CN112175072B

CN112175072B - Monoclonal antibody ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein and application thereof

Info

Publication number: CN112175072B
Application number: CN202011009238.2A
Authority: CN
Inventors: 吴海波; 吴南屏; 姚航平; 杨帆; 肖一鑫
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2022-06-28
Anticipated expiration: 2040-09-23
Also published as: CN112175072A

Abstract

The invention provides a monoclonal antibody ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein and application thereof. The heavy chain amino acid sequence of the antibody 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 H5 subtype avian influenza virus by using the monoclonal antibody as a probe through a colloidal gold immunochromatographic test strip and an enzyme-linked immunosorbent assay is established. The invention provides an effective tool for the auxiliary diagnosis of H5 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 ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein and application thereof

Technical Field

The invention belongs to the field of biotechnology, and relates to preparation and application of a monoclonal antibody against H5 subtype avian influenza virus hemagglutinin protein, which is characterized in that a hybridoma cell line secreting the monoclonal antibody against hemagglutinin protein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain to prepare the monoclonal antibody ZJU5-01 against hemagglutinin protein, the monoclonal antibody is identified as IgG2a and kappa type, and the application of the antibody is realized by affinity purification, an immunization method and other technologies.

Background

The H5 subtype avian influenza virus contains subclasses of H5N1, H5N2, H5N6, H5N8, etc. Outbreaks of H5 subtype avian influenza in asia and america have destroyed the asian market for live poultry and have had a serious negative impact on the economy of each country. From 12 months 2014 to 6 months 2015, nearly 50 million poultry were slaughtered in the united states experiencing the most severe highly pathogenic avian influenza event. After 2014, avian influenza virus subtype H5 was also found in korea, germany, uk, egypt and the netherlands, which caused some economic loss to the local poultry industry. By 9 months 2019, world health organization reported 861 laboratory confirmed cases of H5N1 avian influenza virus infection with 455 deaths. The H5N6 avian influenza virus caused 19 infections and 13 deaths in china and south-east asia. The H5 subtype avian influenza has attracted great attention and importance globally due to its high pathogenic potential. Currently, H5 subtype avian influenza continues to spread in birds to date, and is a great threat to human health and the development of the poultry farming industry.

The classical method of detecting influenza viruses is by virus isolation followed by serological detection of hemagglutinin and neuraminidase. In recent years, molecular detection methods have been developed enormously, 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 detecting viruses, 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 H5 subtype avian influenza virus more early and more widely and control the spread of epidemic situation.

In conclusion, the development of H5 subtype avian influenza virus monoclonal antibody has great significance for the establishment of a rapid sensitive detection method for the prevention and control of viruses. Based on the background, the project selects H5 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 H5 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 myeloma cells to create a hybridoma cell line that secretes homogeneous antibodies, also known as monoclonal antibody technology. 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 H5 subtype avian influenza virus, which can recognize H5 subtype avian influenza virus. The monoclonal antibody subtype is IgG2a and kappa type, is named ZJU5-01, and can specifically recognize avian influenza virus. The DNA sequence of the heavy chain variable region of the antibody is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 2; the light chain variable region DNA sequence is shown in SEQ ID No.3, and the light chain variable region amino acid sequence is shown in SEQ ID No. 4.

SEQ ID No.1

Heavy chain:DNA sequence(363bp)

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

GAGGTTCAGCTGCAACAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTTTATATATTGGGTCAATCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTTTAACAGCAGACACATCCTCCAACACAGCCTTCCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTTCTAGAGGGGGGGATTACGACGTAGGCAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID No.2

Heavy chain:Amino acid sequence(121AA)

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

EVQLQQSGAELVKPGASVKLSCTASGFNIKDTFIYWVNQRPEQGLEWIGRIDPANGNTKYDPKFQGKATLTADTSSNTAFLQLSSLTSEDTAVYYCSRGGDYDVGNAMDYWGQGTSVTVSS

SEQ ID No.3

Light chain:DNA sequence(339bp)

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

GACATTGTGATGACACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCCTTTATTTAGTACCAATCAAAAGAACTACGTGGCCTGGTTCCAGCAGAAACCAGGGCAGTCTCCCAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAATATTATACTTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

SEQ ID No.4

Light chain:Amino acid sequence(113AA)

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

DIVMTQSPSSLAVSVGEKVTMSCKSSQSPLFSTNQKNYVAWFQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYFCQQYYTYPLTFGAGTKLELK

The monoclonal antibody is produced by a hybridoma cell. The hybridoma cell for producing the monoclonal antibody is a hybridoma cell line ZJU5-01 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 ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein.

The second purpose of the invention provides a preparation method of an anti-H5 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 H5 subtype avian influenza virus hemagglutinin protein. The hemagglutinin protein is synthesized according to the hemagglutinin protein sequence expression of H5N6(A/Duck/Guangdong/GD01/2014) 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 method was used. The mice selected in step (1) were sacrificed to obtain spleen lymphocytes. Collecting SP2/0 cells in 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 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-H5 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 8-10 weeks BALB/C healthy mice, each abdominal inoculation containing 5 × 10⁶The 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: purification of monoclonal antibodies in ascites in mice by protein G agar gel affinity purification

(8) The invention obtains a hybridoma line which can generate the hemagglutinin protein of the anti-H5 subtype avian influenza virus, namely, the hybridoma cell line ZJU5-01 and ZJU5-01 are cloned for 4 times, continuously cultured for more than six months and stably excreted with antibody. The cell strain is frozen by liquid nitrogen, and after recovery, the cell strain grows well, and the secretion of antibodies is not declined. The titer of ZJU5-01 culture supernatant and the titer of ascites are respectively 1:64 and 1:2048 respectively by enzyme-linked immunosorbent indirect method experiment. Analysis of the monoclonal antibody immunoglobulin subtype showed that the hybridoma cells produced an antibody of the IgG2a type.

The invention provides a hybridoma cell for generating a monoclonal antibody, which is a mouse hybridoma cell line ZJU5-01 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 ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein.

Another object of the present invention is to provide uses and methods of use of monoclonal antibody ZJU 5-01.

The application of the monoclonal antibody ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein in preparing H5 subtype avian influenza virus detection products. The monoclonal antibody ZJU5-01 can be used for detecting body fluid, allantoic fluid or other environmental samples containing H5 subtype avian influenza virus by preparing colloidal gold immunochromatographic test strips and enzyme-linked immunosorbent direct method.

In addition, the invention provides a colloidal gold immunochromatographic test strip, which comprises a monoclonal antibody ZJU5-01 of hemagglutinin protein of the H5 subtype avian influenza virus, and the colloidal gold immunochromatographic test strip is applied to the detection of the H5 subtype avian influenza virus.

The invention has the advantage of providing the monoclonal antibody for resisting H5 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 H5 subtype avian influenza samples in clinic and laboratories.

Drawings

FIG. 1 is an immunoglobulin subtype analysis of monoclonal antibody ZJU 5-01.

FIG. 2 shows the specificity of detecting H5 subtype avian influenza virus with colloidal gold test strip.

FIG. 3 shows the sensitivity of the colloidal gold test strip for detecting H5 subtype avian influenza virus.

FIG. 4 shows the specificity of enzyme linked immunosorbent assay for detecting H5 subtype avian influenza virus.

FIG. 5 shows the sensitivity of enzyme-linked immunosorbent assay for detecting H5 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 H5

(1) Immunization of mice: for the first immunization, the H5 subtype avian influenza virus hemagglutinin holoprotein and the adjuvant are uniformly mixed 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 H5 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) taking trace tail blood on the 35 th day to carry out enzyme-linked immunosorbent assay determination, wherein the highest antibody titer reaches 1:32000, and selecting the mouse tail vein with the highest antibody titer to carry out boosting immunization once, and carrying out 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 10 ⁸Spleen lymphocytes of each immunized mouse and 3X 10⁷Mouse myeloma cells SP2/0 mix. Mixing the two cells, centrifuging and removing supernatant, gently rubbing the centrifuge tube with palm to loosen cell masses, slowly adding polyethylene glycol pre-warmed at 37 ℃ into the fusion tube, slightly shaking the centrifuge tube during the process, sucking the cells into the fusion tube, standing for 90 seconds, then blowing the cells into the centrifuge tube, adding 1 ml of DMEM medium in the 1 st minute according to the principle of slow first and fast later, adding 2 ml of DMEM medium in the 2 nd minute, adding 7 ml of DMEM medium in the 3 rd minute, and gradually adding 40 ml of DMEM medium pre-warmed at 37 ℃ in the later 1 minute. Low speed centrifugation at 800 rpm for 10 minutes. Then adding hypoxanthine-guanine-phosphoribosyl transferase culture medium containing 20% bovine serum, inoculating to 96-well culture plate containing feeder cells with glass dropper, spreading 2-4 plates for each fusion,the culture was carried out 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 H5 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 (H5 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 to react for 5 minutes in a dark place at room temperature; the reaction is stopped by 2 mol of R per liter of sulfuric acid; the optical density value is measured at 450 nm, and the positive is obtained by dividing the measured value by the negative value which is more than or equal to 2.1.

(5) Cloning of hybridoma cells: the cloning culture of hybridoma is carried out by limiting dilution method, and after the hybridoma cells positive for antibody detection are selected for proper proliferation, the cells are accurately counted. Diluting the cell suspension into 10 cell suspensions per milliliter by using a complete DMEM medium, inoculating the cell suspensions into a 96-well culture plate of the existing feeder cells, observing the growth condition of the cells after 10 days, detecting the antibody level in supernatant, selecting a culture well which has the highest antibody titer and shows the growth of a single clone cell, carrying out limited dilution again, continuously carrying out limited dilution for more than 4 times, and continuously carrying out generation for more than 20 generations to obtain the hybridoma cell strain which stably and efficiently expresses the anti-H5 subtype avian influenza virus monoclonal antibody.

(6) Preparing monoclonal antibody ascites: healthy BALB/C mice were selected for 8-10 weeks, and inoculated with 5X 10 of the inoculum per abdomen⁶The abdomen of the mouse is obviously enlarged after the positive hybridoma cell PBS buffer solution is inoculated with the cell for 7-10 days, the abdomen symptoms of the health condition of the mouse are closely observed, and the ascites of the mouse is collected when the ascites is as much as possible.

(7) Purification of monoclonal antibodies: the monoclonal antibodies in the ascites were purified by affinity purification (protein G agar gel). 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 ℃. ② the affinity purification column pre-filled with protein G agar gel is fully washed with binding buffer solution of 5 times of the volume of the column bed. 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 identification of the monoclonal antibody is carried out by adopting a mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad company. The purified monoclonal antibody is diluted properly and detected, and the operation is strictly carried out according to the instruction of a kit. The test result shows that the monoclonal antibody secreted by the ZJU5-01 hybridoma cell is IgG2a and kappa type.

The results are shown in FIG. 1.

Example 2 qualitative detection of H5 subtype avian influenza Virus with the monoclonal antibody

The monoclonal antibody of the hemagglutinin protein of the anti-H5 subtype avian influenza virus can be used for qualitatively detecting the H5 subtype avian influenza virus, and the identification method can be realized by the following method:

h5 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 markers: firstly, determining the optimal pH value of the colloidal gold: taking 9 centrifuge tubes, adding 1 ml 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 for 1 hour at room temperature; 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 of ZJU5-01 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 colloidal gold solution with the optimal pH value, and respectively adding the colloidal gold solution into centrifuge tubes, wherein each centrifuge tube is 100 microliters; sequentially adding 2, 4, 6, 8, 10 and 12 microliter of 0.1 milligram per milliliter of ZJU5-01 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 ZJU5-01 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 2 milliliters to prepare a ZJU5-01 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 2F11 resisting H5 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 H5 subtype avian influenza virus by using a colloidal gold test strip: 70 samples of allantoic fluid of known viruses were tested with colloidal gold test strips, including H1, H2, H3, H4, H5, H6, H7, H9, H10, avian influenza virus subtype H11, as well as Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV), Avian Paramyxovirus (APMV). The detection result shows that the colloidal gold test strip has better specificity to the H5 subtype avian influenza virus.

The results are shown in FIG. 2.

(10) Determining the sensitivity of the colloidal gold test strip for detecting H5 subtype avian influenza virus: diluting H5 subtype avian influenza virus to 2⁷、2⁶、2⁵、2⁴、2³、2²、2¹、20、2^-1And (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 H5 subtype avian influenza virus, otherwise does not contain H5 subtype avian influenza virus. The detection result shows that the sensitivity of the colloidal gold test strip for detecting the H5 subtype avian influenza virus is 1 hemagglutinin unit.

The results are shown in FIG. 3.

(11) Comparison experiments of the colloidal gold test strip with other detection methods: the detection method of the avian influenza virus H5 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 colloidal gold test strip reaches 98.2%, and the colloidal gold test strip detection method has the advantages of rapidness, specificity, convenience and the like, and has good clinical application prospects.

H5 enzyme-linked immunosorbent assay:

(1) pretreatment of the antibody: diluting the purified ZJU5-01 antibody to 4 micrograms per milliliter with 0.01 mol per liter of phosphate buffer solution, and diluting another antibody 2F11 of the H5 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 2F11 by using a horseradish peroxidase labeling kit, adding 10 microliters of reaction starting solution into 2 milligrams per milliliter of 100 antibody 2F11 solution, and gently mixing; then 100 micrograms of horseradish peroxidase is added, mixed gently and evenly, kept stand for 2 hours at 37 ℃, and can be shaken gently twice in the period; adding 10 microliters of reaction stop solution, gently mixing uniformly, and standing for 1 hour at room temperature; storing at 4 deg.C in dark place;

(3) enzyme label plate coating: adding 10 microliter 4 microgram per milliliter ZJU5-01 antibody into 10 milliliter carbonate buffer solution with 0.01 molar per liter pH9.6, and mixing evenly; 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) And (3) 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 elisa plate after incubation, and washing for 5 times according to the step (4);

(9) and (3) secondary antibody incubation: diluting the horseradish peroxidase labeled antibody 2F11 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 2F11 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 developing solution into the wells of the ELISA plate in the step (10), and keeping the temperature at room temperature

Carrying out light reaction for 5 minutes;

(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 H5 subtype avian influenza virus by an H5 coupled immunoadsorption method: 34 samples of allantoic fluid of known viruses including H1, H2, H3, H4, H5, H6, H7, H9, H10, H11 subtype avian influenza virus, Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV), Avian Paramyxovirus (APMV) were assayed by ELISA direct method. The detection result shows that the combined immunoadsorption method has better specificity to the H5 subtype avian influenza virus.

The results are shown in FIG. 4.

(15) Determining the sensitivity of H5 enzyme-linked immunosorbent assay for detecting H5 subtype avian influenza virus: sequentially diluting H5 subtype avian influenza virus to 2⁷、2⁶、2⁵、2⁴、2³、2²、2¹、20、2^-1Hemagglutinin units, 100. mu.l of the sample was tested in steps (1) to (13). And (4) judging a result: blank holes and negative holes are not developed, so that the enzyme-linked immunosorbent assay is effective, and otherwise, the enzyme-linked immunosorbent assay is ineffective; the positive is judged by dividing the measured value by the negative value of 2.1 or more. The detection result shows that the sensitivity of the enzyme-linked immunosorbent assay for detecting H5 subtype avian influenza virus is 0.5 hemagglutinin unit.

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 H5 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 99.4%, 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-H5 subtype avian influenza virus hemagglutinin protein monoclonal antibody ZJU5-01 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 363

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaggttcagc tgcaacagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg 60

tcctgcacag cttctggctt caacattaaa gacaccttta tatattgggt caatcagagg 120

cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtaa tactaaatat 180

gacccgaagt tccagggcaa ggccacttta acagcagaca catcctccaa cacagccttc 240

ctgcagctca gcagcctgac atctgaggac actgccgtct attactgttc tagagggggg 300

gattacgacg taggcaatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360

tca 363

<210> 2

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Phe Ile Tyr Trp Val Asn Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe

65 70 75 80

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

85 90 95

Ser Arg Gly Gly Asp Tyr Asp Val Gly Asn Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 3

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacattgtga tgacacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60

atgagctgca agtccagtca gagcccttta tttagtacca atcaaaagaa ctacgtggcc 120

tggttccagc agaaaccagg gcagtctccc aaactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttatttct gtcagcaata ttatacttat 300

cctctcacgt tcggtgctgg gaccaagctg gagctgaaa 339

<210> 4

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Pro Leu Phe Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Val Ala Trp Phe Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

Claims

1. A monoclonal antibody ZJU5-01 for resisting H5 subtype avian influenza virus hemagglutinin protein produced by hybridoma cell, the antibody subtype is IgG2a, kappa type, can be specifically combined with H5 subtype avian influenza 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 use of the monoclonal antibody ZJU5-01 of claim 1 for the hemagglutinin protein of avian influenza virus subtype H5 in the preparation of products for the detection of avian influenza virus subtype H5.

3. The use of claim 2, wherein the detection product is used for detecting H5 subtype avian influenza virus in various samples by a colloidal gold immunochromatographic test strip method and an enzyme-linked immunosorbent direct method.

4. A colloidal gold immunochromatographic test strip is characterized in that: a monoclonal antibody ZJU5-01 against hemagglutinin protein of avian influenza virus subtype H5 of claim 1.

5. The use of the colloidal gold immunochromatographic test strip of claim 4 for the detection of H5 subtype avian influenza virus for non-diagnostic purposes.