Monoclonal antibody 1H1 for resisting H6 subtype avian influenza virus hemagglutinin protein and application of monoclonal antibody 1H1

Abstract

The invention belongs to the technical field of biology, and relates to an anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1 and application thereof, cell engineering and antibody engineering technologies are utilized to obtain a hybridoma cell line for secreting a hemagglutinin monoclonal antibody, ascites is induced by mice of the same strain to prepare the anti-hemagglutinin protein monoclonal antibody 1H1, the anti-hemagglutinin protein monoclonal antibody 1H1 is identified as an IgG1, kappa type, and the anti-hemagglutinin protein monoclonal antibody 1H1 is identified as an anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody. And the application of the antibody is realized through technologies of affinity purification, immunization and the like. The monoclonal antibody has the advantages that the preparation method is simple and easy to implement, more importantly, the monoclonal antibody prepared by the method can have multiple purposes, an effective tool is provided for auxiliary diagnosis of H6 subtype avian influenza virus infection in a clinical sample, and the monoclonal antibody can be popularized and applied to multiple detection technologies and clinical and experimental research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to an anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1 and an application thereof. The invention utilizes cell engineering and antibody engineering technologies to obtain a hybridoma cell line that secretes the hemagglutinin monoclonal antibody, induces ascites in mice of the same strain, prepares the anti-hemagglutinin protein monoclonal antibody 1H1, identifies it as IgG1 and κ type, and then realizes the application of the antibody through affinity purification, immunization and other technologies. Background Art

[0002] Avian influenza virus is a type of virus with high variability and potential cross-species transmission, posing a major threat to the poultry industry and public health security. As one of the important subtypes, the H6 subtype of avian influenza virus has attracted widespread attention due to its widespread prevalence and potential zoonotic risk.

[0003] At present, the diagnostic method of H6 subtype avian influenza virus infection mainly includes virus separation and identification, serological method and molecular biological method, such as reverse transcriptase polymerase chain reaction and real-time quantitative polymerase chain reaction, but these methods are very demanding and time-consuming for technology and laboratory. It is imperative to develop a fast and sensitive H6 subtype virus detection product, which is conducive to promoting the earlier and more extensive discovery of H6 subtype avian influenza virus infection and controlling the spread. Although multiple detection technologies and vaccine development strategies have been proposed, there are still certain limitations in existing vaccines and treatment methods, especially when the virus mutates faster, targeted monoclonal antibodies have become important research directions, and the detection method based on monoclonal antibodies is also widely used in virus detection. Therefore, the present invention is intended to illustrate a specific monoclonal antibody for H6 subtype avian influenza virus, and combine it with immunofluorescence technology, so that H6 subtype avian influenza virus in sample can be detected. Through immunofluorescence technology, the fluorescent signal produced by the specific binding of viral antigens and monoclonal antibodies in the sample can be observed, thereby realizing the rapid and sensitive detection of virus.

[0005] Based on the above background, this project selected the hemagglutinin protein of H6 subtype avian influenza virus as the target antigen, used fusion hybridoma technology to establish a hybridoma cell line that stably secretes monoclonal antibodies against H6 subtype avian influenza virus, and prepared, purified and identified these monoclonal antibodies in large quantities. The successful acquisition of this monoclonal antibody laid a material foundation for the establishment of a diagnostic method for H6 subtype avian influenza virus - diagnosis based on immunofluorescence technology. At the same time, it plays an important role in the study of disease pathogenesis, prognosis and efficacy determination.

[0006] In addition, this monoclonal antibody can also be applied to other immunological techniques, such as immunoblotting, enzyme-linked immunosorbent assay, etc., to provide multiple options for the detection of H6 subtype avian influenza virus and lay a material foundation for diagnosis based on immunological technology. Summary of the invention

[0007] The purpose of the present invention is to provide a monoclonal antibody against the hemagglutinin protein of H6 subtype avian influenza virus, which can identify the H6 subtype avian influenza virus.

[0008] The anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1 has an IgG1 and κ subtype and can specifically bind to the H6 subtype avian influenza virus hemagglutinin protein antigen. The amino acid sequence of the antibody's 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.

[0009] SEQ ID No.1

[0010] Heavy chain:DNA sequence(363bp)

[0011] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0012] CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAA

[0013] GGCTTCTGGCTTCACCTTCACCAGCTCCTTGATACACTGGACAAAACAGAGGCCTGGACAAGGCCTTGA

[0014] GTGGATTGGAGAGATTCATCCTAATAGTGGTAATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACA

[0015] CTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCG

[0016] GTCTATTACTGTACAAGAGGGTATGATGGTTACTACGTACAAGCTATGGACTACTGGGGTCAAGGAACC

[0017] TCAGTCACCGTCTCCTCA

[0018] SEQ ID No.2

[0019] Heavy chain:Amino acid sequence(121AA)

[0020] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0021] QVQLQQPGSVLVRPGASVKLSCKASGFTFTSSLIHWTKQRPGQGLEWIGEIHPNSGNTNYNEKFKGKATLTVDTSSS

[0022] TAYVDLSSLTSEDSAVYYCTRGYDGYYVQAMDYWGQGTSVTVSS

[0023] SEQ ID No.3

[0024] Light chain:DNA sequence(336bp)

[0025] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0026] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA

[0027] GATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCC

[0028] AGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCA

[0029] GTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTG

[0030] CTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAASEQ ID No.4

[0031] Light chain:Amino acid sequence(112AA)

[0032] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0033] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTL

[0034] KISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0035] The second object of the present invention is to provide a method for preparing monoclonal antibodies against the hemagglutinin protein of H6 subtype avian influenza virus, which is achieved by the following steps and technical solutions:

[0036] (1) Animal immunization: Six-week-old BALB / C mice were selected and immunized with purified hemagglutinin protein of H6N1 subtype avian influenza virus (A / chicken / Zhejiang / 1664 / 2017).

[0037] (2) Cultivation of mouse myeloma cells: Mouse myeloma cells SP2 / 0 were cultured and maintained in a good growth state for cell fusion.

[0038] (3) Cell fusion: polyethylene glycol fusion method was used. BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were inoculated into a 96-well culture plate and cultured for one day in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. The mice prepared in (1) were killed and spleen lymphocytes were obtained. The mouse myeloma cells in (2) were collected. The above two cells were mixed and centrifuged, and then polyethylene glycol was used to mediate cell fusion. The fused cells were appropriately diluted, inoculated into feeder cell culture plates, and cultured under appropriate conditions.

[0039] (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.

[0040] (5) Cloning of hybridoma cells: Cloning of hybridoma cells was performed by limiting dilution method. Cells diluted to a certain density were inoculated into 96-well plates so that only one cell grew in each well. The culture supernatant of the wells where cell colonies were formed was taken for enzyme-linked immunosorbent assay to identify positive clones. The culture wells with the highest antibody titer and single cloned cell growth were selected and limited dilution was performed again. The limited dilution was performed for more than 4 times in succession and continuous passage was performed for more than 20 generations to obtain hybridoma cell lines that stably and efficiently expressed monoclonal antibodies against H6N1 subtype influenza virus. The cloned hybridoma cells were subjected to antibody identification and physical and chemical property analysis.

[0041] (6) Induction of monoclonal antibody ascites: 8-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 as much as possible of the ascites was collected, the ascites was centrifuged, the antibody titer was determined, and the monoclonal antibodies in the ascites were purified.

[0042] (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites were purified using Protein G affinity purification.

[0043] (8) The present invention obtains a hybridoma line, namely 1H1, which produces monoclonal antibodies against H6 subtype avian influenza virus. The 1H1 hybridoma cell line was cloned four times and cultured for more than six months, and the secretion of antibodies was stable. The cell line was frozen in liquid nitrogen and grew well after recovery, and the antibody secretion did not decline. The titer of the 1H1 culture supernatant was 1:128 and the titer of the ascites was 1:6400 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 IgG1.

[0044] The present invention provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 1H1 obtained by fusing immune BALB / C mouse spleen cells and mouse myeloma cells SP2 / 0, screening, cloning, passage, repeated freezing and resuscitation, and can stably secrete the monoclonal antibody 1H1 against the hemagglutinin protein of H6 subtype avian influenza virus.

[0045] Another object of the present invention is to provide the use of the monoclonal antibody 1H1 in the preparation of a fluorescent detection product containing H6 subtype avian influenza virus. The detection of H6 subtype avian influenza virus in body fluids, allantoic fluid or other environmental samples is achieved by immunofluorescence detection.

[0046] The present invention provides a monoclonal antibody 1H1 of the hemagglutinin protein of H6 subtype avian influenza virus, and a method for detecting H6 subtype influenza virus by immunofluorescence technology. The present invention has the advantages that 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 H6 subtype avian influenza virus infection in clinical samples, and can be promoted and applied to multiple detection technologies and clinical and experimental research.

[0047] Instruction Manual

[0048] Figure 1 Immunoglobulin subtype analysis of monoclonal antibody 1H1.

[0049] Figure 2 The specificity of immunofluorescence technology for detecting H6 subtype avian influenza virus. DETAILED DESCRIPTION

[0050] 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.

[0051] Example 1. Preparation method of monoclonal antibody against H6 subtype avian influenza virus hemagglutinin protein

[0052] (1) Immunization of mice: For the first immunization, the H6 subtype avian influenza virus hemagglutinin protein and adjuvant were mixed in equal volumes, with a total volume of 600 μl. 0.1 ml (containing 5 μg of H6 subtype avian influenza virus hemagglutinin protein antigen) was injected into the inner thigh muscle of each BALB / C mouse. On the 21st day, a booster shot was given in the same manner. On the 35th day, a small amount of tail blood was collected for ELISA assay. The antibody titer reached 1:16000, and then a booster shot was injected into the tail vein. Cell fusion was performed 3 days later.

[0053] (2) Culture 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 an incubator saturated with 5% carbon dioxide at 37°C. Subculture was performed one day before fusion to ensure that the cells entered the logarithmic growth phase at the time of fusion.

[0054] (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 was taken from mice 3 days after the last booster immunization, and spleen cells were separated by pressure injection. The cells were washed twice by centrifugation and resuspended in culture medium. SP2 / 0 cells were collected, centrifuged, washed twice, and resuspended in culture medium as SP2 / 0 cells to be fused. 100 million spleen lymphocytes from immune mice were mixed with 20 million mouse myeloma cells SP2 / 0 and fused under the action of polyethylene glycol. After the two cells are mixed, they are washed once, centrifuged and the supernatant is discarded. The tube wall is flicked to suspend the cells. 0.9 ml of polyethylene glycol preheated at 37°C is added dropwise to the cell pellet within 90 seconds. The centrifuge tube is gently shaken during this period, but do not blow. Let it stand for 1 minute, and then add 1 ml of serum-free DMEM within the first minute, 2 ml of serum-free DMEM within the second minute, and 7 ml of serum-free DMEM within the third minute according to the principle of slow first and fast later. Then, 40 ml of serum-free DMEM medium preheated at 37°C is gradually added within 1 minute. Centrifuge at a low speed of 1000 rpm for 10 minutes. Then add the culture medium and inoculate it into 96-well culture plates with feeder cells. Generally, 2 plates are spread for each fusion of cells and cultured in a cell incubator.

[0055] (4) Screening of hybridoma cells: Change half of the culture medium (containing hypoxanthine-guanine-phosphoribosyltransferase) every 4 days, and switch to culture medium containing hypoxanthine-phosphoribosyltransferase after 10 days. The fused hybridoma cells are cultured in the selective culture medium containing hypoxanthine-phosphoribosyltransferase for about two weeks. The culture supernatant is aspirated for ELISA to screen positive clones. The positive hybridoma clones are screened indirectly by ELISA. Main steps: ① Dilute the H6 subtype hemagglutinin protein with 0.01 mol / L pH 9.6 carbonate buffer to a concentration of 20 ng / well, add 0.1 ml to each well of a 96-well ELISA plate, and incubate at 4°C overnight; ② Wash the plate three times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ Use 5% bovine serum albumin 0.01 mol / L pH 7.4 phosphate buffer was blocked for 2 hours; ④ the plate was washed as above; ⑤ hybridoma culture supernatant was added, 0.1 ml per well, and positive control (immune mouse serum), negative control (SP2 / 0 culture supernatant) and blank control were set up at room temperature for 2 hours; ⑥ the plate was washed; ⑦ 1:6000 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; ⑨ substrate was added and reacted at room temperature in the dark for 5 minutes; ⑩ 2 mol / L sulfuric acid was used to terminate the reaction; the optical density was measured at 450 nm, and the measured value divided by the negative value ≥ 2.1 was considered positive.

[0056] (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 5 culture wells with the highest antibody titer and single clone cell growth are selected for another limiting dilution. The limiting dilution is carried out for more than 4 times and the cells are continuously subcultured for more than 20 generations to obtain a hybridoma cell line that stably and efficiently expresses monoclonal antibodies against H6 subtype influenza virus.

[0057] (6) Induce ascites: Select 8-week-old healthy BALB / C mice and inoculate 5 million positive hybridoma cells in the abdomen of each mouse. About 7 days after the inoculation of the cells, the abdomen of the mouse will be significantly swollen. Closely observe the health status and abdominal signs of the mouse. When the ascites is as much as possible and the mouse is on the verge of death, collect the ascites and centrifuge it, determine the antibody titer, and purify the monoclonal antibody in the ascites.

[0058] (7) Purification of monoclonal antibodies: Affinity purification (Sepharose cross-linked with Protein G) was used to purify monoclonal antibodies in ascites. ① After diluting the ascites 3 times with cold binding buffer, centrifuge at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② The affinity purification column pre-loaded with Sepharose-Protein G was fully washed with 10 times the column volume of binding buffer. ③ The diluted ascites was loaded onto the column, and the flow rate was controlled at 10 drops per minute. ④ The ascites that flowed through was loaded onto the column again. ⑤ The column was fully washed with 20 times the column volume of binding buffer until the absorbance of the flow-through at 280 nm was less than 0.01. ⑥ The bound monoclonal antibody was eluted with elution buffer, and the flow rate was controlled at 10 drops per minute. The eluate was collected in a collection tube pre-added with 0.1 ml of potassium phosphate buffer (PH7.9). 0.5 ml of the antibody-containing eluate was collected in each tube, and more than 20 tubes were collected in total. ⑦ Detect the absorbance of each tube of eluate at 280 nanometers, and collect the eluate with an absorbance value greater than 0.2. ⑧ Place the collected eluate in a dialysis card and dialyze it in a 0.1 mol / L phosphate buffer at pH 7.4. Change the solution every 6 hours for a total of 24 hours. ⑨ Dilute the dialyzed antibody solution and measure the protein content at 280 nanometers. ⑩ Dispense the purified antibody into small tubes and place them in a low-temperature refrigerator for later use.

[0059] (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 1H1 hybridoma cells was IgG1, κ type.

[0060] See attached for the results Figure 1 .

[0061] Example 2. Qualitative detection of H6 subtype avian influenza virus using the monoclonal antibody

[0062] The monoclonal antibody against the hemagglutinin protein of the H6 subtype avian influenza virus prepared by the present invention can be used to qualitatively detect the H6 subtype avian influenza virus, and the identification method can be achieved by the following method:

[0063] Immunofluorescence detection of H6 subtype avian influenza virus:

[0064] (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;

[0065] (2) Take out the cell plate with cells, discard the culture supernatant, wash once with phosphate buffered saline and set aside;

[0066] (3) Determine the specificity of immunofluorescence detection of H6 subtype avian influenza virus: dilute the virus with virus diluent, including H6N1 (A / chicken / Zhejiang / 1664 / 2017), H6N6 (A / chicken / Zhejiang / 727018 / 2014), H6N2

[0067] (A / chicken / Zhejiang / 727031 / 2014), H9N2(A / chicken / Zhejiang / 221 / 2016), H10N7

[0068] (A / chicken / Zhejiang / 2CP8 / 2014), H7N9 (A / chicken / Zhejiang / ZJU01 / 2013), H1N1 (A / Michigan / 45 / 2015), H3N2 (A / Texas / 50 / 2012), and influenza B virus (B / Phuket / 3073 / 2013), the diluted virus solution was used to infect the cells (multiplicity of infection was 0.5) and cultured in a 37°C incubator saturated with 5% carbon dioxide for 2 h;

[0069] (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;

[0070] (5) Remove the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffer;

[0071] (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;

[0072] (7) Permeabilize cells with 0.5% Triton-X100 at room temperature for 30 min and wash three times with phosphate buffer;

[0073] (8) blocking with phosphate buffer containing 3% bovine serum albumin at room temperature for 1 hour, and discarding the bovine serum albumin solution;

[0074] (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;

[0075] (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;

[0076] (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;

[0077] (12) The experimental results were observed under a fluorescence microscope. Green fluorescence was observed as positive. The test results showed that the anti-H6 subtype avian influenza virus monoclonal antibody 1H1 developed based on this study can detect H6 subtype avian influenza virus with good specificity.

[0078] See attached for the results Figure 2 .

[0079] 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-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1, the monoclonal antibody subtype is IgG1, κ type, can specifically bind to the H6 subtype avian influenza virus hemagglutinin protein antigen, the antibody heavy chain variable region amino acid sequence is shown in SEQ ID No.2, the light chain variable region amino acid sequence is shown in SEQ ID No.

4.

2. The monoclonal antibody 1H1 according to claim 1, characterized in that: The monoclonal antibodies are produced by hybridoma cells.

3. The monoclonal antibody 1H1 according to claim 2, characterized in that: The hybridoma cell producing the monoclonal antibody is a hybridoma cell line 1H1 obtained by fusion, screening, cloning, passage, repeated freezing and recovery of immune BALB / C mouse spleen lymphocytes and mouse myeloma cells SP2 / 0. It can stably secrete anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1.

4. The method for preparing the monoclonal antibody 1H1 according to claim 1, characterized in that: The monoclonal antibody was obtained by the following steps: (1) Mouse immunization: 6-week-old BALB / C mice were selected and immunized with the hemagglutinin protein of the H6N1 subtype avian influenza virus; each mouse was immunized by intramuscular injection into the inner thigh with 5 μg of the hemagglutinin protein of the H6 subtype avian influenza virus mixed with an adjuvant in a ratio of 1:1, and immunized again in the same manner after the 21st day, for a total of 2 times; a small amount of mouse tail blood was collected on the 35th day to measure the antibody titer of the mice, and the mice with the highest immune titer were selected and boosted once by tail vein injection, and cell fusion was performed 3 days later; (2) Cultivation of mouse myeloma cells: Cultivating mouse myeloma cells SP2 / 0 and maintaining them in a good growth state for hybridoma cell fusion; (3) Cell fusion: The polyethylene glycol cell fusion method was used; BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were inoculated into a 96-well culture plate and cultured for one day in a hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. The mice prepared in step (1) were killed to obtain spleen lymphocytes. Collect the SP2 / 0 cells in step (2), mix the two cells and centrifuge them, then use polyethylene glycol to mediate cell fusion, appropriately dilute the fused cells, inoculate them into a culture plate, and culture 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 a suitable 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: clone positive hybridoma cells by limiting dilution method, inoculate the cells diluted to a certain density into a 96-well cell culture plate so that only one cell grows in each well; take the supernatant from the wells where cell colonies are formed for ELISA to screen and identify positive clones; repeat limiting dilution several times until the positive well rate of hybridoma cells reaches 100%; expand the culture of cloned hybridoma cells and perform antibody physical and chemical properties analysis and identification; (6) Preparation of monoclonal antibody ascites: 8-10 week old healthy BALB / C mice were selected, and 5 million positive hybridoma cells were inoculated in each abdomen. Seven days after the inoculation of 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, the antibody titer was determined, and the monoclonal antibodies in the ascites were purified; (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites were purified using Protein G affinity purification.

5. Use of the monoclonal antibody 1H1 against the hemagglutinin protein of H6 subtype avian influenza virus according to claim 1, 2 or 3 in the preparation of H6 subtype avian influenza virus detection products.

6. The use according to claim 5, characterized in that: The product detects H6 subtype avian influenza virus in different 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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