Monoclonal Antibody 1F8 Against H5 Subtype Avian Influenza Virus Hemagglutinin Protein and Its Application

Abstract

This invention belongs to the field of biotechnology and relates to a monoclonal antibody 1F8 against the hemagglutinin protein of H5 subtype avian influenza virus and its applications. This invention provides a monoclonal antibody against the hemagglutinin protein of H5 subtype avian influenza virus, capable of recognizing the H5 subtype avian influenza virus. The monoclonal antibody subtype is IgG2a, κ, named 1F8, and specifically recognizes the hemagglutinin protein of the H5 subtype avian influenza virus. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4. The advantages of this invention are that it provides a monoclonal antibody against the hemagglutinin protein of H5 subtype avian influenza virus. The preparation method is simple and easy to implement. More importantly, the monoclonal antibody prepared by this method can have multiple applications, such as qualitative diagnosis of H5 subtype avian influenza samples in clinical and laboratory settings.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the preparation and application of monoclonal antibodies against hemagglutinin protein of H5 subtype avian influenza virus. It utilizes cell engineering and antibody engineering technology to obtain hybridoma cell lines that secrete monoclonal antibodies against hemagglutinin protein. By inducing ascites in mice of the same strain, monoclonal antibody 1F8 against hemagglutinin protein is prepared and identified as IgG2a, κ type. Then, the application of this antibody is realized through affinity purification, immunization and other techniques. Background Technology

[0002] Influenza A viruses belong to the Orthomyxoviridae family. Based on the antigenic characteristics of hemagglutinin and neuraminidase glycoproteins, influenza A viruses are classified into 18 hemagglutinin subtypes and 11 neuraminidase subtypes. Waterfowl are considered the natural hosts of influenza A viruses, and most influenza A virus subtypes have been found in waterfowl. Notably, waterfowl often do not exhibit severe symptoms when infected with avian influenza viruses, but they provide a suitable environment for viral recombination. The H5 subtype of highly pathogenic avian influenza virus has the potential to cause large-scale avian influenza infections, with a mortality rate exceeding 75% in infected poultry. Since its resurgence in 2003, the highly pathogenic H5N1 strain has spread to multiple mammals, raising ongoing concerns about its cross-species transmission capabilities and potential pathogenicity risks to mammals. Between 2005 and 2024, the cumulative number of poultry killed or culled globally due to highly pathogenic H5N1 outbreaks exceeded 633 million, with 146 million deaths in 2022 alone. In March 2024, the United States first detected the H5N1 subtype of avian influenza virus in dairy cows, followed by reports of human infections, further demonstrating that the virus can undergo adaptive evolution in different mammalian hosts. As of July 1, 2025, according to global surveillance data reported by the World Health Organization, 25 countries had reported a cumulative total of 985 human cases of H5N1 infection, with a case fatality rate of 48%, highlighting the continued threat posed by the virus to global public health. Therefore, establishing rapid and accurate detection methods for the H5 subtype of avian influenza virus is crucial for virus control.

[0003] Isolating the virus from chicken embryos or MDCK cells is currently the recognized classic method for influenza virus detection. In recent years, molecular detection methods have also seen significant development, with real-time quantitative polymerase chain reaction (qPCR) being widely used for laboratory diagnosis of influenza virus infection. However, these methods are technically demanding and time-consuming, requiring advanced laboratory expertise. Due to the development of monoclonal antibody technology, monoclonal antibody-based detection methods are also widely used for virus detection. Therefore, this invention aims to describe a specific monoclonal antibody against the H5 subtype avian influenza virus and combine it with immunofluorescence technology to detect the H5 subtype avian influenza virus in samples. This method offers the advantages of speed and sensitivity, facilitating earlier and wider detection of the H5 subtype avian influenza virus and controlling its spread.

[0004] In summary, developing monoclonal antibodies against H5 subtype avian influenza virus and establishing rapid and sensitive detection methods are of great significance for epidemic prevention and control. Based on this background, this project selected the hemagglutinin protein of H5 subtype avian influenza virus as the target antigen, and used hybridoma technology to establish a hybridoma cell line that stably secretes anti-hemagglutinin protein monoclonal antibodies. These monoclonal antibodies were then prepared, purified, and identified in large quantities. The successful acquisition of this monoclonal antibody lays the material foundation for establishing a novel diagnostic method for H5 subtype avian influenza virus—an immunologically based diagnostic method. It also plays an important role in research on disease pathogenesis, prognosis, and efficacy assessment.

[0005] This invention utilizes hybridoma cell technology. This technology involves fusing B lymphocytes from immunized mice with myeloma cells SP2 / 0 to establish a hybridoma cell line that secretes homogeneous antibodies; it is also known as monoclonal antibody technology. This technology involves a series of methods including animal immunization, cell culture, cell fusion, cell clonal culture, and immunoassay. Summary of the Invention

[0006] The purpose of this invention is to provide a monoclonal antibody against the hemagglutinin protein of H5 subtype avian influenza virus, capable of recognizing H5 subtype avian influenza virus. This monoclonal antibody subtype is IgG2a, κ, named 1F8, and specifically recognizes the hemagglutinin protein of H5 subtype avian influenza virus. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4.

[0007] SEQ ID No.1 Heavy chain: DNA sequence (411 bp) Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3-FR4 ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTGAGGTGAAGCTGATGGAGTCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCT GGCTACACATTCACCACCTACTGG ATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAAT ATTTATCCTGGTAGTGGTACTACT AACTACGATGAGAAGTTCAAGAGCAGGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGT ACAAGATCTGTGACGGGGACCTGGTTTGCTTAC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA SEQ ID No.2 Heavy chain: Amino acid sequence (137 AA)

[0008] Signal peptide-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 MGWSLILLFLVAVATRVLSEVKLMESGSELVRPGASVKLSCKAS GYTFTTYW MHWVKQRPGQGLEWIGN IYPGSGTT NYDEKFKSRATLTVDTSSSTAYMQLSSLTSEDSAVYYC TRSVTGTWFAY WGQGTTVTVSS SEQ ID No.3 Light chain: DNA sequence (384 bp)

[0009] Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 ATGCATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCACC TC AAGTGTAAGTTAC ATACACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTAT AGCACATCC AACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGC CAGCAAAGGAGTAGTTACCCGCTCACG TTCGGTGCTGGGACCAAGCTGGACCTGAAA SEQ ID No. 4 Light chain: Amino acid sequence (128 AA)

[0010] Signal peptide-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 MHFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASPGEKVTITCSAT SSVSY IHWFQQKPGTSPKLWIY STS NLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYC QQRSSYPLT FGAGTKLDLK The second objective of this invention is to provide a method for preparing a monoclonal antibody against the hemagglutinin protein of H5 subtype avian influenza virus, achieved through the following steps and technical solutions: (1) Immunization of animals: 6-8 week old BALB / C mice were selected and immunized with hemagglutinin antigen protein of H5-Re14 influenza virus (A / whooperswan / Shanxi / 4-1 / 2020 (H5N8)).

[0011] (2) Culture of mouse myeloma cells: mouse myeloma cells SP2 / 0 were cultured and kept in good growth condition for cell fusion.

[0012] (3) Cell fusion: Polyethylene glycol-mediated cell fusion was used. The mice selected in step (1) were sacrificed and spleen lymphocytes were obtained. SP2 / 0 cells from step (2) were collected, the two types of cells were mixed and centrifuged, and then fused with polyethylene glycol. The fused cells were appropriately diluted and seeded into 96-well culture plates and cultured under appropriate conditions.

[0013] (4) Screening of hybridoma cells: The above cultures were cultured in hypoxanthine-phosphoribosyltransferase selective medium. When the cell colonies grew to a suitable size, the cell culture supernatant was aspirated for antibody identification, and positive clones were screened.

[0014] (5) Cloning of hybridoma cells: Positive hybridoma cells were cloned using the limiting dilution method. Cells diluted to a certain density were seeded into 96-well cell culture plates, ensuring that only one cell grew in each well. The supernatant from the wells where cell colonies formed was collected for enzyme-linked immunosorbent assay (ELISA) to screen and identify positive clones. The wells with the highest antibody titers and showing single-clonal cell growth were selected and subjected to limiting dilution again. This process was repeated at least four times and passaged for at least 20 generations to obtain a stable and efficient hybridoma cell line expressing monoclonal antibodies against H5 subtype avian influenza virus. The cloned hybridoma cells were then subjected to antibody identification and physicochemical property analysis.

[0015] (6) Preparation of monoclonal antibody ascites fluid: Select 8-10 week old BALB / c healthy mice, and inoculate each mouse with 5×10⁻⁶ antibodies into the abdomen. 6 After inoculating positive hybridoma cells with PBS buffer, mice showed significant abdominal distension 7-10 days later. The health status and abdominal signs of the mice were closely observed. Ascites fluid was collected and centrifuged before the mice were near death, the antibody titer was determined, and the monoclonal antibodies in the ascites fluid were purified.

[0016] (7) Purification of monoclonal antibodies: Monoclonal antibodies in mouse ascites fluid were purified using the protein G agarose gel affinity purification method.

[0017] (8) This invention yielded a hybridoma cell line, 1F8, that produces monoclonal antibodies against the hemagglutinin protein of H5 subtype avian influenza virus. The 1F8 hybridoma cell line underwent four cloning processes and was continuously cultured for over six months, exhibiting stable antibody secretion. After cryopreservation in liquid nitrogen, the cell line grew well upon thawing, and antibody secretion showed no decline. Enzyme-linked immunosorbent assay (ELISA) showed that the titer of the 1F8 culture supernatant was 1:64, and the titer of the ascites fluid was 1:4096. Monoclonal antibody immunoglobulin subtype analysis revealed that the antibody type produced by this hybridoma cell line was IgG2a.

[0018] This invention provides hybridoma cells that produce monoclonal antibodies. The hybridoma cell line 1F8 is obtained by fusing, screening, cloning, and passage of spleen cells from immunized BALB / c mice and mouse myeloma cells SP2 / 0. It can stably secrete monoclonal antibodies 1F8 against hemagglutinin protein of H5 subtype avian influenza virus.

[0019] Another objective of this invention is to provide the detection of the monoclonal antibody 1F8 in body fluids, allantoic fluid, or other environmental samples containing H5 subtype avian influenza virus, achieved by an immunofluorescence method.

[0020] The advantage of this invention lies in providing a monoclonal antibody against the hemagglutinin protein of H5 subtype avian influenza virus. The preparation method is simple and easy to implement. More importantly, the monoclonal antibody prepared by this method can have multiple applications, such as qualitative diagnosis of H5 subtype avian influenza samples in clinical and laboratory settings. Attached Figure Description

[0021] Figure 1 Immunoglobulin subtype analysis of monoclonal antibody 1F8.

[0022] Figure 2 To improve the specificity of immunofluorescence detection of H5 subtype avian influenza virus.

[0023] Figure 3 To assess the sensitivity of immunofluorescence assay for detecting H5 subtype avian influenza virus, virus concentration was measured in hemagglutinin units. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] Example 1. Preparation method of monoclonal antibody against H5 subtype avian influenza virus hemagglutinin protein

[0026] (1) Immunization of mice: For the first immunization, the H5 subtype avian influenza virus hemagglutinin protein and adjuvant were mixed at a 1:1 volume ratio, with a total volume of 0.5 ml. 0.1 ml (containing 100 micrograms of H5 subtype avian influenza virus hemagglutinin protein antigen) was injected intramuscularly into the inner thigh of each BALB / c mouse. A booster immunization was performed in the same manner on day 21. On day 35, a small amount of tail blood was collected for enzyme-linked immunosorbent assay (ELISA). The highest antibody titer reached 1:128000. Mice with the highest antibody titer were selected and injected intravenously via the tail vein for a booster immunization. Cell fusion was performed 3 days later.

[0027] (2) Culture and passage of mouse myeloma cells SP2 / 0: SP2 / 0 myeloma cells from BALB / c mice were cultured and passaged in DMEM medium containing 10% bovine serum and incubated at 37°C in a 5% carbon dioxide incubator. Cells were not passaged the day before fusion to ensure that they entered the logarithmic growth phase at fusion.

[0028] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were seeded into 96-well culture plates and cultured for one day in hypoxanthine-guanine-phosphoribotransferase medium containing 20% ​​bovine serum. Spleens were harvested from mice three days after their last booster immunization, and splenic lymphocytes were isolated using the pressure injection method. After centrifugation and washing, the cells were resuspended in DMEM medium. SP2 / 0 cells were collected, centrifuged, washed, and resuspended in DMEM medium for counting. 3 × 10⁶ cells were then... 8 3 × 10 splenic lymphocytes from immunized mice 7 A mixture of SP2 / 0 mouse myeloma cells was prepared. After centrifugation, the supernatant was discarded. The cell clumps were loosened by gently rubbing the centrifuge tube with the palm of the hand. Pre-warmed polyethylene glycol (PEG) at 37°C was slowly added to the fusion tube while gently agitating the centrifuge tube to draw cells into the fusion tube. After standing for 90 seconds, the cells were blown back into the centrifuge tube. Following a slow-then-fast approach, 1 mL of DMEM medium was added within the first minute, 2 mL within the second minute, and 7 mL within the third minute. Then, 40 mL of pre-warmed DMEM medium at 37°C was gradually added within the next minute. The mixture was centrifuged at 800 rpm for 10 minutes. Then, hypoxanthine-guanine-phosphoribotransferase medium containing 20% ​​bovine serum was added. Cells were seeded into 96-well plates containing feeder cells using a glass dropper. Generally, 2-4 plates were seeded from each fusion. The cells were incubated in a 37°C incubator containing 5% carbon dioxide.

[0029] (4) Screening of hybridoma cells: After 5 days, half of the medium (containing hypoxanthine-guanine-phosphoribotransferase) was changed in the 96-well culture plate. After 10 days, the medium was changed to one containing hypoxanthine-phosphoribotransferase. The fused hybridoma cells were cultured in selective medium containing hypoxanthine-phosphoribotransferase for about two weeks. When the cell colonies grew to an appropriate size (observed under a 10x objective lens, the cell clones should fill an entire field of view), the cell culture supernatant was aspirated for enzyme-linked immunosorbent assay (ELISA) to screen for positive clones. Positive hybridoma clones were screened indirectly using ELISA. Main steps: ① Dilute H5 subtype hemagglutinin protein with 0.01 mol / L pH 9.6 carbonate buffer, then add 0.1 mL to each well of a 96-well microplate, with a protein content of 20 ng / well, and incubate overnight at 4°C; ② Wash the plate 5 times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ Use a solution containing 5% bovine serum albumin at 0.01 mol / L pH 9.6 phosphate buffer. ① Block with phosphate buffer at 7.4 for 2 hours; ② Wash the plate 3 times; ③ Add 0.1 mL of hybridoma culture supernatant per well, and set up a positive control (H5 subtype protein-immunized mouse serum), a negative control (SP2 / 0 culture supernatant), and a blank control, and react at room temperature for 2 hours; ④ Wash the plate 3 times; ⑤ Add 0.1 mL of horseradish peroxidase-labeled goat anti-mouse IgG diluted 1:10000 per well, and react at room temperature for 1 hour; ⑥ Wash the plate 3 times; ⑦ Add chromogenic solution and react at room temperature in the dark for 5 minutes; ⑧ Terminate the reaction with 2 mol / L sulfuric acid; ⑤ Measure the optical density at 450 nm, and a negative value divided by ≥2.1 is considered positive.

[0030] (5) Cloning of hybridoma cells: The cloning culture of hybridoma cells was carried out using the limiting dilution method. After appropriate proliferation of hybridoma cells that tested positive for antibodies, the cells were accurately counted. The cells were diluted with complete DMEM medium to a concentration of 10 cells per milliliter and seeded into 96-well culture plates containing feeder cells, 0.1 milliliter per well. After 10 days, cell growth was observed and the antibody level in the supernatant was detected. The wells with the highest antibody titer and showing single-clonal cell growth were selected and subjected to limiting dilution again. The limiting dilution was performed more than 4 times and passaged for more than 20 generations to obtain a hybridoma cell line that stably and efficiently expresses monoclonal antibodies against H5 subtype avian influenza virus.

[0031] (6) Preparation of monoclonal antibody ascites fluid: Select 8-10 week old BALB / c healthy mice, and inoculate each mouse with 5×10⁻⁶ antibodies into the abdomen. 6 After inoculating mice with positive hybridoma cells in PBS buffer, the abdomen of the mice became significantly enlarged 7-10 days later. The health status and abdominal signs of the mice were closely observed, and the ascites was collected when ascites was as abundant as possible.

[0032] (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites fluid were purified using affinity purification (protein G agarose gel). ① Ascites fluid treatment: The ascites fluid was centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. The supernatant was collected and mixed with 3-4 volumes of binding buffer, and then centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② The affinity purification column pre-loaded with protein G agarose gel was thoroughly washed with 5 column volumes of binding buffer. ③ The diluted ascites fluid was loaded onto the column at a flow rate of 8-10 drops per minute. ④ The ascites fluid that had already passed through the column was loaded onto the column again. ⑤ The purification column was thoroughly washed with 5 column volumes of binding buffer. ⑥ Elute the bound monoclonal antibody with elution buffer at a flow rate of 8-10 drops per minute. Collect the eluent in a collection tube pre-filled with 0.1 mL of potassium phosphate buffer (pH 7.9), collecting 0.5 mL of antibody-containing eluent from each tube. ⑦ Detect the absorbance of each eluent tube at 280 nm and collect the eluent with a protein content greater than 0.1 mg / mL. ⑧ Add antibody eluent to an ultrafiltration centrifuge tube and centrifuge at 10,000 rpm for 10-20 minutes at 4°C until the final volume of antibody eluent is approximately 1 mL. Add 10 mL of 0.1 mol / L phosphate buffer (pH 7.4) and centrifuge at 10,000 rpm for 10-20 minutes at 4°C. Concentrate the antibody to a final volume of approximately 1 mL by a final centrifugation and collect the concentrated antibody solution in a collection tube. ⑨ Dilute the desalted antibody solution and measure the protein content at 280 nm. ⑩ Aliquot the purified antibody into small tubes and store them in a low-temperature freezer for later use.

[0033] (8) Identification of monoclonal antibody subtypes: The Bio-Rad mouse monoclonal antibody immunoglobulin typing kit was used for analysis. The purified monoclonal antibody was appropriately diluted before detection, and the procedure was strictly performed according to the kit instructions. The results showed that the monoclonal antibody secreted by 1F8 hybridoma cells was IgG2a, κ type.

[0034] The results are attached. Figure 1 .

[0035] Example 2. Qualitative detection of H5 subtype avian influenza virus using this monoclonal antibody. The monoclonal antibody against H5 avian influenza virus hemagglutinin protein prepared in this invention can be used for qualitative detection of H5 subtype avian influenza virus. The identification method can be achieved through the following steps:

[0036] Immunofluorescence detection method for H5 subtype avian influenza virus: (1) Prepare MDCK cells one day in advance at a rate of 4 × 10⁶ cells per well. 4 The cells were seeded at a density of 1,000 cells in 48-well plates and kept for use after the cells grew to 70%; (2) Remove the cell plate with the cells, discard the culture supernatant, wash once with phosphate buffer, and set aside; (3) Determining the specificity of the immunofluorescence assay for H5 subtype avian influenza virus: Dilute the virus with phosphate buffer, including H5N1 virus (A / Texas / 37 / 2024), H5N2 virus (A / duck / Zhejiang / 6DK19 / 2013), H5N6 virus (A / duck / Zhejiang / 6D2 / 2013), H5N8 virus (A / duck / Zhejiang / W24 / 2013), H1N1 virus (A / California / 07 / 2009), H2N8 virus (A / duck / Zhejiang / 6D10 / 2013), and H3N2 virus (A Cells were infected with diluted viral solutions of H4N6 virus (A / duck / Zhejiang / 4613 / 2013), H6N1 virus (A / chicken / Zhejiang / 1664 / 2017), H7N9 virus (A / chicken / Zhejiang / ZJU01 / 2013), H9N2 virus (A / chicken / Zhejiang / 329 / 2011), and H10N2 virus (A / duck / Zhejiang / 6D20 / 2013) (multiple of infection 0.5) and incubated for 2 hours in an incubator containing 5% carbon dioxide at 37°C. (4) Remove the cell plate, discard the virus solution, wash the cells twice with phosphate buffer, add 200 μL of virus culture medium to each well, and incubate for 16 hours in a 37°C incubator containing 5% carbon dioxide. (5) Remove the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffer; (6) Add 4% paraformaldehyde to each well of the cell plate to fix the cells, fix at room temperature for 30 minutes, and wash 3 times with phosphate buffer. (7) Permeabilize the cells with 0.5% Triton-X100 for 30 minutes at room temperature, and wash three times with phosphate buffer; (8) Block with phosphate buffer containing 3% bovine serum albumin for 1 hour at room temperature, then discard the bovine serum albumin solution; (9) Dilute the monoclonal antibody to 10 μg / mL with phosphate buffer, add 200 μL to each well, incubate overnight at 4°C, and wash 3 times with phosphate buffer. (10) Dilute the fluorescent secondary antibody to 5 μg / mL with a solution containing 1% bovine serum albumin, add 200 μL to each well, incubate in a 37°C incubator in the dark for 90 minutes, and wash 3 times with phosphate buffer. (11) The cell nuclei were stained with deoxyribonucleic acid fluorescent dye (4',6-diamidinyl-2-phenylindole), incubated at room temperature in the dark for 10 minutes, and washed 3 times with phosphate buffer. (12) Observe the experimental results under a fluorescence microscope. The presence of green fluorescence indicates a positive result. The detection results show that the monoclonal antibody 1F8 against H5 subtype avian influenza virus developed in this study has good specificity for detecting H5 subtype avian influenza virus.

[0037] The results are attached. Figure 2 .

[0038] Determining the sensitivity of immunofluorescence assay for detecting H5 subtype avian influenza virus: (1) Prepare MDCK cells one day in advance at a rate of 4 × 10⁶ cells per well. 4 The cells were seeded at a density of 1,000 cells in 48-well plates and kept for use after the cells grew to 70%; (2) Remove the cell plate with the cells, discard the culture supernatant, wash once with phosphate buffer, and set aside; (3) Sensitivity of H5 subtype avian influenza virus immunofluorescence detection method: H5N2 virus (A / duck / Zhejiang / 6DK19 / 2013) was diluted with phosphate buffer and infected with cells at virus concentrations of 8, 4, 2, 1, 0.5 and 0.25 hemagglutinin units, respectively. Cells were incubated for 2 hours in an incubator containing 5% carbon dioxide and at 37°C. Steps (4)-(12) were repeated. The results showed that the sensitivity of immunofluorescence technology to detect H5 subtype avian influenza virus was 1 H5 subtype avian influenza virus hemagglutinin unit, which has good sensitivity.

[0039] The results are attached. Figure 3 .

[0040] It should be understood that the present invention has been described in conjunction with the preferred embodiments. However, after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A monoclonal antibody 1F8 against the hemagglutinin protein of H5 subtype avian influenza virus, the antibody subtype being IgG2a, κ type, capable of specifically binding to the hemagglutinin protein antigen of H5 subtype avian influenza virus, the amino acid sequence of the heavy chain variable region of the antibody being shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region being shown in SEQ ID No.

4.

2. The monoclonal antibody 1F8 against hemagglutinin protein of H5 subtype avian influenza virus according to claim 1, characterized in that: The monoclonal antibody is produced by hybridoma cells.

3. The method for preparing the monoclonal antibody 1F8 against the hemagglutinin protein of H5 subtype avian influenza virus according to claim 1, characterized in that: This monoclonal antibody was obtained through the following steps: (1) Immunization of mice: BALB / C mice aged 6-8 weeks were selected and immunized with purified H5 subtype avian influenza virus hemagglutinin protein. Each mouse was immunized by intramuscular injection of 50 micrograms of H5 subtype hemagglutinin protein mixed with adjuvant in a 1:1 ratio. The mice were immunized again in the same manner on day 21, for a total of 2 times. On day 35, a small amount of tail blood was collected from the mice to determine the antibody titer. The mice with the highest immune titer were selected and given a booster immunization via tail vein injection. Cell fusion was performed 3 days later. (2) Culture of mouse myeloma cells: Two weeks before the fusion was to be prepared, myeloma cells SP2 / 0 were revived, and mouse myeloma cells SP2 / 0 were cultured and kept in good growth condition for hybridoma cell fusion. (3) Cell fusion: Polyethylene glycol-mediated cell fusion was used; the mice selected in step (1) were sacrificed and spleen lymphocytes were obtained; the spleen lymphocytes were mixed with myeloma cells SP2 / 0 and centrifuged, and then fused with polyethylene glycol. The fused cells were appropriately diluted, seeded into culture plates, and cultured under appropriate conditions. (4) Screening of hybridoma cells: The above culture was cultured in a selective medium containing hypoxanthine-phosphoribosetransferase; when the cell colonies grew to an appropriate size, the culture supernatant was aspirated and antibody identification was performed by enzyme-linked immunosorbent assay (ELISA) to screen positive clones; (5) Clonal culture of hybridoma cells: positive hybridoma cells were cloned using the limiting dilution method. Cells diluted to a certain density were seeded into 96-well cell culture plates so that only one cell grew in each well. The supernatant of the wells that formed cell colonies was used for enzyme-linked immunosorbent assay (ELISA) to screen and identify positive clones. The culture wells with the highest antibody titer and showing single-clonal cell growth were selected and limited dilution was performed again. The single-clonal limiting dilution was performed more than 3 times and passaged for more than 20 generations to obtain a hybridoma cell line that stably and efficiently expresses monoclonal antibodies against H5 subtype avian influenza virus. (6) Preparation of monoclonal antibody ascites: select 8-10 weeks BALB / C healthy mice, each abdominal inoculation containing 5x5 6 positive hybridoma cells PBS buffer, 7-10 days after inoculation of cells, the mouse abdomen is significantly swollen, close observation of the health of the mouse abdomen signs, when the ascites as much as possible, and the mouse is on the verge of death, the ascites is collected, centrifuged, the antibody titer is determined, and the monoclonal antibody in the ascites is purified; (7) Purification of monoclonal antibodies: Monoclonal antibodies in mouse ascites fluid were purified using the protein G agarose gel affinity purification method.

4. The application of the monoclonal antibody 1F8 against the hemagglutinin protein of H5 subtype avian influenza virus as described in claim 1 or 2 in the preparation of H5 subtype avian influenza virus detection products.

5. The application according to claim 4, characterized in that: The H5 subtype avian influenza virus detection product uses immunofluorescence technology to detect H5 subtype avian influenza virus in different samples.

6. The application according to claim 5, characterized in that: The samples are throat swabs, feces, or allantoic fluid.

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