Anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody 2G9 and its application

Abstract

The present invention belongs to the field of biotechnology and relates to anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody 2G9 and its application. Monoclonal antibody 2G9 is identified as IgG2a, κ type, and the application of the antibody is achieved through affinity purification, immunological methods and other technologies. Anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody 2G9, the monoclonal antibody subtype is IgG2a, κ type, named 2G9, can specifically recognize the hemagglutinin protein of H10 subtype 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 present invention provides an effective tool for the auxiliary diagnosis of H10 subtype influenza virus infection in clinical samples, and can be promoted and applied to various detection technologies as well as clinical and experimental research.

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 2G9 and its application. The invention utilizes cell engineering and antibody engineering technologies to obtain a hybridoma cell line that secretes a monoclonal antibody against the hemagglutinin protein. Ascites is induced in mice of the same strain to prepare the anti-hemagglutinin protein monoclonal antibody 2G9, which is identified as IgG2a, κ type. The antibody is then applied through affinity purification, immunization methods and other technologies. Background Art

[0002] Influenza is the most widespread zoonosis worldwide, posing a significant threat to both human and animal health. Influenza A virus, one of the main pathogens of influenza, belongs to the Orthomyxoviridae family and is classified into 18 HA and 11 NA subtypes based on the antigenic properties of their hemagglutinin (HA) and neuraminidase (NA) proteins. Influenza A viruses have a wide host range and have been detected and isolated in multiple species, including humans, poultry, pigs, and horses. Waterfowl are considered the natural reservoir of influenza A viruses, and most influenza A subtypes have been found in waterfowl. In recent years, subtype H10 influenza viruses have become widespread in poultry and have undergone multiple reassortment, resulting in an increasing number of subtype H10 viruses being isolated from poultry. Since 2004, human cases of influenza A virus infection with subtype H10N7 have occurred in Egypt and Australia. In 2013, two deaths in humans due to influenza A virus infection with subtype H10N8 were first reported in China. Subsequently, influenza A virus infection with subtype H10 was also detected in European seals. In June 2021, China's National Health Commission reported a case of human infection with the H10N3 subtype of influenza in Jiangsu Province. In June 2022, the Zhejiang Provincial Health Commission also reported a case of human infection with the H10N3 subtype of influenza in Jinhua, Zhejiang Province. In January 2024, the H10N5 subtype of influenza virus was isolated from samples of influenza deaths in Zhejiang Province. Therefore, the H10 subtype of influenza virus poses a potential pandemic threat to global public health. Currently, the H10 subtype of influenza virus continues to circulate in poultry around the world. Therefore, the establishment of a rapid, accurate, and sensitive method for detecting the H10 subtype of influenza virus and effectively detecting and identifying various poultry samples is of great significance for influenza prevention and control.

[0003] Isolating influenza viruses by chicken embryos or MDCK cells is the most classic method for identifying influenza viruses. In recent years, molecular detection methods have been greatly developed, and real-time quantitative polymerase chain reaction has been widely used in laboratory diagnosis of influenza virus infection. However, these methods have high requirements for personnel operation skills and equipment and are very time-consuming. With the continuous 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 the hemagglutinin protein of H10 subtype influenza virus, and combines it with quantum dot fluorescent microsphere detection technology to detect H10 subtype influenza virus in samples. The above method has the advantages of being fast, sensitive, accurate and economical, which will be conducive to discovering H10 subtype influenza virus earlier and faster, and contribute to the effective control of influenza epidemics.

[0004] In summary, the development of monoclonal antibodies against the 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 this background, this project selected the H10 subtype influenza virus hemagglutinin protein as the target antigen. Using fusion hybridoma technology, a hybridoma cell line stably secreting anti-hemagglutinin monoclonal antibodies was established. These monoclonal antibodies were then mass-produced, purified, and characterized. The successful acquisition of these monoclonal antibodies laid the material foundation for the development of a novel diagnostic method for the H10 subtype influenza virus—the quantum dot fluorescent microsphere immunochromatographic strip. It also plays a significant role in research into disease pathogenesis, prognosis, and therapeutic efficacy.

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

[0006] The purpose of the present invention is to provide an anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody capable of identifying H10 subtype influenza virus.

[0007] The anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody 2G9 has an IgG2a, κ type, and is named 2G9. It can specifically recognize the hemagglutinin protein of the H10 subtype influenza virus. 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.

[0008] The monoclonal antibody is produced by hybridoma cells. The hybridoma cell line 2G9, obtained by fusion, screening, cloning, and stable passage of immune BALB / C mouse spleen lymphocytes and mouse myeloma cells SP2 / 0, can stably secrete the monoclonal antibody 2G9 against the hemagglutinin protein of the H10 subtype influenza virus.

[0009] SEQ ID No.1

[0010] Heavy chain:DNA sequence(375bp)

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

[0012] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGT

[0013] TCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGA

[0014] AAGGGTCTGGAATGGCTGACACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAA

[0015] GAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACTAGTGTGG

[0016] ACACTGCAGATACTGCCACATACTACTGTGCTCGAAGCCCCCTACGGACTACGGTAGTAGCTGG

[0017] GGTGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0018] SEQ ID No. 2

[0019] Heavy chain:Amino acid sequence(125AA)

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

[0021] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLTHIYWDDDKRYNPSLKSRLTISKDT

[0022] SRNQVFLKITSVDTADTATYYCARSPPTDYGSSWGVMDYWGQGTSVTVSS

[0023] SEQ ID No.3

[0024] Light chain:DNA sequence(366bp)

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

[0026] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGACAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTTTACCTTCACAAACTATGGAATGAACTGGGTGAGGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATACACACTTACAATGGAGAGCCAGCATATGCTGAAGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAGAATGAGGACACGGCTACATATTTCTGTGCAAGAGGCTATGATTACGCCGAGGGTTACTTTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0027] SEQ ID No.4

[0028] Light chain:Amino acid sequence(122AA)

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

[0030] QIQLVQSGPELKKPGQTVKISCKASGFTFTNYGMNWVRQAPGKGLKWMGWIHTYNGEPAYAEDFKGRFAFS

[0031] LETSASTAYLQINNLKNEDTATYFCARGYDYAEGYFAMDYWGQGTSVTVSS

[0032] The second object of the present invention is to provide a method for preparing monoclonal antibodies against H10 subtype influenza virus hemagglutinin protein, 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) Culture 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: Polyethylene glycol-mediated cell fusion was used. The mice selected in step (1) were sacrificed and spleen lymphocytes were obtained. The SP2 / 0 cells from step (2) were collected, the two cells were mixed and centrifuged, and then the cells were fused using polyethylene glycol. The fused cells were appropriately diluted, inoculated into 96-well culture plates, 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 so that only one cell grows 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 succession, 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. The cloned hybridoma cells are subjected to antibody identification and physical and chemical property analysis.

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

[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 has obtained a hybridoma line, namely 2G9, which produces monoclonal antibodies against the hemagglutinin protein of the H10 subtype influenza virus. The 2G9 hybridoma cell line was cloned three times and cultured continuously for 6 months, with stable antibody secretion. The cell line was frozen in liquid nitrogen and grew well after recovery. The titer of the 2G9 culture supernatant was 1:128 and the titer of the ascites fluid was 1:2048 as measured by enzyme-linked immunosorbent assay. Monoclonal antibody immunoglobulin subtype analysis showed that the antibody produced by the hybridoma cells was IgG2a.

[0041] The present invention provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 2G9 obtained by fusing immune BALB / C mouse spleen cells and mouse myeloma cells SP2 / 0, screening, cloning and passage, and can stably secrete the monoclonal antibody 2G9 against the H10 subtype influenza virus hemagglutinin protein.

[0042] Another object of the present invention is to provide the use of monoclonal antibody 2G9 in the preparation of H10 subtype influenza virus detection products.

[0043] The detection of the monoclonal antibody 2G9 in poultry throat swabs, feces, allantoic fluid or other environmental samples containing H10 subtype influenza virus is achieved through quantum dot fluorescent microsphere immunochromatography technology.

[0044] The present invention provides a monoclonal antibody 2G9 against the H10 subtype influenza virus hemagglutinin protein and its use in quantum dot fluorescent microsphere immunochromatographic strip detection. BALB / C mice are immunized with purified H10 subtype influenza virus hemagglutinin protein, and B lymphocytes from the immunized BALB / C mice are collected and fused with mouse myeloma cells. Positive clones are identified using an enzyme-linked immunosorbent assay to establish a hybridoma cell line 2G9 that secretes a monoclonal antibody against the H10 subtype influenza virus hemagglutinin protein. Positive cells are expanded, cultured, and injected into the peritoneal cavity of mice of the same strain to induce the production of antibody-containing ascites. The ascites is collected and affinity-purified with the antibody to obtain the monoclonal antibody 2G9 against the H10 subtype influenza virus hemagglutinin protein. The heavy chain amino acid sequence of the antibody is shown in SEQ ID No. 1, and the light chain amino acid sequence is shown in SEQ ID No. 2. The monoclonal antibody is further analyzed and identified for its physical and chemical properties, and a method for detecting H10 subtype influenza virus using quantum dot fluorescent microsphere immunochromatographic strip technology using the monoclonal antibody as a probe is established. The present invention provides an effective tool for auxiliary diagnosis of H10 subtype influenza virus infection in clinical samples and can be widely applied to various detection technologies as well as clinical and experimental research.

[0045] The present invention has the advantage of providing a monoclonal antibody against the hemagglutinin protein of the 2G9 subtype influenza virus. The preparation method is simple and easy, and more importantly, the monoclonal antibody prepared by this method can be used in a variety of applications, such as qualitative diagnosis of H10 subtype influenza samples in clinical and laboratory settings.

[0046] Figures in the specification

[0047] Figure 1 Immunoglobulin subtype analysis of monoclonal antibody 2G9.

[0048] Figure 2 The specificity of quantum dot immunochromatographic strip in detecting H10 subtype influenza virus.

[0049] Figure 3 The sensitivity of quantum dot immunochromatographic strip in detecting H10 subtype influenza virus. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0051] Example 1. Preparation of Monoclonal Antibodies Against H10 Subtype Influenza Virus Hemagglutinin Protein

[0052] (1) Immunization of mice: For the first immunization, H10 subtype influenza virus hemagglutinin protein and adjuvant were mixed evenly in a 1:1 ratio, 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 enzyme-linked immunosorbent assay. The highest antibody titer reached 1:32,000. The mouse with the highest antibody titer was selected for a booster immunization via tail vein injection, and cell fusion was performed 3 days later.

[0053] (2) Culture and Passaging of Mouse Myeloma SP2 / 0 Cells: SP2 / 0 myeloma cells from BALB / C mice were cultured and passaged in DMEM medium containing 10% bovine serum in a 37°C incubator containing 5% carbon dioxide. Passaging is usually not performed the day before fusion to ensure that the cells enter 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 in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum for one day. Spleens were obtained from mice 3 days after the last booster immunization. Splenic lymphocytes were isolated by pressure injection, 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 from the immunized mice were mixed with 30 million mouse myeloma SP2 / 0 cells. The two cell types were mixed, centrifuged, and the supernatant discarded. The centrifuge tube was swirled to loosen the cell clumps. Polyethylene glycol pre-warmed at 37°C was slowly added to the fusion tube, gently shaking the tube during this process. The cells were aspirated into the fusion tube. After 90 seconds of quiescence, the cells were blown into the centrifuge tube. Following a slow-to-accelerate 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. Over the next minute, 40 ml of DMEM pre-warmed at 37°C was gradually added. The tube was centrifuged at 800 rpm for 10 minutes. Hypoxanthine-guanine-phosphoribosyltransferase medium supplemented with 20% bovine serum was then added. The cells were seeded into 96-well culture plates containing feeder cells using a glass pipette. Two plates were typically plated for each fusion. The cells were cultured in a 37°C incubator containing 5% carbon dioxide.

[0055] (4) Screening of hybridoma cells: After 5 days, the 96-well culture plate was half-replaced with a culture medium (containing hypoxanthine-guanine-phosphoribosyltransferase) and after 10 days, the culture medium containing hypoxanthine-phosphoribosyltransferase was used instead. The fused hybridoma cells were cultured in a selective culture medium containing hypoxanthine-phosphoribosyltransferase for approximately two weeks. When the cell colonies grew to an appropriate size (observed under a 10x objective lens, the size of the cell clones should be such that they filled one 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 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 microtiter plate, with a protein amount of 20 ng per well, and incubate at 4°C overnight; ② Wash the plate five times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ Wash the plate with 0.01 mol / L pH 7.4 phosphate buffer containing 5% bovine serum albumin; ④ Block with 7.4% phosphate buffer for 2 hours; ④ Wash the plate three times; ⑤ Add hybridoma culture supernatant, 0.1 ml per well, and set up a positive control (H10 subtype protein immune mouse serum), a negative control (SP2 / 0 culture supernatant) and a blank control, and react at room temperature for 2 hours; ⑥ Wash the plate three times; ⑦ Add 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG, 0.1 ml per well, and react at room temperature for 1 hour; ⑧ Wash the plate three times; ⑨ Add color development solution and react at room temperature in the dark for 5 minutes; ⑩ Terminate the reaction with 2 mol / L sulfuric acid; Measure the optical density at 450 nm, and divide the measured value by the negative value by ≥2.1 to define it as positive.

[0056] (5) Cloning of hybridoma cells: The cloning culture of hybridoma cells was carried out according to the limiting dilution method. After appropriate proliferation of hybridoma cells in antibody test positive, the cells were accurately counted. The cell suspension was diluted with complete DMEM medium to 10 cells per ml and inoculated into a 96-well culture plate containing feeder cells, with 0.1 ml per well. After 10 days, the cell growth was observed and the antibody level in the supernatant was detected. The culture well with the highest antibody titer and single clone cell growth was selected and limited dilution was performed again. The limiting dilution was repeated for more than 3 times and the cells were continuously passaged for more than 20 generations to obtain a hybridoma cell line that stably and efficiently expressed monoclonal antibodies against H10 subtype influenza virus.

[0057] (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 into 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.

[0058] (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites were purified by affinity purification (protein G agar gel). 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 agar gel thoroughly with 5 times the column volume of binding buffer. ③ Load the diluted ascites onto the column, controlling the flow rate to 10 drops per minute. ④ Repeat the loading of the ascites that has passed through the column onto the column once more. ⑤ Wash the purification column thoroughly with 5 times the column volume of binding buffer. ⑥ Elute the bound monoclonal antibody with elution buffer at a controlled flow rate of 10 drops per minute. Collect the eluate into a collection tube pre-filled with 0.1 ml of potassium phosphate buffer (pH 7.9), collecting 0.5 ml of antibody-containing eluate per tube. ⑦ Measure the absorbance of each eluate at 280 nm and collect the eluate with a protein content greater than 0.1 mg / ml. ⑧ Add the antibody eluate to an ultrafiltration centrifuge tube and centrifuge at 10,000 rpm at 4°C for 20 minutes to a final volume of approximately 1 ml. Add 10 ml of 0.1 M phosphate buffer, pH 7.4, and centrifuge at 10,000 rpm at 8°C for 20 minutes. Concentrate the antibody by a final centrifugation to a final volume of approximately 1 ml and transfer the concentrated antibody solution to 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 in a freezer until ready for use.

[0059] (8) Monoclonal antibody subtype identification: 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 carried out strictly according to the kit instructions. The test results showed that the monoclonal antibody secreted by the 2G9 hybridoma cells was IgG2a, κ type.

[0060] See attached for the results Figure 1 .

[0061] Example 2. Rapid detection of H10 subtype influenza virus using the monoclonal antibody

[0062] The monoclonal antibody against the hemagglutinin protein of the H10 subtype influenza virus prepared by the present invention can be used to rapidly detect the H10 subtype influenza virus. The identification method can be achieved by the following method:

[0063] H10 subtype influenza virus immunochromatographic strip based on quantum dot fluorescent microspheres:

[0064] (1) Solution preparation: ① Activation buffer: Take a 100 ml glass bottle, add 0.488 g of N-morpholinoethanesulfonic acid, and add 100 ml of ultrapure water, and adjust the pH to 6.0; ② Preparation of coupling agent: Weigh 10 mg of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dissolve them in 1 ml of activation buffer, and prepare them before use; ③ Glass fiber membrane activation solution: Take a 100 ml glass bottle, add 1.5 ml of Tween 20, 4 g of sucrose, and 5 g of bovine serum ④ Blocking solution: add 0.2 ml Tween-20 and 1 g bovine serum albumin to 10 ml activation buffer, mix thoroughly, and store at 4°C; ⑤ Antibody diluent: add 0.1 ml Tween 20 and 0.5 g bovine serum albumin to 10 ml activation buffer, mix thoroughly, and store at 4°C; ⑥ Quantum dot resuspension solution: add 0.1 ml Tween 20 and 0.5 g bovine serum albumin to 10 ml phosphate buffer, and adjust the pH to 7.2.

[0065] (2) Preparation of quantum dot markers: ① Take 40 μL of water-soluble carboxyl quantum dots and add them to a centrifuge tube containing 1 ml of activation buffer, and mix them by ultrasonication for 10 minutes; ② Centrifuge the centrifuge tube at 15,000 rpm for 30 minutes, discard the supernatant after centrifugation, add 400 μL of activation buffer, mix them by ultrasonication for 10 minutes, and add 15 μL of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in a ratio of 1:1, mix them by pipetting, and activate them in a horizontal shaker at room temperature in the dark for 30 minutes; ③ After activation, centrifuge the centrifuge tube at 15,000 rpm for 20 minutes, discard the supernatant after centrifugation, and add 400 μL of Activate the buffer solution and mix it ultrasonically for 10 minutes; ④ Add 0.2 mg of labeled antibody (monoclonal antibody 2G9 against H10 subtype influenza virus) to the above solution and incubate it in the dark on a horizontal shaker at room temperature for 1 hour; ⑤ After the incubation, add 410 μl of blocking solution and incubate it in the dark on a horizontal shaker at room temperature for 1 hour; ⑥ After the blocking is completed, centrifuge the centrifuge tube at 15,000 rpm for 30 minutes at 4°C, discard the supernatant after centrifugation, add 1 ml of phosphate buffer solution and mix, centrifuge at 15,000 rpm for 20 minutes to remove unreacted antibody molecules; ⑦ After the centrifugation, discard the supernatant, add 150 μl of quantum dot buffer solution, mix it ultrasonically for 10 minutes, and set aside.

[0066] (3) Preparation of glass fiber membrane: Soak the carrier glass cellulose membrane in the glass fiber membrane activation solution for 30 minutes and dry it in an oven at 37°C. After drying, spray the quantum dots in step 7 of (2) onto the glass fiber membrane.

[0067] (4) Preparation of nitrocellulose membrane: Goat anti-mouse IgG antibody and another monoclonal antibody 1E8 against influenza virus subtype H10 were diluted to 1 mg / ml and 2 mg / ml, respectively, and drawn on the control line and test line of the nitrocellulose membrane to prepare a coating membrane, which was then dried in a 37°C oven overnight.

[0068] (5) Assembly of the test card: First, install the nitrocellulose membrane coated with the detection monoclonal antibody and the quality control secondary antibody on a dedicated support plate, and then install the sample pad, glass fiber membrane, and absorbent pad in sequence. Leave a section of the glass fiber membrane and absorbent pad on the nitrocellulose membrane, and leave a section of the sample pad on the glass fiber membrane. Ensure that each part is connected to each other so that the sample can flow smoothly. Cut the strips to prepare the detection quantum dot immunochromatographic strips. Each chromatographic strip is integrated into a plastic shell, and the fluorescence intensity excited by 365nm ultraviolet light is detected using a fluorescent immunochromatographic scanner.

[0069] (6) Determining the specificity of quantum dot immunochromatographic strips for detecting H10 subtype influenza virus: Quantum dot immunochromatographic strips were used to detect different viral allantoic fluid samples, including H1, H2, H3, H4, H5, H6, H7, H9, and H11 subtype influenza viruses, as well as several avian respiratory viruses such as Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, and avian paramyxovirus. The test results showed that the quantum dot immunochromatographic strips had good specificity for H10 subtype influenza virus.

[0070] See attached for the results Figure 2 .

[0071] (7) Determine the sensitivity of the quantum dot immunochromatographic strip in detecting H10 subtype influenza virus: dilute H10 subtype influenza virus to 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, and 0.0156 hemagglutination units, add 80 microliters of the sample to be tested to the sample pad area of ​​the immunochromatographic strip, and observe whether purple bands appear on the quality control line and the test line under 365 nm ultraviolet light after 15 minutes. Result judgment: The appearance of a purple band on the quality control line indicates that the immunochromatographic strip is valid, otherwise it is invalid. The appearance of a purple band on the test line indicates that the sample contains H10 subtype influenza virus, otherwise it does not contain H10 subtype influenza virus. At the same time, a fluorescent immunochromatographic scanner can also be used to detect the fluorescence intensity of 365 nm ultraviolet excitation. The absorbance value of the quality control line is greater than 1000, which is valid, otherwise it is invalid; the absorbance value of the test line is greater than 800, which is a positive result, and less than 800, which is a negative result. The test results showed that the sensitivity of the quantum dot immunochromatographic strip in detecting H10 subtype influenza virus was 0.125 hemagglutination units.

[0072] See attached for the results Figure 3 .

[0073] (8) Comparative experiment between quantum dot immunochromatographic strips and other detection methods: A comparative analysis was conducted using the nucleic acid detection method of influenza virus H10 subtype, and clinical samples such as poultry throat swabs and feces were detected and analyzed. The specificity of the quantum dot immunochromatographic strips reached 100%. The quantum dot immunochromatographic strip detection method has the advantages of being fast, specific, and convenient, especially for various poultry samples, and has good clinical application prospects.

[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 2G9, the antibody subtype is IgG2a, κ type, can specifically bind to the H10 subtype influenza virus hemagglutinin protein antigen, the antibody heavy chain variable region amino acid sequence is shown in SEQ ID No. 2, and the light chain variable region amino acid sequence is shown in SEQ ID No.

4.

2. Use of the monoclonal antibody 2G9 according to claim 1 in the preparation of an H10 subtype influenza virus detection product, wherein the H10 subtype influenza virus is one or more of H10N2, H10N3, H10N5, H10N7 and H10N8.

3. The use according to claim 2, characterized in that: The product detects H10 subtype influenza virus in samples through quantum dot fluorescent microsphere immunochromatography technology.

4. The use according to claim 3, characterized in that: The sample is a poultry throat swab, feces or allantoic fluid.

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