CN115850460A - Monoclonal antibody 2H6 against hemagglutinin protein of strain Y influenza B virus and its application in detection - Google Patents

Abstract

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

Description

Monoclonal Antibody 2H6 against Hemagglutinin Protein of Type Y Influenza Virus and Its Detection Applications

technical field

The invention belongs to the field of biotechnology, and relates to the preparation and application of anti-influenza B virus hemagglutinin protein monoclonal antibody, and uses cell engineering and antibody engineering technology to obtain a hybridoma cell line that secretes anti-hemagglutinin protein monoclonal antibody , through the induction of ascites in mice of the same strain, the monoclonal antibody 2H6 against the hemagglutinin protein was prepared, identified as IgG1, κ type, and then the application of the antibody was realized through affinity purification and immunization methods.

Background technique

Currently, influenza A, B, and C viruses can infect humans, and influenza A and B viruses cause seasonal influenza outbreaks every year. Since 2000, two lineages of influenza B viruses, B/Victoria (V lineage) and B/Yamagata (Y lineage), have emerged and began to circulate simultaneously or alternately in each influenza season. Usually, the clinical manifestations of influenza B virus are self-limiting disease, which is mainly mild, but the elderly, infants and some patients with chronic diseases are susceptible to influenza and progress to life-threatening severe acute respiratory disease . Studies have shown that influenza B virus is more likely to infect young people (≤65 years old) than influenza A virus. Although influenza A virus is considered to be the greatest public health problem, the high morbidity and mortality rate of influenza B virus cannot be underestimated. For example, influenza-related complications - myositis in children and young adults more common in flow patients.

Although studies have shown that the antigenic drift 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 ability of influenza B viruses to generate new strains through genetic recombination. However, in the 2012/2013 and 2017/2018 seasonal influenza epidemic seasons, a large number of influenza B virus infection cases were detected in most European countries, accounting for more than 50% of the total influenza outbreak cases. In addition, there are differences in the epidemiology and transmission risk among different lineages of influenza B viruses, and the clinical manifestations caused by Y-lineage influenza B viruses are more severe than those of V-lineages.

Isolating viruses from chicken embryos or MDCK cells is currently recognized as a classical influenza virus detection method. In recent years, molecular detection methods have also been greatly developed, and real-time quantitative polymerase chain reaction has been widely used in laboratory diagnosis of influenza virus infection. However, these methods are technically and laboratory demanding and time consuming. Due to the development of monoclonal antibody technology, detection methods based on monoclonal antibodies are also widely used in virus detection.

In summary, the development of monoclonal antibodies against influenza B virus of the Y strain and the establishment of rapid and sensitive detection methods are of great significance for the prevention and control of the virus. Based on the above background, this project selects the hemagglutinin protein of the Y-series influenza B virus as the target antigen, uses fusion hybridoma technology to establish a hybridoma cell line that stably secretes the monoclonal antibody against the hemagglutinin protein, and prepares, purifies, and These monoclonal antibodies were identified. The successful acquisition of the monoclonal antibody lays a material foundation for the establishment of a new diagnostic method for Y-series influenza B virus—diagnosis based on immunological techniques. At the same time, it plays an important role in the research of disease pathogenesis, prognosis and curative effect determination.

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

Contents of the invention

The purpose of the present invention is to provide a monoclonal antibody of the hemagglutinin protein of the Y-series influenza B virus, which can recognize the Y-series influenza B virus. The subtype of the monoclonal antibody is IgG1 and κ, named 2H6, and can specifically recognize the hemagglutinin protein of the Y-series influenza B 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.

SEQ ID No.1

Heavy chain: DNA sequence(366bp)

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

CAGATCCAGTTGGTGCAATCTGGACCTGAGCTGAAGAAACCTGGAGAGACAGTCAAGATCTCCTG

CAAGGCTTCTGGGTATACAATCACAGGCTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGG

TTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGA

CGGTTTGCCTTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAAT

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

FFTTISSVQSEDLAVYYCQQHYSIPPTFGGGTKLELK

The second object of the present invention is to provide a method for preparing the monoclonal antibody against the hemagglutinin protein of the Y-series influenza B virus, and the monoclonal antibody is produced by hybridoma cells. The hybridoma cell producing the monoclonal antibody is a hybridoma cell line 2H6 obtained after fusion, screening, cloning, and stable passage of immunized BALB/C mouse spleen lymphocytes and mouse myeloma cell SP2/0. Secreted monoclonal antibody 2H6 against hemagglutinin protein of strain Y influenza B virus.

Specifically, it is realized through the following steps and technical solutions:

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

(2) Culture of mouse myeloma cells: culture mouse myeloma cells SP2/0 and keep them in a good growth state for cell fusion.

(3) Cell fusion: the cell fusion method mediated by polyethylene glycol was used. The mice selected in step (1) were sacrificed to obtain spleen lymphocytes. Collect the SP2/0 cells in step (2), mix and centrifuge the above two kinds of cells, and then use polyethylene glycol to mediate cell fusion. The fused cells are properly diluted, inoculated into 96-well culture plates, and cultured under appropriate conditions.

(4) Screening of hybridoma cells: the above culture was cultured in a hypoxanthine-phosphoribosyltransferase selective medium. When the cell colony grows to a suitable size, absorb the cell culture supernatant for antibody identification and screen positive clones.

(5) Cloning of hybridoma cells: Positive hybridoma cells were cloned by the limiting dilution method, and the cells diluted to a certain density were inoculated into 96-well cell culture plates so that only one cell grew in each well. The supernatant of the wells forming cell colonies was taken for enzyme-linked immunosorbent assay, and positive clones were screened and identified. Select the culture well with the highest antibody titer and grow as a single clonal cell, perform limiting dilution again, and perform more than 4 consecutive limiting dilutions, and pass for more than 20 generations in succession to obtain a stable and high-efficiency expression of anti-Y strain influenza B virus monoclonal Antibody hybridoma cell lines, the cloned hybridoma cells for antibody identification and physical and chemical properties analysis.

(6) Preparation of monoclonal antibody ascites: select 8-10 week old BALB/C healthy mice, and inoculate each abdomen with PBS buffer containing 5×10 ⁶ positive hybridoma cells, and the abdomen of the mice is obviously enlarged 7-10 days after the cells are inoculated , closely observe the abdominal signs of the health status of the mice, wait until there is as much ascites as possible, and before the mice are about to die, collect the ascites and centrifuge to measure the antibody titer, and purify the monoclonal antibody in the ascites;

(7) Purification of monoclonal antibodies: use protein G agar gel affinity purification method to purify monoclonal antibodies in mouse ascites

(8) The present invention obtains a hybridoma line that produces monoclonal antibody against the hemagglutinin protein of the Y-series influenza virus, that is, 2H6, and the 2H6 hybridoma cell line is cloned four times, continuously cultivated for more than six months, and secretes antibodies stably . The cell line was cryopreserved in liquid nitrogen and grew well after resuscitation, with no decline in antibody secretion. The titer of 2H6 culture supernatant was 1:128 and the titer of ascites was 1:2048 measured by enzyme-linked immunosorbent assay indirect method. The immunoglobulin subtype analysis of the monoclonal antibody showed that the antibody type produced by the hybridoma cells was IgG1.

The invention provides a hybridoma cell producing monoclonal antibody, which is a mouse hybridoma cell line obtained after fusion, screening, cloning and passage of immune BALB/C mouse splenocytes and mouse myeloma cell SP2/0 2H6, can stably secrete monoclonal antibody 2H6 against the hemagglutinin protein of Y-series influenza B virus.

Another object of the invention is to provide the application of the monoclonal antibody 2H6 in the preparation of Y-series influenza B virus detection products.

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

The advantage of the present invention is that it provides a monoclonal antibody against hemagglutinin protein of Y-series influenza B virus. The preparation method is simple and practicable, and more importantly, the monoclonal antibody prepared by the method can have multiple uses, such as performing qualitative diagnosis on clinical and laboratory influenza B samples.

The invention provides a specific monoclonal antibody against the Y-series influenza B virus, and combines it with immunofluorescence technology to detect the Y-series influenza B virus in samples. The above-mentioned method has the advantages of being fast, sensitive and cheap, which can promote earlier and more extensive discovery of Y-series influenza B viruses and control the spread of the epidemic.

Instructions attached

Figure 1 shows the immunoglobulin subtype analysis of monoclonal antibody 2H6.

Figure 2 shows the specificity of immunofluorescence detection of Y-series influenza B virus.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. 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.

Example 1. Preparation method of monoclonal antibody against influenza B virus hemagglutinin protein

(1) Immunization of mice: For the first immunization, the hemagglutinin protein of strain Y influenza B virus and the adjuvant were mixed uniformly in a volume ratio of 1:1, and the total volume was 0.5 ml. Each BALB/C mouse 0.1 ml (containing 100 micrograms of influenza B virus hemagglutinin protein antigen) was intramuscularly injected into the inner thigh. On the 21st day, the immunization was boosted once in the same way. On the 35th day, a small amount of tail blood was collected for enzyme-linked immunosorbent assay. The antibody titer was up to 1:256000. The mouse with the highest antibody titer was injected into the tail vein for a booster immunization, and cell fusion was carried out 3 days later.

(2) Cultivation and passage of mouse myeloma cell SP2/0: the SP2/0 myeloma cell line from BALB/C mice is cultured and passaged with DMEM medium of 10% bovine serum, and is contained in 5% carbon dioxide at 37 Cultivate in an incubator.

(3) Cell fusion: BALB/c mouse peritoneal macrophages were used as feeder cells, and BALB/c mouse peritoneal macrophages were inoculated in a 96-well culture plate one day before fusion, containing 20% bovine serum hypoxanthine -Guanine-phosphoribosyltransferase medium cultured for one day. Spleen was taken from the mouse 3 days after the last booster immunization, spleen lymphocytes were separated by pressure water injection, and the cells were washed by centrifugation and then resuspended in DMEM medium. The SP2/0 cells were collected, centrifuged, washed and resuspended with DMEM medium for counting. Mix 2.5×10 ⁸ splenic lymphocytes of immunized mice with 2.5×10 ⁷ mouse myeloma cells SP2/0. Mix the two kinds of cells, discard the supernatant by centrifugation, gently rub the centrifuge tube with the palm of your hand to loosen the cell block, slowly add polyethylene glycol pre-warmed at 37°C with the fusion tube, and gently shake the centrifuge tube to suck the cells Fusion tube, after standing still for 90 seconds, blow the cells into the centrifuge tube, and then add 1 ml of DMEM medium in the first minute, and add 2 ml of DMEM medium in the second minute according to the principle of slow first and then fast. Add 7 ml of DMEM medium within 3 minutes, and gradually add 40 ml of 37°C pre-warmed DMEM medium within 1 minute. Centrifuge at 800 rpm for 10 minutes at low speed. Then add hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% bovine serum, inoculate them into 96-well culture plates with feeder cells with glass droppers, and generally spread 2-4 cells per fusion Plates were incubated in a 37°C incubator containing 5% carbon dioxide.

(4) Screening of hybridoma cells: Change the medium (containing hypoxanthine-guanine-phosphoribosyltransferase) once in half of the 96-well culture plate after 5 days, and change to the medium containing hypoxanthine-phosphoribosyltransferase after 10 days. The fused hybridoma cells were cultured for about two weeks in selective medium containing hypoxanthine-phosphoribosyltransferase. When the cell colony grows to an appropriate size (observed under a 10x objective lens, the size of the cell clone should occupy a field of view), the supernatant of the cell culture is aspirated for enzyme-linked immunosorbent assay to screen positive clones. Positive hybridoma clones were screened by ELISA indirect method. Main steps: ① Dilute influenza B virus hemagglutinin protein with 0.01 moles per liter of pH9.6 carbonate buffer, then add 0.1 milliliters per well to 96-well microtiter plate, the protein amount is 20 nanograms per well, overnight at 4°C; ②Wash the plate 5 times with 0.01 mole per liter of pH 7.4 phosphate buffer (containing Tween 20); ③block with 0.01 mole per liter of pH 7.4 phosphate buffer containing 5% bovine serum albumin for 2 4. wash the plate 3 times; 5. add the hybridoma culture supernatant, 0.1 milliliters per well, set positive control (influenza B virus hemagglutinin protein immunized mouse serum) and negative control (SP2/0 culture supernatant ) and blank control, react at room temperature for 2 hours; ⑥ wash the plate 3 times; ⑦ add horseradish peroxidase-labeled goat anti-mouse IgG diluted 1:10000, 0.1 ml per well, react at room temperature for 1 hour; ⑧ wash the plate 3 times; ⑨Add the chromogenic solution and react in the dark for 5 minutes at room temperature; ⑩2 mol per liter of sulfuric acid to terminate the reaction; measure the optical density value at 450 nm, and divide the measured value by negative ≥ 2.1 to be positive.

(5) Cloning of hybridoma cells: The cloning and culturing of hybridoma cells was carried out according to the limiting dilution method, and the hybridoma well cells positive for antibody detection were selected for proper proliferation, and the cells were accurately counted. Dilute the cell suspension into 10 per milliliter with complete DMEM medium and inoculate it into a 96-well culture plate with feeder cells, 0.1 milliliter per well, observe the cell growth after 10 days, and detect the antibody level in the supernatant, select The culture wells with the highest antibody titer and single clonal cell growth were subjected to limited dilution again, and more than 4 consecutive limited dilutions were performed, and continuous passage for more than 20 generations was obtained to obtain stable and high-efficiency expression of anti-Y strain influenza B virus monoclonal antibody hybridoma cell lines.

(6) Preparation of monoclonal antibody ascites: select 8-10 week old BALB/C healthy mice, and inoculate each abdomen with PBS buffer containing 5×10 ⁶ positive hybridoma cells, and the abdomen of the mice is obviously enlarged 7-10 days after the cells are inoculated , closely observe the abdominal signs of the health status of the mice, and collect the ascites of the mice when ascites is as much as possible.

(7) Purification of monoclonal antibody: the monoclonal antibody in ascitic fluid was purified by affinity purification (protein G agar gel). ① Treatment of ascites: Centrifuge the ascites at 10,000 rpm at 4°C for 15 minutes, remove the sediment, collect the supernatant, mix it with 3-4 times the volume of binding buffer, and centrifuge at 10,000 rpm at 4°C for 15 minutes Remove sediment. Centrifuge at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② Fully wash the affinity purification column prepacked with protein G agar gel with 5 times the column bed volume of binding buffer. ③Put the diluted ascitic fluid on the column, and control the flow rate to 8-10 drops per minute. ④Put the ascites that has passed the column on the column again. ⑤ Fully wash the purification column with 5 times the column bed volume of binding buffer. ⑥ Elute the bound monoclonal antibody with elution buffer, control the flow rate of 8-10 drops per minute, collect the eluate in a collection tube pre-added with 0.1 ml of potassium phosphate buffer (pH7.9), each tube Collect 0.5 mL of antibody-containing eluate. 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 antibody eluent to the ultrafiltration centrifuge tube, and centrifuge at 10,000 rpm at 4°C for 10-20 minutes until the final volume of antibody eluent is about 1 ml. Add and add 10 ml of 0.1 mole per liter of pH 7.4 phosphate buffer, centrifuge at 10,000 rpm for 10-20 minutes at 8°C, concentrate the antibody to a final volume of about 1 ml by centrifuging for the last time, and pipette the antibody concentrate into a collection tube middle. ⑨ After diluting the desalted antibody solution, measure the protein content at 280 nm. ⑩ Dispense the purified antibody into small tubes and store in a low-temperature refrigerator for later use.

(8) Subtype identification of the monoclonal antibody: analysis was performed using the Mouse Monoclonal Antibody Immunoglobulin Typing Kit from Bio-Rad. The purified monoclonal antibody was properly diluted for detection, and the operation was performed strictly according to the kit instructions. The test results showed that the monoclonal antibodies secreted by 2H6 hybridoma cells were IgG1 and κ types.

The results are shown in Figure 1.

Embodiment 2. Carry out the qualitative detection of influenza B virus with this monoclonal antibody

The anti-Y series influenza B virus hemagglutinin protein monoclonal antibody prepared by the present invention can be used for qualitative detection of Y series influenza B virus, and the identification method can be realized by the following methods:

Immunofluorescence detection of influenza B virus:

(1) MDCK cells were seeded in a 48-well plate at a density of 4×10 ⁴ cells per well one day in advance, and the cells were grown to 70% for later use;

(2) Take out the cell plate on which the cells have been spread, discard the culture supernatant, wash it once with phosphate buffered saline, and set aside;

(3) Determine the specificity of the influenza B virus immunofluorescence detection method for detecting the Y-series influenza B virus: dilute the virus with phosphate buffer, including B/Phuket/3073/2013 (Y-series), B/Shang Hai/361 /2002(Y series), B/Florida/4/2006(Y series), B/Hubei-Wujiagang/158/2009(Y series), B/Massachusetts/2/2012(Y series), B/Malaysia/2506 /2004 (V series), B/Brisbane/60/2008 (V series), B/Washington/02/2019 (V series), A/Michigan/45/2015 (Influenza A H1N1 virus) and A/Texas/ 50/2012 (Influenza A H3N2 virus), the diluted virus liquid infected cells (the multiplicity of infection was 0.5), and cultured in a 37°C incubator containing 5% carbon dioxide saturated for 2 hours;

(4) Take out the cell plate, discard the virus solution, wash the cells twice with phosphate buffered saline, then add 200 microliters of virus culture solution to each well, and incubate in a 37°C incubator containing 5% carbon dioxide saturation for 16 hours ;

(5) Take out the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffered saline;

(6) Add 4% paraformaldehyde to each well of the cell plate to fix the cells, fix at room temperature for 30 minutes, and wash 3 times with phosphate buffer;

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

(8) Block with phosphate buffer containing 3% bovine serum albumin, block for 1 hour at room temperature, and discard the bovine serum albumin solution;

(9) Dilute the monoclonal antibody to 10 micrograms per milliliter with phosphate buffered saline, add 200 microliters to each well, incubate overnight at 4°C, and wash 3 times with phosphate buffered saline;

(10) Dilute the fluorescent secondary antibody to 5 micrograms per milliliter with a solution containing 1% bovine serum albumin, add 200 microliters to each well, incubate in a 37°C incubator in the dark for 90 minutes, and wash 3 times with phosphate buffer;

(11) Stain the nuclei with a DNA fluorescent dye (4',6-diamidino-2-phenylindole), incubate at room temperature in the dark for 10 minutes, and wash 3 times with phosphate buffer;

(12) Observe the experimental results under a fluorescent microscope, if green fluorescence is observed, it is positive. The detection results showed that the anti-series Y influenza B virus monoclonal antibody 2H6 developed based on this study had good specificity in detecting Y series influenza B viruses.

It should be understood that the present invention is described in conjunction with the best embodiment, but after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present application. The scope 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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