Monoclonal antibody 1F3 against hemagglutinin protein of H10 subtype avian influenza virus and its application in detection

Abstract

The present application belongs to the field of biotechnology, and relates to anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3 and application thereof in detection. The present application is obtained by using cell engineering and antibody engineering technology, and is a hybridoma cell line secreting anti-hemagglutinin protein monoclonal antibody. The anti-hemagglutinin protein monoclonal antibody 1F3 is prepared by inducing ascites of the same strain of mice, and is identified as IgG1, κ type. The application of the antibody is realized by affinity purification, immunization method and other technologies. The present application has the advantages that an anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody is provided. The preparation method is simple and easy to implement, and more importantly, the monoclonal antibody prepared by the method can have multiple uses, such as qualitative diagnosis of H10 subtype avian influenza samples in clinics and laboratories.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3 and its application in detection. The monoclonal antibody 1F3 against hemagglutinin protein is obtained by using cell engineering and antibody engineering technology, and is prepared by inducing ascites of a mouse of the same strain. The monoclonal antibody 1F3 is identified as IgG1, κ type, and is applied by affinity purification and immunization methods. BACKGROUND

[0002] Influenza A virus belongs to Orthomyxoviridae, and is divided into 18 HA and 11 NA subtypes according to the antigenic properties of hemagglutinin and neuraminidase glycoproteins. Waterfowl is considered to be the natural host of influenza A virus, and most of the influenza A virus subtypes have been found in waterfowl (such as domestic ducks). Notably, domestic ducks usually do not show severe symptoms when infected with low pathogenic avian influenza virus, but they provide a suitable environment for the recombination of avian influenza virus. In recent years, H10 subtype avian influenza viruses have been increasingly isolated from birds, such as H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8 and H10N9.

[0003] Isolation of viruses in chicken embryos or MDCK cells is currently a recognized classic method for detecting influenza viruses. In recent years, molecular detection methods have also been greatly developed, and real-time quantitative polymerase chain reaction has been widely used in laboratory diagnosis of influenza virus infection. However, these methods require high technical and laboratory requirements and are time-consuming. Due to the development of monoclonal antibody technology, detection methods based on monoclonal antibodies are also widely used in virus detection. Therefore, the present application aims to describe a specific monoclonal antibody against H10 subtype avian influenza virus, which can be combined with immunofluorescence technology to detect H10 subtype avian influenza virus in samples. The above method has the advantages of rapidness, sensitivity and low cost, which can promote the earlier and more extensive detection of H10 subtype avian influenza virus and control the spread of the epidemic.

[0004] In summary, the development of H10 subtype avian influenza virus monoclonal antibody and the establishment of a rapid and sensitive detection method are of great significance for the prevention and control of viruses. Based on the above background, the present application selects H10 subtype avian influenza virus hemagglutinin protein as the target antigen, establishes a hybridoma cell line that stably secretes anti-hemagglutinin protein monoclonal antibody by using fusion hybridoma technology, and prepares, purifies and identifies a large number of these monoclonal antibodies. The successful obtaining of the monoclonal antibody lays a material foundation for establishing a new type of H10 subtype avian influenza virus diagnostic method based on immunological technology. At the same time, it plays an important role in the research of disease pathogenesis, prognosis and efficacy determination, etc.

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

[0006] The purpose of the present application is to provide an anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody that can recognize H10 subtype avian influenza virus. The monoclonal antibody subtype is IgG1, κ type, named 1F3, which can specifically recognize the hemagglutinin protein of H10 subtype avian influenza virus.

[0007] The heavy chain amino acid sequence of the anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3 is shown in SEQ ID No. 2, and the light chain amino acid sequence is shown in SEQ ID No. 4.

[0008] The anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3 is produced by hybridoma cells; the hybridoma cell line 1F3 for producing the monoclonal antibody is obtained by fusion, screening, cloning and stable passage of the spleen lymphocytes of immunized BALB / C mice and mouse myeloma cells SP2 / 0, and can stably secrete the anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3.

[0009] The second purpose of the present application is to provide a preparation method of the anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody, which is realized by the following steps and technical solutions:

[0010] (1) Immunization of animals: select 6-8 week old BALB / C mice, and immunize the mice with purified H10N3 subtype avian influenza virus (A / Duck / Human / S11205 / 2012) hemagglutinin protein.

[0011] (2) Culture of mouse myeloma cells: culture mouse myeloma cells SP2 / 0 and keep them in good growth state for cell fusion.

[0012] (3) Cell fusion: adopt polyethylene glycol mediated cell fusion method. The mice selected in step (1) are sacrificed to obtain spleen lymphocytes. The SP2 / 0 cells in step (2) are collected, the above two cells are mixed and centrifuged, and then polyethylene glycol mediated cell fusion is adopted. The fused cells are appropriately diluted, inoculated into 96-well culture plates, and cultured under appropriate conditions.

[0013] (4) Screening of hybridoma cells: the above culture is cultured in hypoxanthine-phosphoribosyl transferase selective medium. When the cell colonies grow to a suitable size, the cell culture supernatant is aspirated for antibody identification, and positive clones are screened.

[0014] (5) Cloning of hybridoma cells: the positive hybridoma cells are cloned by limited dilution method, and cells diluted to a certain density are inoculated into 96-well cell culture plates to allow only one cell to grow in each well. The supernatant of the well in which a cell colony is formed is taken for enzyme-linked immunosorbent assay to screen and identify positive clones. The culture well with the highest antibody titer and single clone cell growth is selected, and limited dilution is performed again, and the limited dilution is continuously performed for more than 4 times, and the hybridoma cell strain stably expressing the anti-H10 subtype avian influenza virus monoclonal antibody is obtained by continuous passage for more than 20 generations. The cloned hybridoma cells are subjected to antibody identification and physicochemical property analysis.

[0015] (6) Preparation of monoclonal antibody ascites: 8-10-week-old BALB / C healthy mice are selected, and each mouse is inoculated with 5×10 6 positive hybridoma cells in PBS buffer solution. After 7-10 days of inoculation, the mouse abdomen is significantly swollen, and the health status of the mouse is closely observed. When the ascites is as much as possible and the mouse is on the verge of death, the ascites is collected and centrifuged, the antibody titer is determined, and the monoclonal antibody in the ascites is purified;

[0016] (7) Purification of monoclonal antibody: the protein G agarose gel affinity purification method is used to purify the monoclonal antibody in the mouse ascites

[0017] (8) The present application obtains a hybridoma cell line producing an anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody, i.e. 1F3. The 1F3 hybridoma cell line is cloned for 4 times, continuously cultured for more than 6 months, and stably secretes antibodies. The cell strain is stored in liquid nitrogen, and grows well after resuscitation, and the antibody secretion does not decline. The enzyme-linked immunosorbent indirect method experiment shows that the culture supernatant titer of 1F3 is 1:64, and the ascites titer is 1:2048. The monoclonal antibody immunoglobulin subtype analysis shows that the antibody produced by the hybridoma cell is IgG1.

[0018] The present application provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 1F3 obtained by fusion, screening, cloning and passage of the spleen cells of an immunized BALB / C mouse and mouse myeloma cells SP2 / 0, and can stably secrete an anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3.

[0019] Another object of the present application is to provide detection of the monoclonal antibody 1F3 in a body fluid, allantoic fluid or other environmental sample containing an H10 subtype avian influenza virus, which is achieved by preparing a colloidal gold immunochromatography test strip and an enzyme-linked immunosorbent method.

[0020] The present application has the advantage of providing a monoclonal antibody against H10 subtype avian influenza virus hemagglutinin protein. The preparation method is simple and easy to operate, and more importantly, the monoclonal antibody prepared by the method can have various uses, such as qualitative diagnosis of H10 subtype avian influenza samples in clinics and laboratories.

[0021] Drawings

[0022] Figure 1 Analysis of immunoglobulin subtype of monoclonal antibody 1F3.

[0023] Figure 2 Specificity of immunofluorescence technique for detecting H10 subtype avian influenza virus.

[0024] Figure 3 Biological sequence of monoclonal antibody 1F3. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0026] Example 1. Preparation method of monoclonal antibody against H10 subtype avian influenza virus hemagglutinin protein

[0027] (1) Immunization of mice: For the first immunization, H10 subtype avian influenza virus hemagglutinin protein was mixed with adjuvant at a volume ratio of 1:1 and uniformly mixed, with a total volume of 0.5 milliliters. Each BALB / C mouse was injected with 0.1 milliliters (containing 100 micrograms of H10 subtype avian influenza virus hemagglutinin protein antigen) into the medial thigh muscle. On the 21st day, the same method was used for booster immunization once. On the 35th day, micro-amount of tail blood was collected for enzyme-linked immunosorbent assay to determine that the antibody titer reached 1:32000. The mouse with the highest antibody titer was selected for intravenous injection of the tail vein for booster immunization once, and cell fusion was performed 3 days later.

[0028] (2) Culture and passage of mouse myeloma cells SP2 / 0: SP2 / 0 myeloma cell strain from BALB / C mice was cultured and passed with 10% bovine serum DMEM medium and cultured in a 37°C incubator containing 5% carbon dioxide. Usually, no passage is performed one day before fusion to ensure that the cells are in the logarithmic growth phase at the time of fusion.

[0029] (3) Cell fusion: BALB / C mouse peritoneal macrophage was used as feeder cell. One day before fusion, BALB / c mouse peritoneal macrophage was inoculated into 96-well culture plate, and cultured in hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum for one day. Three days after the last boost, the spleen of the mouse was taken out, and the spleen lymphocytes were isolated by pressure water injection method. After centrifugal washing, the cells were resuspended in DMEM medium. SP2 / 0 cells were collected, centrifuged, washed, and resuspended in DMEM medium for counting. 3x10 8 immune mouse spleen lymphocytes were mixed with 3x10 7 mouse myeloma cells SP2 / 0. The two kinds of cells were mixed, centrifuged, and the supernatant was discarded. The cell mass was loosened by gently rubbing the centrifuge tube with the palm, and polyethylene glycol pre-warmed at 37°C was slowly added. The centrifuge tube was gently shaken during the process, and the cells were sucked into the fusion tube. After 90 seconds, the cells were blown into the centrifuge tube, and then 1 ml of DMEM medium was added in the first minute, 2 ml of DMEM medium was added in the second minute, 7 ml of DMEM medium was added in the third minute, and 40 ml of DMEM medium pre-warmed at 37°C was gradually added in the following 1 minute. Low-speed centrifugation at 800 rpm for 10 minutes was performed. Then, hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum was added, and the 96-well culture plate with feeder cells was inoculated with a glass dropper. Generally, 2-4 plates of cells were inoculated per fusion, and cultured in a 37°C incubator containing 5% carbon dioxide.

[0030] (4) Screening of hybridoma cells: half of the medium in 96-well plates is changed after 5 days (containing hypoxanthine-guanine-phosphoribosyl transferase) once, and the medium containing hypoxanthine-phosphoribosyl transferase is used after 10 days. The hybridoma cells after fusion are cultured in the selective medium containing hypoxanthine-phosphoribosyl transferase for about two weeks. When the cell colonies grow to a proper size (observed under 10 times objective, the cell colony size is suitable to fill one field), the cell culture supernatant is taken for enzyme-linked immunosorbent assay to screen positive clones. The positive hybridoma clones are screened by enzyme-linked immunosorbent assay indirect method. Main steps: ① H10 subtype hemagglutinin protein is diluted in 0.01 mol / L pH 9.6 carbonate buffer, then 0.1 mL is added to each well of 96-well enzyme-labeled plate, and the protein amount is 20 ng per well, and the plate is incubated at 4°C overnight; ② the plate is washed 5 times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ the plate is blocked for 2 hours with 0.01 mol / L pH 7.4 phosphate buffer containing 5% bovine serum albumin; ④ the plate is washed 3 times; ⑤ the hybridoma culture supernatant is added to each well, 0.1 mL, and positive control (H10 subtype protein immunized mouse serum), negative control (SP2 / 0 culture supernatant) and blank control are set, and the plate is incubated at room temperature for 2 hours; ⑥ the plate is washed 3 times; ⑦ 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG is added to each well, 0.1 mL, and the plate is incubated at room temperature for 1 hour; ⑧ the plate is washed 3 times; ⑨ color developing solution is added, and the plate is incubated at room temperature for 5 minutes; ⑩ the reaction is terminated with 2 mol / L sulfuric acid, and the optical density value is measured at 450 nm, and the value is divided by the negative value, and the positive value is ≥2.1.

[0031] (5) Cloning of hybridoma cells: the cloning of hybridoma cells is carried out according to the limited dilution method. The hybridoma cell holes with positive antibody detection are selected for proper proliferation, and the cells are accurately counted. The cells are diluted in complete DMEM medium to 10 cells per mL to inoculate the 96-well culture plate with feeder cells, 0.1 mL per well, and the cell growth is observed after 10 days, and the antibody level in the supernatant is detected. The culture hole with the highest antibody titer and single clone cell growth is selected for limited dilution again, and the limited dilution is continuously carried out for more than 4 times, and the hybridoma cell strain stably and highly expressing anti-H10 subtype avian influenza virus monoclonal antibody is obtained after continuous passage for more than 20 generations.

[0032] (6) Preparation of monoclonal antibody ascites: 8-10 week old BALB / C healthy mice are selected, and 5×10 6 positive hybridoma cells in PBS buffer are inoculated into the abdomen of each mouse. The mouse abdomen is significantly swollen after 7-10 days of inoculation, and the health status of the mouse abdomen is closely observed. The mouse ascites is collected when the ascites is as much as possible.

[0033] (7) Purification of the monoclonal antibody: The monoclonal antibody in the ascites was purified by affinity purification (protein G agar gel). ① Treat the ascites: centrifuge the ascites at 10000 rpm for 15 minutes at 4°C, remove the precipitate, collect the supernatant, mix it with 3-4 times the volume of binding buffer, and centrifuge it at 10000 rpm for 15 minutes at 4°C to remove the precipitate. Centrifuge it at 10000 rpm for 15 minutes at 4°C to remove the precipitate. ② The affinity purification column preloaded with protein G agar gel is washed with 5 times the column bed volume of binding buffer. ③ Dilute the ascites and load it onto the column, controlling the flow rate at 8-10 drops per minute. ④ Repeat the loading of the ascites that has passed through the column once. ⑤ Wash the purification column with 5 times the column bed volume of binding buffer. ⑥ Elute the bound monoclonal antibody with elution buffer, controlling the flow rate at 8-10 drops per minute, and collect the eluate in a collection tube pre-added with 0.1 ml of potassium phosphate buffer (pH 7.9), collecting 0.5 ml of the antibody-containing eluate per tube. ⑦ Detect the absorbance of each tube of eluate at 280 nm, and collect the eluate with a protein content of greater than 0.1 mg / ml. ⑧ Add the antibody eluate to an ultrafiltration centrifuge tube, centrifuge it at 10000 rpm for 10-20 minutes at 4°C to a final volume of about 1 ml of antibody eluate. Add 10 ml of 0.1 M phosphate buffer at pH 7.4, and centrifuge it at 10000 rpm for 10-20 minutes at 8°C. Finally, concentrate the antibody to a final volume of about 1 ml, and collect the concentrated antibody in a collection tube. ⑨ Dilute the desalted antibody solution, and measure the protein content at 280 nm. ⑩ Divide the purified antibody into small tubes, and store them in a low-temperature refrigerator for use.

[0034] (8) Subtype identification of the monoclonal antibody: Use the mouse monoclonal antibody immunoglobulin typing kit from Bio-Rad to analyze. Dilute the purified monoclonal antibody and perform the detection, strictly following the instructions in the kit. The test results show that the monoclonal antibody secreted by the 1F3 hybridoma cells is of the IgG1, κ type.

[0035] The results are shown in the attached Figure 1 .

[0036] Example 2. Qualitative detection of H10 subtype avian influenza virus using the monoclonal antibody

[0037] The anti-H10 avian influenza virus hemagglutinin protein monoclonal antibody prepared in the present application can be used for qualitative detection of H10 subtype avian influenza virus, and the identification method can be achieved by the following method:

[0038] H10 subtype avian influenza virus immunofluorescence detection method:

[0039] (1) Seed MDCK cells at a density of 4×10 4 cells per well in a 48-well plate one day in advance, and let the cells grow to 70% confluence for use.

[0040] (2) Take out the cell plate with cells, discard the culture supernatant, and wash once with phosphate buffer solution for standby;

[0041] (3) Determine the specificity of H10 subtype avian influenza virus immunofluorescence detection method for detecting H10 subtype avian influenza virus: dilute the virus with phosphate buffer solution, including H10N2 (A / duck / Zhejiang / 6D20 / 2013), H10N3 (A / chicken / Zhejiang / 8615 / 2016), H10N7 (A / chicken / Zhejiang / 528189 / 2016), H10N8 (A / chicken / Zhejiang / 102615 / 2016), H1N2 virus (A / duck / Zhejiang / D1 / 2013), H2N8 virus (A / duck / Zhejiang / 6D10 / 2013), H3N2 virus (A / duck / Zhejiang / 4613 / 2013), H4N6 virus (A / duck / Zhejiang / 409 / 2013), H5N1 virus (A / goose / Zhejiang / 97 / 2014), H6N1 virus (A / chicken / Zhejiang / 1664 / 2017), H7N9 virus (A / chicken / Zhejiang / ZJU01 / 2013) and H9N2 virus (A / chicken / Zhejiang / 221 / 2016) diluted virus solution to infect cells (multiplicity of infection is 0.5), and culture in a 37°C incubator containing 5% carbon dioxide for 2 hours;

[0042] (4) Take out the cell plate, discard the virus solution, and wash the cells twice with phosphate buffer solution, then add 200 microliters of virus culture solution to each well, and incubate in a 37°C incubator containing 5% carbon dioxide for 16 hours;

[0043] (5) Take out the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffer solution;

[0044] (6) Add 4% paraformaldehyde to fix the cells in each well of the cell plate, and fix at room temperature for 30 minutes, then wash 3 times with phosphate buffer solution;

[0045] (7) Permeabilize the cells with 0.5% Triton-X100 at room temperature for 30 minutes, and wash 3 times with phosphate buffer solution;

[0046] (8) Block with 3% bovine serum albumin in phosphate buffer solution at room temperature for 1 hour, and discard the bovine serum albumin solution;

[0047] (9) Dilute the monoclonal antibody to 10 microgram per milliliter with phosphate buffer solution, add 200 microliters to each well, incubate at 4°C overnight, and wash 3 times with phosphate buffer solution;

[0048] (10) Dilute the fluorescent secondary antibody to 5 microgram per milliliter with 1% bovine serum albumin solution, add 200 microliters to each well, incubate in a 37°C incubator for 90 minutes in the dark, and wash 3 times with phosphate buffer solution;

[0049] (11) Stain the cell nucleus with deoxyribonucleic acid fluorescent dye (4', 6-diamidino-2-phenylindole), incubate at room temperature in the dark for 10 minutes, and wash 3 times with phosphate buffer solution;

[0050] (12) Observe the experimental results under a fluorescence microscope, and green fluorescence indicates a positive result. The results show that the anti-H10 subtype avian influenza virus monoclonal antibody 1F3 developed in the present research has good specificity for detecting H10 subtype avian influenza virus.

[0051] It should be understood that the present application is described in conjunction with the preferred embodiments, however, those skilled in the art can make various modifications or changes to the present application after reading the above description of the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto.

Claims

1. An anti-H10 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1F3, the antibody subtype is IgG1, κ type, can specifically bind to the H10 subtype avian influenza virus hemagglutinin protein antigen, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

2. Use of the monoclonal antibody 1F3 against the hemagglutinin protein of H10 subtype avian influenza virus according to claim 1 in the preparation of a product for detecting H10 subtype avian influenza virus, wherein the H10 subtype avian influenza virus is H10N2, H10N3, H10N7 or H10N8, and the detection is a qualitative detection.

3. The use according to claim 2, characterized in that: The detection product detects H10 subtype avian influenza virus in different samples through immunofluorescence technology.

4. A H10 subtype avian influenza virus detection kit comprising the monoclonal antibody 1F3 against the hemagglutinin protein of H10 subtype avian influenza virus according to claim 1.

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