CN112724249B

CN112724249B - Monoclonal antibody ZJU9-01 for resisting H9 subtype avian influenza virus hemagglutinin protein and application thereof

Info

Publication number: CN112724249B
Application number: CN202110237370.7A
Authority: CN
Inventors: 吴海波; 肖一鑫; 杨帆; 吴南屏; 姚航平
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2021-03-03
Filing date: 2021-03-03
Publication date: 2022-05-17
Anticipated expiration: 2041-03-03
Also published as: CN112724249A

Abstract

The invention provides a monoclonal antibody ZJU9-01 for resisting H9 subtype avian influenza virus hemagglutinin protein and application thereof. The heavy chain amino acid sequence of the monoclonal antibody ZJU9-01 of the H9 subtype avian influenza virus resisting hemagglutinin protein is shown as SEQ ID No.2, and the light chain amino acid sequence is shown as SEQ ID No. 4. The monoclonal antibody is further analyzed and identified in physical and chemical properties, and a method for detecting H9 subtype avian influenza virus by using a colloidal gold immunochromatographic test strip and an enzyme-linked immunosorbent assay on the basis of the monoclonal antibody is established. The invention provides an effective tool for the auxiliary diagnosis of H9 subtype avian influenza virus infection in clinical samples, and can be popularized and applied to various detection technologies and clinical and experimental researches.

Description

Monoclonal antibody ZJU9-01 for resisting H9 subtype avian influenza virus hemagglutinin protein and application thereof

Technical Field

The invention belongs to the field of biotechnology, and relates to an anti-H9 subtype avian influenza virus hemagglutinin protein monoclonal antibody ZJU9-01 and preparation and application thereof, wherein a hybridoma cell line secreting a monoclonal antibody against hemagglutinin protein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by a mouse of the same strain, the monoclonal antibody ZJU9-01 against hemagglutinin protein is prepared and identified as IgG1 and kappa type, and the application of the antibody is realized by affinity purification, an immunization method and other technologies.

Background

Most avian influenza viruses have been spread widely and continuously in waterfowl populations. Of these, the H5N1 and H7N9 subtypes of avian influenza viruses have received considerable attention because of the serious harm they pose to humans, but another subtype of avian influenza, H9N2, has also spread widely in poultry in many parts of the world. The H9N2 subtype avian influenza virus was first discovered in turkeys in 1966, then in waterfowl in 1988, and then in ducks and chickens, and has become the most widespread and prevalent avian influenza virus strain in asia, with an H9N2 virus isolation rate of about 5% in the live avian market.

Although infection with the H9N2 virus is mainly restricted to poultry, it has been reported that the H9N2 virus has been isolated in swine many times. In the last two decades, laboratory-diagnosed cases of H9N2 infection have been reported and immunocompromised individuals are more susceptible to infection. The subtype H9 avian influenza virus is a potential epidemic risk to humans. At present, the H9 subtype avian influenza virus still continuously spreads in birds, and the establishment of a method for quickly detecting the H9 subtype avian influenza virus is urgent.

The classical method of influenza virus detection is by viral isolation. In recent years, molecular detection methods have been greatly developed, and real-time quantitative polymerase chain reaction has been widely used for 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, monoclonal antibody-based detection methods are also widely used for virus detection, such as antigen capture enzyme-linked immunosorbent assay and colloidal gold immunochromatographic test strips. The method has the advantages of rapidness, sensitivity and low cost, and can promote the discovery of H9 subtype avian influenza virus more early and more widely and control the spread of epidemic situation.

In conclusion, the development of H9 subtype avian influenza virus monoclonal antibody has great significance for disease prevention and control by establishing a rapid and sensitive detection method. Based on the background, the project selects H9 subtype avian influenza virus hemagglutinin protein as a target antigen, adopts a fusion hybridoma technology to establish a hybridoma cell line which stably secretes monoclonal antibodies against the hemagglutinin protein, and prepares, purifies and identifies the monoclonal antibodies in large quantities. The successful acquisition of the monoclonal antibody lays a material foundation for establishing a novel H9 subtype avian influenza 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 the myeloma cell SP2/0 to create a hybridoma cell line that secretes homogeneous antibodies, also known as the 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 monoclonal antibody for resisting hemagglutinin protein of H9 subtype avian influenza virus, which can recognize H9 subtype avian influenza virus. The monoclonal antibody subtype is IgG1 and kappa type, is named ZJU9-01, and can specifically recognize hemagglutinin protein of avian influenza virus.

The heavy chain amino acid sequence of the antibody is shown as SEQ ID No.2 (the DNA sequence is shown as SEQ ID No.1), and the light chain amino acid sequence is shown as SEQ ID No.4 (the DNA sequence is shown as SEQ ID No. 3).

SEQ ID No.1

Heavy chain:DNA sequence(336bp)

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

GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGC AAGGCTTCTGGATACACCTTCACTGACTACTACATGAAGTGGGTGAAGCAGAGCCATGGAAAGAGC CTTGAGTGGATTGGAGAAATTAATCCTAAGAATGGTGATACTTTCTACAACCAGAAATTCAAGGGC AAGGCCACATTGACTGTAGACAAATCCTCAAGCACAGCCTACATGCAGCTCAATAGCCTGACATCTG AGGACTCTGCAGTCTATTACTGTGAAAGCCAGAGGGGGTCCGGCCAAGGGACTCTGGTCACTGTCT CTGCA

SEQ ID No.2

Heavy chain:Amino acid sequence(112AA)

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

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGEINPKNGDTFYNQKFKGK ATLTVDKSSSTAYMQLNSLTSEDSAVYYCESQRGSGQGTLVTVSA

SEQ ID No.3

Light chain:DNA sequence(336bp)

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

GACGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGGACAACCAGCCTCCATCTCTT GCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGC CAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTT CACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGG AGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA AAA

SEQ ID No.4

Light chain:Amino acid sequence(112AA)

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

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGS GSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK

The invention provides a hybridoma cell for generating a monoclonal antibody, which is a mouse hybridoma cell line ZJU9-01 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 ZJU9-01 for resisting H9 subtype avian influenza virus hemagglutinin protein.

The invention provides a preparation method of an anti-H9 subtype avian influenza virus hemagglutinin protein monoclonal antibody, which is realized by the following steps and technical scheme:

(1) immunization of animals: BALB/C mice 6-8 weeks old were selected and immunized with the purified H9 subtype avian influenza virus hemagglutinin protein. The hemagglutinin protein is synthesized according to the hemagglutinin protein sequence expression of H9N2 avian influenza virus.

(2) Culture of mouse myeloma cells: mouse myeloma cell SP2/0 was 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 the 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 stably and efficiently expressing the anti-H9 subtype avian influenza virus monoclonal antibody, and carrying out antibody identification and physicochemical property analysis on the cloned hybridoma cells.

(6) Preparing monoclonal antibody ascites: selecting BALB/C healthy mice for 8-10 weeks, inoculating PBS buffer solution containing 5 multiplied by 106 positive hybridoma cells to each abdomen, obviously expanding the abdomen of the mice 7-10 days after inoculating the cells, closely observing the abdominal symptoms of the health condition of the mice, collecting ascites and centrifuging until the ascites is as much as possible and the mice are close to death, measuring the titer of the antibody, 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 line for producing the monoclonal antibody of the hemagglutinin protein of the H9 subtype avian influenza virus, namely the hybridoma cell line ZJU9-01 and ZJU9-01, is cloned for 4 times, is continuously cultured for more than six months, 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 titer of the culture supernatant of ZJU9-01 is 1:128 and the titer of ascites is 1:4096 by enzyme-linked immunosorbent indirect method. Analysis of the monoclonal antibody immunoglobulin subtype showed that the hybridoma cells produced the antibody type IgG 1.

The other purpose of the invention is to provide the detection (non-diagnosis purpose) of the monoclonal antibody ZJU9-01 in body fluid, allantoic fluid or other environmental samples containing H9 subtype avian influenza virus, which is realized by preparing a colloidal gold immunochromatographic test strip and an enzyme-linked immunosorbent assay.

In addition, the invention provides another colloidal gold immunochromatographic test strip which contains a monoclonal antibody ZJU9-01 of hemagglutinin protein of the H9 subtype avian influenza virus and is used for detecting the H9 subtype avian influenza virus.

The invention has the advantage of providing the monoclonal antibody for resisting H9 subtype avian influenza virus hemagglutinin protein. 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 H9 subtype avian influenza samples in clinic and laboratories.

Drawings

FIG. 1 is an immunoglobulin subtype analysis of monoclonal antibody ZJU 9-01.

FIG. 2 shows the specificity of detecting H9 subtype avian influenza virus with colloidal gold test strip.

FIG. 3 shows the sensitivity of the colloidal gold test strip for detecting H9 subtype avian influenza virus.

FIG. 4 shows the specificity of enzyme-linked immunosorbent assay for detecting H9 subtype avian influenza virus.

FIG. 5 shows the sensitivity of enzyme-linked immunosorbent assay for detecting H9 subtype avian influenza virus.

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 antibody against hemagglutinin protein of avian influenza virus subtype H9

(1) Immunization of mice: for the first immunization, the hemagglutinin holoprotein of the H9 subtype avian influenza virus is uniformly mixed with the adjuvant in the volume of 1:1, and the total volume is 0.5 ml. 0.1 ml of BALB/C mouse (containing 100 micrograms of H9 subtype avian influenza virus hemagglutinin whole 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) collecting trace tail blood on the 35 th day for enzyme-linked immunosorbent assay determination, wherein the antibody titer reaches 1:32000 to the maximum, and selecting the mouse tail with the highest antibody titer for intravenous injection for boosting immunization once, and performing cell fusion after 3 days.

(2) Culture passages of mouse myeloma cell SP 2/0: SP2/0 myeloma cell line from BALB/C mice was subcultured in DMEM medium containing 10% bovine serum and cultured in an incubator at 37 ℃ containing 5% carbon dioxide. The day prior to fusion is usually not passaged to ensure that the cells enter log phase growth upon fusion.

(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. Will be 3X 10⁸Spleen lymphocytes of each immunized mouse and 3X 10 cells⁷Mouse 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 1ml 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 hypoxanthine-guanine-phosphoribosyltransferase medium 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 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 (observed under a 10-fold objective lens, the cell colony size is preferably full of one field), the cell culture supernatant is sucked to carry out an 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: 0.01 mol per liter of pH9.6 carbonate buffer solution is used for diluting H9 subtype 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 ℃; 0.01 mol phosphate buffer solution (containing Tween 20) with pH value of 7.4 per liter is used for washing the plate for 5 times; ③ sealing for 2 hours by using phosphate buffer solution containing 0.01 mol of 5 percent bovine serum albumin and pH7.4 per liter; washing the plate for 3 times; adding hybridoma culture supernatant into each well of 0.1 ml, setting positive control (H9 subtype protein immune mouse serum), negative control (SP2/0 culture supernatant) and blank control, and reacting at room temperature for 2 hr; sixthly, washing the plate for 3 times; seventhly, adding 0.1 ml of horse radish peroxidase-labeled goat anti-mouse IgG diluted by 1:10000 into each hole, and reacting for 1 hour at room temperature; washing the plate for 3 times; ninthly, adding color development liquid 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 cloning culture of the hybridoma cells is carried out according to a limiting dilution method, and after the hybridoma cells positive in antibody detection are selected for proper proliferation, the cells are accurately counted. Diluting the cell suspension into 10 per milliliter by using a complete DMEM culture medium, inoculating the cell suspension into a 96-well culture plate with 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 grows in a single clone cell, carrying out limited dilution again, continuously carrying out limited dilution for more than 4 times, and continuously carrying out generation for more than 20 generations to obtain the hybridoma cell strain which stably and efficiently expresses the anti-H9 subtype avian influenza virus monoclonal antibody.

(6) Preparing monoclonal antibody ascites: selecting BALB/C healthy mice of 8-10 weeks, inoculating PBS buffer solution containing 5 x 106 positive hybridoma cells to each abdomen, expanding the abdomen of the mice obviously 7-10 days after inoculating the cells, closely observing the abdominal symptoms of the health condition of the mice, and collecting the ascites of the mice 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). Treating ascites: the ascites fluid was centrifuged at 10000rpm at 4 ℃ for 15 minutes to remove the precipitate, the supernatant was collected, mixed with 3 to 4 times the volume of the binding buffer, and centrifuged at 10000rpm at 4 ℃ for 15 minutes to remove the precipitate. The precipitate was removed by centrifugation at 10000rpm for 15 minutes at 4 ℃. Second, the affinity purification column pre-loaded with protein G agar gel is washed thoroughly with 5 column bed volumes of binding buffer. Thirdly, the diluted ascites is put on a column, and the flow rate is controlled to be 8-10 drops per minute. Fourthly, the ascites which passes through the column is repeatedly loaded on the column once. Fifthly, the purification column is fully washed by binding buffer solution with 5 times of the volume of the column bed. Sixthly, eluting the combined monoclonal antibody by using an elution buffer solution, controlling the flow rate to be 8-10 drops per minute, collecting the eluent in a collecting pipe which is pre-added with 0.1 ml of potassium phosphate buffer solution (PH7.9), and collecting 0.5 ml of eluent containing the antibody in each pipe. Seventhly, detecting the absorbance of each tube of eluent at 280 nm, and collecting the eluent with the protein content of more than 0.1 mg per ml. Adding antibody eluent into an ultrafiltration centrifugal tube, and centrifuging at 4 ℃ and 10000rpm for 10-20 minutes until the final volume of the antibody eluent is about 1 ml. Adding 10 ml of 0.1 mol/L phosphate buffer solution with pH7.4, centrifuging at 8 deg.C 10000rpm for 10-20 min, centrifuging to concentrate antibody to final volume of about 1ml, and sucking the concentrated antibody solution into a collecting tube. Ninthly, measuring the protein content at 280 nm after diluting the antibody solution after desalting. 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 ZJU9-01 hybridoma cell is IgG1 and kappa type.

The results are shown in FIG. 1.

Example 2 qualitative detection of H9 subtype avian influenza Virus with the monoclonal antibody

The monoclonal antibody of the hemagglutinin protein of the anti-H9 subtype avian influenza virus can be used for qualitatively detecting the H9 subtype avian influenza virus, and the identification method can be realized by the following method:

h9 immunochromatographic colloidal gold test strip:

(1) preparing a colloidal gold solution: taking 1 glass bottle of 100 ml, adding 49.5 ml of ultrapure water, then adding 0.5 ml of 1% chloroauric acid to prepare 50 ml of 0.01% chloroauric acid solution, heating to boil, then adding 1.8 ml of 1% trisodium citrate solution at one time under the condition of continuous stirring, continuously stirring and heating, continuously heating while observing the change of the solution color (gray blue is changed into purple and then changed from purple to red), stopping heating after the solution color is not changed any more, naturally cooling to room temperature, fixing the volume to 50 ml with ultrapure water, and storing at 4 ℃ in a dark place for later use;

(2) optimizing the conditions of the colloidal gold marker: firstly, determining the optimal pH value of the colloidal gold: taking 9 centrifuge tubes, adding 1ml of colloidal gold solution into each centrifuge tube, sequentially adding 0, 5, 10, 15, 20, 25, 30, 40 and 50 microliters of 0.1 mol/L potassium carbonate solution, uniformly mixing, and standing at room temperature for 1 hour; sequentially taking 100 microliters of liquid from each centrifuge tube, putting the liquid into a new centrifuge tube, respectively adding 3 microliters of 1 milligram per milliliter ZJU9-01 antibody, uniformly mixing, and standing for 15 minutes; adding 20 microliter of 10% sodium chloride solution into each hole, mixing uniformly, standing for 2 hours, observing the color of the colloidal gold, and keeping the lowest red pH value to be the optimal pH value of the colloidal gold solution. Taking 600 microliters of the colloidal gold solution with the optimal pH value, and respectively adding the colloidal gold solution into centrifuge tubes, wherein each tube is 100 microliters; sequentially adding 2, 4, 6, 8, 10 and 12 microliter of 0.1 milligram per milliliter of ZJU9-01 antibody, uniformly mixing, and standing for 15 minutes; adding 20 microliter of 10% sodium chloride solution, mixing uniformly, standing at room temperature for 2 hours, observing the color of the colloidal gold, keeping the red minimum protein amount as the minimum stable protein amount of the antibody, and increasing the minimum protein amount by 20 percent on the basis to obtain the optimal protein amount of the gold-labeled antibody;

(4) preparation of colloidal gold marker: taking 20 ml of the colloidal gold solution in the step (1), stirring the colloidal gold solution by an electromagnetic stirrer at 250 rpm according to the optimal conditions selected in the step (3), dropwise adding 2 ml of 1 mg/ml ZJU9-01 antibody, and reacting for 10 minutes; dropwise adding 2 ml of 10% bovine serum albumin, and continuously stirring for reaction for 10 minutes; centrifuging the gold-labeled antibody solution at 4 ℃ at 12000 rpm for 30 minutes, removing the supernatant, collecting the precipitate, and diluting the precipitate with a gold-labeled antibody diluent to a constant volume of 1.5 ml to prepare a ZJU9-01 antibody colloidal gold marker;

(5) preparing a colloidal gold film: soaking the carrier glass cellulose membrane in the colloidal gold marker solution, and naturally airing at room temperature for later use;

(6) preparation of nitrocellulose membrane: diluting goat anti-mouse IgG antibody and another monoclonal antibody 2A7 resisting H9 subtype avian influenza virus to 1 milligram per milliliter, respectively marking on a quality control line and a detection line of a nitrocellulose membrane, preparing a coating, and naturally airing at room temperature;

(7) sample pad pretreatment: soaking the glass cellulose membrane in the sample pad treatment solution, and naturally airing at room temperature;

(8) assembling the detection card: firstly, installing the nitrocellulose membrane coated with the detection monoclonal antibody and the quality control secondary antibody on a special supporting plate, and then sequentially installing a gold label pad, a sample pad and a water absorption pad, wherein one section of the gold label pad and one section of the water absorption pad are arranged on the nitrocellulose membrane, and one section of the sample pad is arranged on the gold label pad, so that the mutual connection of each part is ensured, and the sample can smoothly flow. And cutting into strips to prepare the test strip.

(9) Determining the specificity of detecting H9 subtype avian influenza virus by using a colloidal gold test strip: the allantoic fluid samples of different viruses are detected by colloidal gold test strips, and comprise H1, H2, H3, H4, H5, H6, H7, H9, H10 and H11 subtype avian influenza viruses, influenza B viruses, Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV) and avian paramyxovirus (APMV-4). The detection result shows that the colloidal gold test strip has better specificity to the H9 subtype avian influenza virus.

The results are shown in FIG. 2.

(10) Determining the sensitivity of the colloidal gold test strip for detecting H9 subtype avian influenza virus: diluting H9 subtype avian influenza virus to 2⁶、2⁵、2⁴、2³、 2²、2¹And (3) in a hemagglutinin unit, dripping 100 microliters of a sample to be detected into a sample pad area of the test strip, and observing whether a red strip appears on a quality control line and a detection line. And (4) judging a result: the red strip appearing on the quality control line indicates that the test strip is effective, otherwise, the test strip is ineffective. The red strip appears in the detection line, which indicates that the sample contains H9 subtype avian influenza virus, otherwise does not contain H9 subtype avian influenza virusA virus. The detection result shows that the sensitivity of the colloidal gold test strip for detecting H9 subtype avian influenza virus is 4 hemagglutinin units.

The results are shown in FIG. 3.

(11) Comparison experiments of the colloidal gold test strip with other detection methods: the detection method for detecting the avian influenza virus H9 subtype nucleic acid is adopted for comparative analysis, and clinical samples are detected and analyzed, so that the specificity of the colloidal gold test strip reaches 98.2 percent, and the detection method of the colloidal gold test strip has the advantages of rapidness, specificity, convenience and the like, and has good clinical application prospect.

H9 enzyme-linked immunosorbent assay:

(1) pretreatment of the antibody: diluting the purified ZJU9-01 antibody to 4 micrograms per milliliter with 0.01 mol per liter of phosphate buffer solution, and diluting another antibody 2A7 of the H9 subtype avian influenza virus to 2 milligrams per milliliter with 0.01 mol per liter of phosphate buffer solution for later use;

(2) horse radish peroxidase-labeled detection antibody: labeling the antibody 2A7 by using a horseradish peroxidase labeling kit, adding 10 microliters of reaction starting solution into 2 milligrams per milliliter of 100 antibody 2A7 solution, and gently mixing; then 100 micrograms of horseradish peroxidase are added, the mixture is mixed lightly and is kept stand for 2 hours at 37 ℃, and the mixture can be shaken lightly twice in the period; adding 10 microliter of reaction termination solution, gently mixing, and standing for 1 hour at room temperature; storing at 4 deg.C in dark for use;

(3) enzyme label plate coating: adding 10 microliter of 4 microgram per milliliter of ZJU9-01 antibody into 10 milliliters of 0.01 mol per liter of pH9.6 carbonate buffer solution, and mixing uniformly; taking a 96-well enzyme label plate, adding 100 microliters of antibody-coating solution into each well, and standing overnight at 4 ℃;

(4) washing: removing the antibody-coating solution after coating, washing with a phosphate Tween buffer solution, adding 400 microliters of the phosphate Tween buffer solution into each well, removing the washing solution, adding 400 microliters of the phosphate Tween buffer solution, repeatedly washing for 5 times, and patting dry the enzyme-labeled plate;

(5) and (3) sealing: adding 200 microliters of 5% bovine serum albumin phosphate solution into the coated ELISA plate, and standing for 2 hours at room temperature;

(6) washing: washing the enzyme label plate after the sealing is finished, and washing for 5 times according to the step (4);

(7) sample incubation: adding 100 microliters of sample into the wells of the ELISA plate in the step (6), and incubating for 1 hour at 37 ℃;

(8) washing: washing the enzyme label plate after incubation, wherein the step refers to the step (4) for 5 times;

(9) and (3) secondary antibody incubation: diluting the horseradish peroxidase labeled antibody 2A7 in the step (2) by 500 times to 4 micrograms per milliliter with a phosphate Tween buffer solution; adding 100 microliters of horseradish peroxidase labeled antibody 2A7 solution into the wells of the ELISA plate in the step (8), and incubating for 30 minutes at 37 ℃;

(10) washing: washing the enzyme label plate after incubation, wherein the step refers to the step (4) for 5 times;

(11) color development: adding 100 microliters of tetramethylbenzidine color development solution into the wells of the ELISA plate in the step (10), and reacting for 5 minutes at room temperature in a dark place;

(12) and (4) terminating: adding 100 microliters of 2 mol/L sulfuric acid per hole into the enzyme label plate to terminate the reaction;

(13) reading a plate: measuring the optical density value at 450 nm, and dividing the measured value by the negative value to be more than or equal to 2.1 to obtain the positive value;

(14) determining the specificity of detecting the H9 subtype avian influenza virus by an H9 combined immunoadsorption method: the detection of allantoic fluid samples of known viruses including avian influenza virus subtypes H1, H2, H3, H4, H5, H6, H7, H9, H10, H11, influenza B virus, Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV), avian paramyxovirus (APMV-4) is carried out by enzyme-linked immunosorbent assay. The detection result shows that the combined immunoadsorption method has better specificity to the H9 subtype avian influenza virus.

The results are shown in FIG. 4.

(15) Determining the sensitivity of H9 enzyme-linked immunosorbent assay for detecting H9 subtype avian influenza virus: sequentially diluting H9 subtype avian influenza virus to 2⁶、 2⁵、2⁴、2³、2²、2¹Hemagglutinin units, 100. mu.l of the sample was assayed in steps (1) to (13). And (4) judging a result: blank well and femaleThe non-color development of the sex pore indicates that the enzyme-linked immunosorbent assay is effective, otherwise, the enzyme-linked immunosorbent assay is ineffective; the positive result is determined by dividing the measured value by the negative value by 2.1 or more. The detection result shows that the sensitivity of the enzyme-linked immunosorbent assay for detecting H9 subtype avian influenza virus is 4 hemagglutinin units.

The results are shown in FIG. 5.

(16) The enzyme-linked immunosorbent assay and other detection methods are compared and tested: the detection method of the avian influenza virus H9 subtype nucleic acid detection kit (Shanghai river biology Co., Ltd.) is adopted for comparative analysis, clinical samples are detected and analyzed, the specificity of the combined immunoadsorption method detection method reaches 93.5%, the combined immunoadsorption method detection method has the advantages of being rapid, specific, convenient and fast, and the like, and has good clinical application prospects.

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.

Sequence listing

<110> Zhejiang university medical college affiliated to the first hospital

<120> anti-H9 subtype avian influenza virus hemagglutinin protein monoclonal antibody ZJU9-01 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata caccttcact gactactaca tgaagtgggt gaagcagagc 120

catggaaaga gccttgagtg gattggagaa attaatccta agaatggtga tactttctac 180

aaccagaaat tcaagggcaa ggccacattg actgtagaca aatcctcaag cacagcctac 240

atgcagctca atagcctgac atctgaggac tctgcagtct attactgtga aagccagagg 300

gggtccggcc aagggactct ggtcactgtc tctgca 336

<210> 2

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Lys Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Glu Ser Gln Arg Gly Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ala

100 105 110

<210> 3

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacgttgtga tgacccagac tccactcact ttgtcggtga ccattggaca accagcctcc 60

atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120

ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240

agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttccg 300

tacacgttcg gaggggggac caagctggaa ataaaa 336

<210> 4

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Claims

1. An anti-H9 subtype avian influenza virus hemagglutinin protein monoclonal antibody ZJU9-01, the antibody subtype is IgG1, kappa type, can be specifically combined with H9 subtype avian influenza virus hemagglutinin protein antigen; 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.

2. The monoclonal antibody ZJU9-01 of claim 1, wherein: the monoclonal antibodies are produced by hybridoma cells.

3. The monoclonal antibody ZJU9-01 of claim 2, wherein: the hybridoma cell for producing the monoclonal antibody is a hybridoma cell line ZJU9-01 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 ZJU9-01 for resisting H9 subtype avian influenza virus hemagglutinin protein.

4. The use of the monoclonal antibody ZJU9-01 against hemagglutinin protein of avian influenza virus subtype H9 as claimed in claim 1 or 2 for the preparation of a product for detecting avian influenza virus subtype H9.

5. Use according to claim 4, characterized in that: the detection product detects H9 subtype avian influenza virus in a sample through a colloidal gold immunochromatographic test strip method and an enzyme-linked immunosorbent assay.

6. A colloidal gold immunochromatographic test strip is characterized in that: comprises the monoclonal antibody ZJU9-01 against the H9 subtype avian influenza virus hemagglutinin protein of claim 1 or 2.

7. The use of the colloidal gold immunochromatographic test strip of claim 6 for the detection of H9 subtype avian influenza virus for non-diagnostic purposes.