Monoclonal antibody panel directed against hemagglutinin protein of avian influenza virus h5 subtype and uses thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a combination of monoclonal antibodies for avian influenza virus H5 subtype hemagglutinin protein and application thereof. The combination can specifically recognize avian influenza virus H5-HA protein, wherein the heavy chain and light chain variable region complement determining region (CDR) sequences of 2B8 and 6B5 are clear (as shown in SEQ ID NO. 1-12, respectively), which ensures the high affinity and specific binding capacity of the antibody combination. The combination is applied to the construction of a colloidal gold immunochromatographic detection test strip, and provides a stable and reliable biological recognition tool for rapid and accurate detection of avian influenza virus H5. The colloidal gold detection test strip constructed based on the combination has a detection sensitivity as high as 1 ng / mL, and is simple to operate without the need of complex instruments, and can be judged within 20 minutes, thereby providing reliable technical support for early warning, on-site rapid screening and monitoring of H5 subtype avian influenza.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies against the hemagglutinin protein of the H5 subtype of avian influenza virus and its applications. Background Technology

[0002] Avian influenza virus (AIV) is an RNA virus belonging to the Orthomyxoviridae family. It is a type A influenza virus that infects birds, is widespread and transmissible in poultry, and can infect mammals across species under certain conditions. It is a significant pathogen that seriously threatens the poultry industry and global public health security. Among them, highly pathogenic avian influenza virus H5 subtypes (such as H5N1) are highly pathogenic and have a high mortality rate. In addition to causing devastating damage to the poultry industry, there are also reports of avian influenza virus infecting humans and dairy cattle across species, posing a potential risk of triggering a zoonotic pandemic.

[0003] Hemagglutinin (HA) is the most important glycoprotein on the surface of influenza viruses, playing a crucial role in viral adsorption and invasion of host cells. Significant antigenic differences exist between different HA subtypes. Based on HA protein differences, influenza A viruses are further divided into Group 1 (including subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) and Group 2 (including subtypes H3, H4, H7, H10, H14, and H15). Among these, the H5 and H7 subtypes cause highly pathogenic avian influenza viruses, which are classified as Category A animal diseases by the World Organisation for Animal Health (OIE) and as Category I animal diseases in my country. Highly pathogenic avian influenza viruses spread rapidly in poultry, with a mortality rate that can reach 100%. Some strains can cross species boundaries and infect humans, leading to high mortality rates in severe cases. Therefore, rapid and accurate detection of specific HA subtypes, especially the highly pathogenic H5 subtype, is crucial for avian influenza outbreak early warning, prevention and control, and epidemiological investigation.

[0004] When poultry are infected with the H5 subtype avian influenza virus, they often exhibit symptoms such as lethargy, decreased appetite, difficulty breathing, a sharp drop in egg production, and even mass mortality, causing significant economic losses to the poultry industry. Furthermore, there is currently a lack of effective clinical cures for H5 subtype avian influenza; once an outbreak occurs, the only recourse is often passive measures such as culling and quarantine, resulting in extremely high economic losses and control costs. Therefore, establishing early, rapid, and accurate detection methods for the H5 subtype avian influenza virus is crucial for timely detection and precise control of outbreaks, reducing the risk of transmission and economic losses.

[0005] Currently, while various detection methods exist for H5 subtype avian influenza virus, limitations remain in terms of field applicability, detection stability, and result consistency. In particular, some immunological detection methods, due to insufficient antibody specificity or diverse sources, are prone to cross-reactivity or batch-to-batch variations, affecting the accuracy and reliability of the detection. Hemagglutinin (HA) protein is a key surface antigen determining the subtype specificity of avian influenza virus, exhibiting distinct subtype characteristics in its sequence and spatial structure, making it an ideal target for developing highly specific immunoassay methods. However, effectively avoiding the recognition of conserved structural regions of the HA protein during antibody screening to obtain subtype-specific monoclonal antibodies remains a pressing technical problem to be solved in this field. Summary of the Invention

[0006] This invention successfully prepared a monoclonal antibody that specifically recognizes the HA protein of H5 subtype avian influenza virus using hybridoma technology, and constructed a colloidal gold immunochromatographic test strip based on this antibody. Verification showed that the antibody exhibited good detection sensitivity and specificity against the H5 subtype antigen, meeting the application requirements for rapid on-site screening, and providing a reliable core antibody raw material basis for the development of diagnostic reagents for H5 subtype avian influenza virus.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include: A monoclonal antibody ensemble targeting the hemagglutinin protein of avian influenza virus subtype H5, the monoclonal antibody ensemble comprising monoclonal antibody 2B8 and monoclonal antibody 6B5. The heavy chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0008] In some embodiments, the heavy chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3. The light chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6. The heavy chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.9. The light chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.12.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO. 14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.16.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO. 18.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO. 20.

[0012] Secondly, the use of the monoclonal antibody combination provided in this application in the preparation of a tool for specifically recognizing the hemagglutinin protein of the H5 subtype of avian influenza virus.

[0013] In some embodiments, the tool includes colloidal gold test strips, reagents, kits, and antibody chips.

[0014] The tool is used to detect avian influenza virus H5 hemagglutinin protein in samples selected from any of the following: environmental samples, swab samples, cell culture supernatants, and tissue homogenates, and the identification is not used for disease diagnosis.

[0015] Environmental samples refer to non-biological samples collected from poultry activity or processing sites during avian influenza virus surveillance. These mainly include wastewater from poultry washing, swabs from cutting board surfaces, poultry feces, swabs from cages, and swabs from the ground. These samples are usually collected from places such as live poultry markets, poultry farms, slaughterhouses, farmers' markets, and transport vehicles.

[0016] Swab samples refer to samples collected from live poultry, such as the oropharynx, cloaca, nasal cavity, and trachea, using sterile cotton swabs or special sampling swabs in avian influenza virus testing.

[0017] In some embodiments, the colloidal gold test strip uses monoclonal antibody 2B8 as the capture antibody and monoclonal antibody 6B5 as the labeling antibody.

[0018] In some embodiments, the colloidal gold test strip includes a nitrocellulose membrane, a gold-labeled conjugate pad, a sample pad, and absorbent paper attached to a backing plate.

[0019] In some embodiments, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 2B8, the control line is coated with goat anti-mouse IgG, and the gold-labeled conjugate pad is coated with monoclonal antibody 6B5.

[0020] Beneficial effects: This application provides a monoclonal antibody suite targeting the hemagglutinin protein of avian influenza virus H5 subtype, specifically a monoclonal antibody combination for detecting / recognizing the avian influenza virus H5 hemagglutinin protein. The monoclonal antibody combination consists of monoclonal antibody 2B8 and monoclonal antibody 6B5, capable of specifically recognizing the avian influenza virus H5-HA protein. The complementarity-determining region (CDR) sequences of the heavy and light chain variable regions of 2B8 and 6B5 are clearly defined (as shown in SEQ ID NO. 1-12, respectively), ensuring high affinity and specific binding ability of the antibody combination. This combination is applied to the construction of colloidal gold immunochromatographic test strips, providing a stable and reliable biorecognition tool for the rapid and accurate detection of avian influenza virus H5. The colloidal gold test strip constructed based on this combination has a detection sensitivity of up to 1 ng / mL, is easy to operate, requires no complex instruments, and can be interpreted within 20 minutes, providing reliable technical support for early warning, rapid on-site screening, and monitoring of H5 subtype avian influenza. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Identification results for the purification of H5 monoclonal antibody; Figure 2 This is a schematic diagram of colloidal gold assembly; Figure 3 The results show the specificity of H5-HA monoclonal antibody colloidal gold test strips for multiple avian influenza HA subtypes and other viral recombinant proteins. Figure 4 This refers to the specific detection results of the H5-HA monoclonal antibody colloidal gold test strip for virus or vaccine antigens. Figure 5 The results are from the sensitivity test of the test strips; Figure 6 This is the result of antibody binding identification. Detailed Implementation

[0023] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0024] The detection method described in this application is not intended for disease diagnosis and treatment.

[0025] Example 1 1. Screening of H5-HA monoclonal antibodies 1.1 Mouse Immunization Mice were immunized with H5-HA recombinant protein (A / Anhui / 1 / 2005 HA, 11048-V08B, Sinopharm). The H7 recombinant protein (A / Shanghai / 1 / 2013 HA, 40104-V08B, Sinopharm) with the same His tag was used as a retro-screening antigen for monoclonal antibody screening. Although there are certain structural differences between different HA subtypes, they still contain some conserved epitopes. Therefore, introducing a representative subtype from Group 2 as a retro-screening antigen during the monoclonal antibody screening process can exclude antibodies that recognize the conserved structural regions of HA, thus obtaining monoclonal antibodies that specifically recognize the H5 subtype HA protein. Furthermore, given that the H7 subtype and H5 subtype are both important highly pathogenic avian influenza virus-related subtypes, choosing H7 as the retro-screening antigen has strong specificity.

[0026] Specifically, purified recombinant H5 protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. In weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites, mixed with an equal volume of Freund's incomplete adjuvant at the same dose. In week 6, mice were immunized by direct injection of insulin (10 μg / mouse) into the spleen. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg of recombinant H5 protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0027] 1.2 Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for H5-HA recombinant protein were selected by indirect ELISA. Due to the presence of the His tag on the immunogen, the background components were screened using recombinant expression of the His-tagged H7 protein to select specific cell lines targeting the H5 protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0028] 1.3 Screening of positive clones using indirect ELISA: H5-HA recombinant protein and control recombinant protein (H7, His tag) were coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a concentration of 1 μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 2% sucrose + 3% BSA per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and blotted dry. 50 μL of cell culture supernatant was added, and the plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewand, 1001SA) to stop the reaction. Measure the OD450nm value using a microplate reader. Select positive cell lines that react with the H5-HA recombinant protein but not with the control recombinant protein H7-HA for subsequent experiments.

[0029] Table 1: Screening Results of Monoclonal Antibodies

[0030] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0031] 2. Purification of monoclonal antibodies Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl (pH 9) and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS (pH 7.4) for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0032] The identification method is the same as the indirect ELISA detection described above. The purified antibody is diluted to 1 μg / ml, and the antibody-antibody binding reaction is identified. The results are as follows: Figure 1 As shown. According to Figure 1 It can be seen that the selected monoclonal antibody specifically binds to the H5-HA protein and does not react with the irrelevant antigen H7-HA, indicating that the monoclonal antibody has good specificity and can be used for subsequent testing.

[0033] 3. Colloidal gold pairing of H5 monoclonal antibodies Preparation of antibody-colloidal gold labeled complex.

[0034] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL (optimal), 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. After mixing, 5 μg of the H5 monoclonal antibody to be labeled was added to each tube, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and incubation at room temperature was continued for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.05M Tris + 1% BSA + 2% Sucrose + 0.2% NaCl + 0.5% N100, pH 9) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0035] 4. Preparation of test strips coated with different H5 monoclonal antibodies The selected H5 monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20mm × 300mm). Diluted monoclonal antibodies (diluent for scribing antibodies: 0.01M PB + 0.5% BSA + 0.05% Tween-20, pH 7.8, diluted to 1 mg / mL) were sprayed horizontally in a linear pattern using a scribing instrument. Each line was 0.8 μL / cm, forming a detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL in 0.01M PBS (pH 7.4), were then sprayed horizontally in a linear pattern at 6mm intervals, forming a control line (C line).

[0036] 5. Screening of paired monoclonal antibodies Nitrocellulose membranes streaked with different H5 monoclonal antibodies were individually paired with different colloidal gold-labeled monoclonal antibodies. H5 protein was diluted to 20 ng / mL for detection, while a mixed recombinant protein of H7 and H9 was diluted to 20 ng / mL as a negative control antigen for detection. Combinations showing the strongest color development for H5 protein and not reacting with the control antigen were screened. The screening results are shown in Table 2. Therefore, the optimal pairing for detecting recombinant H5 protein was determined to be 2B8 streaking and 6B5 gold labeling.

[0037] Table 2: Results of screening paired monoclonal antibodies using H5 recombinant protein

[0038] - indicates negative, meaning no color develops; + / ++ / +++ indicates positive, meaning a color reaction occurs. The more + signs there are, the deeper the color, indicating a stronger positive reaction.

[0039] Table 2 shows the screening results using H5-HA recombinant protein diluted to a concentration of 20 ng / ml as a positive antigen. The results for negative antigen and blank dilution were all negative and are not shown in Table 2. The results showed that the combination of 2B8 monoclonal antibody streaking and 6B5 monoclonal antibody labeled with gold produced the deepest staining of H5-HA recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody 2B8 as the capture antibody and monoclonal antibody 6B5 as the labeling antibody can specifically recognize H5-HA recombinant protein.

[0040] 6. Preparation and assembly of colloidal gold test strips Preparation of gold-labeled pads: A 6mm x 300mm glass fiber membrane RB65 was treated with Tris-HCl containing 0.5% BSA and 0.5% NP40 at pH 8. The prepared colloidal gold-labeled antibody was then uniformly dropped onto the glass fiber at a rate of 1200μL / pad. After air drying, it was dried at 37℃ for 2 hours for later use. See Figure 2 , Figure 2 This is a schematic diagram of the colloidal gold assembly. A 60mm x 300mm PVC backing plate is used as a support. Sample pads, gold-labeled pads (also called gold-labeled conjugation pads), nitrocellulose membranes, and absorbent paper are attached to this backing plate. The nitrocellulose membrane is coated with two lines and dried at 37℃ for 12 hours before use. The nitrocellulose membrane is coated with a detection line (monoclonal antibody 2B8) and a control line (goat anti-mouse IgG). The gold-labeled conjugation pad is coated with monoclonal antibody 6B5. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings. The sample pads are exposed at the sample application wells of the plastic casings, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip is now assembled.

[0041] 7. Test strip specificity test Recombinant protein samples: H1 recombinant protein (A / California / 04 / 2009 HA), H3 recombinant protein (A / Perth / 6 / 2009 HA), H5 recombinant protein (A / Anhui / 1 / 2005 HA, 11048-V08B, Yiqiao Shenzhou), H7 recombinant protein (A / Shanghai / 1 / 2013 HA, 40104-V08B, Yiqiao Shenzhou), H9 recombinant protein (A / chicken / Hong Kong / G9 / 1997 HA, 40036-V08H, Yiqiao Shenzhou), influenza A virus NP protein (expressed by the inventor using E. coli, FluA-NP), and Newcastle disease virus NP protein (expressed by the inventor using E. coli, NDV-NP) were diluted to 100 ng / mL with sample diluent for detection.

[0042] Virus or vaccine samples: inactivated PR8 mouse lung-adapted strain (H1N1), avian influenza H9 subtype inactivated vaccine (NJ01 strain, Shandong Huahong), Newcastle disease virus (NDV) live vaccine (LaSota strain, Qingdao Yibang), and infectious bronchitis (IBV) live vaccine (H120 strain, Qingdao Yibang). The vaccines were reconstituted according to the instructions and then diluted 10-fold with the sample diluent before testing.

[0043] Add 80 μL of the diluted sample to the sample well of the test strip. Simultaneously, add another 80 μL of the diluent to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid.

[0044] Figure 3 , Figure 4 The results showed that the test strip could detect the H5 recombinant protein very well, and there was no cross-reaction with other recombinant proteins such as H1, H3, H7, H9, influenza A NP, Newcastle disease virus NP, as well as non-target antigens such as PR8 strain, avian influenza H9 vaccine, Newcastle disease vaccine, and infectious bronchitis virus vaccine, indicating that the test strip has good specificity.

[0045] The amino acid sequence of the NP protein of influenza A virus is shown in SEQ ID NO.21: MASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWMRELILYDKEEIRRVWRQANNGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETMDSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDS。

[0046] The nucleotide of the influenza A virus NP protein is as shown in SEQ ID NO.22:

[0047] The amino acid profile of the Newcastle disease virus NP protein is shown in SEQ ID NO. 23: MSSVFDEYEQLLASQTRPNGSHGGGEKGSTLKVEVPVFTLNSDDPEDRWNFAVFCLRIAVSEDANKPLRQGALISLLCTHSQVMRNHVALAGRQNEATLAILEIDGFSNGVPQFNNRSGVSE ERAQRFMMIAGSLPRACSNGTPFVTAGVEDDAPEDITTDTLERILSIQVQVWVTVAKAMTAYETADESETRRINKYMQQGRVQKRCILHPVCRSAIQLTIRQSLAVRIFLVSELKRGRNTAGGT STYYNLVGDVDSYIRNTGLTAFFLTLKYGINTKTSVLALSSLSGDIQKMKQLMRLYRMKGENAPYMTLLGDSDQMSFAPAEYAQLYSFAMGMASVLDKGTVKYQFARDFMSTSFWRLGVEYA QAQGSSINEDMAAELKLTPAVRRGLAAAAQRVSEDASNMDLPTQQAGVLTGLSDNTPPAQPGGSKPQGSADGNEGETQFLDLMRAVANSMRDAPNSAQGSSQPAPPPTPGGNQDNDTDWGY.

[0048] The NP protein nucleotides of Newcastle disease virus are shown in SEQ ID NO. 24:

[0049] 8. Sensitivity test of test strips The H5 recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.1 ng / mL before detection. Figure 5 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 1 ng / mL, while the blank dilution, i.e. the sample dilution (0.01M Tris + 2% Sucrose + 0.9% NaCl + 0.1% Tween-20 + 0.1% N100 + 0.5% BSA, pH 8.4) (0 ng / mL), did not show color development, indicating that the test strip card's limit of detection for H5 recombinant protein is 1 ng / mL.

[0050] 9. Monoclonal antibody binding activity assay Based on the selection of potential paired antibodies using colloidal gold, the selected paired monoclonal antibodies and other murine unrelated monoclonal antibodies were serially diluted (to concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL, respectively) using the aforementioned indirect ELISA method to evaluate their binding activity with the H5-HA recombinant protein. Influenza A virus NP murine monoclonal antibody 1B5 was used as a negative control to exclude the influence of non-specific binding. Results are shown below. Figure 6 . Figure 6 In the middle, "Ctrl" represents the negative control influenza A virus NP mouse monoclonal antibody 1B5 (inventor's commercially available product, M100014).

[0051] 10. Gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0052] Standard gold monoclonal antibody 6B5 Light chain variable region nucleotide sequence: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 6B5 is shown in SEQ ID NO.20: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCGACCTG GCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0053] Light chain variable region amino acid sequence: The amino acid sequence of the variable region of the light chain of the monoclonal antibody 6B5 is shown in SEQ ID NO.16.

[0054] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSDLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK.

[0055] Light chain CDR area annotation: The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 6B5 is shown in SEQ ID NO.10: CDR-L1: SASSVSYMY; The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 6B5 is shown in SEQ ID NO.11: CDR-L2: RTSDLAS; The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 6B5 is shown in SEQ ID NO.12: CDR-L3: QQYHSYPLT.

[0056] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.19: GAGGTGCAGCTGCAGCAGTCTGGACCTGGCCTGGTGGCACCTCACAGAGCCTGTCCATCACATGCACTGTCTCTGGGTTCTCATTAGCCATATATAGTGTTCACTGGGTTCGCCAGCCTCCTGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGGTGGTGGAAGCACAGACTATAA TTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAAGAGGGGACTATGGTAACTACGAGTTTGCTTACTGGGGCCGAGGGACTCTGGTCACTGTCTCTGCA.

[0057] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.15; EVQLQQSGPGLVAPSQSLSITCTVSGFSLAIYSVHWVRQPPGKGLEWLGMIWGGGGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARRGDYGNYEFAYWGRGTLVTVSA.

[0058] Heavy chain CDR region annotation: The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 6B5 is shown in SEQ ID NO.7: CDR-H1: IYSVH; The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 6B5 is shown in SEQ ID NO. 8: CDR-H2: MIWGGGSTDYNSALKS; The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 6B5 is shown in SEQ ID NO.9: CDR-H3:RGDYGNYEFAY.

[0059] Membrane-scraped monoclonal antibody 2B8 Light chain variable region nucleotide sequence: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 2B8 is shown in SEQ ID NO.18.

[0060] GACATCCAGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAGGTGTAAGTTACATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTC TGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCCGAAGATGGTGCCACTTATTACTGCCAGCAAAGGAGTAGTTACCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTG.

[0061] Light chain variable region amino acid sequence: The amino acid sequence of the variable region of the light chain of the monoclonal antibody 2B8 is shown in SEQ ID NO.14: DIQMTQSPAIMSASPGEKVTITCSASSGVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDGATYYCQQRSSYPPTFGAGTKLELKRTV.

[0062] Light chain CDR area annotation: The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody 2B8 is shown in SEQ ID NO.4: CDR-L1: SASGVSYMH; The amino acid sequence of the light chain variable region of the monoclonal antibody 2B8, which contains CDR-L2, is shown in SEQ ID NO. 5: CDR-L2: STSNLAS; The amino acid sequence of the light chain variable region CDR-L3 of the monoclonal antibody 2B8 is shown in SEQ ID NO. 6: CDR-L3: QQRSSYPPT.

[0063] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO.17: GAAGTGCAGCTGTTGGAGACTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAGCCATTAGTGATGGTGGTAGTTACACCTACTATCC AGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTATTTCTGTGCAAGAGATCACTCCGGTAATAGTTACTATGTTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0064] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO.13: EVQLLETGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVAAISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYFCARDHSGNSYYVLDYWGQGTSVTVSS.

[0065] Heavy chain CDR region annotation: The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 2B8 is shown in SEQ ID NO.1: CDR-H1: DYYMY; The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 2B8 is shown in SEQ ID NO.2: CDR-H2: AISDGGSYTYYPDSVKG; The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 2B8 is shown in SEQ ID NO.3: CDR-H3: DHSGNSYYVLDY.

Claims

1. A monoclonal antibody combination targeting the hemagglutinin protein of avian influenza virus H5 subtype, characterized in that, The monoclonal antibody combination includes monoclonal antibody 2B8 and monoclonal antibody 6B5. The heavy chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination against the hemagglutinin protein of avian influenza virus H5 subtype according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 2B8 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 6B5 includes three complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

3. The monoclonal antibody combination against the H5 subtype hemagglutinin protein of avian influenza virus according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO.

14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination against the H5 subtype hemagglutinin protein of avian influenza virus according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2B8 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination against the hemagglutinin protein of avian influenza virus H5 subtype according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6B5 is shown in SEQ ID NO.

20.

6. Use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for specifically recognizing the hemagglutinin protein of avian influenza virus H5 subtype.

7. The use according to claim 6, characterized in that, The tools include colloidal gold test strips, reagents, kits, and antibody chips.

8. The use according to claim 7, characterized in that, The colloidal gold test strip uses monoclonal antibody 2B8 as the capture antibody and monoclonal antibody 6B5 as the labeling antibody.

9. The use according to claim 8, characterized in that, The colloidal gold test strip includes a nitrocellulose membrane, a gold-labeled conjugate pad, a sample pad, and absorbent paper attached to a backing plate.

10. The use according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 2B8, the control line is coated with goat anti-mouse IgG, and the gold-labeled binding pad is coated with monoclonal antibody 6B5.

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