Monoclonal antibody 1E1 for resisting H10 subtype influenza virus hemagglutinin protein and application of monoclonal antibody 1E1

Abstract

The invention belongs to the technical field of biology, and relates to an anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody and application thereof, cell engineering and antibody engineering technologies are utilized to obtain a hybridoma cell line for secreting the monoclonal antibody of the anti-hemagglutinin protein, ascites is induced by mice of the same strain to prepare the monoclonal antibody 1E1 of the anti-hemagglutinin protein, and the monoclonal antibody 1E1 of the anti-hemagglutinin protein is applied to preparation of the H10 subtype influenza virus hemagglutinin protein monoclonal antibody. The antibody is identified as an IgG2a, kappa type, and the application of the antibody is realized through technologies such as affinity purification and immune methods. The antibody can specifically recognize hemagglutinin protein of the H10 subtype influenza virus, the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID No.2, and the amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID No.4. The invention provides an effective tool for auxiliary diagnosis of H10 subtype influenza virus infection in a clinical sample, and can be popularized and applied to various detection technologies and clinical and experimental researches.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to an anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody and an application thereof. The present invention utilizes cell engineering and antibody engineering technologies to obtain a hybridoma cell line that secretes a monoclonal antibody against the hemagglutinin protein, induces ascites in mice of the same strain, prepares a monoclonal antibody 1E1 against the hemagglutinin protein, identifies it as IgG2a, κ type, and then realizes the application of the antibody through affinity purification, immunization methods and other technologies. Background Art

[0002] Influenza A virus belongs to the family Orthomyxoviridae. According to the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, influenza A virus is divided into 18 HA and 11 NA subtypes. Waterfowl are considered to be the natural host of influenza A virus, and most influenza A virus subtypes have been found in waterfowl. In recent years, H10 subtype influenza viruses have been increasingly isolated from poultry, such as H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9. H10 subtype influenza viruses pose a potential pandemic threat to human health. Therefore, establishing a rapid and accurate method for detecting H10 subtype influenza viruses is of great significance for influenza prevention and control.

[0003] Isolating viruses in chicken embryos or MDCK cells is a current classical method for detecting influenza viruses. In recent years, molecular detection methods have also been greatly developed, and real-time quantitative polymerase chain reaction has been widely used in laboratory diagnosis of influenza virus infection, but these methods are very demanding and time-consuming for technology and laboratories. Due to the development of monoclonal antibody technology, detection methods based on monoclonal antibodies are also widely used in virus detection. Therefore, the present invention aims to illustrate a specific monoclonal antibody for H10 subtype influenza virus, and combines it with immunofluorescence technology, and can detect H10 subtype influenza virus in samples. The above method has the advantages of fast, sensitive and cheap, which will be conducive to earlier and more extensive discovery of H10 subtype influenza virus, effectively controlling virus diffusion.

[0004] In summary, the development of monoclonal antibodies for H10 subtype influenza virus and the establishment of rapid and sensitive detection methods are of great significance for the prevention and control of the virus. Based on the above background, this project selected the H10 subtype influenza virus hemagglutinin protein as the target antigen, used fusion hybridoma technology to establish a hybridoma cell line that stably secretes anti-hemagglutinin protein monoclonal antibodies, and prepared, purified and identified these monoclonal antibodies in large quantities. The successful acquisition of this monoclonal antibody lays a material foundation for the establishment of a new H10 subtype influenza virus diagnostic method - diagnosis based on immunofluorescence technology. At the same time, it plays an important role in the study of disease pathogenesis, prognosis and efficacy determination.

[0005] The present invention uses hybridoma cell technology. This technology fuses B lymphocytes of immune mice with myeloma cells SP2 / 0 to establish a hybridoma cell line that secretes homogeneous antibodies, also known as monoclonal antibody technology. This technology involves a series of methods such as animal immunization, cell culture, cell fusion, cell cloning culture and immunoassay. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody, which can identify H10 subtype influenza virus. The monoclonal antibody subtype is IgG2a, κ type, named 1E1, can specifically identify the hemagglutinin protein of H10 subtype influenza virus, the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID No.2, and the light chain variable region amino acid sequence is shown in SEQ ID No.4.

[0007] SEQ ID No.1

[0008] Heavy chain:DNA sequence(375bp)

[0009] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0010] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGG

[0011] TTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAATGGCTGACACA

[0012] CATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGA

[0013] AACCAGGTATTCCTCAAGATCACTAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGAAGCCCCCCTAC

[0014] GGACTACGGTAGTAGCTGGGGTGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCASEQ ID No.2

[0015] Heavy chain:Amino acid sequence(125AA)

[0016] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0017] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLTHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDT

[0018] ADTATYYCARSPPTDYGSSWGVMDYWGQGTSVTVSS

[0019] SEQ ID No.3

[0020] Light chain:DNA sequence(318bp)

[0021] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0022] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCGTCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAG

[0023] CTCGAGTATAACTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCA

[0024] AACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATG

[0025] GAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTGGGACCAAGC

[0026] TGGAGCTGAAA

[0027] SEQ ID No.4

[0028] Light chain:Amino acid sequence(106AA)

[0029] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0030] QIVLTQSPAIMSASPGEKVTMTCSASSSITYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQ

[0031] QWSSNPLTFGAGTKLELK

[0032] The second object of the present invention is to provide a method for preparing anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibodies, which is achieved by the following steps and technical solutions:

[0033] (1) Animal immunization: 8-week-old BALB / C mice were selected and immunized with purified hemagglutinin protein of H10N7 subtype influenza virus (A / chicken / Zhejiang / 2CP8 / 2014).

[0034] (2) Cultivation of mouse myeloma cells: Mouse myeloma cells SP2 / 0 were cultured and maintained in a good growth state for cell fusion.

[0035] (3) Cell fusion: Cell fusion mediated by polyethylene glycol is used. The mice selected in step (1) are killed and spleen lymphocytes are obtained. The SP2 / 0 cells in step (2) are collected, the two cells are mixed and centrifuged, and then the cells are fused by polyethylene glycol. The fused cells are appropriately diluted, inoculated into a 96-well culture plate, and cultured under appropriate conditions.

[0036] (4) Screening of hybridoma cells: The above culture was cultured in a 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.

[0037] (5) Cloning of hybridoma cells: clone positive hybridoma cells by limiting dilution method, inoculate the cells diluted to a certain density into 96-well cell culture plates, and make only one cell grow in each well. Take the supernatant from the wells where cell colonies are formed for enzyme-linked immunosorbent assay to screen and identify positive clones. Select the culture wells with the highest antibody titer and single clone cell growth, perform limiting dilution again, perform limiting dilution more than 3 times in a row, and continuously passage for more than 20 generations to obtain hybridoma cell lines that stably and efficiently express monoclonal antibodies against H10 subtype influenza virus. Perform antibody identification and physical and chemical property analysis on the cloned hybridoma cells.

[0038] (6) Preparation of monoclonal antibody ascites: 10-week-old healthy BALB / C mice were selected and 5 million positive hybridoma cells were inoculated in each abdomen. About 10 days after the inoculation of the cells, the abdomen of the mice was significantly swollen. The health status and abdominal signs of the mice were closely observed. When the ascites was as much as possible and the mice were on the verge of death, the ascites was collected and centrifuged to determine the antibody titer and purify the monoclonal antibodies in the ascites.

[0039] (7) Purification of monoclonal antibodies: Monoclonal antibodies were purified from mouse ascites using protein G agarose gel affinity purification.

[0040] (8) The present invention obtains a hybridoma line, namely 1E1, which produces monoclonal antibodies against the hemagglutinin protein of subtype H10 influenza virus. The 1E1 hybridoma cell line was cloned three times and cultured for 6 months. The secretion of antibodies was stable. The cell line was frozen in liquid nitrogen and grew well after recovery. The titer of the 1E1 culture supernatant was 1:128 and the titer of the ascites was 1:2048 as measured by the enzyme-linked immunosorbent assay indirect method. The monoclonal antibody immunoglobulin subtype analysis showed that the antibody type produced by the hybridoma cell was IgG2a.

[0041] The invention provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 1E1 obtained by fusing, screening, cloning and passage of immune BALB / C mouse spleen cells and mouse myeloma cells SP2 / 0, and can stably secrete the monoclonal antibody 1E1 of H10 subtype influenza virus hemagglutinin protein.

[0042] Another object of the present invention is to provide the use of the monoclonal antibody 1E1 in the preparation of a fluorescent detection product for H10 subtype influenza virus.

[0043] Detection of influenza virus subtype H10 in body fluids, allantoic fluid, or other environmental samples is achieved by immunofluorescence.

[0044] The present invention provides a monoclonal antibody 1E1 of the hemagglutinin protein of H10 subtype influenza virus, which is a method for detecting H10 subtype influenza virus by immunofluorescence technology as a probe. The advantages of the present invention are: the preparation method is simple and easy, and more importantly, the monoclonal antibody prepared by the method can have multiple uses, providing an effective tool for auxiliary diagnosis of H10 subtype influenza virus infection in clinical samples, and can be promoted and applied to multiple detection technologies and clinical and experimental research.

[0045] Instruction Manual

[0046] Figure 1 Immunoglobulin subtype analysis of monoclonal antibody 1E1.

[0047] Figure 2 The specificity of immunofluorescence technology for detecting H10 subtype influenza virus. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0049] Example 1. Preparation of monoclonal antibodies against H10 subtype influenza virus hemagglutinin protein

[0050] (1) Immunization of mice: For the first immunization, the H10 subtype influenza virus hemagglutinin protein and adjuvant were mixed evenly in a volume ratio of 1:1, with a total volume of 0.5 ml. 0.1 ml (containing 5 μg of H10 subtype influenza virus hemagglutinin protein antigen) was injected into the inner thigh muscle of each BALB / C mouse. On the 21st day, a booster immunization was performed in the same manner. On the 35th day, a small amount of tail blood was collected for ELISA. The highest antibody titer reached 1:32000. The mouse with the highest antibody titer was selected for a booster immunization by tail vein injection, and cell fusion was performed 3 days later.

[0051] (2) Culture and subculture of mouse myeloma cells SP2 / 0: SP2 / 0 myeloma cells from BALB / C mice were cultured and subcultured in DMEM medium containing 10% bovine serum in a 37°C incubator containing 5% carbon dioxide. Subculture was usually not performed one day before fusion to ensure that the cells entered the logarithmic growth phase at the time of fusion.

[0052] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mouse peritoneal macrophages were inoculated into 96-well culture plates and cultured for one day in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. The spleen of the mouse was taken 3 days after the last booster immunization, and the spleen lymphocytes were separated by pressure injection. The cells were washed by centrifugation and resuspended in DMEM culture medium. SP2 / 0 cells were collected, centrifuged, washed, resuspended in DMEM culture medium, and counted. 300 million spleen lymphocytes of immune mice were mixed with 30 million mouse myeloma cells SP2 / 0. The two cells were mixed, centrifuged and the supernatant was discarded. The centrifuge tube was rubbed to loosen the cell clumps. Polyethylene glycol preheated at 37°C was slowly added into the fusion tube. The centrifuge tube was gently shaken during this period. The cells were sucked into the fusion tube. After standing for 90 seconds, the cells were blown into the centrifuge tube. Then, according to the principle of slow first and fast later, 1 ml of DMEM medium was added within the first minute, 2 ml of DMEM medium was added within the second minute, and 7 ml of DMEM medium was added within the third minute. Then, 40 ml of DMEM medium preheated at 37°C was gradually added within 1 minute. The cells were centrifuged at a low speed of 800 rpm for 10 minutes. Then, hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum was added, and the cells were inoculated into 96-well culture plates with feeder cells using a glass dropper. Generally, two plates were spread for each fusion of cells and cultured in a 37°C incubator containing 5% carbon dioxide.

[0053] (4) Screening of hybridoma cells: After 5 days, the 96-well culture plate was replaced with half of the medium (containing hypoxanthine-guanine-phosphoribosyltransferase) and after 10 days, the culture medium containing hypoxanthine-phosphoribosyltransferase was used. The fused hybridoma cells were cultured in the selective culture medium containing hypoxanthine-phosphoribosyltransferase for about two weeks. When the cell colonies grew to an appropriate size (observed under a 10x objective, the size of the cell clones should be sufficient to fill a field of view), the cell culture supernatant was aspirated for ELISA to screen positive clones. The ELISA method was used to indirectly screen positive hybridoma clones. Main steps: ① dilute H10 subtype hemagglutinin protein with 0.01 mol / L pH 9.6 carbonate buffer, then add 0.1 ml per well to a 96-well ELISA plate, with a protein amount of 20 ng per well, 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 0.01 mol / L pH 7.4 phosphate buffer containing 5% bovine serum albumin to 7.4 phosphate buffer was blocked for 2 hours; ④ the plate was washed 3 times; ⑤ the hybridoma culture supernatant was added, 0.1 ml per well, and a positive control (H10 subtype protein immune mouse serum), a negative control (SP2 / 0 culture supernatant) and a blank control were set up, and the reaction was carried out at room temperature for 2 hours; ⑥ the plate was washed 3 times; ⑦ 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG was added, 0.1 ml per well, and the reaction was carried out at room temperature for 1 hour; ⑧ the plate was washed 3 times; ⑨ the color developing solution was added and the reaction was carried out at room temperature in the dark for 5 minutes; ⑩ 2 mol / L sulfuric acid was used to terminate the reaction; the optical density value was measured at 450 nm, and the measured value divided by the negative value ≥ 2.1 was considered positive.

[0054] (5) Cloning of hybridoma cells: The cloning culture of hybridoma cells is carried out according to the limiting dilution method. After appropriate proliferation, the cells are accurately counted. The cell suspension diluted with complete DMEM medium to 10 / ml is inoculated into a 96-well culture plate with feeder cells, 0.1 ml per well. After 10 days, the cell growth is observed and the antibody level in the supernatant is detected. The culture well with the highest antibody titer and single clone cell growth is selected and limited dilution is performed again. The limiting dilution is performed for more than 3 times continuously and the cells are continuously passaged for more than 20 generations to obtain a hybridoma cell line that stably and efficiently expresses monoclonal antibodies against H10 subtype influenza virus.

[0055] (6) Preparation of monoclonal antibody ascites: 10-week-old healthy BALB / C mice were selected and phosphate-buffered saline containing 5 million positive hybridoma cells was inoculated in each abdomen. Ten days after cell inoculation, the abdomen of the mice was significantly swollen. The health status and abdominal signs of the mice were closely observed. The ascites of the mice were collected when as much as possible.

[0056] (7) Purification of monoclonal antibodies: Affinity purification (protein G agarose gel) was used to purify monoclonal antibodies in ascites. ① Treatment of ascites: Centrifuge the ascites at 4°C and 10,000 rpm for 15 minutes, remove the precipitate, collect the supernatant, mix it with 3 volumes of binding buffer, and centrifuge it at 4°C and 10,000 rpm for 15 minutes to remove the precipitate. Centrifuge it at 4°C and 10,000 rpm for 15 minutes to remove the precipitate. ② Wash the affinity purification column pre-loaded with protein G agarose gel thoroughly with 5 column volumes of binding buffer. ③ Load the diluted ascites onto the column and control the flow rate to 10 drops per minute. ④ Repeat the loading of the ascites that has passed through the column once. ⑤ Wash the purification column thoroughly with 5 column volumes of binding buffer. ⑥ Elute the bound monoclonal antibody with elution buffer, control the flow rate to 10 drops per minute, collect the eluate in a collection tube pre-added with 0.1 ml of potassium phosphate buffer (pH7.9), and collect 0.5 ml of antibody-containing eluate in each tube. ⑦ Detect the absorbance of each tube of eluate at 280 nanometers, and collect the eluate with a protein content greater than 0.1 mg per milliliter. ⑧ Add the antibody eluate to the ultrafiltration centrifuge tube, centrifuge at 4°C and 10,000 rpm for 20 minutes until the final volume of the antibody eluate is about 1 ml. Add 10 ml of 0.1 mol / L pH7.4 phosphate buffer, centrifuge at 8°C and 10,000 rpm for 20 minutes, and concentrate the antibody to a final volume of about 1 ml by centrifugation for the last time, and draw the antibody concentrate into the collection tube. ⑨ Dilute the desalted antibody solution and measure the protein content at 280 nanometers. ⑩ Dispense the purified antibody into small tubes and place in a low-temperature refrigerator for standby use.

[0057] (8) Identification of monoclonal antibody subtypes: The mouse monoclonal antibody immunoglobulin typing kit from Bio-Rad was used for analysis. The purified monoclonal antibody was appropriately diluted and tested, and the operation was strictly carried out according to the instructions of the kit. The test results showed that the monoclonal antibody secreted by the 1E1 hybridoma cells was IgG2a, κ type.

[0058] See attached for the results Figure 1 .

[0059] Example 2. Qualitative detection of H10 subtype influenza virus using the monoclonal antibody

[0060] The anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody prepared by the present invention can be used to qualitatively detect H10 subtype influenza virus, and the identification method can be achieved by the following method:

[0061] H10 subtype influenza virus immunofluorescence detection method:

[0062] (1) MDCK cells were seeded in a 48-well plate at a density of 40,000 cells per well one day in advance and the cells were grown to 70% for later use;

[0063] (2) Take out the cell plate with cells, discard the culture supernatant, wash once with phosphate buffered saline and set aside;

[0064] (3) Specificity of immunofluorescence detection of H10 subtype influenza virus: Viruses were diluted with phosphate buffer, including H10N2 (A / duck / Zhejiang / 6D20 / 2013), H10N3 (A / chicken / Zhejiang / 8615 / 2016), H10N5 (A / Zhejiang / ZJU01 / 2023), H10N7 (A / chicken / Zhejiang / 2CP8 / 2014), H1N1 (A / Michigan / 45 / 2015), H2N8 (A / duck / Zhejiang / 6D10 / 2013), The cells were infected with diluted virus solutions of H3N2 (A / Texas / 50 / 2012), H4N6 (A / duck / Zhejiang / 409 / 2013), H5N1 (A / goose / Zhejiang / 97 / 2014), H6N1 (A / chicken / Zhejiang / 1664 / 2017), H7N9 (A / chicken / Zhejiang / ZJU01 / 2013), and H9N2 (A / chicken / Zhejiang / 221 / 2016) (multiplicity of infection 0.5) and cultured in a 37°C incubator saturated with 5% carbon dioxide for 2 h;

[0065] (4) Remove the cell plate, discard the virus solution, wash the cells twice with phosphate buffer, add 200 μl of virus culture solution to each well, and culture in a 37°C incubator saturated with 5% carbon dioxide for 24 hours;

[0066] (5) Remove the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffer;

[0067] (6) Add 4% paraformaldehyde to each well of the cell plate to fix the cells at room temperature for 30 minutes and wash three times with phosphate buffered saline;

[0068] (7) Permeabilize cells with 0.5% Triton-X100 at room temperature for 30 min and wash three times with phosphate buffer;

[0069] (8) blocking with phosphate buffer containing 3% bovine serum albumin at room temperature for 1 hour, and discarding the bovine serum albumin solution;

[0070] (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 three times with phosphate buffer;

[0071] (10) Dilute the fluorescent secondary antibody to 5 μg / mL with 1% bovine serum albumin solution, add 200 μL to each well, incubate in a dark incubator at 37°C for 90 min, and wash three times with phosphate buffered saline;

[0072] (11) The cell nuclei were stained with a fluorescent DNA dye (4',6-diamidino-2-phenylindole), incubated at room temperature in the dark for 10 min, and washed three times with phosphate buffer;

[0073] (12) The experimental results were observed under a fluorescence microscope. Green fluorescence was considered positive. The test results showed that the anti-H10 subtype influenza virus monoclonal antibody 1E1 developed in this study had good specificity for detecting H10 subtype influenza virus.

[0074] It should be understood that the present invention is described in conjunction with the best embodiment. However, after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody 1E1, the antibody subtype is IgG2a, κ type, can specifically bind to the H10 subtype influenza virus hemagglutinin protein antigen, the amino acid sequence of the antibody heavy chain variable region 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.

2. The monoclonal antibody 1E1 according to claim 1, characterized in that: The monoclonal antibodies are produced by hybridoma cells.

3. The monoclonal antibody 1E1 according to claim 2, characterized in that: The hybridoma cell producing the monoclonal antibody is a hybridoma cell line 1E1 obtained by fusing, screening, cloning and stably propagating spleen lymphocytes of immune BALB / C mice and mouse myeloma cells SP2 / 0, and can stably secrete the monoclonal antibody 1E1 of H10 subtype influenza virus hemagglutinin protein.

4. The method for preparing the monoclonal antibody 1E1 against the hemagglutinin protein of H10 subtype influenza virus according to claim 1, characterized in that The monoclonal antibody was obtained by the following steps: (1) Mouse immunization: 8-week-old BALB / C mice were selected and immunized with purified hemagglutinin protein of H10 subtype influenza virus: each mouse was immunized with 5 μg of H10 subtype influenza virus hemagglutinin protein mixed with adjuvant in a 1:1 ratio and injected into the inner thigh muscle. After 21 days, the mice were immunized again in the same manner. On the 35th day, tail blood was collected to measure the antibody titer of the mice. The mice with the highest immune titer were selected and boosted once by tail vein injection. Cell fusion was performed 3 days later. (2) Cultivation of mouse myeloma cells: Two weeks before the fusion, myeloma cells SP2 / 0 were revived, cultured and maintained in a good growth state for hybridoma cell fusion; (3) Cell fusion: using polyethylene glycol-mediated cell fusion method; killing the mice selected in step (1) to obtain spleen lymphocytes; mixing spleen lymphocytes with myeloma cells SP2 / 0 cells and centrifuging them, and then merging the cells through polyethylene glycol-mediated cell fusion; appropriately diluting the fused cells, inoculating them into a culture plate, and culturing them under appropriate conditions; (4) Screening of hybridoma cells: The above culture is cultured in a selective medium containing hypoxanthine-phosphoribosyltransferase; when the cell colonies grow to an appropriate size, the culture supernatant is aspirated for antibody identification by enzyme-linked immunosorbent assay to screen positive clones; (5) Cloning and culture of hybridoma cells: positive hybridoma cells were cloned by limiting dilution method, and the cells diluted to a certain density were inoculated into 96-well cell culture plates so that only one cell grew in each well; the supernatant of the wells where cell colonies were formed was taken for enzyme-linked immunosorbent assay to screen and identify positive clones; the culture wells with the highest antibody titer and single clone cell growth were selected and limited dilution was performed again. Monoclonal limiting dilution was performed for more than 3 times in succession, and the cells were continuously passaged for more than 20 generations to obtain hybridoma cell lines that stably and efficiently expressed monoclonal antibodies against H10 subtype influenza virus; (6) Preparation of monoclonal antibody ascites: 10-week-old healthy BALB / C mice were selected and 5 million positive hybridoma cells were inoculated in each abdomen. About 10 days after the inoculation of cells, the abdomen of the mouse was obviously swollen. Before the mouse was on the verge of death, the ascites was collected and centrifuged to determine the antibody titer and purify the monoclonal antibody in the ascites; (7) Purification of monoclonal antibodies: Monoclonal antibodies were purified from mouse ascites using protein G agarose gel affinity purification.

5. Use of the monoclonal antibody 1E1 against the hemagglutinin protein of H10 subtype influenza virus according to claim 1, 2 or 3 in the preparation of a fluorescent detection product for H10 subtype influenza virus.

6. The use according to claim 5, characterized in that: The product detects H10 subtype influenza virus in samples through immunofluorescence technology.

7. The use according to claim 6, characterized in that: The sample is body fluid or allantoic fluid.

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