / C female mice.

[0081] 2. Cellular immunity

[0082] Cellular immunization was initiated on the third day after the fifth immunization in BALB / c female mice. The process of cellular immunization specifically includes the following steps:

[0083] (1) Preparation of spleen cell suspension: On the third day after the fifth immunization of BALB / c female mice, blood was collected by removing the eyeballs of BALB / c female mice, and the serum of BALB / c female mice was separated as a positive control for antibody detection; at the same time, BALB / c female mice were killed by cervical dislocation, and their spleens were taken to prepare spleen cell suspension.

[0084] (2) Preparation of myeloma cell suspension: Myeloma cells were revived two weeks in advance (to ensure that the myeloma cells are in the logarithmic growth phase when used) to obtain myeloma cell suspension;

[0085] (3) Preparation of feeder cells: One day before cell fusion, peritoneal macrophages and spleen cells from blank BALB / c female mice were taken and cultured in 96-well plates to obtain cell plates containing feeder cells (cell concentration of 1×10⁻⁶). 4 / pore), to obtain feeder layer cells;

[0086] (4) Cell fusion process: Polyethylene glycol (PEG) was used to mediate cell fusion. Spleen cell suspension and myeloma cell suspension were taken and mixed in serum-free 1640 medium at a cell number ratio of 5:1. The mixture was centrifuged at 1200 rpm for 5 min and the supernatant was removed.

[0087] Gently tap the bottom of the centrifuge tube with your finger to loosely mix the two types of cells. Place the tube in a beaker containing 37°C water and incubate. Add 1 mL of 50% PEG1500 (pH 8.0) fusion cells over 1 minute while shaking. After adding the cells, let the tube stand for 30 seconds. Add serum-free 1640 medium to terminate the fusion. Centrifuge at 800 rpm for 5 minutes. Resuspend the pellet in HAT medium and aliquot it into cell plates containing feeder cells to obtain cell plates containing fusion cells and feeder cells. Incubate the plates in a 37°C, 5% CO2 cell culture incubator.

[0088] The following reagents are needed to prepare 500ml of HAT medium: 100mL of fetal bovine serum (FBS), 5mL of penicillin-streptomycin antibiotics for cell culture (100×), 10mL of HAT medium additive (50×), and 385mL of 1640 medium.

[0089] 3. Screening of positive hybridoma cells

[0090] The cell plates containing the fused cells-feeder layer cells were cultured until day 4, at which point the medium was partially changed, and then cultured until day 7, at which point the medium was completely changed. When the fused cells covered 10-50% of the bottom of the wells, positive wells were screened using a conventional indirect ELISA method, specifically including the following steps:

[0091] (1) Plate coating: The novel coronavirus N protein recombinantly expressed by Escherichia coli was used as the coating antigen. It was diluted to 2 μg / ml with 0.05 mol / L CB buffer (Na2CO3 31.8 g, NaHCO3 58.8 g, ultrapure water to 2 L) at pH 9.6. 100 μL / well was added to the microplate and coated overnight at 4℃. After drying, it was blocked with 1% gelatin-PBS buffer at 300 μL / well. After blocking overnight at 4℃, it was dried and ready for use.

[0092] (2) Detection: 100 μL of cell culture supernatant from cell plate containing fused cell-feeder layer cells was added to ELISA plate and incubated at 37°C for 60 min. The plate was then washed with 0.01 mol / L PBST buffer containing Tween-20 and blotted dry. 100 μL of HRP-labeled goat anti-mouse secondary antibody was added to each well and incubated at 37°C for 60 min. The plate was then washed and blotted dry. 100 μL of TMB chromogenic solution was added to each well and the plate was incubated at 37°C in the dark for 10 min. The reaction was terminated by adding 50 μL of 1 mol / L HCl to each well.

[0093] Meanwhile, BALB / c female mouse serum obtained from blood collection and separation from the eyeballs during cell immunization was used as a positive control to screen for fusion cells with high antibody titers, which were identified as positive hybridoma cells.

[0094] 4. Cloning of positive hybridoma cells

[0095] Positive hybridoma cells obtained from cell plates containing fusion cell-feeder layer cells originate from more than two hybridoma cell lines; therefore, the antibodies secreted by the selected hybridoma cells are heterogeneous. To obtain completely homogeneous monoclonal antibodies, the positive hybridoma cells need to be cloned.

[0096] The day before cloning, feeder cells were prepared and plated according to step (1) of cell immunization to obtain a cell plate containing feeder cells. The positive hybridoma cells obtained by screening were suspended in HT medium and mixed by pipetting. The cells were then seeded into the cell plate containing feeder cells. The cells in the wells of the cell plate were diluted to 1 cell per well using HT medium. The cells were then placed in a humidified environment at 37°C and 5% CO2 for 7 to 10 days. When clonal cells were visible to the naked eye, antibodies could be detected.

[0097] The following reagents are needed to prepare 500 mL of HT medium: 100 mL of fetal bovine serum (FBS), 5 mL of penicillin-streptomycin antibiotics for cell culture, 10 mL of HT medium additives, and 385 mL of 1640 medium.

[0098] Under an inverted microscope, wells showing only a single clone were marked, resulting in 12 monoclonal antibody hybridoma cell lines obtained through preliminary screening. The supernatant was then used for further functional screening. These 10 monoclonal antibody hybridoma cell lines were named as shown in Table 2.

[0099] Table 2.

[0100]

[0101]

[0102] Example 2.

[0103] This embodiment provides a method for preparing antibodies. In this method, ascites fluid is prepared using 10 cell lines obtained in Example 1, and the ascites fluid is purified to obtain antibodies. The specific steps include:

[0104] (1) Preparation of ascites fluid: 8-10 week old Balb / c mice were intraperitoneally injected with a specific adjuvant for ascites fluid. On the 10th day after injection, the monoclonal antibody hybridoma cell lines shown in Table 1 (1x10⁻¹⁰) were used to prepare the ascites fluid. 6 (1 cell / mouse) was injected into the peritoneal cavity of Balb / c mice, and the ascites fluid of the Balb / c mice was collected with a medical syringe 12 days later.

[0105] (2) Purification of ascites fluid: The ascites fluid of Balb / c mice collected in (1) was poured into a centrifuge tube, centrifuged at 12500 rpm for 20 min, the supernatant was collected and filtered through a 0.22 μm filter membrane and purified by Protein A affinity chromatography. The names of the antibodies prepared by each cell line are shown in Table 2.

[0106] Example 3.

[0107] In this embodiment, horseradish peroxidase (HRP) labeling was performed on 10 strains from Example 2, specifically including the following steps:

[0108] (1) The purified antibody was diluted to 2 mg / mL with 0.05 mol / L CB buffer at pH 9.6 to obtain an antibody protein solution; the dialysis membrane was soaked twice with 0.05 mol / L CB buffer at pH 9.6, 1 mL of antibody protein solution was transferred to the dialysis membrane, and the membrane was dialyzed at 4°C with stirring using 0.05 mol / L CB buffer at pH 9.6. The buffer was changed every 1 h, and the membrane was dialyzed a total of 5 times.

[0109] (2) Dissolve HRP in ultrapure water to prepare an HRP solution with a concentration of 20 mg / mL, and dissolve NaIO4 in ultrapure water to prepare a NaIO4 solution with a concentration of 20 mg / mL; after vortexing to fully dissolve, mix the HRP solution and NaIO4 solution at a volume ratio of 1:1, that is, slowly add the NaIO4 solution to the HRP solution, immediately wrap the centrifuge tube with tin foil, and activate HRP at 4℃ for 30 min in the dark;

[0110] (3) Slowly add ethylene glycol dropwise to the HRP-activated centrifuge tube while gently shaking (add 1 μL of ethylene glycol for every 1 mg of HRP), and continue at 4°C in the dark for 30 min to terminate HRP activation; then add the terminated HRP solution to the antibody dialysis membrane (add 1 mg of HRP and 1 mg of NaIO4 for every 1 mg of antibody), and couple overnight at 4°C in 0.05 mol / L CB buffer with a pH of 9.6; the next day, change the 0.05 mol / L CB buffer with a pH of 9.6 and continue dialysis for 2 h. After dialysis, transfer the coupled dialysate to a centrifuge tube to obtain the antibody-HRP coupled solution.

[0111] (4) Prepare a 20 mg / mL NaBH4 solution with pure water and add it to the antibody-HRP conjugate solution obtained in (3). The amount of NaBH4 solution added is 2 μL for every 1 mg of HRP. React at 4℃ for 2 h, and mix by inverting the solution several times every 0.5 h to obtain the conjugate protein. Then, use 50% ammonium sulfate (i.e., an equal volume of saturated ammonium sulfate mixed with the obtained conjugate protein solution at a 1:1 ratio) to precipitate the conjugate protein. Precipitate at 4℃ for 15 min, and then centrifuge at 10000 rpm for 10 min to obtain the HRP-antibody conjugate protein precipitate. The specific naming of the HRP-antibody conjugate protein is shown in Table 2.

[0112] Example 4.

[0113] In this embodiment, 10 antibodies from Example 2 and 10 HRP-antibody conjugate proteins from Example 3 were paired and screened using a double-antibody sandwich ELISA method to identify antibodies that can specifically recognize different epitopes of canine coronavirus N protein.

[0114] In the double-antibody sandwich ELISA method, antibodies are used as capture antibodies and HRP-antibody-conjugate protein is used as labeling antibodies. The specific steps are as follows: 10 antibodies are coated onto the ELISA plate as capture antibodies. Then, the novel coronavirus N protein is added to the wells of the ELISA plate. After incubation, unbound novel coronavirus N protein is washed away. Next, HRP-antibody-conjugate protein is added as labeling antibody. After incubation, unbound HRP-antibody-conjugate protein is washed away. Finally, chromogenic solution is added for color development, and the absorbance is measured at a wavelength of 280 nm using a spectrophotometer. The detection results are shown in Table 3.

[0115] Table 3.

[0116]

[0117]

[0118] The test results show that the antibody pair 3A5 and 1G4 has high titers and can be used for qualitative and / or quantitative detection of canine coronavirus and its N protein or RDB of N protein.

[0119] Example 5.

[0120] In this embodiment, the 3A5 and 1G4 cell lines prepared in Example 1 were cultured on a scale-up basis, and 5 × 10⁶ cells were collected. 6 Cells / mL were collected in centrifuge tubes, the supernatant was aspirated, and the cells were frozen and sent to Nanjing Detai Biotechnology Co., Ltd. on dry ice for hybridoma cell sequencing. The sequencing results are shown in Table 4.

[0121] Table 4.

[0122]

[0123] Example 6.

[0124] The kit provided in this embodiment includes colloidal gold chromatography strips, which are prepared using 3A5 antibody as the capture antibody and 1G4 antibody as the labeling antibody through the following steps:

[0125] S1. Preparation of the test pad: Weigh 2.901g of Na2HPO4·12H2O, 0.2914g of NaH2PO4·2H2O, 8.5g of NaCl, and 25g of sucrose, and dissolve them in 1000mL of ultrapure water to prepare a coating buffer. Take 1mg / mL of 3A5 antibody and spray it onto the nitrocellulose membrane at a spraying rate of 1.2μL / cm to obtain a test line with a length of 30cm. Take 1mg / mL of DNP antibody and spray it onto the nitrocellulose membrane at a spraying rate of 1.2μL / cm to obtain a control line with a length of 30cm, and the distance between the control line and the test line is 5mm. The nitrocellulose membrane coated with the test line and control line is the test pad. Attach the test pad to a PVC backing and dry it in an oven at 50℃ for 24±2h.

[0126] S2. Labeling of the gold conjugate: Weigh 0.36 g of Tris, 1 g of BSA, 100 μL of Tween-20, 2 g of sucrose, and 100 μL of Proclin 300 and dissolve them in 100 mL of ultrapure water to obtain a reconstituted solution. Place 100 mL of 0.01% colloidal gold solution in a clean container, add 1–3 mL of 0.2 M K2CO3 solution and stir until homogeneous. Then add 500 μg–1500 μg of 1G4 antibody and stir for 15 min. Add 1000 μL of 20% BSA solution and stir. After blocking for 10 min, centrifuge at 8000–10000 r / min for 20 min, discard the supernatant, and reconstitute the precipitate with the reconstituted solution to obtain the labeled antibody. Store at 4 °C for later use.

[0127] S3. Preparation of conjugate pads: Weigh 0.362g of Tris, 0.3g of sodium caseinate, 0.2g of PEG20000, 100μL of Tween-20, 3g of sucrose, and 100μL of Proclin 300 and dissolve them in 100mL of ultrapure water to obtain a gold-labeled conjugate solution; add a certain amount of labeled antibody to the gold-labeled conjugate solution and mix well. The concentration of labeled antibody in the solution is 15wt%. Coat the above solution evenly on a glass fiber membrane at a coating rate of 35mL / sheet and dry it in an oven at 50℃ for 24h to obtain the conjugate pads.

[0128] S4. Preparation of sample pads: Weigh 2.901g of Na2HPO4·12H2O, 0.2914g of NaH2PO4·2H2O, 8.5g of NaCl, 25g of sucrose, 5g of BSA, and 1mL of proclin300 and dissolve them in 1000mL of ultrapure water to obtain the sample pad treatment solution; evenly spread 32mL of sample pad treatment solution per plate onto a glass fiber membrane (25cm×30cm), and dry it in an oven at 50℃ for 24±2h to obtain the sample pads.

[0129] S5. Assembly of the gold-labeled paper strip: Under conditions of temperature of 18-28℃ and humidity of 10-30%, the sample pad and conjugate pad are sequentially attached to the end of the PVC board near the detection line, with the sample pad partially overlapping the conjugate pad and the conjugate pad partially overlapping the detection pad; the absorbent paper is attached to the end of the PVC board away from the detection line, with the absorbent paper partially overlapping the detection pad. After assembling the large board, it is cut into thin strips with a width of 3mm to obtain the colloidal gold chromatography paper strip. In order to facilitate subsequent detection, the assembled colloidal gold chromatography paper strip is installed inside the test strip card shell.

[0130] Example 7.

[0131] This embodiment provides a kit for preparing colloidal gold chromatography strips according to the method provided in Example 6. The difference is that 1G4 antibody is used as the capture antibody and 3A5 antibody is used as the labeling antibody in the colloidal gold chromatography strip, while other conditions are the same.

[0132] Comparative example.

[0133] This comparative example provides a kit for preparing colloidal gold chromatography strips according to the method provided in Example 6, except that canine coronavirus monoclonal antibody 5B1 is used as the capture antibody and canine coronavirus monoclonal antibody 13H5 is used as the labeling antibody in the colloidal gold chromatography strips, while other conditions are the same.

[0134] Test example.

[0135] Sensitivity and specificity tests were performed on the colloidal gold chromatography strips provided in Examples 6 and 7, with a comparative example used as a control, as detailed below:

[0136] (1) Sensitivity test: Different concentrations of inactivated canine coronavirus were detected using diluent (diluent composition: 10mM PBS, 0.3% PVP40), and the test results are shown in Table 5.

[0137] Table 5. Sensitivity Test Results

[0138] Dilution factor Example 6 Example 7 Comparative Example 100 times Strong positive Strong positive Positive 500 times Positive Positive Weak positive 1000 times Positive Positive Weak positive 5000 times Weak positive Weak positive Negative 10000 times Weak positive Negative Negative 20,000 times Weak positive Negative Negative diluent Negative Negative Negative

[0139] Depend on Figure 1 As shown in Table 5, when the virus solution was diluted 20,000 times, the detection result of the colloidal gold chromatography strip provided in Example 6 was still positive; Figure 2 As shown in Table 5, when the virus solution was diluted 5000 times, the colloidal gold chromatography strip provided in Example 7 still showed a positive result; while the... Figure 3As shown in Table 5, when the virus solution was diluted 5000 times, the colloidal gold chromatography strip provided in the comparative example could not detect canine coronavirus in the solution and was negative. That is, the antibody pair of 1G4 antibody and 3A5 antibody used in Examples 6 and 7 has higher sensitivity for canine coronavirus than the antibody pairs commonly used in the prior art. Furthermore, when the antibody pair uses 3A5 antibody as the capture antibody and 1G4 antibody as the labeling antibody, the antibody pair can work synergistically to achieve the best effect and can detect canine coronavirus in the virus solution diluted 20000 times, with higher detection sensitivity.

[0140] (2) Specific detection: Inactivated canine coronavirus liquid, recombinant N protein, feline coronavirus liquid, porcine transmissible gastroenteritis virus liquid, canine parvovirus liquid, rotavirus liquid, canine brucellosis liquid, streptococcus liquid, Escherichia coli liquid, Vero cell lysate, newborn calf serum, MEM medium, 1% BSA and 1% skim milk powder were tested. The test results are shown in Table 6.

[0141] Table 6. Specific detection results

[0142] Detection material Example 6 Example 7 Comparative Example Inactivated canine coronavirus liquid Strong Yang Strong Yang Zhongyang Recombinant N protein Strong Yang Strong Yang Zhongyang Feline coronavirus liquid Negative Negative Negative Porcine transmissible gastroenteritis virus fluid Negative Negative Negative Canine parvovirus fluid Negative Negative Negative Rotavirus fluid Negative Negative Negative Brucella canis liquid Negative Negative Negative Streptococcal liquid Negative Negative Negative Escherichia coli liquid Negative Negative Negative Vero cell lysis buffer Negative Negative Negative Newborn calf serum Negative Negative Weak Yang MEM culture medium Negative Negative Negative 1% BSA Negative Negative Negative 1% skim milk powder Negative Negative Negative

[0143] The test results show that the colloidal gold chromatography strips provided in Examples 6 and 7 showed strong positive results for the detection of inactivated canine coronavirus liquid and recombinant N protein, while the detection results for other substances were negative. Compared with the monoclonal antibody provided in the comparative example, which showed a weak positive result for the detection of newborn calf serum, the 1G4 antibody and 3A5 antibody provided in Examples 6 and 7 of this invention have higher detection specificity for canine coronavirus. When applied to actual non-diagnostic canine coronavirus detection, they have higher detection accuracy and efficiency.

[0144] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. An antibody pair or its antigen-binding fragment, characterized in that, The antibody pair or its antigen-binding fragment comprises: a first antibody or its antigen-binding fragment with amino acid sequences of complementarity-determining regions 1-3 of the heavy chain variable region as shown in SEQ ID NO:1-3, and amino acid sequences of complementarity-determining regions 1-3 of the light chain variable region as shown in SEQ ID NO:4-6. The amino acid sequences of the heavy chain variable region complementarity-determining regions 1-3 are shown in SEQ ID NO:7-9, and the amino acid sequences of the light chain variable region complementarity-determining regions 1-3 are shown in SEQ ID NO:10-12, respectively, for the second antibody or its antigen-binding fragment.

2. The antibody pair or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the first antibody or its antigen-binding fragment is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; The amino acid sequence of the heavy chain variable region of the second antibody or its antigen-binding fragment is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

3. The antibody pair or its antigen-binding fragment according to claim 1, characterized in that, The first antibody or its antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fv, single-chain antibody, chimeric antibody and monoclonal antibody.

4. The antibody pair or its antigen-binding fragment according to claim 1, characterized in that, The second antibody or its antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fv, single-chain antibody, chimeric antibody and monoclonal antibody.

5. A nucleic acid molecule, characterized in that, Encodes the antibody pair or antigen-binding fragment thereof as described in any one of claims 1 to 4.

6. A cell line that secretes any one of the antibody pairs or antigen-binding fragments of claims 1 to 4, characterized in that, The cell line is a hybridoma cell line and / or an Escherichia coli cell line containing a gene library of antibody pairs or their antigen-binding fragments.

7. A method for preparing the antibody pair or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The method includes: culturing the cell line of claim 6 under suitable conditions, and recovering monoclonal antibody pairs or antigen-binding fragments thereof from the cell culture.

8. A reagent kit, characterized in that, Includes the antibody pair or antigen-binding fragment thereof as described in any one of claims 1 to 4.

9. The reagent kit according to claim 8, characterized in that, The first antibody or its antigen-binding fragment further includes a detectable marker selected from one or more of a radioactive isotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.

10. The reagent kit according to claim 8, characterized in that, The second antibody or its antigen-binding fragment further includes a detectable marker selected from one or more of a radioactive isotope, a fluorescent dye, biotin, colloidal gold, and an enzyme.

11. The reagent kit according to claim 8, characterized in that, The first antibody or its antigen-binding fragment is a capture antibody, and the second antibody or its antigen-binding fragment is a labeling antibody.

12. The use of the antibody pair or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the cell line according to claim 6, or the kit according to any one of claims 8 to 11 in the preparation of a canine coronavirus detection formulation.

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