H10 subtype avian influenza virus monoclonal antibody and its application
The monoclonal antibody 8F4, prepared using a eukaryotic expression system, solved the problems of insufficient specificity and affinity in the detection of H10 subtype avian influenza virus, and achieved efficient virus detection and monitoring.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LONGHU LAB
- Filing Date
- 2026-06-18
- Publication Date
- 2026-07-31
AI Technical Summary
Existing monoclonal antibodies for H10 subtype avian influenza virus lack high specificity and high affinity, making it difficult to meet the needs of rapid detection and monitoring.
A monoclonal antibody 8F4 against the HA1 protein of H10 subtype avian influenza virus was prepared. The recombinant HA1 protein was expressed in HEK293F cells using a eukaryotic expression system. BALB/c mice were immunized with the antibody, which was secreted by the hybridoma cell line 8F4. After purification, a monoclonal antibody with high titer and high affinity was obtained.
It achieves specific identification and efficient detection of H10 subtype avian influenza virus, with a titer of 1:2.048×10⁶, and is suitable for early diagnosis, animal quarantine and epidemiological investigation.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological detection technology, specifically relating to the application of monoclonal antibodies against H10 subtype avian influenza virus. Background Technology
[0002] The H10 subtype avian influenza virus (H10 AIV), a member of the influenza virus family, can spread widely among poultry and wild birds. In recent years, numerous cases of human infection with the H10 subtype avian influenza virus have been reported globally, drawing widespread attention in the public health field. Although the virus has low pathogenicity in poultry and rarely causes large-scale outbreaks in farmed animals, it is prone to genetic mutations during cross-species transmission, potentially leading to new strains with altered antigenicity or enhanced pathogenicity, posing a potential threat to public health security.
[0003] Monoclonal antibody technology provides an important means for the rapid detection of avian influenza viruses. Monoclonal antibodies targeting major avian influenza virus subtypes such as H5, H7, and H9 have been widely studied and applied. In recent years, with the continued spread of H10 subtype avian influenza virus in birds and mammals, research on monoclonal antibodies targeting the H10 subtype has also attracted attention. Previous studies have prepared monoclonal antibodies targeting the HA protein of the H10 subtype avian influenza virus and constructed fluorescent immunochromatographic test strips. However, the types of monoclonal antibodies currently available for the high-specificity and high-affinity detection of the H10 subtype avian influenza virus remain limited. Developing more high-performance anti-H10 subtype HA protein monoclonal antibodies is of great significance for improving the monitoring system and rapid on-site detection of H10 subtype avian influenza. Summary of the Invention
[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a monoclonal antibody 8F4 that can specifically recognize the H10 subtype avian influenza virus. This monoclonal antibody can achieve specific recognition of the H10 subtype avian influenza virus.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] A monoclonal antibody against the HA1 protein of H10 subtype avian influenza virus, wherein the monoclonal antibody is monoclonal antibody 8F4, which can specifically bind to H10 subtype avian influenza virus, including a light chain variable region and a heavy chain variable region.
[0007] The nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody 8F4 is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2; the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 8F4 is shown in SEQ ID NO.3, and the amino acid sequence it encodes is shown in SEQ ID NO.4.
[0008] The heavy chain variable region of the monoclonal antibody 8F4 includes four variable region frames FR-H1, FR-H2, FR-H3 and FR-H4, and three complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, with corresponding amino acid sequences as shown in SEQ ID NO.7-SEQ ID NO.13, respectively.
[0009] The light chain variable region of the monoclonal antibody 8F4 includes four variable region frames FR-L1, FR-L2, FR-L3 and FR-L4, and three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the corresponding amino acid sequences of which are shown in SEQ ID NO.14-SEQ ID NO.20, respectively.
[0010] The application of the monoclonal antibody in the preparation of a detection reagent or kit for H10 subtype avian influenza virus.
[0011] The beneficial effects of this invention are:
[0012] 1. This invention utilizes a eukaryotic expression system (HEK293F cells) to express the recombinant HA1 protein from H10 AIV. Verification showed that H10 AIV-positive serum can specifically recognize the recombinant HA1 protein. The purified recombinant HA1 protein was used as an immunogen to immunize BALB / c mice using immunological methods to prepare anti-HA1 protein monoclonal antibodies. The resulting antibodies specifically recognize and bind to the HA1 protein. Compared to the prokaryotic expression system, the eukaryotically expressed HA1 protein has a spatial conformation and post-translational modifications closer to the native protein, exhibiting stronger immunogenicity and higher biological activity, providing a superior molecular basis for subsequent monoclonal antibody preparation and quantum dot labeling.
[0013] 2. This invention provides a monoclonal antibody against H10 subtype avian influenza virus, secreted by hybridoma cell line 8F4. This antibody exhibits high affinity and specificity. ELISA testing shows an antibody titer greater than 1:2.048×10⁻⁶. 6 It has high potency and good stability, and can be used to prepare detection reagents for H10 subtype avian influenza virus.
[0014] 3. The monoclonal antibody of the present invention can be applied to the early diagnosis, animal quarantine and inspection and epidemiological investigation of H10 subtype avian influenza, and has important promotional value. Attached Figure Description
[0015] Figure 1 The results of SDS-PAGE identification of HA1 protein before and after purification;
[0016] Where lane M: protein marker; lane 1: HA1 protein before purification; lanes 2 and 3: HA1 protein after purification;
[0017] Figure 2 Western blot identification results for purified HA1 protein;
[0018] Where lane M: protein marker; lane 1: purified HA1 protein;
[0019] Figure 3 These are the purification and identification results of the monoclonal antibody of this invention;
[0020] Lane 1 contains 8F4 ascites fluid; Lane 2 contains purified 8F4 monoclonal antibody.
[0021] Figure 4 This is the regression curve of the affinity constant of the monoclonal antibody of the present invention;
[0022] Figure 5 The results of HI titer determination of the monoclonal antibody of the present invention;
[0023] Figure 6 The results of Western blot detection of the monoclonal antibody of this invention are shown, where M is the protein molecular weight marker; lane 1: H10 AIV HA1 recombinant protein; lane 2: pcDNA3.1 empty vector;
[0024] Figure 7 This is the result of the monoclonal antibody specificity detection of the present invention. Detailed Implementation
[0025] The specific embodiments of the present invention will be further described in detail below with reference to examples.
[0026] Unless otherwise specified, the instruments and equipment involved in the embodiments are all conventional instruments and equipment, and the biochemical reagents involved are all obtained from commercial sources; the experimental methods involved are all conventional molecular biology and immunology methods.
[0027] Example 1: Expression and purification of H10 AIV HA1 protein
[0028] The HA protein of avian influenza virus is a major glycoprotein on the viral envelope. The HA1 subunit contains all the major antigenic sites of the HA gene and can induce the production of neutralizing antibodies, making it a leading candidate antigen for diagnostic reagent development. Mammalian cells possess the ability to facilitate proper protein folding and post-translational modifications, mimicking human protein glycosylation patterns, and are the most widely used host for biopharmaceuticals. Therefore, HA1 glycoprotein was expressed in HEK293F cells. The specific preparation process of the recombinant HA1 protein is as follows.
[0029] 1. Construction of eukaryotic expression vectors
[0030] Based on the nucleotide sequence of the HA1 protein in the H10 subtype avian influenza virus strain (accession number: KP861987.1) from Genebank, a signal peptide was added upstream of the HA1 gene to synthesize an optimized HA1 nucleotide sequence, as shown in SEQ ID NO.5. The optimized HA1 gene sequence was cloned into the pcDNA3.1 vector to construct the eukaryotic recombinant expression vector pcDNA3.1-HA1, whose nucleotide sequence is shown in SEQ ID NO.6.
[0031] 2. Expression and purification of recombinant HA1 protein.
[0032] The recombinant plasmid was transfected into HEK293F cells and cultured in a 37 ℃, 5% CO2 incubator. After 96 h of culture, the culture supernatant was collected, centrifuged, filtered, and used for nickel column affinity chromatography. The filtered supernatant was incubated with the nickel column at 4 ℃ for 2 h, followed by fractional elution with a gradient imidazole elution buffer, and residual protein was washed with a high concentration of imidazole. The eluent was collected, dialyzed, and concentrated to obtain the purified product.
[0033] SDS-PAGE analysis (see) Figure 1 ) and Western blot identification (H10 AIV positive serum) (see Figure 2 The results confirmed that the obtained protein was a recombinant HA1 protein.
[0034] Example 2: Preparation of Monoclonal Antibodies
[0035] 1. Animal immunization
[0036] An equal volume of Freund's complete adjuvant was added to the recombinant HA1 immunogen from Example 1, and after emulsification, a Freund's complete adjuvant immunogen was prepared for the first immunization. Two female BALB / c mice aged 4-8 weeks were immunized with the Freund's complete adjuvant immunogen at a dose of 10 μg / mouse by subcutaneous injection at multiple sites on the back. Subsequently, every 2 weeks, BALB / c mice were immunized again with an equal volume of Freund's incomplete adjuvant emulsified with HA1 protein, using the same method and dose, for a total of 3 immunizations. One week later, blood was collected from the tail vein to determine the specific antibody titer of HA1 protein. Mice with higher titers were selected, and BALB / c mice were given a super-immunization with adjuvant-free recombinant HA1 protein via intraperitoneal injection 3-4 days before cell fusion, at a dose of 20 μg / mouse.
[0037] 2. Cell fusion and monoclonal antibody preparation
[0038] Using the polyethylene glycol fusion method, spleen cells from super-immunized mice were fused with mouse myeloma cells SP2 / 0 at a cell ratio of 8:1. The fused cells were initially screened using HAT selective medium. Twelve days after fusion, positive hybridoma cells were initially screened using HA1 recombinant protein as the coating antigen by indirect ELISA, and a positive hybridoma cell line (named 8F4) was obtained.
[0039] 3. Subcloning of hybridoma cells
[0040] Dilute the above-mentioned positive hybridoma cells to 10 cells / mL using HT-added 1640 complete medium. Add 100 μL to each well of a 96-well plate pre-coated with 100 μL of feeder cells and incubate at 37°C, 5% CO2 for 6–8 days. Screen for positive hybridoma cells using indirect ELISA, and perform 2–3 subcloning cycles until a stable hybridoma cell line 8F4 secreting anti-HA1 protein monoclonal antibody is obtained. Expand the obtained positive hybridoma cell line to a cell number of 1 × 10⁻⁶ cells / mL. 6 ~2×10 6 / The tubes are frozen.
[0041] 4. Stability identification of hybridoma cell lines
[0042] The obtained positive hybridoma cell line 8F4 was cultured continuously for 3 months and repeatedly frozen and thawed in liquid nitrogen. The titer of the cell supernatant was then tested. The results showed that the titer of the cell supernatant remained stable after multiple freeze-thaw cycles, indicating that the positive hybridoma cell line 8F4 has good stability.
[0043] 5. Preparation of monoclonal antibodies
[0044] Multiparous female BALB / c mice were selected and injected intraperitoneally with 500 μL of sterile paraffin. One week later, they were injected intraperitoneally again with monoclonal hybridoma cells 8F4 at a dose of 2 × 10⁻⁶. 5 One week later, after the mouse abdomen swelled, ascites fluid was extracted, centrifuged, and the supernatant was collected. Antibody purification was performed using Protein A affinity chromatography to obtain purified monoclonal antibody 8F4. Figure 3 ).
[0045] Example 3: Performance Identification of Monoclonal Antibody 8F4
[0046] 1. Titer determination of monoclonal antibody 8F4
[0047] The titer of monoclonal antibodies was determined using an indirect ELISA method, as follows:
[0048] (1) Coating: The purified H10 AIV HA1 recombinant protein was diluted to four concentration gradients of 2 μg / mL with CBS, and 100 μL was plated in each well (n=3). The plates were incubated at 4℃ for 12 h. The plates were washed 5 times with PBST.
[0049] (2) Blocking: Add 5% skim milk to the microplate and incubate at 37°C for 3 h; discard the blocking solution after incubation and repeat the washing process above.
[0050] (3) Primary antibody: Set the serum of immunized mice as positive and the serum of non-immunized mice as negative. Dilute the monoclonal antibody serially twofold from the initial concentration of 1:1000, 100 μL / well, and incubate at 37°C for 1 h. Repeat the washing process above after the incubation.
[0051] (4) Secondary antibody: HRP-labeled goat anti-mouse IgG was diluted 1:5000 in 5% skim milk, and then 100 μL of the dilution buffer was added to each well. The mixture was incubated at 37°C for 1 h. After incubation, the unbound secondary antibody was removed by washing 5 times.
[0052] (5) Color development: Mix equal volumes of color development solution A and solution B to prepare TMB color development solution. Add 100 μL to each well and react at room temperature in the dark for 6 min;
[0053] (6) Termination: After the color development is complete, immediately add 100 μL of stop solution to each well;
[0054] (7) Reading: After termination, place the sample into the microplate reader and measure the absorbance at a wavelength of 450 nm. Calculate the ratio of positive wells to negative wells (P / N).
[0055] Indirect ELISA results showed that the titer of 8F4 could reach 1:2.048×10⁻⁶. 6 .
[0056] 2. Determination of affinity constant
[0057] The affinity constant of monoclonal antibody 8F4 was determined using an indirect ELISA method, as follows:
[0058] (1) Using H10 AIV HA1 recombinant protein as the coating agent, dilute to 1 μg / mL and 2 μg / mL with CBS buffer respectively, add to 96-well microplate at a volume of 100 μL / well, and coat overnight at 4℃; use BCA kit to determine the protein concentration of purified monoclonal antibody.
[0059] (2) The purified monoclonal antibody was serially diluted twice from the initial concentration at a ratio of 1:1000. Subsequent operations such as secondary antibody incubation, substrate color development, reaction termination and plate washing were performed in accordance with the monoclonal antibody titer determination procedure.
[0060] (3) Plot the reciprocal of antibody concentration on the x-axis, OD 450 Using the reciprocal of the values as the ordinate, linear regression was performed on the experimental data at the two coating concentrations to obtain the corresponding regression equations, and a double reciprocal curve was plotted. (See...) Figure 4 The equations of the two curves are Y = 0.002016X + 0.4449, R² = 0.9914 and Y = 0.001559X + 0.3352, R² = 0.9989.
[0061] (4) Based on the intercept of the regression equation (i.e., 1 / ODmax), calculate the maximum response OD value (ODmax) for the two coating concentrations. Substitute the ordinate corresponding to 1 / 2 ODmax into the respective regression equations to calculate the corresponding antibody mass concentration, convert it to molar concentration, and then substitute it into the following formula to calculate the affinity constant of the monoclonal antibody. The affinity constant of the monoclonal antibody 8F4 is 2.621 × 10⁻⁶. 9 L / mol.
[0062] Kaff=(n-1) / 2(n[Ab′]t-[Ab]t) (1)
[0063] In the formula, n = [Ag]t / [Ag′]t, where [Ag]t corresponds to a high coating concentration and [Ag′]t corresponds to a low coating concentration; [Ab′]t and [Ab]t represent 50% OD. 450 The corresponding molar concentration of the monoclonal antibody.
[0064] 3. Assay for the hemagglutination inhibitory activity of monoclonal antibody 8F4
[0065] The specific steps for determining hemagglutination inhibition activity using HI are as follows:
[0066] (1) Before the experiment, the hemagglutination titer of H10 AIV virus solution was determined by endpoint titration using 1% chicken red blood cells. The virus solution was diluted to 4 hemagglutination units (HAU) / 25μL for later use.
[0067] (2) Take a 96-well V-type blood coagulation plate, add 25 μL PBS to wells 1-11, and add 50 μL PBS to well 12 as a red blood cell control;
[0068] (3) Add the obtained purified monoclonal antibody 8F4 to the first well of the corresponding row (each monoclonal antibody occupies one row), add 25 μL of monoclonal antibody to each well, mix thoroughly by pipetting, then transfer 25 μL from the first well to the second well, and perform serial 2-fold dilutions to the tenth well. After mixing in the tenth well, discard 25 μL, and add 25 μL of PBS to the eleventh well to ensure that the final volume of each well is consistent.
[0069] (4) Add 25 μL of 4 HAU H10 AIV virus solution to each of wells 1 to 11, shake to mix, and incubate at room temperature for at least 20 min. Well 11 is the virus control (PC).
[0070] (5) Add 25 μL of 1% chicken red blood cell suspension to each well, gently shake to mix thoroughly, and let stand at room temperature for 20-40 min. When the red blood cells in the control wells form obvious button-shaped precipitates, the results can be judged.
[0071] (6) The highest monoclonal antibody dilution that can completely inhibit erythrocyte aggregation is used as the endpoint for determining HI titer.
[0072] The HI titer assay results showed that the monoclonal antibody 8F4HI had a titer of 2. -10 It has strong hemagglutination inhibitory activity (see...) Figure 5 ).
[0073] 4. Monoclonal antibody specificity identification
[0074] (1) Western blot identification
[0075] Samples of recombinant H10 AIV HA1 protein and supernatant obtained from transfecting HEK293F cells with empty vector plasmid (negative control) were prepared and subjected to SDS-PAGE. The primary antibody was a monoclonal antibody (8F4), and the secondary antibody was HRP-labeled goat anti-mouse IgG. An ECL chromogenic kit was used for development. Western blot was used to identify the specificity of the antibodies. The H10 AIV HA1 recombinant protein was used as the detection antigen, and the culture supernatant of pcDNA3.1 empty vector plasmid served as the negative control (lane 2).
[0076] from Figure 6 It can be seen that the monoclonal antibody 8F4 showed a clear band in lane 1 (45 kDa), while the negative control (lane 2) showed no band, indicating that the monoclonal antibody can specifically bind to the H10 AIV HA1 recombinant protein and has good reactivity.
[0077] (2) Indirect ELISA identification
[0078] The binding specificity of monoclonal antibody 8F4 to five recombinant proteins—H10 AIV HA1, H3 AIV HA1, H9N2 HA1, IBDV VP2, and IBV S—was evaluated using an indirect ELISA method. The five recombinant proteins were used as coating antigens, with the monoclonal antibody diluted 1:1000 as the primary antibody and HRP-labeled goat anti-mouse IgG diluted 1:5000 as the secondary antibody. The procedure was performed according to the indirect ELISA protocol.
[0079] The results showed that the monoclonal antibody 8F4 specifically bound to the H10 AIV HA1 recombinant protein only, and did not react with other common avian virus proteins such as H3N8 AIV HA1, H9N2 HA1, IBDV VP2, and IBV S. Figure 7 The results confirmed that the monoclonal antibody 8F4 of the present invention has strong specificity.
[0080] Example 4: Sequencing of Monoclonal Antibodies
[0081] Total RNA was extracted from the positive hybridoma cell line 8F4 and reverse transcribed into cDNA. Using cDNA as a template, universal primers were designed based on the constant region sequence to amplify the variable region sequence of the monoclonal antibody 8F4, and the sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing.
[0082] The nucleotide sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 8F4 are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively. The key amino acid sequences of the variable region are shown in Table 1 and Table 2.
[0083] Table 1. Amino acid sequence of the heavy chain variable region of monoclonal antibody 8F4
[0084]
[0085] Table 2. Amino acid sequence of the light chain variable region of monoclonal antibody 8F4
[0086]
[0087] Example 5: Application of monoclonal antibodies in the detection of H10 subtype avian influenza virus antigens
[0088] The monoclonal antibody 8F4 of this invention can be used to establish immunological detection methods such as ELISA, HI, and immunochromatographic strips for the antigen detection of H10 subtype avian influenza virus.
[0089] As can be seen from the above embodiments, the present invention provides a monoclonal antibody against the HA1 recombinant protein of H10 subtype avian influenza virus and its variable region sequence, providing a gene template for subsequent antibody modification and affinity maturation.
[0090] The gene sequences of the heavy chain variable region and light chain variable region that have been correctly sequenced are used to construct expression vectors through genetic engineering technology. Single-chain antibodies scFv and Fab fragments are recombinantly expressed in prokaryotic systems, and full-length monoclonal antibody chimeric antibodies and humanized antibodies are recombinantly expressed in eukaryotic systems (such as CHO cells) to meet the needs of clinical diagnosis and treatment.
[0091] Partial sequence in the instruction manual
[0092] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 8F4 (SEQ ID NO.1):
[0093] ATGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACCTTCACTGACTACTACATGAAGTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGATAC TTTCTACAACCAGAAATTCAAGGACATGGCCACATTGACTGTAGACAAATCATCCACCACAGCCTACATGCAACTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTACATCAAGGGGATACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCATAA.
[0094] The amino acid sequence of the heavy chain variable region of monoclonal antibody 8F4 (SEQ ID NO.2):
[0095] MLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNNGDTFYNQKFKDMATLTVDKSSSTTAYMQLNSLTSEDSAVYYCTSRGYWGQGTTVTVSS*.
[0096] The nucleotide sequence of the light chain variable region of monoclonal antibody 8F4 (SEQ ID NO.3):
[0097] ATGGATTGTTCTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCCACAAAGTT TCCAATCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTACGGTGGAGGCACCAAGTTGGAAATCAAATAA.
[0098] The amino acid sequence of the light chain variable region of monoclonal antibody 8F4 (SEQ ID NO.4):
[0099] MDVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTYGGGTKLEIK*.
[0100] HA1 protein nucleotide sequence (SEQ ID NO.5):
[0101]
[0102] Nucleotide sequence of pcDNA3.1-HA1 recombinant expression vector (SEQ ID NO.6):
[0103]
Claims
1. A monoclonal antibody against H10 subtype avian influenza virus HA1 protein, characterized in that, The monoclonal antibody is monoclonal antibody 8F4, which can specifically bind to the H10 subtype avian influenza virus, including the light chain variable region and the heavy chain variable region. The nucleotide sequence of the variable region of the 8F4 heavy chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.
2. The nucleotide sequence of the variable region of the light chain of monoclonal antibody 8F4 is shown in SEQ ID NO.3, and the amino acid sequence it encodes is shown in SEQ ID NO.
4.
2. The monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody 8F4 includes four variable region frames FR-H1, FR-H2, FR-H3 and FR-H4, and three complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, with corresponding amino acid sequences as shown in SEQ ID NO.7-SEQ ID NO.13, respectively. The light chain variable region of the monoclonal antibody 8F4 includes four variable region frames FR-L1, FR-L2, FR-L3 and FR-L4, and three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the corresponding amino acid sequences of which are shown in SEQ ID NO.14-SEQ ID NO.20, respectively.
3. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a detection reagent or kit for H10 subtype avian influenza virus.