Monoclonal antibody assemblies specifically recognizing hemagglutinin protein of avian influenza virus H6 subtype and their applications

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a monoclonal antibody combination that specifically recognizes the hemagglutinin protein of the H6 subtype of avian influenza virus and its applications. The antibody combination consists of monoclonal antibodies 2D12 and 4G3, targeting different epitopes of the H6 hemagglutinin protein, and possesses clearly defined heavy and light chain variable region complementarity-determining region sequences, as shown in SEQ ID NO. 1-12, respectively. The antibody combination exhibits high specificity and affinity, effectively avoiding cross-reactivity with other influenza subtypes such as H1, H5, H7, and H9, as well as common avian pathogens. The colloidal gold test strip constructed based on this antibody combination is highly sensitive and easy to operate, suitable for in vitro detection and epidemiological screening of the H6 subtype of avian influenza virus. It solves the false positive problem caused by cross-reactivity in existing technologies, providing a reliable technical means for the monitoring and quarantine of the H6 subtype of virus.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies that specifically recognize the hemagglutinin protein of the H6 subtype of avian influenza virus and its applications. Background Technology

[0002] Avian influenza virus (AIV) belongs to the genus *Influenza A* of the family Orthomyxoviridae and is an important zoonotic pathogen. Its genome consists of eight segmented single-stranded negative-sense RNA molecules that encode various structural and non-structural proteins. Based on the antigenic differences of two major glycoproteins on the viral envelope—hemagglutinin (HA) and neuraminidase (NA)—it can be divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). Further based on the sequence characteristics and structural differences of the HA proteins, these 18 subtypes can be divided into two evolutionary branches: Group 1 (including H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18) and Group 2 (including H3, H4, H7, H10, H14, H15). Waterfowl, wild birds, and migratory birds are considered natural hosts of AIV, but some subtypes, such as H5, H6, H7, H9, and H10, have been shown to be able to cross the species barrier and infect humans, posing a continued threat to public health and the safety of aquaculture production.

[0003] Based on their pathogenicity, AIVs can be divided into highly pathogenic avian influenza viruses (HPAIV) and low pathogenic avian influenza viruses (LPAIV). The amino acid sequence of the hemagglutinin cleavage site directly affects its pathogenicity. The molecular characteristics of the H6 subtype AIV are consistent with LPAIV; its HA cleavage site is mostly a single-base motif, lacking multi-base insertion sequences. It can only be recognized and cleaved by trypsin-like proteases in the respiratory and digestive tracts, thus limiting the site of viral replication. Therefore, H6 subtype avian influenza virus infection usually presents as asymptomatic, making it difficult to detect in a timely manner.

[0004] The H6 subtype of avian influenza virus was first isolated in North American poultry and has since continued to circulate in wild birds, waterfowl, and poultry worldwide, gradually becoming a widespread and long-term stable low-pathogenic subtype. Although its pathogenicity is low, this subtype exhibits strong genetic variation, with dynamic gene recombination occurring between it and other co-circulating AIV subtypes, resulting in 16 different genotypes. A / teal / HK / W312 / 97 (H6N1) has been identified as a potential precursor strain of A / HongKong / 156 / 1997 (H5N1), and some H6N× strains have been identified as donor strains for H5 and H9N2 subtype avian influenza viruses. These gene reassortment events may promote the emergence of novel influenza viruses, thus posing a potential threat to animal and human health.

[0005] Because H6 subtype infections are mostly asymptomatic and difficult to detect during poultry farming, the virus can continue to spread in flocks and provide conditions for reassortment of other subtypes, increasing the complexity of the epidemic. Therefore, establishing rapid and specific detection methods for the H6 subtype is of great significance for early detection and risk warning.

[0006] Currently, the detection of avian influenza virus mainly relies on viral nucleic acid detection and antigen immunological detection methods. Nucleic acid detection has high sensitivity, but it has strict requirements for experimental conditions and operation. In contrast, viral antigen immunological detection has a relatively simple operation procedure and is more suitable for on-site screening and routine monitoring. However, some existing immunological detection products still have insufficient specificity and are susceptible to cross-reactivity between different subtypes.

[0007] Hemagglutinin (HA) protein is a major antigenic component on the surface of avian influenza viruses, playing a crucial role in viral adsorption and invasion of host cells, and is also a key antigen determining viral subtype specificity. While HA proteins differ in structure and antigenic epitopes among different subtypes, they still exhibit a degree of homology within the same evolutionary group, particularly in certain conserved regions where they share similar antigenic characteristics. Therefore, when detecting specific subtypes, it is necessary to utilize their specific epitopes for identification while avoiding cross-reaction interference from conserved regions. Summary of the Invention

[0008] This invention provides a monoclonal antibody ensemble that specifically recognizes the hemagglutinin protein of the H6 subtype of avian influenza virus and its application. The antibody ensemble consists of monoclonal antibodies 2D12 and 4G3, targeting different epitopes of the H6 hemagglutinin protein, and possesses clearly defined heavy and light chain variable region complementarity-determining region sequences, as shown in SEQ ID NO. 1-12, respectively. The antibody ensemble exhibits high specificity and affinity, effectively avoiding cross-reactivity with other influenza subtypes such as H1, H5, H7, and H9, as well as common avian pathogens. The colloidal gold test strip constructed based on this antibody ensemble is highly sensitive and easy to operate, suitable for in vitro detection and epidemiological screening of the H6 subtype of avian influenza virus. It solves the false positive problem caused by cross-reactivity in existing technologies, providing a reliable technical means for the monitoring and quarantine of the H6 subtype of virus.

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, this application provides a monoclonal antibody ensemble that specifically recognizes the hemagglutinin protein of the H6 subtype of avian influenza virus, the monoclonal antibody ensemble comprising monoclonal antibody 2D12 and monoclonal antibody 4G3.

[0011] The heavy chain variable region of the monoclonal antibody 2D12 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0012] The light chain variable region of the monoclonal antibody 2D12 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0013] The heavy chain variable region of the monoclonal antibody 4G3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0014] The light chain variable region of the monoclonal antibody 4G3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0015] In some embodiments, the heavy chain variable region of the monoclonal antibody 2D12 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.

[0016] The light chain variable region of the monoclonal antibody 2D12 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.

[0017] The heavy chain variable region of the monoclonal antibody 4G3 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.

[0018] The light chain variable region of the monoclonal antibody 4G3 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.

[0019] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO. 14.

[0020] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.16.

[0021] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO. 18.

[0022] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO. 20.

[0023] 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 H6 subtype of avian influenza virus.

[0024] In some embodiments, the tool includes colloidal gold test strips, reagents, kits, and antibody chips; the tool is used to detect avian influenza virus H6 hemagglutinin protein in samples selected from any of environmental samples, swab samples, cell culture supernatants, and tissue homogenates, and the identification is not used for disease diagnosis.

[0025] 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.

[0026] 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.

[0027] In some embodiments, the colloidal gold test strip uses monoclonal antibody 2D12 as the capture antibody and monoclonal antibody 4G3 as the labeling antibody.

[0028] 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.

[0029] In some embodiments, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 2D12, the control line is coated with goat anti-mouse IgG, and the gold-labeled binding pad is coated with monoclonal antibody 4G3.

[0030] Beneficial effects:

[0031] This application provides a monoclonal antibody combination that specifically recognizes the hemagglutinin protein of the H6 subtype of avian influenza virus. The monoclonal antibody combination consists of monoclonal antibody 2D12 and monoclonal antibody 4G3, which can specifically recognize the hemagglutinin (HA) protein of the H6 subtype of avian influenza virus. The complementarity-determining region (CDR) sequences of the heavy chain and light chain variable regions of 2D12 and 4G3 are clearly defined (as shown in SEQ ID NO.1-12, respectively), ensuring the high affinity and specific binding ability of the antibody combination. This antibody combination can effectively avoid cross-reactions with other influenza subtypes such as H1, H5, H7, and H9, as well as common avian pathogens, significantly improving the accuracy and sensitivity of detection. The colloidal gold test strip constructed based on the antibody combination is simple and rapid to operate, suitable for on-site screening of the H6 subtype of avian influenza, and solves the problem that existing immunoassay products are easily interfered with by cross-reactions between different subtypes. It provides a reliable technical means for the early detection and risk warning of the H6 subtype of avian influenza virus, and at the same time provides a stable and reliable biometric tool for the rapid and accurate detection of the H6 avian influenza virus. Attached Figure Description

[0032] 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.

[0033] Figure 1 The image shows the identification results of the purified monoclonal antibody.

[0034] Figure 2 This is a schematic diagram of colloidal gold assembly.

[0035] Figure 3 The results show the specificity of H6-HA monoclonal antibody colloidal gold test strips for multiple avian influenza HA subtypes and other viral recombinant proteins.

[0036] Figure 4 This refers to the specific detection results of the H6-HA monoclonal antibody colloidal gold test strip for virus or vaccine antigens.

[0037] Figure 5 This is a graph showing the sensitivity test results of the test strips;

[0038] Figure 6 This is a graph showing the results of antibody binding identification. Detailed Implementation

[0039] 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.

[0040] Developing specific antibodies targeting the HA protein of H6 subtype avian influenza virus has practical application value in improving the subtype recognition capability of immunoassay methods. This invention utilizes hybridoma technology to prepare monoclonal antibodies that specifically recognize the HA protein of H6 subtype avian influenza virus and applies them to a colloidal gold immunochromatographic detection system. Verification showed that the monoclonal antibody combination exhibited good detection sensitivity and specificity against H6 subtype avian influenza virus-related antigens, meeting the application requirements for rapid detection and on-site screening, and providing a raw material basis for the development of diagnostic reagents for H6 subtype avian influenza virus.

[0041] The avian influenza virus H6 hemagglutinin protein, also known as the avian influenza virus H6-HA protein, or simply H6-HA protein, includes recombinant H6-HA protein and natural H6-HA protein.

[0042] The detection method described in this application is not intended for disease diagnosis and treatment.

[0043] Example 1

[0044] 1. Screening of H6-HA monoclonal antibodies

[0045] 1.1 Mouse Immunization

[0046] Mice were immunized with H6 recombinant protein (A / chicken / Guangdong / C273 / 2011, 40398-V08B, Sinopharm). Monoclonal antibody screening was performed using H7 recombinant protein (A / Shanghai / 1 / 2013 HA, 40104-V08B, Sinopharm) and H9 recombinant protein (A / chicken / Hong Kong / G9 / 1997 HA, 40036-V08H, Sinopharm) with the same His tag as the reverse screening antigens. H6 and H9 belong to Group 1, and the HA protein structure shows high homology and certain similarities in conserved regions. Therefore, H9-HA was selected as the same group reverse screening antigen to exclude antibodies that recognize the conserved HA domain. Meanwhile, the H7 subtype belongs to Group 2 and differs significantly from H6 in the overall structure and epitope distribution of the HA protein. Introducing H7-HA as a cross-group reverse screening antigen can further eliminate non-specific antibodies with broad-spectrum recognition capabilities for the HA protein. Furthermore, the H9 subtype is widespread in poultry, posing a high risk of cross-interference in actual testing. Including H9-HA in the reverse screening system helps avoid screening for antibodies that cross-react with common prevalent subtypes. Through combined H9 and H7 reverse screening, antibodies are rigorously screened based on both structural homology and antigenic differences, thereby obtaining monoclonal antibodies specific to the epitopes of the H6 subtype.

[0047] Specifically, purified H6-HA recombinant 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 20 μg / mouse. In weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. In week 6, the spleen of mice was directly injected with insulin at a dose of 5 μg / mouse. 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 H6-HA recombinant protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0048] 1.2 Screening of hybridoma cells

[0049] 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 up the bottom of the well, clones positive for both H6-HA recombinant protein were obtained by indirect ELISA. Since the immunogen contained a His tag, background components needed to be screened to identify specific cell lines targeting H6-HA recombinant protein. Positive cells were cloned to monoclonal status using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0050] 1.3 Screening of positive clones using indirect ELISA method

[0051] H6-HA, H7-HA, and H9-HA recombinant proteins 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 IgG (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. Incubate at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with H6-HA recombinant protein but not with control H7-HA and H9-HA recombinant proteins were selected for subsequent experiments.

[0052] Table 1: Screening results of monoclonal antibodies

[0053]

[0054] 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.

[0055] 2. Purification and Identification of Monoclonal Antibodies

[0056] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, dilute it 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), and filter it through a 0.22 μm filter. Pump the filtered sample slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. Connect the column to a protein purification instrument and wash with binding buffer for 5-10 column volumes until the UV absorption peak flattens. Then elute with elution buffer (0.1 M glycine, pH 2.7), collect the elution peak, and adjust the collected sample to neutral with 1 M Tris-HCl (pH 9). Transfer the solution to a dialysis bag (MW: 8000-14000) and dialyze in 20 mM PBS (pH 7.4) at 2-8°C for 16 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody.

[0057] The purified monoclonal antibody was diluted 1 μg / ml, and its binding activity with H6-HA recombinant protein and irrelevant antigen H9 recombinant protein was detected by indirect ELISA. See below for specific results. Figure 1 .according to Figure 1 It can be seen that the selected monoclonal antibody specifically binds to the H6-HA protein and does not react with irrelevant antigens H7-HA and H9-HA proteins, indicating that the monoclonal antibody has good specificity and can be used for subsequent testing.

[0058] 3. Preparation of test strips coated with different monoclonal antibodies against H6-HA

[0059] The selected H6-HA monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (antibody dilution solution: 0.01 M PB + 0.1% BSA + 0.5% Trehalose + 0.2% Tween-20, pH 7.8) were horizontally scribed in a linear fashion on each membrane, with a scribing volume of 0.8 μL / cm, forming the detection line (T line). Goat anti-mouse IgG antibody, diluted in 0.01 M PBS at pH 7.4 to a concentration of 1 mg / mL, was then scribed horizontally in a linear fashion at a volume of 0.8 μL / cm onto the nitrocellulose membrane, forming the control line (C line).

[0060] 4. Colloidal gold pairing of H6-HA monoclonal antibodies

[0061] Preparation of antibody-colloidal gold labeled complex.

[0062] 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, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. The optimal conjugation effect was ultimately found at 5 μL. After mixing, 5 μg of the H6-HA monoclonal antibody to be labeled was added, 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 + 0.15M NaCl + 0.5% N100 + 5% Sucrose + 1% BSA + 0.1% PC300, pH 8.4) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0063] 5. Screening of paired monoclonal antibodies

[0064] Nitrocellulose membranes marked with different monoclonal antibodies against H6-HA were individually paired with different colloidal gold-labeled monoclonal antibodies. The H6-HA recombinant protein was diluted to 20 ng / mL for detection, while the H7 and H9 recombinant proteins were diluted to 20 ng / mL as negative antigens for detection. Combinations showing the strongest color development for H6-HA protein and not reacting with the control protein were selected. The screening results are shown in Table 2.

[0065] Table 2: Results of screening paired monoclonal antibodies using H6-HA recombinant protein

[0066]

[0067] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0068] Table 2 shows the screening results using H6-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 2D12 monoclonal antibody streaking combined with 4G3 monoclonal antibody labeled with gold produced the deepest staining of H6-HA recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-2D12 as the capture antibody and monoclonal antibody 4G3 as the labeling antibody can specifically recognize H6-HA recombinant protein.

[0069] 6. Preparation and assembly of colloidal gold test strips

[0070] Preparation of gold-labeled pads: Using a 6mm×300mm glass fiber membrane, the prepared colloidal gold-labeled antibody was evenly dropped onto the glass fiber at a rate of 1200μL / strip, allowed to air dry naturally, and then dried at 37℃ for 2 hours for later use.

[0071] See Figure 2 , Figure 2 This is a schematic diagram of the colloidal gold assembly. A 60mm × 300mm PVC backing plate is used as a support. Sample pads, gold-labeled pads (also called gold-labeled binding pads), nitrocellulose membranes, and absorbent paper are attached to the 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 2D12 scratch) and a control line (goat anti-mouse IgG). The gold-labeled pad is coated with monoclonal antibody 4G3. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with a colloidal gold plastic casing. The sample pad is exposed at the sample application well of the plastic casing, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip is now assembled.

[0072] 7. Test strip specificity test

[0073] Recombinant protein samples: H1 recombinant protein (A / California / 04 / 2009 HA, 11055-V08B, Sinopharm), H5 recombinant protein (A / Anhui / 1 / 2005 HA, 11048-V08B, Sinopharm), H6 recombinant protein, H7 recombinant protein, H9 recombinant protein (A / chicken / Hong Kong / G9 / 1997 HA, 40036-V08H, Sinopharm), H10 recombinant protein (A / Jiangxi-Donghu / 346 / 2013 HA, 40359-V08B, Sinopharm), 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.

[0074] The amino acid sequence of the NP protein of influenza A virus is shown in SEQ ID NO.21:

[0075] MASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWMRELILYDKEEIRRVWRQAN NGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPG NAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETM DSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDS.

[0076] The NP protein nucleotides of influenza A virus are shown in SEQ ID NO.22:

[0077]

[0078] The amino acid profile of the Newcastle disease virus NP protein is shown in SEQ ID NO. 23:

[0079] MSSVFDEYEQLLASQTRPNGSHGGGEKGSTLKVEVPVFTLNSDDPEDRWNFAVFCLRIAVSEDANKPLRQGALISLLCTHSQVMRNHVALAGRQNEATLAILEIDGFSNGVPQFNNRSGVSE ERAQRFMMIAGSLPRACSNGTPFVTAGVEDDAPEDITTDTLERILSIQVQVWVTVAKAMTAYETADESETRRINKYMQQGRVQKRCILHPVCRSAIQLTIRQSLAVRIFLVSELKRGRNTAGGT STYYNLVGDVDSYIRNTGLTAFFLTLKYGINTKTSVLALSSLSGDIQKMKQLMRLYRMKGENAPYMTLLGDSDQMSFAPAEYAQLYSFAMGMASVLDKGTVKYQFARDFMSTSFWRLGVEYA QAQGSSINEDMAAELKLTPAVRRGLAAAAQRVSEDASNMDLPTQQAGVLTGLSDNTPPAQPGGSKPQGSADGNEGETQFLDLMRAVANSMRDAPNSAQGSSQPAPPPTPGGNQDNDTDWGY.

[0080] The NP protein nucleotides of Newcastle disease virus are shown in SEQ ID NO. 24:

[0081]

[0082] Virus or vaccine antigen samples: inactivated PR8 mouse lung-adapted strain (H1N1), avian influenza H5+H7 inactivated vaccine (H5-Re8 strain, H7-Re1 strain, Shandong Huahong), avian influenza H9 subtype inactivated vaccine (NJ01 strain, Shandong Huahong), Newcastle disease virus (NDV) live vaccine (LaSota strain, Qingdao Yibang), infectious bronchitis virus (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.

[0083] 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.

[0084] like Figure 3 , Figure 4 As shown, the test strip can effectively detect recombinant H6-HA protein, but does not react with other HA subtype recombinant proteins such as H1, H5, H7, H9, and H10, as well as NP protein of influenza A virus and Newcastle disease virus. It also shows no cross-reactivity with H1N1 mouse lung-adapted virus, avian influenza H5+H7 bivalent inactivated vaccine, avian influenza H9 subtype inactivated vaccine, Newcastle disease virus vaccine, and infectious bronchitis virus vaccine, indicating that the test strip has good specificity.

[0085] 8. Sensitivity test of test strips

[0086] The H6-HA recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 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 + 1% BSA + 0.9% NaCl + 0.1% Tween 20, pH 8.0) (0 ng / mL), did not show color development, indicating that the limit of detection for H6-HA recombinant protein on the test strip is 1 ng / mL.

[0087] 9. Monoclonal antibody binding activity assay

[0088] Based on the selection of paired antibodies using colloidal gold, the selected paired monoclonal antibodies and other murine-derived 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 H6-HA recombinant protein. Results are shown below. Figure 6 , Figure 6 In the text, “Ctrl” represents the negative control mouse monoclonal antibody 1B5 for the NP protein of influenza A virus (inventor's commercially available product, M100014).

[0089] This invention utilizes hybridoma technology to screen and obtain a pair of monoclonal antibodies capable of specifically recognizing the H6-HA recombinant expression antigen. This combined antibody pair can efficiently recognize the H6-HA target protein derived from a recombinant baculovirus expression system, exhibiting good specificity and sensitivity. This invention applies the monoclonal antibody combination to an immunoassay platform, constructing a rapid test strip or test card based on colloidal gold immunochromatography technology. This test strip exhibits high sensitivity to the H6-HA recombinant expression antigen and shows no cross-reactivity with other proteins, making it suitable for rapid, on-site detection.

[0090] 10. Gene sequence of monoclonal antibodies

[0091] 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 PCR in three rounds. The PCR products were purified by gel excision 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.

[0092] The sequence of the membrane-scraped monoclonal antibody 2D12:

[0093] Light chain variable region nucleotide sequence:

[0094] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 2D12 is shown in SEQ ID NO.18:

[0095] GACATCCAGATGATTCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTGTGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACAAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATC CACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGCAAGCAATCTTATGATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0096] Light chain variable region amino acid sequence:

[0097] The amino acid sequence of the variable region of the light chain of the monoclonal antibody 2D12 is shown in SEQ ID NO.14:

[0098] DIQMIQSPSSLAVSAGEKVTVSCKSSQSLLNSRTRKNYLAWYQQKQGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYDLYTFGGGTKLEIKRTV.

[0099] Light chain CDR area annotation:

[0100] The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody 2D12 is shown in SEQ ID NO.4:

[0101] CDR-L1: KSSQSLLNSRTRKNYLA;

[0102] The amino acid sequence of the light chain variable region CDR-L2 of the monoclonal antibody 2D12 is shown in SEQ ID NO.5:

[0103] CDR-L2: WASTRES;

[0104] The amino acid sequence of the light chain variable region CDR-L3 of the monoclonal antibody 2D12 is shown in SEQ ID NO. 6:

[0105] CDR-L3: KQSYDLYT.

[0106] Heavy chain variable region nucleotide sequence:

[0107] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO.17:

[0108] CAGGCTTATCTACAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTCTGGCTACACATTCACCACCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGTACT AACTACGATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGATAACTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0109] Heavy chain variable region amino acid sequence:

[0110] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO.13:

[0111] QAYLQQSGSELVRPGASVKLSCKASGYTFTTYWMHWVKQRPGQGLEWIGNIYPGSGSTNYDEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTRDNYYFDYWGQGTTLTVSS.

[0112] Heavy chain CDR region annotation:

[0113] The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 2D12 is shown in SEQ ID NO.1:

[0114] CDR-H1: TYWMH;

[0115] The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 2D12 is shown in SEQ ID NO.2:

[0116] CDR-H2: NIYPGSGSTNYDEKFKS;

[0117] The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 2D12 is shown in SEQ ID NO.3:

[0118] CDR-H3: DNYYFDY.

[0119] Gold monoclonal antibody 4G3 sequence

[0120] Light chain variable region nucleotide sequence:

[0121] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 4G3 is shown in SEQ ID NO.20:

[0122] AAATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTTATAGTTTTATGAACTGGTACCAACAGAGACCAGGACAGCCACCCAAACTCCTCATCTATGGAACTTCCAAT CTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCCGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCACCAGAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTACGGTG.

[0123] Light chain variable region amino acid sequence:

[0124] The amino acid sequence of the variable region of the light chain of the monoclonal antibody 4G3 is shown in SEQ ID NO.16:

[0125] NIVLTQSPASLAVSLGQRATISCKASQSVDFDGYSFMNWYQQRPGQPPKLLIYGTSNLES

[0126] GIPARFSGRGSGTDFTLNIHPVEEEDAATYYCHQSNEDPYTFGGGTKLEIKRTV.

[0127] Light chain CDR area annotation:

[0128] The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody 4G3 is shown in SEQ ID NO.10:

[0129] CDR-L1: KASQSVDFDGYSFMN;

[0130] The amino acid sequence of the light chain variable region CDR-L2 of the monoclonal antibody 4G3 is shown in SEQ ID NO.11:

[0131] CDR-L2: GTSNLES;

[0132] The amino acid sequence of the light chain variable region CDR-L3 of the monoclonal antibody 4G3 is shown in SEQ ID NO.12:

[0133] CDR-L3: HQSNEDPYT.

[0134] Heavy chain variable region nucleotide sequence:

[0135] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.19:

[0136] CAGCGTGAGCTGCAGCAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAATTACACCTACTAT CCAGACAGTGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGTCCTTGTATTACTGTGCAAGGCCCTATGGTAACTACTTGTACTACCTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

[0137] Heavy chain variable region amino acid sequence:

[0138] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.15:

[0139] QRELQQSGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISSGGNYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTSLYYCARPYGNYLYYLDYWGQGTTLTVSS.

[0140] Heavy chain CDR region annotation:

[0141] The heavy chain variable region of the monoclonal antibody 4G3 includes the amino acid sequence of CDR-H1 as shown in SEQ ID NO.7:

[0142] CDR-H1: SYDMS;

[0143] The heavy chain variable region of the monoclonal antibody 4G3 includes the amino acid sequence of CDR-H2 as shown in SEQ ID NO. 8:

[0144] CDR-H2: TISSGGNYTYYPDSVKG;

[0145] The heavy chain variable region of the monoclonal antibody 4G3 includes the amino acid sequence of CDR-H3 as shown in SEQ ID NO. 9:

[0146] CDR-H3: PYGNYLYYLDY.

Claims

1. A monoclonal antibody combination that specifically recognizes the hemagglutinin protein of avian influenza virus H6 subtype, characterized in that, The heavy chain variable region of the monoclonal antibody 2D12 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 2D12 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 4G3 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 4G3 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.

2. The monoclonal antibody combination specifically recognizing the hemagglutinin protein of avian influenza virus H6 subtype according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO.

14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.

16.

3. The monoclonal antibody combination specifically recognizing the hemagglutinin protein of avian influenza virus H6 subtype according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D12 is shown in SEQ ID NO.

18.

4. The monoclonal antibody combination specifically recognizing the hemagglutinin protein of avian influenza virus H6 subtype according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4G3 is shown in SEQ ID NO.

20.

5. Use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for specifically recognizing the H6 subtype hemagglutinin protein of avian influenza virus, The tools include colloidal gold test strips, reagents, kits, and antibody chips.

6. The use according to claim 5, characterized in that, The colloidal gold test strip uses monoclonal antibody 2D12 as the capture antibody and monoclonal antibody 4G3 as the labeling antibody.

7. The use according to claim 6, 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.

8. The use according to claim 7, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 2D12, the control line is coated with goat anti-mouse IgG, and the gold-labeled binding pad is coated with monoclonal antibody 4G3.

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