Monoclonal antibody, hybridoma cell secreting monoclonal antibody and application

Abstract

The invention discloses a monoclonal antibody, a hybridoma cell secreting the monoclonal antibody and application of the hybridoma cell, and belongs to the field of peptides. The invention aims to provide a monoclonal antibody and a hybridoma cell secreting the monoclonal antibody. The hybridoma cell secreting the monoclonal antibody disclosed by the invention is a hybridoma cell 3G3, and the preservation number of the hybridoma cell 3G3 in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No.46345. The monoclonal antibody and the BVDV antibody detection kit based on the monoclonal antibody and the blocking ELISA method can be used for detecting a neutralizing antibody of the BVDV, and have the characteristics of strong specificity, good sensitivity, high sensitivity, high accuracy and the like.

Description

Technical Field

[0001] The present invention belongs to the field of peptides, and particularly relates to monoclonal antibodies, hybridoma cells secreting monoclonal antibodies, and their applications. Background Art

[0002] Bovine viral diarrhea (BVD) is a serious infectious disease caused by bovine viral diarrhea virus (BVDV). It mainly affects cattle and sheep, and can also infect pigs and wild ruminants. The disease spreads rapidly and is globally distributed, causing serious economic losses to the cattle industry. BVDV is a single-stranded positive-strand RNA virus of the genus Pestivirus in the family Flaviviridae. According to the 5'UTR sequence, it is mainly divided into two genotypes: BVDV-1 and BVDV-2, which seriously endanger the healthy development of the cattle industry. In recent years, inactivated vaccines have been widely used. How long after vaccination can immunized cattle produce neutralizing antibodies at an effective protection level and the duration of neutralizing antibodies at an effective protection level are important indicators for evaluating the immune effect of vaccines.

[0003] Currently, the classic methods for detecting BVDV neutralizing antibodies mainly include the virus neutralization test (VNT) and enzyme-linked immunosorbent assay (ELISA). Compared with the VNT method, ELISA has become the preferred method for evaluating BVDV neutralizing antibodies because of its simple operation, rapidity, and suitability for large-scale detection. In existing research, blocking ELISA based on the NS3 protein, competitive ELISA based on the E0 protein, and indirect ELISA based on the whole virus have been developed. The blocking ELISA based on the NS3 protein can detect serum antibodies against the NS3 protein and is suitable for differentiating wild virus-infected and inactivated vaccine-immunized cattle for culling infected cattle to implement purification. The competitive ELISA based on the E0 protein and the indirect ELISA based on the whole virus can detect serum antibodies against the E0 protein and total serum antibodies against the virus, respectively, and are both suitable for epidemiological investigations of bovine viral diarrhea virus and screening for bovine viral diarrhea virus-infected cattle in non-immunized farms, but are not suitable for detecting neutralizing antibody levels.

[0004] With the progress of molecular biology techniques, monoclonal antibodies (mAb) have become key tools for research and diagnosis. After being infected with BVDV or vaccinated, neutralizing antibodies against BVDV are produced in the body. Currently, there is no commercial ELISA detection kit available for evaluating the immunogenicity of BVDV vaccines. Existing blocking ELISA based on NS3 protein, competitive ELISA based on E0 protein, and indirect ELISA kit based on whole virus mainly detect NS3, E0, and whole virus antibodies, and only qualitatively judge the negativity or positivity of NS3, E0 antibodies or total virus antibodies, but cannot specifically evaluate the neutralizing antibody level of bovine viral diarrhea and cannot be used to evaluate the immunogenicity of vaccines. Therefore, there is an urgent need for an ELISA kit that is sensitive, specific, and high-throughput for detecting neutralizing antibodies of inactivated BVDV vaccines to replace the virus neutralization test method that is cumbersome, time-consuming, and has poor repeatability. Summary of the Invention

[0005] The object of the present invention is to provide a monoclonal antibody and a hybridoma cell secreting the monoclonal antibody.

[0006] The monoclonal antibody provided by the present invention is a monoclonal antibody secreted by the hybridoma cell 3G3, and the preservation number of the hybridoma cell 3G3 in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 46345.

[0007] The present invention also provides the application of the monoclonal antibody in the preparation of a product for detecting neutralizing antibodies of bovine viral diarrhea virus. The product can be a kit.

[0008] The present invention also provides the application of the monoclonal antibody in detecting neutralizing antibodies of bovine viral diarrhea virus.

[0009] The present invention also provides the application of the monoclonal antibody in the preparation of a product for detecting bovine viral diarrhea virus.

[0010] In the above text, the products can all be kits.

[0011] The present invention also provides a kit containing the monoclonal antibody.

[0012] The kit can be used to detect BVDV antibodies. Specifically, the BVDV can be bovine viral diarrhea virus type 1 (BVDV-1) or bovine viral diarrhea virus type 2 (BVDV-2).

[0013] Furthermore, the kit is a BVDV antibody detection kit based on the blocking ELISA method, and the monoclonal antibody is labeled with horseradish peroxidase.

[0014] The above-mentioned kit may also contain E2 protein, BVDV antibody-positive serum, BVDV antibody-negative serum, sample diluent and blocking solution; The sample diluent may be PBST; The blocking solution may be composed of a solvent and a solute. The solvent is PBST, and the solute and its concentration in the blocking solution are 2.5 g / 100 mL gelatin and 5 g / 100 mL sucrose respectively.

[0015] The sample diluent can be used to dilute the serum to be tested and the monoclonal antibody labeled with horseradish peroxidase.

[0016] The BVDV antibody-positive serum can be obtained by the following method: Immunize healthy calves with bovine viral diarrhea / mucosal disease inactivated vaccine (type 1, NM01 strain). When the neutralizing antibody titer detected by the neutralization test is not less than 1:1024, collect the serum, inactivate it at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, and filter and sterilize it with a 0.22 μm pore size filter membrane.

[0017] The BVDV antibody-negative serum can be obtained by the following method: Collect the serum of healthy calves negative for BVDV neutralizing antibody, inactivate it at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, and filter and sterilize it with a 0.22 μm pore size filter membrane.

[0018] In the above-mentioned kit, the E2 protein may be the E2 protein shown by positions 28-371 of SEQ ID No. 2, or the recombinant E2 protein shown by SEQ ID No. 2.

[0019] The above-mentioned kit may also contain a 96-well plate, washing solution, substrate solution A, substrate solution B, termination solution and / or sealing film; The washing solution is PBST; The substrate solution A: TMB two-component chromogenic solution A (Beijing Solarbio Science & Technology Co., Ltd., product number PR1210); The substrate solution B: TMB two-component chromogenic solution B (Beijing Solarbio Science & Technology Co., Ltd., product number PR1210); The termination solution is 1 M HCl aqueous solution.

[0020] The present invention also provides a hybridoma cell, which is hybridoma cell 3G3 that secretes the monoclonal antibody. The preservation number of hybridoma cell 3G3 in the China General Microbiological Culture Collection Center is CGMCC No. 46345.

[0021] The present invention also provides the use of the hybridoma cells in the preparation of a monoclonal antibody against BVDV, or in the preparation of a product for detecting neutralizing antibodies against bovine viral diarrhea virus, or in the preparation of a product for detecting bovine viral diarrhea virus.

[0022] The product may be a reagent or a kit.

[0023] The monoclonal antibody of the present invention and the BVDV antibody detection kit prepared by labeling the monoclonal antibody with horseradish peroxidase based on the blocking ELISA method have the following advantages: (1) Strong specificity: The monoclonal antibody can specifically recognize bovine viral diarrhea virus type 1 and type 2, and does not cross-react with classical swine fever virus, bovine herpesvirus type 1, bovine parainfluenza virus type 3, and bovine nodular dermatitis virus. Using the kit of the present invention to detect BVDV antibody-positive serum and other bovine infectious disease-positive sera, the detection results of other bovine infectious disease-positive sera (bovine foot-and-mouth disease virus antibody-positive serum, bovine herpesvirus type 1 antibody-positive serum, bovine brucella antibody-positive serum, bovine pathogenic Escherichia coli antibody-positive serum, and bovine mycoplasma antibody-positive serum) are all negative. (2) Good sensitivity: Using the kit of the present invention to detect 28 BVDV antibody weakly positive serum samples identified by neutralization test, the results show that the positive coincidence rate with the neutralization test is 100%. (3) High sensitivity: Dilute the BVDV antibody-positive serum (neutralization titer 1:4096) in a 4-fold gradient, and use the kit of the present invention to detect it simultaneously with the neutralization test. The sensitivity of the method of the present invention can reach a dilution of 1:1024, which is consistent with the results of the neutralization test. (4) Good consistency with the detection results of other methods: Use the kit of the present invention, the neutralization test, and the detection kit of IDEXX Laboratories, Inc. (referred to as the IDEXX kit) to detect 218 bovine serum samples simultaneously, and compare the coincidence rates among the three detection methods. The results show that: the positive coincidence rate of the kit of the present invention with the neutralization test is 98.65%, the negative coincidence rate is 92.86%, and the total coincidence rate is 96.79%; compared with the IDEXX kit, the kit of the present invention has a better coincidence rate with the neutralization test and higher specificity. (5) High detection efficiency: Using the monoclonal antibody and kit of the present invention, the detection process takes less than 1.5 hours, the operation is more convenient, and the detection time is greatly shortened. (6) High accuracy: Using the kit of the present invention to detect batch samples and perform ROC curve analysis. When the PI value is 24.72%, the diagnostic sensitivity is 93.33%, the diagnostic specificity is 93.33%, and the area under the ROC curve is 0.9856, indicating that the kit of the present invention has high accuracy. (7) Evaluation of vaccine immunization effect: The serum detection result of the kit of the present invention shows that there is a certain corresponding relationship between the PI value and the neutralizing antibody titer. Therefore, the negative and positive of the neutralizing antibody in the serum sample and the range of the neutralizing titer of the positive serum can be judged according to the PI value, and further, according to the lowest neutralizing antibody standard of the immunoprotected cattle obtained in the challenge protection test, it can be judged whether the antibody level of the serum sample can provide effective immune protection for the immunized calves, thus providing a scientific and reliable test method and product for evaluating the vaccine immunization effect.In summary, the monoclonal antibody secreted by the hybridoma cells of the present invention has the advantages of good sensitivity and strong specificity, and the detection operation is simple and rapid. The monoclonal antibody of the present invention can semi-quantitatively detect the neutralizing antibody level of bovine viral diarrhea virus in serum, providing a rapid and reliable detection method for the evaluation of the immune effect of BVDV vaccine, and having a high coincidence rate with the detection results of the traditional VNT method.

[0024] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0025] Description of Biological Material Deposit Abbreviation of Depositary Institution: CGMCC Name of Depositary Institution: General Microbiology Center, China Committee for Culture Collection of Microorganisms Address of Depositary Institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101 Date of Deposit: April 18, 2025 Accession Number Registered by the Deposit Center: CGMCC No. 46345 Taxonomic Nomenclature: Hybridoma Cells ( Mus musculus ) Strain Number: 3G3 Brief Description of Drawings

[0026] Figure 1 . Schematic diagram for the preparation of plasmid pFastBac Dual-E2. HBM: HBM tag; His: His tag; E2: E2 gene sequence; pH: polyhedrin (PH) promoter; MCS: Multiple Cloning Site (MCS), i.e., the position where the target fragment is inserted.

[0027] Figure 2 . Identification results of Sf9 cells infected with recombinant baculovirus. The left figure is the nuclear staining result, the middle figure is the detection result of recombinant E2 protein, and the right figure is the superposition result of the two figures.

[0028] Figure 3. Western blot identification results of Sf9 cells infected with rBV-E2. A: Cell culture supernatant sample group. Lane M is the protein molecular weight standard, lane 1 is the culture supernatant of normal Sf9 cells, lane 2 is the culture supernatant of Sf9 cells infected with baculovirus BV, and lane 3 is the culture supernatant of Sf9 cells infected with recombinant baculovirus rBV-E2. B: Cell lysate supernatant sample group. Lane M is the protein molecular weight standard, lane 1 is the lysate supernatant of normal Sf9 cells, lane 2 is the lysate supernatant of Sf9 cells infected with baculovirus BV, and lane 3 is the lysate supernatant of Sf9 cells infected with recombinant baculovirus rBV-E2. The arrow indicates the target protein.

[0029] Figure 4 . Purification and detection results of recombinant E2 protein. A: Coomassie brilliant blue staining results of SDS-PAGE. Lane M is the protein molecular weight standard, and lane 1 is the detection result of the target protein. B: Western blot detection results. Lane M is the protein molecular weight standard, and lane 1 is the detection result of the target protein. The arrow indicates the target protein.

[0030] Figure 5 . Detection results of mouse serum titer after the third immunization.

[0031] Figure 6 . Detection results of the antibody secretion stability of 3G3 hybridoma cells.

[0032] Figure 7 . Specificity detection results of 3G3 monoclonal antibody. A: MDBK cells without virus infection as a control. B: Results of BVDV-1 JL strain. C: Results of BVDV-2 HLJ-10 strain. D: Results of CSFV. E: Results of BoHV-1. F: Results of BPIV-3. G: Results of LSDV.

[0033] Figure 8 . Western blot verification results of 3G3 monoclonal antibody. Lane M is the protein molecular weight standard, lane 1 is the negative control, and lane 2 is the detection result of the target protein.

[0034] Figure 9 . ROC curve for determining the critical value of the kit. Specific implementation mode

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. In the following examples, at least three repeated experiments are set for quantitative tests.

[0036] Bovine viral diarrhea virus (BVDV) standard strain NADL: China Institute of Veterinary Drug Control (CVCC AV67).

[0037] The bovine viral diarrhea virus type 1 JL strain (BVDV-1 JL strain) in the following examples is described in the article "Liu Mengyao, Wang Zhanhui, Wu Hao, Gu Yue, Wu Wenxue. Establishment of a quadruple real-time fluorescence quantitative RT-PCR detection method for bovine astrovirus, bovine viral diarrhea virus type 1, bovine coronavirus and bovine rotavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(07): 1942-1952". The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0038] The bovine viral diarrhea virus type 2 HLJ-10 strain (BVDV-2 HLJ-10 strain) in the following examples is described in the article "Liu Mengyao, Wang Zhanhui, Wu Hao, Gu Yue, Wu Wenxue. Establishment of a quadruple real-time fluorescence quantitative RT-PCR detection method for bovine astrovirus, bovine viral diarrhea virus type 1, bovine coronavirus and bovine rotavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(07): 1942-1952". The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0039] Classical swine fever virus (CSFV) in the following examples: China Institute of Veterinary Drug Control (CVCC AV280).

[0040] The bovine herpesvirus 1 (BoHV-1) in the following examples is described in the article "Pengpeng Wang, Shulin Huang, Chengwu Hao, Zhanhui Wang, Haoran Zhao, Mengyao Liu, Xinrui Tian, Letu Ge, Wenxue Wu, and Chen Peng. Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1. Vaccines 2021, 9, 1006. https: / / doi.org / 10.3390 / vaccines9091006". The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0041] The Bovine Parainfluenza Virus Type 3 (BPIV-3) SD0835 strain in the following embodiments is described in the article "Liu Mengyao, Wang Zhanhui, Wu Hao, Gu Yue, Wu Wenxue. Establishment of a quadruple real-time fluorescence quantitative RT-PCR detection method for bovine astrovirus, bovine viral diarrhea virus type 1, bovine coronavirus, and bovine rotavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(07): 1942-1952". The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0042] The Lumpy Skin Disease Virus (LSDV) in the following embodiments is described in the article "Xie S, Cui L, Liao Z, etal. Genomic analysis of lumpy skin disease virus asian variants and evaluation of its cellular tropism. NPJ Vaccines. 2024 Mar 21;9(1):65. doi:10.1038 / s41541-024-00846-8". The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0043] DH10Bac competent cells: Beijing Bomed Gene Technology Co., Ltd.

[0044] pFastBac Dual vector: Thermo Fisher product.

[0045] MAb against BVDV E2 protein (i.e., Bovine Viral Diarrhea Virus Type 1&2 (BVDV-1&2) MAb Ascites E2 gp53 IgG2b Isotype): VMRD Inc., product number 348.

[0046] HRP-conjugated Goat anti-Mouse IgG(H+L) antibody: Abbkine Scientific Co., Ltd., product number AS003.

[0047] HRP-conjugated Rabbit anti-Goat IgG(H+L) antibody: Abbkine Scientific Co., Ltd., product number AS029.

[0048] FITC-labeled goat anti-mouse IgG (H+L) antibody: Shanghai Beyotime Biotechnology Co., Ltd., catalog number A0568.

[0049] Mouse monoclonal antibody isotyping reagents (i.e., Mouse monoclonal Antibody Isotyping Reagents): Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number ISO2. This kit contains goat anti-mouse IgG1 specific antibody, goat anti-mouse IgG2a specific antibody, goat anti-mouse IgG2b specific antibody, goat anti-mouse IgG3 specific antibody, goat anti-mouse IgA specific antibody, and goat anti-mouse IgM specific antibody.

[0050] 6-8-week-old female BALB / c mice: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0051] BVDV antibody-positive serum: Immunize healthy calves with bovine viral diarrhea / mucosal disease inactivated vaccine (type 1, NM01 strain). When the neutralizing antibody titer detected by the neutralization test is not less than 1:1024, collect the serum, inactivate it at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, filter and sterilize it with a 0.22 μm pore size filter membrane, and store it at -70 °C or below. Among them, the bovine viral diarrhea / mucosal disease inactivated vaccine (type 1, NM01 strain) is a product of Tiankang Biopharmaceutical Co., Ltd., batch number 2023007.

[0052] BVDV antibody-negative serum: Collect the serum of healthy calves with negative BVDV neutralizing antibody detection, inactivate it at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, filter and sterilize it with a 0.22 μm pore size filter membrane, and store it at -70 °C or below.

[0053] Bovine foot-and-mouth disease virus antibody-positive serum: Immunize healthy calves with bovine foot-and-mouth disease O-type and A-type bivalent inactivated vaccine (O / MYA98 / BY / 2010 strain + Re-A / WH / 09 strain) according to the instructions. After the antibody turns positive detected by the bovine and sheep foot-and-mouth disease virus VP1 structural protein antibody ELISA diagnostic kit, collect the serum, inactivate it at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, filter and sterilize it with a 0.22 μm pore size filter membrane, and store it at -70 °C or below. Among them, the bovine foot-and-mouth disease O-type and A-type bivalent inactivated vaccine (O / MYA98 / BY / 2010 strain + Re-A / WH / 09 strain) is a product of Shenlian Biopharmaceuticals (Shanghai) Co., Ltd., batch number: 20240101.

[0054] Bovine herpesvirus 1 type antibody-positive serum: Healthy calves were immunized with a bovine herpesvirus 1 type (BoHV-1) Us9 gene-deleted vaccine (a bovine herpesvirus 1 type gene-deleted attenuated strain in Chinese Patent Application 202310884644.0 (Construction and application of a bovine herpesvirus 1 type Us9 gene-deleted vaccine strain)). When the neutralizing antibody titer detected by the neutralization test was not less than 1:32, serum was collected, inactivated at 56 °C for 30 min, added with thimerosal sodium at a final concentration of 0.01%, filtered and sterilized with a 0.22 μm pore size filter membrane, and stored at -70 °C or below.

[0055] Bovine Brucella antibody-positive serum: Healthy cattle were immunized with a live Brucella vaccine (strain A19). After the antibody turned positive detected by a bovine Brucella indirect ELISA antibody detection kit (Beijing Mingrida Technology Development Co., Ltd., batch number 20231109), it was inactivated at 56 °C for 30 min, added with thimerosal sodium at a final concentration of 0.01%, filtered and sterilized with a 0.22 μm pore size filter membrane, and stored at -70 °C or below. Among them, the live Brucella vaccine (strain A19) was a product of Harbin Veterinary Research Institute Co., Ltd., batch number: 2024002.

[0056] Bovine pathogenic Escherichia coli antibody-positive serum: Bovine pathogenic Escherichia coli O5 serotype B4 strain (Zhang Yun. Construction and biological characteristics study of a galE gene-deleted strain of bovine Escherichia coli. 2017) was cultured in LB broth medium (Beijing Aoboxing Biological; product number: 02-136) to 10 10 CFU / ml. After inactivating with 30% formaldehyde, an inactivated vaccine was prepared by emulsifying with Freund's adjuvant and used to immunize healthy calves intramuscularly in the neck at 2 ml / head. The second immunization was given at the same dose and route after 14 days. When the Escherichia coli serum antibody titer detected by the tube agglutination test was not less than 1:40, serum was collected, inactivated at 56 °C for 30 min, added with thimerosal sodium at a final concentration of 0.01%, filtered and sterilized with a 0.22 μm pore size filter membrane, and stored at -70 °C or below.

[0057] Mycoplasma bovis antibody-positive serum: Mycoplasma bovis strain PD (Zhao Haoran; Zhang Yunke; Wang Zhanhui; Liu Mengyao; Wang Pengpeng; Wu Wenxue; Peng Chen. MBOVPG45_0375 Encodes an IgG-Binding Protein and MBOVPG45_0376 Encodes an IgG-Cleaving Protein in Mycoplasma bovis. Front Vet Sci. 2021 Apr 15;8:644224) was cultured in PPLO medium (BD, catalog number: 0175457) to 10 10 CFU / ml. After inactivating with BEI, it was emulsified with Freund's adjuvant to prepare an inactivated vaccine, and healthy calves were immunized intramuscularly in the neck at 2 ml / head. The second immunization was given at the same dose and route 14 days later. After the serum antibody turned positive detected by a commercial Mycoplasma bovis ELISA antibody detection kit (Beijing Biaochi Zehui Biotechnology Co., Ltd., production batch number E53202401), it was inactivated at 56 °C for 30 min, added with thimerosal sodium at a final concentration of 0.01%, filtered and sterilized with a 0.22 μm pore size filter membrane, and stored at -70 °C or below.

[0058] Baculovirus extraction kit: Shanghai Shangbao Biotech.

[0059] The specific operation steps of the neutralization test are as follows: (1) Transfer MDBK cells to a 96-well plate one day in advance to ensure that the cells used in the experiment are in the logarithmic growth phase.

[0060] (2) Incubate the serum in a 56 °C water bath for 30 min for inactivation.

[0061] (3) Dilute the serum to be tested, BVDV antibody-negative serum, and positive serum from 1:4 and 1:8 to 1:256 successively with maintenance medium, add the serum to be tested, negative, and positive sera to the wells of a 96-well cell culture plate, 120 μL per well, and repeat 2 wells for each group. Set a blank control well with only maintenance medium added.

[0062] (4) Add 120 μL of a virus suspension of 200 TCID 50 / 0.1 mL to each well. The virus used is BVDV-1 JL strain.

[0063] (5) Use maintenance medium to make 4 serial 10-fold dilutions of a virus solution of 10 4 TCID 50 / 0.1 mL, with 4 wells for each dilution, add 120 μL of virus suspension to each well, and make a virus regression control.

[0064] (6) Place the culture plate in a 37 °C CO2 incubator for 1 hour.

[0065] (7) Discard the medium in the 96-well plate and wash twice with PBS. Pipette 100 μL of the above mixture and add it to the monolayer-grown MDBK cells, with 4 replicates per group.

[0066] (8) Observe with an inverted microscope and record the number of cell wells with cytopathic effect, and calculate its 50% protective dose (PD 50 ) according to the Reed-Muench method, and then calculate the neutralization titer of the serum.

[0067] PBST: Take 10 packets of PBS phosphate buffer powder (produced by Beijing Solarbio Science & Technology Co., Ltd., specification: dissolve and dilute to 2 L / packet), add 20 L of deionized water to dissolve thoroughly, add 10 ml of Tween-20, autoclave at 121 °C for 30 minutes. After placing at room temperature, add Proclin 300 at a final concentration of 0.04%, mix well and dispense aseptically in aliquots.

[0068] Example 1. Expression and purification of BVDV recombinant E2 protein 1. Construction of pFastBac Dual-E2 plasmid Based on the codon-optimized BVDV NADL strain E2 gene sequence and the DNA fragment containing the HBM tag and His tag, prepare the HBM-His-E2 fusion gene (its sequence is SEQ ID No.1).

[0069] In SEQ ID No.1, positions 1-16 show the upstream homologous arm, positions 17-79 show the DNA sequence of the HBM signal peptide, positions 80-97 show the DNA sequence of the His tag, positions 98-1132 show the optimized E2 gene sequence, and positions 1133-1148 show the downstream homologous arm.

[0070] Linearize the pFastBac Dual vector by double digestion (restriction enzymes SalI and PstI) to obtain a linearized vector; use homologous recombinase to perform homologous recombination of the linearized vector with the BM-His-E2 fusion gene, and denote the resulting recombinant vector with the correct sequence as pFastBac Dual-E2 plasmid ( Figure 1 ).

[0071] The pFastBac Dual-E2 plasmid is a recombinant plasmid obtained by replacing the DNA fragment between the SalI and PstI recognition sequences of the vector pFastBac Dual with the DNA fragment shown at positions 17 - 1132 of SEQ ID No.1. This plasmid drives the expression of the target gene by the polyhedrin (PH) promoter and can express the HBM-His-E2 fusion protein (i.e., recombinant E2 protein) shown in SEQ ID No.2.

[0072] In SEQ ID No.2, positions 1 - 21 show the HBM signal peptide, positions 22 - 27 show the His tag, and positions 28 - 371 show the E2 protein.

[0073] 2. Construction and Identification of Recombinant Bacmid rBacmids-E2 Transfer the plasmid pFastBac Dual-E2 into DH10Bac competent cells. The transfer steps are as follows: Take DH10Bac competent cells, add 1 μg of the plasmid pFastBac Dual-E2 after ice-bathing until melted, and continue ice-bathing for 30 min. Then heat-shock at 42°C for 1 min, ice-bathe for 2 min, add 900 μL of SOC medium, and incubate with shaking at 37°C and 225 rpm for 4 h. After incubation, centrifuge the cells to recover and resuspend them, and spread them on an LB plate containing Kan, Gen, Tet, X-gal, and IPTG, and incubate inverted at 37°C for 24 h. Select white monoclonal colonies and inoculate them into an LB liquid medium containing Kan, Gen, and Tet, and incubate with shaking at 37°C and 200 rpm until the bacterial solution becomes turbid. Take a sample and identify the recombinant bacmid by PCR. Send the positive clone for sequencing confirmation. Take the confirmed positive bacteria and extract the recombinant bacmid rBacmids-E2 using a bacmid extraction kit according to the instructions.

[0074] 3. Rescue of Recombinant Baculovirus rBV-E2 Take Sf9 cells, add 5 volumes of IT SFM 03 medium, centrifuge at 1,000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with the medium, and adjust the cell density to 1×10 6 cells / mL, and culture them in suspension at 27°C and 125 rpm for 3 days. After subculturing 3 times, use them for the rescue experiment. The density is 4×10 5Sf9 cells at a density of

[0075] According to the method in Step 2, replace "pFastBac Dual-E2" with "pFastBac Dual", and keep other steps unchanged to obtain Bacmids. According to the method in Step 3, replace "rBacmids-E2" with "Bacmids", and keep other steps unchanged to obtain the recombinant empty vector baculovirus BV.

[0076] 4. Identification of the expression of recombinant E2 protein (1) Indirect immunofluorescence analysis Inoculate the recombinant baculovirus strain rBV-E2 and the recombinant empty vector baculovirus BV into Sf9 cells cultured in a 24-well plate respectively. After 3 days of culture, discard the culture supernatant and fix the cells. Add cell fixative and let it stand at room temperature for 15 min. After discarding the fixative, wash the cells twice with PBST, add PBS containing 0.1% Triton-100, 200 μL per well, and let it stand at room temperature for 10 min for permeabilization. After discarding the permeabilization solution, wash the cells twice with PBST, add 200 μL of PBST containing 5% skim milk powder, and incubate at 37°C for 1 h for blocking. After discarding the blocking solution, wash the cells twice with PBST, add the monoclonal antibody against BVDV E2 protein diluted 1:500 as the primary antibody, and incubate at 37°C for 2 h. After discarding the primary antibody, wash the cells three times with PBST, add the FITC-labeled goat anti-mouse IgG (H+L) antibody diluted 1:1,000 as the secondary antibody, and let it stand in an incubator at 37°C for 1 h. Finally, discard the secondary antibody, wash the cells three times with PBST, and observe the results under a fluorescence microscope. As Figure 2 shown, green fluorescence was detected in Sf9 cells infected with the recombinant baculovirus rBV-E2, while there was no fluorescence in the control group infected with the recombinant empty vector baculovirus BV, indicating that the recombinant E2 protein was successfully expressed in Sf9 cells.

[0077] (2) Western blot analysis The recombinant baculovirus strain rBV-E2 or the recombinant empty vector baculovirus BV was inoculated into Sf9 cells. After 3 days of culture, the cell culture supernatant and cell pellet precipitate were collected by centrifugation at 1,000 rpm for 10 min. The cell pellet precipitate was lysed and then centrifuged at 12,000 rpm for 10 min to collect the cell lysate supernatant. The cell culture supernatant and cell lysate supernatant were used as the test samples. Normal Sf9 cells were processed in the same way as a blank control. The test samples were subjected to SDS-PAGE and then detected by Western blot. The detection antibodies for E2 were the monoclonal antibody against BVDV E2 protein diluted 1:5,000 and the HRP-labeled goat anti-mouse IgG (H+L) antibody diluted 1:8,000. The blocking solution was PBST containing 5% skim milk powder. The results were as Figure 3 shown. The recombinant E2 protein was correctly expressed, and a protein with an expected molecular weight of approximately 50 kDa was obtained. The E2 protein was detected in both the cell culture supernatant and cells, indicating that it could be secreted extracellularly.

[0078] 5. Large-scale expression of recombinant E2 protein Take High Five cells, add 5 volumes of ITS SFM 03 medium, centrifuge at 1,000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with ITS SFM 03 medium and transfer it to a shake flask, adjust the cell density to 1×10 6 cells / mL, and suspend and culture at 27°C and 125 rpm for 2 days and passage 3 times. Inoculate High Five cells at 1×10 6 cells / mL into a 125 mL shake flask with a working volume of 30 mL. When the cell density reaches 3×10 6 cells / mL, inoculate the recombinant baculovirus rBV-E2 at an MOI of 3, culture at 27°C and 125 rpm for 2 days. After the culture is completed, centrifuge at 2,000 rpm for 10 min to remove the cell pellet, and then centrifuge at 12,000 rpm for 30 min to remove cell debris and impurities. Harvest the supernatant as the recombinant E2 protein sample. The sample was preliminarily purified by the saturated ammonium sulfate method, and the recombinant E2 protein with His tag was further purified by Ni + affinity chromatography. The purity of the purified sample was detected by SDS-PAGE, Coomassie brilliant blue staining and Western blot. The results were as Figure 4 shown. High-purity recombinant E2 protein was obtained after two-step purification.

[0079] Example 2. Preparation and purification of monoclonal antibody against BVDV E2 protein 1. Mouse immunization Four 6-week-old female BALB / c mice were selected. Among them, three mice were immunized with the purified recombinant E2 protein antigen obtained in Example 1, and the remaining one mouse was used as a negative control without immunization. Each mouse in the immunized group was subcutaneously injected with 200 μL of the emulsion (prepared by emulsifying the antigen with Freund's complete adjuvant or Freund's incomplete adjuvant) at multiple points. The immunization protocol was as follows: The first immunization was performed on day 0, with 50 μg of the antigen subcutaneously injected at multiple points and Freund's complete adjuvant used; the second immunization was performed on day 14 and the third immunization on day 28, with 100 μg of the antigen subcutaneously injected at multiple points each time and Freund's incomplete adjuvant used; the booster immunization (the fourth immunization) was performed on day 35, with 100 μg of the antigen intraperitoneally injected without adjuvant. The negative control mice were only injected with the same volume of Freund's complete adjuvant or Freund's incomplete adjuvant.

[0080] 2. Detection of serum antibody titer An indirect ELISA method for screening hybridoma cells was established using the mouse serum after the fourth immunization.

[0081] (1) The recombinant E2 protein was diluted to 0.5 μg / mL with the antigen coating solution (0.05 mol / L carbonate buffer with a pH value of 9.6 ± 0.2). 100 μL was added to each well and coated overnight at 4°C (about 8 h), and then washed 3 times with PBST.

[0082] (2) 200 μL of 5% skim milk solution was added to each well and blocked at 37°C for 2 h, and then washed 3 times with PBST.

[0083] (3) The mouse serum samples were diluted in a gradient from 1:200 to 1:25600. Three replicate wells were set for each sample, with 100 μL per well, incubated at 37°C for 1 h, and then washed 3 times with PBST.

[0084] (4) The HRP-labeled goat anti-mouse IgG (H+L) antibody was diluted 1:5000 with 2.5% skim milk. 100 μL was added to each well, incubated at 37°C for 1 h, and then washed 3 times with PBST.

[0085] (5) 100 μL of TMB chromogenic solution was added to each well and left to stand at room temperature in the dark for 10 min.

[0086] (6) 50 μL / well of ELISA stop solution (1 M HCl) was added to terminate the color development, and the absorbance at 450 nm was measured within 5 min after the color development was terminated.

[0087] (7) Calculate the P / N value (the ratio of the absorbance of the serum of the immunized group mice to the absorbance of the serum of the negative control group mice). When P / N > 2, it is judged as positive (indicating that the immunized group mice have successfully produced E2 antibodies), otherwise it is negative. The mouse with the highest antibody titer was selected for the cell fusion experiment to prepare monoclonal antibodies. As Figure 5As shown, the immune effects of mice No. 2 and No. 3 are better, and the titer of mouse No. 3 is the highest.

[0088] 3. Cell Fusion and Screening Resuscitate SP2 / 0 cells 30 days before fusion, subculture them until the logarithmic growth phase, collect cells in good condition for standby, and collect the culture supernatant as a negative control at the same time. Prepare feeder cells 24 hours before fusion. Dilute the peritoneal cells of healthy BALB / c mice to about 1×10 5 cells / mL and inoculate them into 96-well plates for culture. The splenocytes for fusion are taken from BALB / c mice on the 3rd day after booster immunization. After anesthesia and blood collection, obtain the spleen aseptically and prepare a splenocyte suspension for standby. Mix the splenocytes and SP2 / 0 cells at a ratio of 2:1, use 50% PEG1450 to induce fusion, slowly resuspend the cells after fusion and inoculate them into 96-well plates containing feeder cells, and culture them in an incubator at 37°C and 5% CO2. Observe cell growth on the 4th day after fusion, and detect the antibody activity of the supernatant by ELISA on the 7th day to screen for positive clones. Use HAT medium at the initial stage of culture, change half of the medium every 3 - 4 days; change to HT medium between the 2nd and 3rd rounds of subcloning, and finally change to DMEM medium to screen and expand stable hybridoma cell lines.

[0089] According to the indirect ELISA method in step "2. Detection of Serum Antibody Titer", use recombinant E2 protein or BVDV-1 JL strain as the coating antigen to detect the culture supernatant of hybridoma cells. Optimize the coating conditions (concentration of recombinant E2 protein or dilution of BVDV virus solution) by the checkerboard titration method, with the maximum positive / negative absorbance ratio (P / N value) and the OD of positive serum 450 close to 1.0 being the best. Determine as positive when the P / N value > 2. The results of indirect ELISA show that the P / N value is the largest when the BVDV-1 JL virus solution with a titer of 10 7.0 TCID 50 is diluted 1:8 for coating (Table 1); the P / N value is the largest when coating 0.5 μg / mL of recombinant E2 protein (Table 2). Based on this, establish an indirect ELISA method to detect the monoclonal antibody supernatant secreted by hybridoma cells and screen for positive monoclonal cells. Perform limited dilution subcloning on hybridoma cells that are positive in both detections, and obtain a cell line that stably secretes monoclonal antibodies after 2 - 4 times of cloning. After screening by the indirect ELISA method and four times of subcloning, 7 single hybridoma cells against BVDV are obtained, named 2H5, 3G3, 5H7, 7C12, 7D6, 8E1, and 9F3 respectively.

[0090]

[0091]

[0092] 4. Preparation and Purification of Monoclonal Antibodies The screened hybridoma cell lines were inoculated into T175 culture flasks at a density of 5×10 5 cells / mL, and 30 mL of DMEM medium containing 10% fetal bovine serum was added. After culturing until the medium turned yellow, the supernatant was collected, centrifuged at 1000 rpm for 10 min, and the supernatant was transferred and stored frozen for later use. The antibody was purified by the caprylic acid-ammonium sulfate method: The supernatant was mixed with acetate buffer in a ratio of 1:2, 33 μL / mL of caprylic acid was added, and the mixture was stirred at room temperature for 30 min and then left standing at 4°C for 4 h until the final concentration reached 45%. After standing for 4 hours, it was centrifuged again. The precipitate was resuspended with PBS, centrifuged at 12,000 rpm for 5 min to remove impurities, ultrafiltered to remove ammonium sulfate and the solution was exchanged 3 times to obtain the purified monoclonal antibodies of each hybridoma cell, and the concentration was determined by the BCA method. The monoclonal antibodies obtained from hybridoma cells 2H5, 3G3, 5H7, 7C12, 7D6, 8E1, and 9F3 were designated as 2H5 monoclonal antibody, 3G3 monoclonal antibody, 5H7 monoclonal antibody, 7C12 monoclonal antibody, 7D6 monoclonal antibody, 8E1 monoclonal antibody, and 9F3 monoclonal antibody, respectively.

[0093] 5. Identification of Monoclonal Antibodies (1) Subclass Identification of Monoclonal Antibodies An indirect ELISA experiment was performed using a mouse monoclonal antibody typing reagent to determine the class of the monoclonal antibody. The coated antigen was recombinant E2 protein, which was co-incubated with the supernatant of the monoclonal antibody secreted by the hybridoma cells. The detection antibodies were goat anti-mouse IgG1 specific antibody, goat anti-mouse IgG2a specific antibody, goat anti-mouse IgG2b specific antibody, goat anti-mouse IgG3 specific antibody, goat anti-mouse IgA specific antibody, and goat anti-mouse IgM specific antibody, and the enzyme-labeled antibody was HRP-labeled rabbit anti-goat IgG (H+L) antibody. Detection was carried out according to the indirect ELISA method established in step "2. Detection of Serum Antibody Titer", and the OD 450 nm absorbance value was read. The results showed that the monoclonal antibodies of 2 hybridoma cell lines (3G3, 7D6) were IgG1, the monoclonal antibodies of 2 hybridoma cell lines (8E1, 9F3) were IgG2a, and the monoclonal antibodies of 3 hybridoma cell lines (2H5, 5H7, 7C12) were all IgM.

[0094]

[0095] (2) Titer Determination of Monoclonal Antibodies The enzyme-linked immunosorbent assay (ELISA) plate was coated with recombinant E2 protein, and HRP-labeled goat anti-mouse IgG (H+L) antibody was used as the enzyme-labeled secondary antibody. After the purified monoclonal antibodies secreted by each hybridoma cell were serially diluted, detection was carried out according to the ELISA method in step "2. Detection of Serum Antibody Titer", and the P / N value was calculated (P is the OD of the sample to be tested450nm value, and N is the OD value of the negative control 450nm value (the negative control is DMEM medium). The titer of the monoclonal antibody is the maximum dilution multiple when P / N is greater than 2. The results showed that the titer of the IgG1 monoclonal antibody 3G3 was the highest, with a detection limit up to 1:51200, and the titer of the monoclonal antibody 7D6 was 1:25600 (Table 4); the titer of the IgG2a monoclonal antibody 9F3 reached 1:25600, and the titer of the monoclonal antibody 8E1 was 1:6400 (Table 5); the titers of the IgM antibodies (Table 6) were 2H5 (1:1600), 5H7 (1:800), and 7C12 (1:800) respectively, all lower than that of the 3G3 monoclonal antibody. Therefore, the 3G3 monoclonal antibody secreted by the hybridoma cell line 3G3 was used for subsequent experiments.

[0096]

[0097]

[0098]

[0099] The hybridoma cell line 3G3 was deposited on April 18, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 46345.

[0100] (3) Determination of the passage stability of the hybridoma cell line The obtained hybridoma cell line 3G3 was continuously passaged in vitro for 10 generations, and the cell supernatants were collected respectively and detected by the established indirect ELISA method. The stability of the antibody secreted by the hybridoma cell line was determined. As Figure 6 shown, after continuous passage 10 times, the 3G3 cell line could still stably secrete monoclonal antibody, showing good stability.

[0101] (4) Identification of the specificity of the monoclonal antibody Viruses to be tested: BVDV-1 JL strain, BVDV-2 HLJ-10 strain, bovine herpesvirus type 1 (BoHV-1), bovine parainfluenza virus type 3 (BPIV-3) SD0835 strain, lumpy skin disease virus (LSDV), classical swine fever virus (CSFV). MDBK cells were infected with BVDV-1 JL strain, BVDV-2 HLJ-10 strain, BoHV-1, BPIV-3, and LSDV respectively, and PAM cells were infected with CSFV. After 48 h of infection, the specificity of the monoclonal antibody was identified by indirect immunofluorescence (IFA) using the 3G3 monoclonal antibody as the primary antibody and the FITC-labeled goat anti-mouse IgG (H+L) antibody as the secondary antibody. The specific operation is as follows: Prepare 5×10 5Cell suspension at [number] cells / mL was inoculated in 24-well plates and cultured at 37 °C in 5% CO₂ for 12 h. The virus to be tested was inoculated onto the cell monolayer at an MOI of 0.1 and the cells were incubated for 2 h. The inoculum was discarded, and the adherent cells were washed 3 times with PBS to remove the non-infected virus. Cell maintenance medium was added, and the cells were cultured in an incubator at 37 °C in 5% CO₂. After 48 h, the culture medium was discarded, and 250 μL of cell fixative was added to each well and left standing at room temperature for 15 min. The fixative was discarded, and the cells were washed 3 times with PBS. Then, 250 μL of 0.1% Triton-100 was added and the cells were permeabilized at room temperature for 10 min. The permeabilization solution was discarded, and the cells were washed 3 times with PBS. Next, 250 μL of 2% BSA was added and the cells were blocked at 37 °C for 30 min. The blocking solution was discarded, and 250 μL of the purified 3G3 monoclonal antibody diluted 1:500 with PBST was added and incubated overnight at 4 °C. The primary antibody was discarded, and the cells were washed 3 times with PBS. Then, 250 μL of FITC-labeled goat anti-mouse IgG (H+L) antibody diluted 1:1000 with PBST was added as the secondary antibody and incubated for 1 h at room temperature in the dark. The secondary antibody was discarded, and the cells were washed 3 times with PBS. Then, 250 μL of DAPI was added and incubated for 5 min at room temperature in the dark. The cells were washed 3 times with PBS and observed under a fluorescence microscope.

[0102] The results were as Figure 7 shown. The 3G3 monoclonal antibody had specific fluorescence reactions with BVDV-1 JL strain ( Figure 7 B in Figure 7 ) and BVDV-2 HLJ-10 strain ( Figure 7 C in Figure 7 ), but the fluorescence intensity of BVDV-1 JL strain was higher than that of BVDV-2 HLJ-10 strain. The 3G3 monoclonal antibody did not react with CSFV ( Figure 7 D in Figure 7 ), BoHV-1 ( Figure 7 E in Figure 7 ), BPIV-3 ( Figure 7 F in Figure 7 ), LSDV ( Figure 7 G in Figure 7 ). The above results indicated that the 3G3 monoclonal antibody had good specificity for detecting BVDV and could bind to both BVDV-1 JL strain and BVDV-2 HLJ-10 strain.

[0103] (5) Western blot analysis The purified recombinant E2 protein obtained in Example 1 was subjected to Western blot analysis using the 3G3 monoclonal antibody (diluted 1:2000) as the primary antibody and HRP-labeled goat anti-mouse IgG (H+L) antibody (diluted 1:5000) as the secondary antibody. As Figure 8 shown, the 3G3 murine monoclonal antibody (i.e., 3G3 monoclonal antibody) could bind to the E2 protein, and the band size was approximately 50 kDa.

[0104] Example 3. Establishment of a blocking ELISA method for detecting BVDV neutralizing antibodies 1. Horseradish peroxidase-labeled monoclonal antibody Use commercially available "aldehyde-activated horseradish peroxidase" to label the monoclonal antibody against E2 protein (i.e., 3G3 monoclonal antibody) with horseradish peroxidase (HRP). The steps are as follows: (1) Take 1 mg of 3G3 monoclonal antibody and add it to the tube containing activated HRP. Mix well and add 100 μL of carbonate buffer.

[0105] (2) Incubate at room temperature in the dark for 4 h, shaking on a mixer.

[0106] (3) Add sodium borohydride at a concentration of 4 mg / mL at 1 / 10 of the reaction volume.

[0107] (4) Vortex to mix well and incubate on a mixer at room temperature in the dark for 30 min.

[0108] (5) Add 100 μL of 0.5 M Tris-HCl to the reaction tube.

[0109] (6) Vortex to mix well and incubate on a mixer at room temperature in the dark for 30 min.

[0110] (7) Replace the buffer with PBS in a 1 mL ultrafiltration tube and concentrate as much as possible to reduce the volume, i.e., obtain horseradish peroxidase-labeled 3G3 monoclonal antibody.

[0111] (8) Add the preservative ProClin300 and 30 - 50% glycerol to the antibody, mix well and store at -20°C.

[0112] 2. Determination of the optimal antigen coating concentration and the optimal serum dilution ratio Dilute the recombinant E2 protein (prepared in Example 1) with a concentration of 25 mg / mL to 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL respectively with carbonate buffer (0.05 mol / L, pH 9.6 ± 0.2) as the coating antigen, and coat the enzyme-linked immunosorbent assay (ELISA) plate at a volume of 100 μL / well. Dilute the positive serum and negative serum of BVDV antibody with PBST at ratios of 1:5, 1:10, 1:25, 1:50, 1:100, 1:250, and 1:500. Use HRP-labeled 3G3 monoclonal antibody to perform blocking ELISA with the positive serum and negative serum of BVDV antibody. Through the checkerboard titration test, with the highest blocking rate PI value (PI = 1 - OD value of the positive serum well of BVDV antibody / OD value of the negative serum well of BVDV antibody) 450nm value 450nmThe concentrations or dilutions corresponding to the (value) are used as the optimal antigen coating concentration and serum dilution, respectively. The results show that the optimal antigen coating concentration is 0.01 - 0.05 μg / well, and the optimal serum dilution is 1:10, Table 7.

[0113]

[0114] 3. Determination of the optimal blocking solution Prepare PBST solutions containing 5% sheep serum + 5% sucrose (add 5 mL sheep serum and 5 g sucrose to every 95 mL PBST), 2% trehalose + 5% sucrose (add 2 g trehalose and 5 g sucrose to every 100 mL PBST), 2.5% gelatin + 5% sucrose (add 2.5 g gelatin and 5 g sucrose to every 100 mL PBST), and 1.5% ovalbumin + 5% sucrose (add 1.5 g ovalbumin and 5 g sucrose to every 100 mL PBST) as blocking solutions. Prepare a 0.25 μg / mL recombinant E2 protein solution using carbonate buffer (0.05 mol / L, pH 9.6 ± 0.2), and coat the ELISA plate at a volume of 100 μL / well. Block the plate with these 4 blocking solutions at 37°C for 1.5 h. Use BVDV antibody positive serum and negative serum (diluted 1:10) for blocking ELISA with HRP-labeled 3G3 monoclonal antibody. Determine the optimal blocking solution according to the blocking rate PI value (PI = 1 - OD 450 nm value of the BVDV antibody positive serum well / OD 450 nm value of the BVDV antibody negative serum well). The results (Table 8) show that when using the PBST solution containing 2.5% gelatin + 5% sucrose as the blocking solution, the OD 450nm value of the negative serum is relatively high and the PI value is the largest. Therefore, the PBST solution containing 2.5% gelatin + 5% sucrose is the optimal blocking solution.

[0115]

[0116] 4. Determination of the optimal dilution of the enzyme-labeled monoclonal antibody Prepare a 0.25 μg / mL recombinant E2 protein solution using carbonate buffer (0.05 mol / L, pH 9.6 ± 0.2), and coat the ELISA plate at a volume of 100 μL / well. Block the plate with the optimal blocking solution. Dilute the HRP-labeled 3G3 monoclonal antibody with PBST at 1:50, 1:60, 1:70, and 1:80 respectively, and perform blocking ELISA with BVDV antibody positive serum and BVDV antibody negative serum (diluted 1:10). Determine according to the blocking rate PI value (PI = 1 - OD of the positive serum well 450nm value / OD of negative serum wells 450 The optimal dilution of the enzyme-labeled monoclonal antibody was determined by the PI value (). The results (Table 9) showed that there was no significant difference in the PI value when the concentration of the enzyme-labeled monoclonal antibody was 0.2 - 1.0 μg / mL. Therefore, the optimal concentration of the enzyme-labeled monoclonal antibody was 0.2 - 1.0 μg / mL.

[0117]

[0118] 5. Selection of sample diluent PBST solution containing 15 mg / mL BSA, PBST solution containing 5% (v / v) sheep serum, and PBST solution were prepared respectively as sample diluents. A recombinant E2 protein solution at 0.25 μg / mL was prepared using carbonate buffer (0.05 mol / L, pH 9.6 ± 0.2) and coated on an enzyme-labeled plate at 100 μL / well. The best blocking solution was used for blocking. Three sample diluents were used to dilute the BVDV antibody-positive serum and antibody-negative serum at a ratio of 1:10 respectively, and were subjected to blocking ELISA with HRP-labeled 3G3 monoclonal antibody (0.2 μg / mL). According to the blocking rate PI value (PI = 1 - OD of positive control well 450nm value / OD of negative control well 450nm value), the optimal sample diluent was determined. The results (Table 10) showed that when PBST was used as the sample diluent, the PI value was relatively large and the difference was not significant. Therefore, the optimal sample diluent was PBST.

[0119]

[0120] 6. Preparation of BVDV antibody detection kit based on blocking ELISA According to the above, the composition of the BVDV antibody detection kit based on blocking ELISA was determined as follows: 1) Antigen-coated plate, that is, a 96-well enzyme-labeled plate coated with a dilution of recombinant E2 protein in carbonate buffer at 0.01 - 0.05 μg / well and blocked with a blocking solution (PBST solution containing 2.5% gelatin + 5% sucrose); 2) BVDV antibody-positive serum; 3) BVDV antibody-negative serum; 4) Sample diluent, that is, PBST, used to dilute the serum to be tested and the enzyme-labeled monoclonal antibody. The serum to be tested can be diluted at a ratio of 1:10; 5) Enzyme-labeled monoclonal antibody, that is, 3G3 monoclonal antibody labeled with horseradish peroxidase. The working concentration of the enzyme-labeled monoclonal antibody is 0.2 - 1.0 μg / mL; 6) Blocking solution, that is, PBST solution containing 2.5% gelatin + 5% sucrose (add 2.5 g of gelatin and 5 g of sucrose to every 100 mL of PBST); 7) Washing solution, i.e., PBST; 8) Substrate Solution A, i.e., TMB two-component chromogenic solution A (Solarbio Science & Technology Co., Ltd., Beijing, Catalog No. PR1210); 9) Substrate Solution B, i.e., TMB two-component chromogenic solution B (Solarbio Science & Technology Co., Ltd., Beijing, Catalog No. PR1210); 10) Stop solution, i.e., 1 M HCl aqueous solution; 11) Sealing film.

[0121] The operation steps of the BVDV antibody detection kit based on the blocking ELISA method are as follows: (1) Sample preparation: Take whole animal blood. After the blood coagulates, centrifuge at 3000 r / min for 10 minutes to collect the supernatant. Serum can also be collected by allowing the blood to clot and then naturally separating the serum. The serum is required to be clear and free of hemolysis.

[0122] (2) Sample dilution: Dilute the serum to be tested, BVDV antibody positive serum, and BVDV antibody negative serum 1:10 with the sample diluent.

[0123] (3) Kit preparation: Restore the kit to room temperature before use. Gently rotate or shake the liquid reagents to mix well.

[0124] (4) Numbering: Take out the antigen-coated plate (which can be disassembled and used in multiple batches according to the number of samples), and record the positions of the samples in a record book.

[0125] (5) Sample addition: Add the diluted BVDV antibody positive serum, BVDV antibody negative serum, and serum to be tested to the antigen-coated plate in sequence, 100 μL per well. Gently mix the samples in the wells (do not overflow), seal with a sealing film, and incubate at 37°C for 1 hour.

[0126] (6) Incubation: Seal the enzyme-labeled plate with a film and incubate in a 37°C constant temperature incubator for 1 h.

[0127] (7) Plate washing: Discard the liquid in the wells, add 200 μL of the diluted washing solution to each well, mix gently, then discard the washing solution. Repeat the washing 5 times. After the last washing, discard the washing solution and pat dry on absorbent paper.

[0128] (8) Addition of enzyme-labeled monoclonal antibody: Add 100 μL / well of the enzyme-labeled monoclonal antibody to the sample wells. The enzyme-labeled monoclonal antibody is diluted to 0.2 - 1.0 μg / mL with PBST. Each plate can detect 90 samples, with 3 positive control wells and 3 negative control wells set. Replace the pipette tips after each sample addition, and accurately record the positions of each sample on the plate.

[0129] (9) Incubation: Seal with a sealing film and incubate at 37°C for 1 hour.

[0130] (10) Wash the plate, discard the liquid in the wells, and repeat step (7).

[0131] (11) Color development: After mixing liquid A and liquid B of the substrate in equal proportions, add 100 μL to each well, cover with a sealing film, and incubate at 37 °C in the dark for 10 minutes for color development.

[0132] (12) Termination: Add 50 μL of the termination solution to each well (measure the results within 10 minutes).

[0133] (13) Measurement: Set the wavelength of the microplate reader at 450 nm, zero with the blank well, and then measure the OD value of each well.

[0134] (14) Result determination: Calculate the PI value. The PI value = (1 - OD value of the sample / OD value of the BVDV antibody-negative serum) × 100%. 450nm value / OD value of the BVDV antibody-negative serum 450nm ) × 100%.

[0135] 7. Determination of the cut-off value of the kit Using the above kit, detect 30 BVDV antibody-positive sera and 30 BVDV antibody-negative sera according to the operating steps of the kit, measure the OD values of all samples, and calculate the PI value. The results are shown in Table 11. The PI value = (1 - OD value of the sample / average OD value of the negative control) × 100%. The average OD value of the negative control = (OD value of the first well of the negative control + OD value of the second well of the negative control) / 2. The negative control refers to the BVDV antibody-negative serum in the kit. 450nm value, and calculate the PI value. The PI value = (1 - OD value of the sample / OD value of the negative control 450nm value / average OD value of the negative control 450nm ) × 100%. The average OD value of the negative control 450nm average = (OD value of the first well of the negative control 450nm value + OD value of the second well of the negative control 450nm value) / 2. The negative control refers to the BVDV antibody-negative serum in the kit.

[0136] Among them, the preparation of 30 BVDV antibody-positive sera: Immunize healthy calves with the inactivated vaccine against bovine viral diarrhea / mucosal disease (type 1, NM01 strain). Collect the serum when the neutralizing antibody titer detected by the neutralization test is not lower than 1:1024. Inactivate at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, filter and sterilize with a 0.22 μm pore size filter membrane, and store at -70 °C or below.

[0137] The preparation of 30 BVDV antibody-negative sera: Collect the sera of healthy calves with negative BVDV neutralizing antibody detection. Inactivate at 56 °C for 30 min, add thimerosal sodium with a final concentration of 0.01%, filter and sterilize with a 0.22 μm pore size filter membrane, and store at -70 °C or below.

[0138]

[0139] The ROC curve analysis was performed using GraphPad Prism 8.0.2 software to finally determine the critical values of negative and positive sera. Based on the serum background results and the detection results of this kit, the corresponding relationship between the PI values of the samples tested and sensitivity and specificity was analyzed. With the true positive rate (sensitivity) as the ordinate and the false positive rate (100 - specificity) as the abscissa, an ROC curve was plotted. Each point on the curve represents the sensitivity and specificity corresponding to that PI value. Using the area under the ROC curve value (AUC) as the evaluation criterion, the accuracy of the diagnostic method was evaluated.

[0140] When AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. When AUC is between 0.5 - 0.7, there is relatively low accuracy. When AUC is between 0.7 - 0.9, there is a certain degree of accuracy. When AUC is above 0.9, there is high accuracy.

[0141] When the critical value was 24.72%, the diagnostic sensitivity was 93.33%; the diagnostic specificity was 93.33%; the area under the ROC curve was 0.9856, indicating that this kit has high accuracy ( Figure 9 )). For the convenience of result determination of the kit, the integer 25% was taken as the critical value. Finally, the blocking rate of 25% was determined as the critical value of the kit of the present invention, that is, the determination condition of the kit: when the blocking rate value is less than 25%, the sample is determined to be negative for BVDV neutralizing antibody. When the PI value is greater than or equal to 25%, the sample is determined to be positive for BVDV neutralizing antibody.

[0142] Example 4. Performance test of the BVDV antibody detection kit based on the blocking ELISA method 1. Sensitivity test The BVDV antibody detection kit based on the blocking ELISA method prepared in Example 3 (referred to as the kit of the present invention) was used to detect 28 weakly positive sera of BVDV antibodies. As shown in Table 12, the PI values of the 28 serum samples were all between 25% and 35%, and the positive coincidence rate between the kit and the neutralization test was 100%.

[0143] Among them, the preparation of weakly positive sera of BVDV antibodies: Take the 28 positive sera of BVDV antibodies prepared in the step of "7. Determination of the kit critical value", dilute them with BVDV antibody - negative serum and then detect the neutralization test titer. Take the sera with neutralization titers of 1:4 and 1:8 as the weakly positive sera of BVDV antibodies.

[0144]

[0145] 2. Coincidence rate test A total of 218 inactivated bovine viral diarrhea vaccine immune serum samples were detected using neutralization test, the kit of the present invention, and the IDEXX bovine viral diarrhea virus antibody detection kit of IDEXX Corporation of the United States (referred to as the IDEXX kit). The operation steps of the kit of the present invention refer to Example 3, and the operation steps of the IDEXX kit refer to the kit instruction manual.

[0146] The results showed that among the 218 sera, 148 sera were positive for BVDV antibody and 70 sera were negative for BVDV antibody in the neutralization test. 151 sera were positive for BVDV antibody and 67 sera were negative for BVDV antibody in the detection by the kit of the present invention: 150 sera were positive for BVDV antibody and 68 sera were negative for BVDV antibody in the detection by the IDEXX kit.

[0147] Comparison of the detection results of the kit of the present invention with the neutralization test: The kit of the present invention detected 151 positive samples, among which 146 were consistent with the neutralization test; 67 negative samples were detected, among which 65 were consistent with the neutralization test (Table 13). Therefore, the total coincidence rate of the kit of the present invention with the neutralization test was 96.79%, among which the positive coincidence rate was 98.65% and the negative coincidence rate was 92.86%.

[0148]

[0149] Comparison of the detection results of the IDEXX kit with the neutralization test: The IDEXX kit detected 150 positive samples, among which 144 were consistent with the neutralization test; 68 negative samples were detected, among which 64 were consistent with the neutralization test (Table 14). Therefore, the total coincidence rate of the IDEXX kit with the neutralization test was 95.41%, among which the positive coincidence rate was 97.30% and the negative coincidence rate was 91.43%.

[0150]

[0151] Both the positive coincidence rate and the negative coincidence rate of the kit of the present invention were higher than those of the IDEXX kit. Among the 218 sera detected, 144 positive and 67 negative samples were jointly detected by the two kits. The positive coincidence rate between the kit of the present invention and the IDEXX kit was 96.0%, the negative coincidence rate was 98.53%, and the total coincidence rate was 96.79% (Table 15).

[0152]

[0153] 3. Sensitivity test The BVDV antibody-positive serum (neutralization titer 1:4096) was serially diluted 4-fold, and the positive sera at each dilution were detected using 3 batches of the kit of the present invention and the IDEXX kit according to the instructions respectively. The results are shown in Table 16. The PI values of the positive sera at dilutions of 1:4 to 1:1024 detected repeatedly by the kit of the present invention were all greater than 25%, showing positive results, and were consistent with the detection results of the IDEXX kit.

[0154]

[0155] 4. Specificity test Six bovine infectious disease positive sera were detected using the kit of the present invention, including 5 other bovine infectious disease positive sera (bovine foot-and-mouth disease virus antibody-positive serum, bovine herpesvirus 1 antibody-positive serum, bovine Brucella antibody-positive serum, bovine pathogenic Escherichia coli antibody-positive serum, and bovine Mycoplasma antibody-positive serum) and 1 BVDV antibody-positive serum. The results showed that the detection results of the 5 other infectious disease positive sera by the kit were all negative (Table 17), indicating that the kit of the present invention has very good specificity.

[0156]

[0157] 5. Evaluation of vaccine immune effect Twelve newly born calves negative for BVDV nucleic acid and antibody were selected and immediately transferred to the experimental animal house without contact with the cows and without feeding colostrum. They were fed milk replacer and forage until 2 months old. After re-detection and found negative for BVDV nucleic acid and antibody again, 9 of them were intramuscularly inoculated in the neck with 4 ml of BVDV-1 JL strain antigen solution at a content of 10 8.0 、10 7.0 、10 6.0 TCID 50 / ml respectively, with 3 heads in each dose group. After 21 days of interval, a second immunization was carried out. Fourteen days after the second immunization, the neutralizing antibody titers of the 12 cows were detected, and they were challenged with 10 6.5 TCID 50 / ml of HTB strain BVDV virus solution through the intranasal and intramuscular routes. The occurrence of any one of the following three conditions was defined as disease: ① body temperature increased by 1℃ and at least 1 temperature measurement, ② diarrhea, ③ leukopenia rate ≥ 30%. Those without any of the three conditions were judged as protected. According to the neutralizing antibody titers of the diseased cows and protected cows at the time of challenge, the lowest neutralizing antibody titer that can provide anti-infection protection after vaccine immunization was determined to be 1:16.

[0158] The sensitivity test shows that there is a certain corresponding relationship between the PI value of the serum detected by the kit of the present invention and the neutralizing antibody level (Table 16). Therefore, the negativity, positivity of the neutralizing antibody in the serum sample and the range of the neutralizing antibody titer of the positive serum can be judged according to the PI value, and further, whether the antibody level of the serum sample can provide effective immune protection for the immunized calves can be judged according to the minimum neutralizing antibody standard of the immune-protected cattle obtained in the challenge protection test, so as to provide a scientific and reliable test method and product for evaluating the immune effect of the vaccine.

[0159] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application is intended to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. The application of some basic features can be made according to the scope of the appended claims below.

Claims

1. Monoclonal antibody, characterized in that: The monoclonal antibody is a monoclonal antibody secreted by hybridoma cell 3G3, and the preservation number of the hybridoma cell 3G3 in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 46345.

2. Use of a monoclonal antibody in the preparation of a product for detecting neutralizing antibodies against bovine viral diarrhea virus, characterized in that: The monoclonal antibody is the monoclonal antibody described in claim 1.

3. Use of monoclonal antibodies in detecting neutralizing antibodies against bovine viral diarrhea virus, characterized in that: The monoclonal antibody is the monoclonal antibody described in claim 1.

4. Use of monoclonal antibody in the preparation of a product for detecting bovine viral diarrhea virus, characterized in that: The monoclonal antibody is the monoclonal antibody described in claim 1.

5. Kit, characterized in that: The kit contains the monoclonal antibody described in claim 1.

6. The kit according to claim 5, wherein: The kit is a BVDV antibody detection kit based on the blocking ELISA method, and the monoclonal antibody is labeled with horseradish peroxidase.

7. The kit according to claim 6, wherein: The kit further contains E2 protein, BVDV antibody positive serum, BVDV antibody negative serum, sample diluent and blocking solution; The sample diluent is PBST; The blocking solution is composed of a solvent and a solute. The solvent is PBST, and the solute and its concentration in the blocking solution are 2.5 g / 100 mL gelatin and 5 g / 100 mL sucrose, respectively.

8. The kit according to claim 7, characterized in that: The E2 protein is the E2 protein shown at positions 28-371 of SEQ ID No. 2, or the recombinant E2 protein shown in SEQ ID No.

2.

9. Hybridoma cells, characterized in that: The hybridoma cell is hybridoma cell 3G3, and the preservation number of the hybridoma cell 3G3 in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 46345.

10. Application of hybridoma cells, characterized in that: The hybridoma cell is hybridoma cell 3G3, and the preservation number of the hybridoma cell 3G3 in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 46345; the application is the application in the preparation of BVDV monoclonal antibody, or the application in the preparation of products for detecting neutralizing antibodies against bovine viral diarrhea virus, or the application in the preparation of products for detecting bovine viral diarrhea virus.

Images