Hybridoma cell strain secreting bovine coronavirus antibody and blocking ELISA kit

Abstract

The invention discloses a hybridoma cell strain secreting a bovine coronavirus antibody and a blocking ELISA kit, and belongs to the field of biological immunoassay. The preservation number of the hybridoma cell strain is CCTCC (China Center For Type Culture Collection) NO: C2025146, the monoclonal antibody secreted by the hybridoma cell strain has a blocking effect, the monoclonal antibody is applied to a BCoV blocking ELISA (Enzyme-Linked Immunosorbent Assay) kit, and the BCoV blocking ELISA kit can be used for specifically detecting an antibody generated after BCoV infects or immunizes cattle. The invention plays an important role in BCoV epidemiological investigation and vaccine immune effect monitoring.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological immune analysis, in particular to a hybridoma cell strain secreting bovine coronavirus antibody and a blocking ELISA kit. BACKGROUND

[0002] Bovine coronavirus (BCoV) is a single-stranded positive-sense RNA virus with a capsid and non-segmented, belonging to the genus of Coronaviridae. β Coronaviridae Embecovirus Subgenus, HCoV-OC43, HCoV-HKU1, porcine hemagglutinating encephalomyelitis virus (PHEV), equine coronavirus (ECoV), caprine coronavirus (cpCoV) and canine respiratory coronavirus (CrCoV) are closely related in evolutionary relationship. The bovine coronavirus particles are polygonal, with a diameter of 65-210 nm, and the genome size is between 27-32 kb. It contains 10 open reading frames (ORFs), which encode 5 major structural proteins, respectively, spike protein S (150 kDa), nucleocapsid protein N (50 kDa), envelope protein E (9 kDa), membrane protein M (25 kDa) and hemagglutinin-esterase protein HE (47 kDa). The S protein is located on the outermost side of the virus particle, and is the largest structural protein of coronavirus, which is related to receptor binding. The N protein is located inside the capsid, and is a relatively conserved protein, which is related to viral RNA binding and viral gene replication and transcription. The E protein is the smallest structural protein, which plays an important role in the assembly and release of virus particles. The M protein can interact with other structural proteins, affecting the budding of the virus.

[0003] BCoV is widely present in the world, and is an important pathogen causing bovine enteric disease and respiratory disease, which can cause diarrhea in calves, winter dysentery in adult cattle and upper and lower respiratory tract infection in calves. Studies have shown that BCoV has up to 96% nucleotide homology with human coronavirus HCoV-OC43 first isolated in 1967, and a coronavirus closely related to BCoV was isolated from the feces of a child with diarrhea in Germany in 1988, indicating that BCoV may have the risk of zoonosis. The zoonotic potential of BCoV means that the virus poses a great threat to human health, but there is currently a lack of specific and high-throughput methods for detecting BCoV.

[0004] Monoclonal antibodies as a tool for detecting and treating animal diseases have revolutionized the technology of disease prevention and control. Monoclonal antibodies have the following advantages over polyclonal antibodies: (1) the antigen epitope targeted is single and specific; (2) it can be produced in unlimited quantities. Monoclonal antibodies can more easily identify and characterize the characteristics of pathogens in animal disease diagnosis. The blocking ELISA detection method established using monoclonal antibodies plays an increasingly important role in the monitoring and prevention of animal infectious diseases. The establishment of a rapid, effective and high-throughput BCoV antibody detection method can provide technical support for the prevention and control of BCoV. SUMMARY

[0005] The purpose of the present application is to provide a hybridoma cell strain secreting bovine coronavirus antibody and a blocking ELISA kit to solve the problems existing in the prior art. A hybridoma cell strain 2C4B3C7D6-B is screened from a hybridoma cell library secreting BCoV monoclonal antibody constructed by immunizing mice with BCoV-NXWZ2301 strain. The monoclonal antibody secreted by the hybridoma cell strain has a blocking effect and can be applied to the preparation of a BCoV blocking ELISA kit. The blocking ELISA kit can specifically detect the antibodies produced after BCoV infection or immunization of cattle, providing technical support for the prevention and control of BCoV.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions: The present application provides a hybridoma cell strain 2C4B3C7D6-B secreting anti-bovine coronavirus monoclonal antibody, the preservation number of the hybridoma cell strain is CCTCC NO: C2025146, the preservation unit is China Center for Type Culture Collection, the preservation date is November 6, 2025, and the preservation address is Wuhan University.

[0007] The present application also provides a monoclonal antibody secreted by the hybridoma cell strain.

[0008] The present application also provides applications of the hybridoma cell strain in any of the following: (1) the application in the preparation of a kit for detecting bovine coronavirus antibody; (2) the application in the preparation of a kit for detecting antibody produced after bovine coronavirus infection or immunization of bovine.

[0009] The application further provides the application of the monoclonal antibody in any of the following: (1) the application in the preparation of a kit for detecting bovine coronavirus antibody; (2) the application in the preparation of a kit for detecting antibody produced after bovine coronavirus infection or immunization of bovine.

[0010] Preferably, the kit is a blocking ELISA kit.

[0011] The application further provides a blocking ELISA kit for detecting antibody produced after bovine coronavirus infection or immunization of bovine, wherein the blocking ELISA kit comprises the monoclonal antibody.

[0012] Preferably, the blocking ELISA kit further comprises a coated enzyme plate, horseradish peroxidase-labeled goat anti-mouse IgG, a substrate, a stop solution, a positive control and a negative control.

[0013] Preferably, the coated enzyme plate is obtained by coating bovine coronavirus as an antigen.

[0014] Preferably, the positive control is bovine coronavirus positive bovine serum, and the negative control is bovine coronavirus negative bovine serum.

[0015] The application discloses the following technical effects: (1) The BCoV immunogen used in the application is a BCoV-NXWZ2301 strain isolated from a clinic, and a hybridoma cell strain 2C4B3C7D6-B is screened from an established hybridoma cell library secreting BCoV monoclonal antibody.

[0016] (2) The monoclonal antibody secreted by the hybridoma cell strain 2C4B3C7D6-B has a blocking effect and can be applied to the preparation of a BCoV blocking ELISA kit.

[0017] (3) The BCoV blocking ELISA kit can specifically detect antibody produced after BCoV infection or immunization of bovine and sheep. 350 clinical serum samples of bovine and sheep are collected, a blocking ELISA method and a neutralization experiment are used to detect BCoV antibody in the serum, and the result shows that the coincidence rate of the blocking ELISA result and the neutralization experiment is 98%. The blocking ELISA method constructed in the application has high accuracy, strong specificity and high sensitivity, and plays an important role in BCoV epidemiological investigation and vaccine immunization effect monitoring. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. These embodiments are described herein for illustrative purposes only and are not intended to limit the scope of the application, which is defined by the appended claims as interpreted in light of the full written specification and the appended drawings.

[0019] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range is inclusive of its end points. Obvious modifications and variations are possible in light of the above teachings.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for the disclosure and

[0021] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations fall within the scope of the application. Further, it is to be understood that the disclosure is for illustrative purposes only and that the scope of the present application will be limited only by the claims and the equivalents thereof.

[0022] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0023] Example 1 I. Preparation of Monoclonal Antibody Hybridoma Cell Strains 1. Preparation of antigen: HRT-18 cells were infected with BCoV virus (NXWZ2301), and the virus was harvested 48 h after the cells showed pathological changes. The cells were removed by centrifugation at 10,000 rpm for 30 min. The supernatant was subjected to ultracentrifugation at 40,000 g for 2 h, and the supernatant was discarded. The precipitate was dissolved with PBS overnight to obtain the ultracentrifugation virus.

[0024] 2. Immunization of Balb / c mice: Balb / c mice were immunized subcutaneously with ultralocal virus in multiple sites, a total of 3 times, with 2 weeks interval between each immunization. The first immunization was performed with 100 μL of ultralocal virus mixed with an equal amount of complete Freund's adjuvant, and the following two immunizations were performed with 100 μL of ultralocal virus mixed with an equal amount of incomplete Freund's adjuvant. Two weeks after the third immunization, blood was collected to detect the titer of the immune serum of the mice, and mice with a titer of >10 6 were selected. Four days before cell fusion, the mice were immunized once again with 100 μL of ultralocal virus intraperitoneally.

[0025] 3. Cell fusion: The PEG cell fusion method was used for cell fusion. Mouse myeloma cells (SP2 / 0) and immune spleen cells of Balb / c mice were mixed at a ratio of 1:5 in terms of cell number, centrifuged at 2000 rpm for 5 min at 25°C, and the supernatant was discarded. An appropriate amount of serum-free RPMI-1640 medium was added for resuspension, and the cells were washed by centrifugation at 2000 rpm for 5 min at 25°C. The supernatant was discarded, and the cells were gently tapped to loosen and evenly distribute. The cells were preheated at 37°C for 1 min, and then 0.8 mL of PEG2000 preheated at 37°C was added while oscillating. After addition, oscillation was continued for 1 min, and then 12 mL of preheated serum-free RPMI-1640 medium was added at a rate of 1, 2, 3, 3, and 3 mL / min, respectively, within 5 min. The cells were incubated at 37°C for 10 min, and then centrifuged at 2000 rpm for 5 min at 25°C. The supernatant was discarded, and RPMI-1640 medium containing 15% fetal bovine serum (FBS) and 10% HAT (H—Hypoxanthine, Hypoxanthine; A—Aminopterin, aminopterin; T—Thymidine, thymidine) was added for resuspension. The cells were then dispensed into 96-well plates pre-coated with feeder cells and cultured in a 5% CO2 incubator. During the culture, the cell growth in the wells was observed. After 7 days of cell fusion, the supernatant was collected for antibody detection when the cells grew to 1 / 10-1 / 5 of the area of the 96-well plate.

[0026] 4. Screening of hybridoma cells: Prepare 96-well plate with HRT-18 in advance, inoculate BCoV-NXWZ2301 into the cell plate, and set up a cell control without inoculation. Take out the inoculated 48 h and 96-well plate with HRT-18 cells, discard the supernatant culture medium, wash 3 times with PBST, dry, and add 100 μL of 4% paraformaldehyde per well for fixation at 4°C overnight. Discard the fixing solution, wash 3 times with PBST, dry, add 200 μL of 1% BSA blocking solution per well, and block at 37°C for 2 h. Discard the blocking solution, wash 3 times with PBST, dry, add 100 μL of cell supernatant per well, and incubate at 37°C for 1 h. Discard the cell supernatant, wash 3 times with PBST, dry, add 100 μL of FITC-labeled goat anti-mouse IgG (1:500 dilution) per well in the dark, and incubate at 37°C for 1 h. Wash 3 times with PBST, leave PBST in the plate for the third time, and observe the results under a fluorescence microscope. Select the hybridoma cell strain with specific green fluorescent signal for subcloning.

[0027] 5. Cloning of hybridoma cells: Stain the selected positive well cell strain with trypan blue, count, dilute to 100 cells / 10 mL of culture medium with RPMI-1640 culture medium containing 15% FBS, add the diluted cell suspension to a 96-well plate with feeder cells in advance, 100 μL per well, and culture in a 37°C, 5% CO2 incubator. Observe the cell strain during the period, and when the growth reaches 1 / 10-1 / 5 of the bottom area of the 96-well plate, take the cell supernatant for IFA detection as described above. Record the positive wells of the single clone cells, and perform the same subcloning for more than 3 times until all the cloned cell strains are positive and the OD values of each well detection are close. Expand the cloned BCoV-specific monoclonal antibody hybridoma cell strain for culture, and freeze it. 450

[0028] 6. Determination of blocking effect of hybridoma cells: ​The enzyme-labeled plate was coated with 0.05 mol / L pH 9.6 carbonate buffer as a coating solution, and 100-fold diluted ultralinear virus as a coating antigen, 100 μL / well, 4°C overnight. PBST was washed 3 times, and dried; 1% BSA blocking solution was added, 200 μL / well, 37°C for 2 h. PBST was washed 3 times, and dried; BCoV positive bovine serum diluted with PBS buffer at a volume ratio of 1:4 and BCoV negative bovine serum diluted at a volume ratio of 1:4 were added to the corresponding wells, 100 μL / well, 37°C for 1 h, PBST was washed 3 times, and dried; hybridoma cell supernatant was added, 100 μL / well, 37°C for 1 h. PBST was washed 3 times, and dried; horseradish peroxidase (HRP) labeled goat anti-mouse IgG diluted with PBS buffer at a volume ratio of 1:25000 was added, 100 μL / well, 37°C for 1 h, PBST was washed 3 times, and dried. Substrate TMB was added, 100 μL / well, color development at room temperature for 15 min in the dark; 50 μL 2 mol / L sulfuric acid was added to each well to stop the reaction. The OD value of the enzyme-labeled plate was determined by an enzyme-labeled instrument 450 value, P is the OD 450 value of each positive serum well, N is the OD 450 value of negative serum, when the OD 450 value of negative serum is ≤0.2 and the OD 450 value of positive serum is ≥2.1, i.e. the ratio of the OD 450 value of positive serum to the OD value of negative serum is ≥2.1, under the premise that the negative control and the positive control are both met, the determination criterion for positive well is P / N≥2.1, and then the positive and negative of the detection well are determined. In the primary screening of BCoV monoclonal antibodies, 43 specific positive cell strains were screened from 700 hybridoma cell wells, and one hybridoma cell 2C4 had better blocking effect. The 2C4 cell strain was further subcloned, and one cell strain 2C4B3C7D6-B with the best blocking effect was screened from 30 positive subcloned cell strains secreting BCoV antibodies (Table 1).

[0029] Table 1 Results of positive well screening by third subcloning blocking ELISA The above hybridoma cell strain 2C4B3C7D6-B was preserved in China Center for Type Culture Collection on November 6, 2025, with a preservation number of CCTCC NO: C2025146, and a preservation address of Wuhan, Wuhan University, China.

[0030] 7. Preparation of ascites: Sterile liquid paraffin was injected intraperitoneally into 10-12 week old Balb / c mice (purchased from the Comparative Medicine Experimental Center of Yangzhou University), 0.3 mL per mouse. Seven days later, hybridoma cell line 2C4B3C7D6-B was injected intraperitoneally into each mouse, 0.2 mL (2 × 10⁻⁶) per mouse. 6 ~3×10 6 (1 hybridoma cell). After 7-10 days, ascites fluid was collected from mice with obvious abdominal distension. The cells were centrifuged at 3000 rpm for 20 min, the supernatant was collected, aliquoted, labeled and stored at -20℃ for later use.

[0031] 8. Monoclonal antibody indirect ELISA titer assay: Using 0.05 mol / L pH 9.6 carbonate buffer as the coating medium, and ultravirus diluted 100-fold with PBS buffer as the coating antigen, 100 μL / well was coated onto the ELISA plate and incubated overnight at 4°C. After washing three times with PBST and patting dry, 200 μL / well of 1% BSA blocking buffer was added and incubated at 37°C for 2 h. After washing three times with PBST and patting dry, 100 μL / well of 2C4B3C7D6-B monoclonal antibody ascites fluid was added to the detection wells, and PBS was added to the control wells, and incubated at 37°C for 1 h. After washing three times with PBST and patting dry, 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted 1:25000 with PBS buffer was added and incubated at 37°C for 1 h. After washing three times with PBST and patting dry, the plates were incubated. Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 15 min. Stop the reaction by adding 50 μL of 2 mol / L sulfuric acid to each well. Measure the OD of the microplate using a microplate reader. 450 Value, P is the OD of each detection well. 450 Value, N is the OD of the control well. 450 Value, when the detection well OD 450 Value and reference well OD 450 The criteria for determining a positive well are a ratio ≥2.1 and a test for monoclonal antibody titer (Table 2).

[0032] Table 2. Monoclonal antibody indirect ELISA titer 9. Monoclonal antibody subtype identification: The detection was performed using an SBA mouse monoclonal antibody typing kit. The specific steps were as follows: 100-fold diluted supernatant virus was immobilized in an ELISA plate with carbonate coating buffer (100 μL / well), and incubated overnight at 4°C. The plate was washed three times with PBST and blotted dry. 100 μL of 1% BSA was added to each well and incubated at room temperature for 1 hour. The plate was washed three times with PBST and blotted dry. 100 μL of monoclonal antibody supernatant was added to each well and incubated at 37°C for 1 hour. The plate was washed three times with PBST and blotted dry. 100 μL of HRP-labeled detection antibody diluted in PBS was added to each well and incubated at room temperature for 1 hour. The plate was washed three times with PBST and blotted dry. 100 μL of TMB chromogenic solution was added to each well and incubated at 37°C in the dark for 15 minutes. Finally, 50 μL of 2 mol / L sulfuric acid stop solution was added to each well, and the OD values ​​were read using an ELISA reader. 450 Numerical values ​​(Table 3).

[0033] Table 3. Results of Monoclonal Antibody Subtype Identification II. Establishment of the blocking ELISA kit 1. By optimizing the antigen coating concentration, the dilution of the serum to be tested, the working concentrations of monoclonal antibody 2C4B3C7D6-B and HRP-goat anti-mouse IgG secondary antibody (Beijing Bio-Sens Biotechnology Co., Ltd.), and the reaction time of the serum to be tested, monoclonal antibody 2C4B3C7D6-B, HRP-goat anti-mouse IgG and substrate TMB, the BCoV-specific monoclonal antibody 2C4B3C7D6-B blocking ELISA detection method and the kit assembly were finally completed.

[0034] 2. Optimization of antigen coating concentration and working concentration of monoclonal antibody 2C4B3C7D6-B: The matrix method was used, with 0.05 mol / L pH 9.6 carbonate buffer as the coating solution. Ultraviral whole virus diluted 20, 40, 80, 160, 320, 640, 1280, and 2560 times was used as the coating antigen, and the plates were coated overnight at 4°C (Costar). After washing three times with PBST, 0.5% BSA was added and the plates were incubated at 37°C for 2 hours. After washing three times with PBST and patting dry, monoclonal antibody 2C4B3C7D6-B diluted 200, 400, 800, 1600, and 3200 times was added sequentially. Each dilution was repeated once, and the average value was used to select the OD value. 450 The reaction conditions with the highest values ​​were taken as the optimal reaction conditions. The results are as follows: the optimal antigen coating concentration was 1:160 dilution; the optimal working concentration of monoclonal antibody 2C4B3C7D6-B was 1:3200 dilution (Table 4).

[0035] Table 4. Selection of antigen coating concentration and working concentration of monoclonal antibody 2C4B3C7D6-B 3. Optimization of serum dilution and reaction time: Coat the ELISA plate with the optimal coating concentration described above. After washing three times with PBST, add 0.5% BSA and incubate at 37°C for 2 hours. Wash three times with PBST, pat dry, and add 50 μL / well of BCoV-positive and BCoV-negative bovine and sheep serum diluted 1:1, 1:3, 1:7, and 1:15, respectively. Repeat each dilution once. Incubate at 37°C for 1 hour, wash three times with PBST, pat dry, and add monoclonal antibody simultaneously before continuing incubation. Select an appropriate serum dilution.

[0036] Coat the ELISA plate with the optimal coating concentration. After washing three times with PBST, add 0.5% BSA and block at 37℃ for 2 hours. Wash three times with PBST and blot dry. Add 50 μL / well of 1:7 diluted BCoV bovine positive and negative sera. Incubate at 37℃ for 30 min, 45 min, 60 min, and 90 min, respectively. Remove the corresponding ELISA strips, wash three times with PBST, blot dry, and add monoclonal antibody before continuing incubation. Take the average value of 8 replicate wells and select the OD of negative serum. 450 The optimal reaction conditions were defined as those with the shortest reaction time, where the PI value was close to 1, the PI value was the highest, and the first two results were similar. The results are as follows: the serum dilution was 1:7; the serum reaction time was 30 min (Tables 5 and 6).

[0037] Table 5 Optimization of serum dilution Table 6 Optimization of serum treatment time 4. Optimization of the action time of monoclonal antibodies: The ELISA plate was coated and blocked according to the above conditions. Serum was added, and after incubation for 30 min, the plate was washed three times with PBST, patted dry, and monoclonal antibody 2C4B3C7D6-B diluted 1:3200 was added. The plate was then incubated at 37°C. At incubation times of 30 min, 45 min, 60 min, and 90 min, the corresponding ELISA strips were removed, washed three times with PBST, and patted dry. Secondary antibody was added to the ELISA strips at all four time points, and incubation continued. Eight replicates were performed at each time point. The PI value under each condition was calculated, and the average value of the replicates was taken. The OD value of negative serum was selected. 450 The optimal reaction conditions were defined as those with the shortest reaction time when the PI value was close to 1, the highest PI value, and the first two results were similar. The results are as follows: the optimal reaction time for monoclonal antibody 2C4B3C7D6-B was 45 min (Table 7).

[0038] Table 7 Optimization of monoclonal antibody action time 5. Optimization of working concentration of enzyme-labeled secondary antibody: Following the optimal reaction conditions described above, the ELISA plates were coated and blocked sequentially. Serum and monoclonal antibody were added, and the plates were incubated at 37°C for 45 min. The plates were then removed, washed three times with PBST, and patted dry. 100 μL / well of HRP-goat anti-mouse IgG enzyme-labeled secondary antibody diluted 1:5000, 1:15000, 1:25000, and 1:35000 were added to each well, with each dilution repeated eight times. The PI value under each condition was calculated, and the average value of the replicates was taken. The OD value of negative serum was selected. 450 The reaction conditions with a value close to 1 and the highest PI value were considered the optimal reaction conditions. The results are as follows: the optimal working concentration of enzyme-labeled secondary antibody HRP-goat anti-mouse IgG was 1:15000 (Table 8).

[0039] Table 8 Optimization of working concentration of enzyme-labeled secondary antibody 6. Optimization of the reaction time of enzyme-labeled secondary antibodies: Following the optimal reaction conditions described above, after adding the enzyme-labeled secondary antibody, the strips were incubated at 37°C. At incubation times of 30 min, 45 min, 60 min, and 90 min, the corresponding enzyme strips were removed, washed three times with PBST, and patted dry. The substrate TMB was added simultaneously to the strips at all four time points for color development. Each time point was repeated eight times. The PI value under each condition was calculated, and the average value of the replicates was taken. The OD value of negative serum was selected. 450 The optimal reaction conditions were defined as those with the shortest reaction time when the PI value was close to 1, the PI value was the highest, and the first two results were similar. The results are as follows: the optimal reaction time for enzyme-labeled secondary antibody HRP-goat anti-mouse IgG was 60 min (Table 9).

[0040] Table 9 Optimization of enzyme-labeled secondary antibody incubation time 7. Optimization of substrate interaction time: The reaction was carried out sequentially under the aforementioned optimal conditions. 100 μL of the substrate TMB was added per well, and the reaction was incubated at room temperature in the dark. The corresponding enzyme-labeled strips were removed at 5 min, 10 min, 15 min, and 20 min, and the reaction was terminated by adding 50 μL of 2M H₂SO₄ per well. Each time point was repeated 8 times. The PI value under each condition was calculated, and the average value of the replicates was taken. The OD value of negative serum was selected. 450 The optimal reaction conditions were defined as those with the shortest reaction time when the PI value was close to 1, the PI value was the highest, and the first two results were similar. The results are as follows: the optimal reaction time for the substrate was 15 min (Table 10).

[0041] Table 10 Optimization of substrate interaction time 8. Determination of the ELISA cutoff value: The blocking rate of BCoV antibody-negative bovine and ovine serum, as determined by blocking ELISA, was calculated. Statistical analysis was performed, and the results were compared with those of neutralization experiments to conduct specificity and sensitivity analysis. Serum samples were considered antibody-positive when the PI ≥ 57%, and antibody-negative when the PI ≤ 57%.

[0042] Verification Example Three hundred and fifty bovine and ovine clinical serum samples were collected, and the BCoV antibodies in the serum were detected using the established blocking ELISA method and neutralization assay, respectively. The concordance rate between the blocking ELISA and neutralization assay results was 98%.

[0043] Table 11 Comparison of results from blocking ELISA and virus neutralization assays The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A hybridoma cell strain secreting an anti-bovine coronavirus monoclonal antibody, characterized in that, The preservation number of the hybridoma cell strain is CCTCC NO: C2025146.

2. A monoclonal antibody secreted by the hybridoma cell strain of claim 1.

3. The use of the hybridoma cell strain of claim 1 in any one of the following: (1) the preparation of a kit for detecting bovine coronavirus antibody; (2) the preparation of a kit for detecting antibody produced after bovine coronavirus infection or immunization of bovine.

4. The use of the monoclonal antibody of claim 2 in any one of the following: (1) the preparation of a kit for detecting bovine coronavirus antibody; (2) the preparation of a kit for detecting antibody produced after bovine coronavirus infection or immunization of bovine.

5. Use according to claim 3 or 4, wherein the compound is ###0002### The kit is a blocking ELISA kit.

6. A blocking ELISA kit for detecting bovine coronavirus infection or antibodies produced after immunization of a bovine, characterized in that, The blocking ELISA kit comprises the monoclonal antibody of claim 2.

7. The blocking ELISA kit of claim 6, wherein, The blocking ELISA kit further comprises a coated enzyme-labeled plate, a horseradish peroxidase-labeled goat anti-mouse IgG, a substrate, a stop solution, a positive control and a negative control.

8. The blocking ELISA kit of claim 7, wherein, The coated enzyme-labeled plate is obtained by coating bovine coronavirus as an antigen.

9. The blocking ELISA kit of claim 7, wherein, The positive control is bovine coronavirus positive bovine serum, and the negative control is bovine coronavirus negative bovine serum.

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