CN115850460A - anti-Y-series influenza B virus hemagglutinin protein monoclonal antibody 2H6 and application thereof in detection - Google Patents

Abstract

The invention provides a monoclonal antibody 2H6 of Y-series influenza B virus hemagglutinin protein and application thereof in detection. The heavy chain amino acid sequence of the monoclonal antibody 2H6 is shown as SEQ ID No.2, and the light chain amino acid sequence is shown as SEQ ID No. 4. The invention provides an effective tool for the auxiliary diagnosis of the influenza B virus infection in clinical samples, and can be popularized and applied to various detection technologies and clinical and experimental researches.

Description

anti-Y-series influenza B virus hemagglutinin protein monoclonal antibody 2H6 and application thereof in detection

Technical Field

The invention belongs to the field of biotechnology, and relates to preparation and application of a monoclonal antibody against hemagglutinin proteins of influenza B viruses, which is characterized in that a hybridoma cell line secreting monoclonal antibodies against hemagglutinin proteins is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, monoclonal antibodies 2H6 against hemagglutinin proteins are prepared and identified as IgG1 and kappa types, and the application of the antibodies is realized by technologies such as affinity purification and immunization methods.

Background

Influenza a, b and c viruses can now infect humans, with influenza a and b viruses causing seasonal influenza outbreaks each year. Since 2000, influenza B virus appeared in two lineages, B/Victoria (V line) and B/Yamagata (Y line), and began to circulate simultaneously or alternately in each influenza season. In general, influenza b virus clinically manifests as a self-limiting disease, mainly mild, but elderly, infants and some patients with chronic diseases are predisposed to influenza and progress to serious acute respiratory diseases that are life threatening. It has been shown that influenza B virus is more susceptible to infection in young people (65 years) than influenza A virus. Although influenza a is considered the biggest public health problem, the high morbidity and mortality of influenza b also does not amount in a small amount, for example, the influenza-associated complication, myositis, is more common in patients with influenza b in children and young adults.

Although studies have shown that the antigenic drift rate of influenza b viruses is slower than that of influenza a viruses. Unlike influenza a viruses, influenza b viruses almost exclusively infect humans, which limits the production of new virus strains by influenza b viruses through genetic recombination. However, in the seasonal influenza epidemics season of 2012/2013 and 2017/2018, a large number of influenza b virus infection cases, accounting for more than 50% of total influenza outbreak cases, are detected in most european countries. In addition, there are differences in epidemiology and transmission risk between different lineages of influenza b virus, and the clinical manifestations caused by the Y-line influenza b virus are more severe than those of the V-line.

Isolation of viruses in chicken embryos or MDCK cells is currently the accepted classical method for detection of 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 are technically and laboratory demanding and time consuming. Monoclonal antibody-based detection methods are also widely used for virus detection due to the development of monoclonal antibody technology.

In conclusion, the development of the Y-series influenza B virus monoclonal antibody and the establishment of a rapid and sensitive detection method have important significance for the prevention and control of viruses. Based on the background, the project selects the Y-series influenza B virus hemagglutinin protein as the target antigen, adopts the fusion hybridoma technology to establish the hybridoma cell line which stably secretes the monoclonal antibody of the hemagglutinin protein, and prepares, purifies and identifies the monoclonal antibodies in large quantity. The successful acquisition of the monoclonal antibody lays a material foundation for establishing a novel Y-series influenza B virus diagnosis method, namely the diagnosis based on immunological technology. Meanwhile, the method plays an important role in the research of the aspects of disease pathogenesis, prognosis, curative effect judgment and the like.

The invention uses hybridoma cell technology. This technique fuses B lymphocytes from immunized mice with myeloma cells SP2/0 to create a hybridoma cell line that secretes homogeneous antibodies, also known as monoclonal antibody technique. The technology relates to a series of methods such as animal immunization, cell culture, cell fusion, cell clone culture, immunoassay and the like.

Disclosure of Invention

The invention aims to provide a Y-series influenza B virus hemagglutinin protein monoclonal antibody which can identify Y-series influenza B virus. The monoclonal antibody subtype is IgG1 and kappa type, is named as 2H6, and can specifically recognize hemagglutinin protein of Y-series influenza B 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.

SEQ ID No.1

Heavy chain:DNA sequence(366bp)

Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

CAGATCCAGTTGGTGCAATCTGGACCTGAGCTGAAGAAACCTGGAGAGACAGTCAAGATCTCCTG

CAAGGCTTCTGGGTATACAATCACAGGCTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGG

TTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGA

CGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAAT

GAGGACATGGCTACATATTTCTGTGCAAGATCGGGAACTACGATACTAGATTACTATTCTATGGACTAC

TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID No.2

Heavy chain:Amino acid sequence(122AA)

Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

QIQLVQSGPELKKPGETVKISCKASGYTITGYGMNWVKQAPGKGLKWMGWINTYTGEPTYTDDFKGRF

AFSLETSASTAYLQINNLKNEDMATYFCARSGTTILDYYSMDYWGQGTSVTVSS

SEQ ID No.3

Light chain:DNA sequence(321bp)

Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

GACATTGTGATGACCCAGTCTCCCAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTATCACCT

GCAAGGCCAGTCAGGATGTGAGTTCTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAA

AGTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGAATCTG

GGACGGATTTCACTTTCACTATCAGCAGTGTGCAGTCTGAAGACCTGGCAGTTTATTACTGTCAGCA

ACATTATAGTATTCCTCCCACGTTCGGTGGTGGGACCAAGCTGGAGCTGAAA

SEQ ID No.4

Light chain:Amino acid sequence(107AA)

Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

DIVMTQSPKFMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKVLIYSASYRYTGVPDRFTGSESGTD

FTFTISSVQSEDLAVYYCQQHYSIPPTFGGGTKLELK

The second object of the present invention is to provide a method for preparing monoclonal antibodies against hemagglutinin protein of Y-series influenza B virus, said monoclonal antibodies being produced by hybridoma cells. The hybridoma cell for producing the monoclonal antibody is a hybridoma cell line 2H6 obtained by fusing, screening, cloning and stably passaging immune BALB/C mouse spleen lymphocytes and mouse myeloma cells SP2/0, and can stably secrete the monoclonal antibody 2H6 for resisting Y-series influenza B virus hemagglutinin protein.

The method is realized by the following steps and technical scheme:

(1) Immunization of animals: 6-8 week-old BALB/C mice were selected and immunized with purified influenza B virus of the Y series (B/Phuket/3073/2013) hemagglutinin protein.

(2) Culture of mouse myeloma cells: mouse myeloma cells SP2/0 were cultured and kept in a good growth state for cell fusion.

(3) Cell fusion: polyethylene glycol mediated cell fusion was used. The mice selected in step (1) were sacrificed to obtain spleen lymphocytes. Collecting SP2/0 cells in the step (2), mixing and centrifuging the two cells, then mediating cell fusion by polyethylene glycol, properly diluting the fused cells, inoculating the cells to a 96-well culture plate, and culturing under proper conditions.

(4) Screening of hybridoma cells: the above culture was cultured in a hypoxanthine-phosphoribosyl transferase selective medium. When the cell colony grows to be proper in size, the cell culture supernatant is sucked for antibody identification, and positive clones are screened.

(5) Cloning of hybridoma cells: positive hybridoma cells were cloned by limiting dilution, and cells diluted to a certain density were seeded into a 96-well cell culture plate to allow only one cell to grow per well. Taking the supernatant from the hole where the cell colony is formed, performing enzyme-linked immunosorbent assay, and screening and identifying positive clones. Selecting a culture hole with the highest antibody titer and growing in a single clone cell, carrying out limiting dilution again, continuously carrying out more than 4 times of limiting dilution, continuously carrying out generation for more than 20 generations to obtain a hybridoma cell strain for stably and efficiently expressing the anti-Y-series influenza B virus monoclonal antibody, and carrying out antibody identification and physicochemical character analysis on the cloned hybridoma cells.

(6) Preparing monoclonal antibody ascites: selecting 8-10 weeks BALB/C healthy mice, each abdominal inoculation containing 5 × 10 ⁶ Individual positive crossTumor cell PBS buffer solution, inoculating cell, after 7-10 days, the mouse abdomen is obviously enlarged, closely observing the abdomen symptom of the health condition of the mouse, when ascites is as much as possible and before the mouse is dying, collecting ascites, centrifuging, measuring the antibody titer, and purifying the monoclonal antibody in the ascites;

(7) Purification of monoclonal antibodies: purification of monoclonal antibodies in mouse ascites Using protein G agar gel affinity purification

(8) The hybridoma cell line for producing the monoclonal antibody of the hemagglutinin protein of the Y-series influenza B virus, namely the 2H6 and 2H6 hybridoma cell line, is cloned for 4 times and cultured for more than six months continuously, and the secreted antibody is stable. The cell strain is frozen and stored by liquid nitrogen, the cell strain grows well after recovery, and the secretion of the antibody is not declined. The ELISA indirect method tests show that the titer of the 2H6 culture supernatant is 1. Analysis of monoclonal antibody immunoglobulin subtype shows that the antibody type produced by the hybridoma cell is IgG1.

The invention provides a hybridoma cell for generating a monoclonal antibody, which is a mouse hybridoma cell line 2H6 obtained by fusing, screening, cloning and passaging immune BALB/C mouse spleen cells and mouse myeloma cells SP2/0 and can stably secrete the monoclonal antibody 2H6 of anti-Y-series influenza B virus hemagglutinin protein.

The invention also aims to provide the application of the monoclonal antibody 2H6 in preparing Y-series influenza B virus detection products.

The detection of the detection product in body fluid, allantoic fluid or other environmental samples containing the influenza B virus is realized by an immunofluorescence detection method.

The invention has the advantage of providing the monoclonal antibody for resisting the hemagglutinin protein of the Y-series influenza B virus. The preparation method is simple and easy to implement, and more importantly, the monoclonal antibody prepared by the method can be used for multiple purposes, such as qualitative diagnosis of influenza B samples in clinic and laboratories.

The invention provides a specific monoclonal antibody aiming at Y-series influenza B virus, and the specific monoclonal antibody is combined with an immunofluorescence technology to detect the Y-series influenza B virus in a sample. The method has the advantages of rapidness, sensitivity and low cost, and can promote the early and extensive discovery of the Y-series influenza B virus and control the spread of epidemic situations.

Drawings

FIG. 1 is an immunoglobulin subtype analysis of monoclonal antibody 2H6.

FIG. 2 shows the specificity of detecting Y-series influenza B virus by immunofluorescence technique.

Detailed Description

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

Example 1 preparation of monoclonal antibodies against influenza B virus hemagglutinin protein

(1) Immunization of mice: for the first immunization, the hemagglutinin protein of the Y-series influenza B virus and the adjuvant 1 are uniformly mixed, and the total volume is 0.5 ml. 0.1 ml of BALB/C mice (containing 100 micrograms of influenza B virus hemagglutinin protein antigen) is injected into the inner thigh of the muscle. The immunization was boosted once on day 21 in the same manner. And (3) taking trace tail blood on the 35 th day to carry out enzyme-linked immunosorbent assay determination, wherein the antibody titer reaches 1 to 256000 to the maximum, and selecting the mouse tail with the highest antibody titer to carry out intravenous injection for boosting immunization once, and carrying out cell fusion after 3 days.

(2) Culture passages of mouse myeloma cells SP 2/0: SP2/0 myeloma cell lines derived from BALB/C mice were subcultured in DMEM medium containing 10% bovine serum, and cultured in an incubator at 37 ℃ containing 5% carbon dioxide.

(3) Cell fusion: BALB/C mouse abdominal cavity macrophages are taken as feeder cells, and are inoculated to a 96-hole culture plate one day before fusion, and are cultured for one day in a hypoxanthine-guanine-phosphoribosyl transferase culture medium containing 20% of bovine serum. Taking the spleen of the mice which are subjected to the last 3 days of boosting immunization, separating spleen lymphocytes by adopting a pressure water injection method, centrifugally washing the cells, and then resuspending the cells by using a DMEM culture solution. SP2/0 cells were collected, centrifuged, washed, resuspended in DMEM medium and counted. Mixing 2.5X 10 ⁸ Spleen lymphocytes of immunized mice and 2.5×10 ⁷ Mouse myeloma cells SP2/0 mix. Mixing the two cells, centrifuging to remove supernatant, gently rubbing the centrifuge tube with palm to loosen cell mass, slowly adding polyethylene glycol with a pre-temperature of 37 ℃ into the fusion tube, gently shaking the centrifuge tube during the process, sucking the cells into the fusion tube, standing for 90 seconds, blowing the cells into the centrifuge tube, adding 1 ml of DMEM medium in 1 minute according to the principle of slow first and fast second, adding 2 ml of DMEM medium in 2 minutes, adding 7 ml of DMEM medium in 3 minutes, and gradually adding 40 ml of DMEM medium with a pre-temperature of 37 ℃ in 1 minute later. Centrifuge at 800 rpm for 10 minutes at low speed. Then, a culture medium of hypoxanthine-guanine-phosphoribosyltransferase containing 20% bovine serum was added, and the cells were inoculated into a 96-well culture plate containing feeder cells by a glass pipette, generally, 2 to 4 plates were spread for each fusion, and cultured in an incubator at 37 ℃ containing 5% carbon dioxide.

(4) Screening of hybridoma cells: the culture plate with 96 wells is changed once after 5 days (containing hypoxanthine-guanine-phosphoribosyl transferase), and the culture plate is changed to culture medium containing hypoxanthine-phosphoribosyl transferase after 10 days. The fused hybridoma cells were cultured in selective medium containing hypoxanthine-phosphoribosyl transferase for approximately two weeks. When the cell colony grows to a proper size (the cell colony is observed under a 10-time objective lens, the cell colony size is suitable for occupying one visual field), the supernatant fluid of the cell culture is extracted to carry out enzyme-linked immunosorbent assay, and positive clones are screened. Screening positive hybridoma clones by adopting an enzyme-linked immunosorbent assay indirect method. The method mainly comprises the following steps: (1) 0.01 mol per liter of pH9.6 carbonate buffer solution is used for diluting influenza B virus hemagglutinin protein, then 0.1 ml per hole is respectively added into a 96-hole enzyme label plate, the protein amount is 20 nanograms per hole, and the mixture stays overnight at 4 ℃; (2) 0.01 mol per liter pH7.4 phosphate buffer (containing Tween 20) plate washing 5 times; (3) blocking with phosphate buffer containing 0.01 mol/l of 5% bovine serum albumin, pH7.4, for 2 hours; (4) washing the plate for 3 times; (5) adding hybridoma culture supernatant, setting positive control (influenza B virus hemagglutinin protein immune mouse serum), negative control (SP 2/0 culture supernatant) and blank control in each 0.1 ml well, and reacting at room temperature for 2 hr; (6) washing the plate for 3 times; (7) adding 1; (8) washing the plate for 3 times; (9) adding color developing solution to react for 5 minutes in a dark place at room temperature; the reaction is stopped by 2 mol of R per liter of sulfuric acid; the optical density value is measured at 450 nm, and the positive is obtained by dividing the measured value by the negative value which is more than or equal to 2.1.

(5) Cloning of hybridoma cells: the hybridoma cells are subjected to cloning culture according to a limiting dilution method, and after hybridoma cells positive in antibody detection are selected and appropriately proliferated, the cells are accurately counted. Diluting the cell suspension into 10 per milliliter by using a complete DMEM medium, inoculating the cell suspension into a 96-well culture plate of the existing feeder cells, observing the growth condition of the cells after 10 days, detecting the antibody level in supernatant, selecting a culture well which has the highest antibody titer and shows the growth of a single clone cell, carrying out limited dilution again, continuously carrying out limited dilution for more than 4 times, and continuously carrying out passage for more than 20 generations to obtain the hybridoma cell strain which stably and efficiently expresses the anti-Y-system influenza B virus monoclonal antibody.

(6) Preparing monoclonal antibody ascites: selecting 8-10 weeks BALB/C healthy mice, each abdominal inoculation containing 5 × 10 ⁶ The abdomen of the mouse is obviously enlarged after the positive hybridoma cell PBS buffer solution is inoculated with the cell for 7-10 days, the abdomen symptoms of the health condition of the mouse are closely observed, and the ascites of the mouse is collected when the ascites is as much as possible.

(7) Purification of monoclonal antibodies: the monoclonal antibodies in the ascites were purified by affinity purification (protein G agar gel). (1) Treating ascites: the ascites was centrifuged at 4 ℃ 10000 rpm for 15 minutes to remove the precipitate, the supernatant was collected and mixed with 3 to 4 times the volume of the binding buffer, and then centrifuged at 4 ℃ 10000 rpm for 15 minutes to remove the precipitate. The precipitate was removed by centrifugation at 10000 rpm for 15 minutes at 4 ℃. (2) The affinity purification column, pre-loaded with protein G agar gel, was washed thoroughly with 5 bed volumes of binding buffer. (3) The diluted ascites fluid was applied to the column, and the flow rate was controlled to 8-10 drops per minute. (4) The ascites fluid which has passed through the column is applied to the column again once. (5) The purification column was washed thoroughly with 5 bed volumes of binding buffer. (6) The bound monoclonal antibody was eluted with an elution buffer, and the flow rate was controlled to 8 to 10 drops per minute, and the eluate was collected in a collection tube pre-loaded with 0.1 ml of potassium phosphate buffer (pH 7.9), and 0.5 ml of the antibody-containing eluate was collected per tube. (7) The absorbance of each tube of eluate was measured at 280 nm and the eluate was collected with a protein content greater than 0.1 mg per ml. (8) Adding antibody eluent into the ultrafiltration centrifugal tube, and centrifuging at 4 ℃ and 10000 rpm for 10-20 minutes until the final volume of the antibody eluent is about 1 ml. Adding 10 ml of 0.1 mol per liter of phosphate buffer solution with pH7.4, centrifuging at 8 ℃ and 10000 rpm for 10-20 minutes, finally centrifuging and concentrating the antibody to about 1 ml of the final volume, and sucking the antibody concentrated solution into a collecting pipe. (9) And (4) diluting the desalted antibody solution, and measuring the protein content at 280 nm. And (c) subpackaging the purified antibody into small tubes, and placing the small tubes in a low-temperature refrigerator for later use.

(8) The subtype of the monoclonal antibody is identified by adopting a mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad company. And (4) properly diluting the purified monoclonal antibody, and detecting, wherein the operation is strictly carried out according to the kit instruction. The test result shows that the monoclonal antibody secreted by the 2H6 hybridoma cell is IgG1 and kappa type.

The results are shown in FIG. 1.

Example 2 qualitative detection of influenza B Virus Using the monoclonal antibody

The monoclonal antibody of the hemagglutinin protein of the Y-series influenza B virus can be used for qualitatively detecting the Y-series influenza B virus, and the identification method can be realized by the following steps:

immunofluorescence assay for influenza B virus:

(1) MDCK cells were plated one day in advance at 4X 10 per well ⁴ Inoculating the density of each cell in a 48-well plate until the cell grows to 70% for later use;

(2) Taking out the cell plate with the cells laid, removing culture supernatant, and washing with phosphate buffer solution for later use;

(3) Determining specificity of detecting Y-series influenza B virus by using an influenza B virus immunofluorescence assay: diluting the virus with phosphate buffer including B/Phuket/3073/2013 (line Y), B/Shang Hai/361/2002 (line Y), B/Florida/4/2006 (line Y), B/Hubei-Wujiaging/158/2009 (line Y), B/Massachusetts/2/2012 (line Y), B/Malaysia/2506/2004 (line V), B/Brisbane/60/2008 (line V), B/Washington/02/2019 (line V), A/Michigan/45/2015 (influenza A H1N 1) and A/Texas/50/2012 (influenza A H3N 2), infecting the diluted virus liquid infected cells (multiplicity of infection is 0.5), culturing for 2 hours in 37 ℃ incubator containing 5% saturated carbon dioxide;

(4) Taking out the cell plate, discarding the virus solution, washing the cells for 2 times by using a phosphate buffer solution, adding 200 microliters of virus culture solution into each hole, and culturing for 16 hours in an incubator at 37 ℃ and saturated by 5 percent of carbon dioxide;

(5) Taking out the cell plate, discarding the culture supernatant, and washing the cells for 1 time by using phosphate buffer;

(6) Adding 4% paraformaldehyde into each hole of the cell plate to fix the cells, fixing for 30 minutes at room temperature, and washing for 3 times by using phosphate buffer;

(7) Permeabilizing the cells with 0.5% Triton-X100 for 30 minutes at room temperature, and washing 3 times with phosphate buffer;

(8) Sealing with phosphate buffer solution containing 3% bovine serum albumin, sealing at room temperature for 1 hr, and removing bovine serum albumin solution;

(9) Diluting the monoclonal antibody to 10 micrograms per milliliter with a phosphate buffer solution, adding 200 microliters to each well, incubating overnight at 4 ℃, and washing 3 times with the phosphate buffer solution;

(10) Diluting the fluorescent secondary antibody to 5 micrograms per milliliter by using a solution containing 1 percent of bovine serum albumin, adding 200 microliters of the fluorescent secondary antibody into each hole, incubating for 90 minutes in an incubator at 37 ℃ in a dark place, and washing for 3 times by using a phosphate buffer solution;

(11) Staining cell nuclei by using deoxyribonucleic acid fluorescent dye (4', 6-diamidino-2-phenylindole), incubating for 10 minutes at room temperature in a dark place, and washing for 3 times by using phosphate buffer;

(12) And observing the experimental result under a fluorescence microscope, wherein the green fluorescence is positive when the experimental result is observed. The detection result shows that the Y-series influenza B virus monoclonal antibody 2H6 developed based on the research has better specificity when being used for detecting the Y-series influenza B virus.

It should be understood that the present invention has been described in connection with the preferred embodiments, but various changes or modifications may be made by those skilled in the art after reading the above disclosure of the present invention, and these equivalents also fall within the scope of the present invention defined by the appended claims.

Claims (7)

1. A monoclonal antibody 2H6 for resisting Y-series influenza B virus hemagglutinin protein, the antibody subtype is IgG1, kappa type, can be specifically combined with influenza B virus hemagglutinin protein antigen, the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID No.2, and the light chain variable region amino acid sequence is shown as SEQ ID No. 4.

2. The monoclonal antibody 2H6 against the hemagglutinin protein of influenza b virus of Y-series according to claim 1, which is characterized in that: the monoclonal antibodies are produced by hybridoma cells.

3. The monoclonal antibody 2H6 against the hemagglutinin protein of influenza b virus of Y-series according to claim 1, which is characterized in that: the hybridoma cell for producing the monoclonal antibody is a hybridoma cell line 2H6 obtained by fusing, screening, cloning and stably passaging immune BALB/C mouse spleen lymphocytes and mouse myeloma cells SP2/0, and can stably secrete the monoclonal antibody 2H6 for resisting Y-series influenza B virus hemagglutinin protein.

4. The method for producing the monoclonal antibody 2H6 against hemagglutinin protein of influenza b virus according to claim 1, wherein: the monoclonal antibody is obtained by the following steps:

(1) Mouse immunization: BALB/C mice 6-8 weeks old were selected and immunized by purified hemagglutinin protein of influenza B virus. Each mouse was immunized by intrafemoral intramuscular injection with 100 μ g of hemagglutinin protein b 1 mixed with adjuvant, and another immunization was performed in the same manner after day 21 for 2 times; collecting trace mouse tail blood on day 35, measuring the antibody titer of the mouse, selecting the mouse with the highest immune titer, injecting the tail vein once for boosting immunity, and performing cell fusion 3 days later.

(2) Culture of mouse myeloma cells: two weeks before the preparation for fusion, the myeloma cells SP2/0 were recovered, and the mouse myeloma cells SP2/0 were cultured and kept in a good growth state for hybridoma cell fusion.

(3) Cell fusion: polyethylene glycol mediated cell fusion method was used. The mice selected in step (1) were sacrificed to obtain spleen lymphocytes. Mixing and centrifuging spleen lymphocytes and myeloma cells SP2/0 cells, then mediating cell fusion through polyethylene glycol, appropriately diluting the fused cells, inoculating the cells to a culture plate, and appropriately culturing the cells under appropriate conditions.

(4) Screening of hybridoma cells: the above culture was cultured in a selective medium containing hypoxanthine-phosphoribosyl transferase. When the cell colony grows to a proper size, the culture supernatant is absorbed and identified by an enzyme-linked immunosorbent assay method to screen positive clones.

(5) Cloning and culturing hybridoma cells: positive hybridoma cells were cloned by limiting dilution, and cells diluted to a certain density were seeded into a 96-well cell culture plate to allow only one cell to grow per well. Taking supernatant from the hole where the cell colony is formed, carrying out enzyme linked immunosorbent assay, and screening and identifying positive clones. And selecting a culture hole with the highest antibody titer and growing in a single clone cell, carrying out limiting dilution again, continuously carrying out more than 3 times of monoclonal limiting dilution, and continuously carrying out generation for more than 20 generations to obtain the hybridoma cell strain capable of stably and efficiently expressing the anti-influenza B virus monoclonal antibody.

(6) Preparing monoclonal antibody ascites: selecting 8-10 weeks BALB/C healthy mice, each abdominal inoculation containing 5 × 10 ⁶ The positive hybridoma cell PBS buffer solution is inoculated with cells, the abdomen of the mouse is obviously expanded after 7-10 days, the abdomen signs of the health condition of the mouse are closely observed, ascites is collected and centrifuged until the ascites is as much as possible and the mouse is dying, the titer of the antibody is determined, and the monoclonal antibody in the ascites is purified;

(7) Purification of monoclonal antibodies: monoclonal antibodies were purified from mouse ascites fluid using protein G agar gel affinity purification.

5. The use of the monoclonal antibody 2H6 against the hemagglutinin protein of influenza b virus according to claim 1 or 2 for the preparation of a Y-series influenza b virus detection product.

6. Use according to claim 5, characterized in that: the detection product detects the influenza B virus in different samples by an immunofluorescence technique.

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

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