Monoclonal antibody combination against hemagglutinin protein of avian influenza virus h9 subtype and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a combination of monoclonal antibodies against hemagglutinin protein of avian influenza virus H9 subtype and application thereof. The combination comprises monoclonal antibodies 5E2 and 1F3, the CDR sequences of the heavy chain and light chain variable regions of which are clear, and are shown as SEQ ID NO. 1-12 respectively, ensuring high affinity and specific recognition of H9-HA protein. The antibody combination is suitable for constructing a colloidal gold immunochromatographic detection test strip, wherein 5E2 is used as a capture antibody for membrane drawing, and 1F3 is used as a labeled antibody for gold labeling. The technical scheme effectively solves the problem of cross-reaction with other HA subtypes (such as H5 and H7) caused by poor antibody specificity of the existing detection means. The sensitivity of the test strip to H9-HA recombinant protein can reach 1 ng / mL, and there is no cross-reaction, so the test strip is suitable for on-site rapid screening and large-scale monitoring of avian influenza virus H9 subtype.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the combination and application of monoclonal antibodies against the H9 subtype hemagglutinin protein of avian influenza virus. Background Technology

[0002] Avian influenza virus (AIV) belongs to the Orthomyxoviridae family and can infect humans, poultry, and various wild animals, with wild waterfowl considered the primary long-term host. The virus can be classified into several subtypes based on antigenic differences in its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Among them, the H9N2 subtype of avian influenza virus is the most widespread and prevalent low-pathogenic avian influenza virus in the world. This subtype is mainly transmitted through the respiratory and digestive routes, can continuously spread among poultry flocks through direct or indirect contact, and has the potential to infect other mammals and humans.

[0003] In nature, the H9 subtype influenza virus can combine with nine known NA subtypes (N1-N9). Of the approximately 9,500 independent H9 hemagglutinin (HA) sequences indexed in public databases, over 75% (approximately 7,200) co-occur with N2 subtype NA sequences. This indicates a significant preferential pairing and co-evolutionary relationship between H9 and N2 gene segments, and this combination exhibits a persistent prevalence.

[0004] Hemagglutinin (HA) is the most important glycoprotein on the surface of influenza viruses, playing a crucial role in viral adsorption and entry into host cells. It is also a major antigen determining viral subtype specificity. Based on the antigenic characteristics of the HA protein, influenza A viruses can be 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). The H9 subtype belongs to Group 1 and shares some structural homology with subtypes such as H5, but its epidemiological characteristics and pathogenic mechanisms differ significantly.

[0005] Compared to the highly pathogenic H5 and H7 subtypes, the H9 subtype of avian influenza virus typically exhibits low pathogenicity, but its potential risks cannot be ignored. On the one hand, the H9 subtype virus can undergo gene reassortment with various subtypes during evolution, providing an important internal gene source for the emergence of highly pathogenic avian influenza viruses (such as H5N1 and H7N9), playing a crucial role as a "gene donor" in viral evolution. On the other hand, this virus can cross interspecies barriers, infecting humans and causing influenza-like symptoms, posing a potential threat to public health. Furthermore, H9N2 virus infection can also impair the immune function of poultry, increasing the risk of secondary infections with other pathogens, thereby further exacerbating economic losses in the poultry industry. Due to its relatively low pathogenicity, insidious infection process, continuous transmission, and active evolution, this virus is easily overlooked in poultry flocks, yet it poses a long-term potential threat. Therefore, the focus of prevention and control efforts against the H9 subtype of avian influenza virus should be on long-term surveillance and early detection.

[0006] Currently, there is no specific treatment for H9 subtype avian influenza virus. Prevention and control mainly rely on vaccination, scientific feeding management, and strict biosecurity measures. However, due to the long-term circulation and continuous mutation of the virus in poultry flocks, the protective effect of vaccines is very limited, making it difficult to achieve long-term effective control of circulating strains. At present, detection methods for H9N2 subtype avian influenza virus mainly include nucleic acid detection and immunological detection. Nucleic acid detection has high sensitivity, but it has high requirements for equipment and operating conditions, making it difficult to meet the needs of rapid screening at the grassroots level and in the field. Immunological detection, especially colloidal gold immunochromatography, is more suitable for large-scale testing due to its simple operation and rapid detection, but its detection performance is highly dependent on the specificity and stability of the antibody. Some existing immunoassay products use polyclonal antibodies or antibody preparations of unknown origin, which are prone to cross-reaction with other HA subtypes (such as H5, H7, etc.), affecting the accuracy of the test results. Therefore, screening for monoclonal antibodies that can specifically recognize the H9 subtype HA protein and applying them to rapid detection systems is of great significance for the accurate identification and effective monitoring of H9 subtype avian influenza virus. Summary of the Invention

[0007] This invention utilizes hybridoma technology to successfully obtain a monoclonal antibody that specifically recognizes the H9-HA protein of avian influenza virus, and applies it to the construction of colloidal gold immunochromatographic test strips. Verification has shown that the monoclonal antibody exhibits good detection sensitivity and specificity against the H9-HA antigen, meeting the application requirements for rapid detection and on-site screening. This provides a raw material basis for the development of diagnostic reagents for the H9 subtype of avian influenza virus, and solves the technical problems of cross-reactivity and insufficient detection sensitivity of the H9 subtype of avian influenza virus compared to other subtypes (such as H5 and H7) due to the lack of antibody specificity in existing detection methods.

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include: This application provides a monoclonal antibody combination against the hemagglutinin protein of avian influenza virus H9 subtype, wherein the monoclonal antibody combination includes monoclonal antibody 5E2 and monoclonal antibody 1F3. The heavy chain variable region of the monoclonal antibody 5E2 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 5E2 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 1F3 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 1F3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0009] In some embodiments, the heavy chain variable region of the monoclonal antibody 5E2 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 5E2 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 1F3 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 1F3 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.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO. 14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.16.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO. 18.

[0012] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO. 20.

[0013] Secondly, this application provides the use of the above-mentioned monoclonal antibody combination in the preparation of a tool for recognizing the hemagglutinin protein of avian influenza virus H9.

[0014] In some embodiments, the tool includes colloidal gold test strips, reagents, kits, and antibody chips.

[0015] The tool is used to identify the H9 avian influenza virus hemagglutinin protein in samples. The samples are selected from any of the following: environmental samples, swab samples, cell culture supernatants, and tissue homogenates, and the identification is not intended for disease diagnosis.

[0016] 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.

[0017] 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.

[0018] In some embodiments, the colloidal gold test strip uses monoclonal antibody 5E2 as the capture antibody and monoclonal antibody 1F3 as the labeling antibody.

[0019] 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.

[0020] In some embodiments, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 5E2, the control line is coated with goat anti-mouse IgG, and the gold-labeled conjugate pad is coated with monoclonal antibody 1F3.

[0021] Beneficial effects: This application provides a monoclonal antibody combination against the hemagglutinin protein of avian influenza virus H9 subtype. The monoclonal antibody combination consists of monoclonal antibody 5E2 and monoclonal antibody 1F3, which can specifically recognize the hemagglutinin (HA) protein of avian influenza virus H9 subtype. The complementarity-determining region (CDR) sequences of the heavy chain and light chain variable regions of 5E2 and 1F3 are clearly defined (as shown in SEQ ID NO.1-12, respectively), ensuring the high affinity and specific binding ability of the antibody combination. It is applied to the construction of colloidal gold immunochromatographic test strips, providing a stable and reliable biorecognition tool for rapid and accurate detection of avian influenza virus H9. This technical solution solves the problem of cross-reactivity with other HA subtypes (such as H5 and H7) caused by the lack of specific antibodies in existing detection methods, thus improving the specificity of the detection. This monoclonal antibody combination exhibits high affinity and sensitivity to the H9-HA recombinant protein. The constructed colloidal gold test strip is easy to operate, provides rapid detection, and has good stability, meeting the application needs of large-scale rapid screening and monitoring of avian influenza virus H9 subtype at the grassroots level and in the field. Attached Figure Description

[0022] 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.

[0023] Figure 1 Identification results for purified monoclonal antibodies; Figure 2 This is a schematic diagram of colloidal gold assembly; Figure 3The results show the specificity of H9-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 H9-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

[0024] 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.

[0025] The detection / identification methods described in this application are not intended for disease diagnosis and treatment.

[0026] Example 1 1. Screening of H9-HA monoclonal antibodies 1.1 Mouse Immunization Mice were immunized with H9-HA recombinant protein (A / chicken / HongKong / G9 / 1997HA, 40036-V08H, Sinopharm). Simultaneously, H5-HA recombinant protein (A / Anhui / 1 / 2005HA, 11048-V08B, Sinopharm) and H7-HA recombinant protein (A / Shanghai / 1 / 2013HA, 40104-V08B, Sinopharm) with the same His tag were used as reverse screening antigens for monoclonal antibody selection. Since the H9 subtype and H5 belong to Group 1, they share some structural homology, while H7 belongs to Group 2 and differs significantly from H9 in its antigenic epitopes. This combined reverse screening effectively eliminated non-specific antibodies recognizing conserved structural regions of the HA protein and avoided obtaining antibodies that cross-react with other highly pathogenic avian influenza subtypes (such as H5 and H7), thus selecting monoclonal antibodies that recognize H9 subtype-specific epitopes.

[0027] Specifically, purified H9-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 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, 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 30 μg H9-HA recombinant protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0028] 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 up the bottom of the well, clones positive for both H9-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 H9-HA recombinant protein. Positive cells were cloned to monoclonal status using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0029] 1.3 Screening of positive clones using indirect ELISA method Recombinant H9-HA, H5-HA, and H7-HA 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 Meikewand, 1001SA) to stop the reaction. Measure the OD450nm value using a microplate reader. Select positive cell lines that react with the H9-HA recombinant protein but not with the control recombinant protein for subsequent experiments.

[0030] Table 1: Screening results of monoclonal antibodies

[0031] 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.

[0032] 2. Purification and Identification of Monoclonal Antibodies 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 PBS, 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 for 16 hours at 2-8°C in 20 mM PBS (pH 7.4). 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.

[0033] The purified monoclonal antibody was diluted 1 μg / ml, and its binding activity with H9-HA recombinant protein and irrelevant antigens H5-HA and H7-HA recombinant protein was detected by indirect ELISA. Specific results are shown in [link to results]. Figure 1 .according to Figure 1 It can be seen that the selected monoclonal antibody specifically binds to the H9-HA protein and does not react with irrelevant antigens H5-HA and H7-HA, indicating that the monoclonal antibody has good specificity and can be used for subsequent testing.

[0034] 3. Colloidal gold pairing of H9-HA monoclonal antibodies Preparation of antibody-colloidal gold labeled complex.

[0035] 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 H9-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 + 0.1% N100 + 3% Sucrose + 2% Trehalose + 1% BSA + 0.1% PC300, pH 8.6) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0036] 4. Preparation of test strips coated with different monoclonal antibodies against H9-HA: The selected H9-HA monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (antibody dilution solution: 0.01 MPB + 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 to 1 mg / mL in 0.01 M PBS (pH 7.4), was then scribed horizontally in a linear fashion at a 6 mm interval, coating the nitrocellulose membrane at a volume of 0.8 μL / cm, forming the control line (C line).

[0037] 5. Screening of paired monoclonal antibodies Nitrocellulose membranes streaked with different monoclonal antibodies against H9-HA were individually paired with different colloidal gold-labeled monoclonal antibodies. H9-HA protein was diluted to 20 ng / mL for detection, while a mixed recombinant protein of H5 and H7 was diluted to 20 ng / mL as a negative antigen for detection. Combinations that showed the strongest color development for H9-HA protein and did not react with the control protein were screened. The screening results are shown in Table 2. Therefore, the optimal pairing for detecting recombinant H9-HA protein was determined to be 5E2 streaking and 1F3 gold labeling.

[0038] Table 2: Results of screening paired monoclonal antibodies using H9-HA recombinant protein

[0039] - 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.

[0040] Table 2 shows the screening results using H9-HA recombinant protein diluted to a concentration of 20 ng / mL as a positive antigen. The detection results for negative antigens and blank dilutions were all negative and are not shown in Table 2. The results showed that the 5E2 monoclonal antibody, when scratched and combined with the 1F3 monoclonal antibody labeled with gold, produced the deepest staining of the H9-HA recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody 5E2 as the capture antibody and monoclonal antibody 1F3 as the labeling antibody can specifically recognize the H9-HA recombinant protein.

[0041] 6. Preparation and assembly of colloidal gold test strips 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. 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 5E2) and a control line (goat anti-mouse IgG). The gold-labeled pad is coated with monoclonal antibody 1F3. 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.

[0042] 7. Test strip specificity test Recombinant protein samples: H1 recombinant protein (A / California / 04 / 2009HA), H3 recombinant protein (A / Perth / 6 / 2009HA, 40035-V08H, Sinocare), H5 recombinant protein (A / Anhui / 1 / 2005HA, 11048-V08B, Sinocare), H6 recombinant protein (A / northernshoveler / California / HKWF115 / 2007HA, 11723-V08H, Sinocare), H7 recombinant protein (A / Shanghai / 1 / 2013HA, 40104-V08B, Sinocare), H9 recombinant protein, 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.

[0043] 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.

[0044] The amino acid sequence of the NP protein of influenza A virus is shown in SEQ ID NO.21: MASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWMRELILYDKEEIRRVWRQANNGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETMDSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDS。

[0045] The nucleotide of the influenza A virus NP protein is shown as SEQ ID NO.22:

[0046] The amino acid profile of the Newcastle disease virus NP protein is shown in SEQ ID NO. 23: MSSVFDEYEQLLASQTRPNGSHGGGEKGSTLKVEVPVFTLNSDDPEDRWNFAVFCLRIAVSEDANKPLRQGALISLLCTHSQVMRNHVALAGRQNEATLAILEIDGFSNGVPQFNNRSGVSE ERAQRFMMIAGSLPRACSNGTPFVTAGVEDDAPEDITTDTLERILSIQVQVWVTVAKAMTAYETADESETRRINKYMQQGRVQKRCILHPVCRSAIQLTIRQSLAVRIFLVSELKRGRNTAGGT STYYNLVGDVDSYIRNTGLTAFFLTLKYGINTKTSVLALSSLSGDIQKMKQLMRLYRMKGENAPYMTLLGDSDQMSFAPAEYAQLYSFAMGMASVLDKGTVKYQFARDFMSTSFWRLGVEYA QAQGSSINEDMAAELKLTPAVRRGLAAAAQRVSEDASNMDLPTQQAGVLTGLSDNTPPAQPGGSKPQGSADGNEGETQFLDLMRAVANSMRDAPNSAQGSSQPAPPPTPGGNQDNDTDWGY.

[0047] The NP protein nucleotides of Newcastle disease virus are shown in SEQ ID NO. 24:

[0048] 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.

[0049] Figure 3 , Figure 4 The results showed that the test strip could produce a clear detection signal for the H9-HA recombinant protein and was positive for avian influenza H9 inactivated vaccine samples containing the NJ01 strain. However, it did not react with other HA subtype recombinant proteins such as H1, H3, H5, H6, and H7, as well as the NP protein of influenza A virus and Newcastle disease virus. There was no cross-reaction with H1N1 mouse lung-adapted virus, avian influenza H5+H7 bivalent inactivated vaccine, Newcastle disease virus vaccine, and infectious bronchitis virus vaccine, indicating that the test strip has good specificity.

[0050] 8. Sensitivity test of test strips The H9-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 (0.01 M Tris + 0.1% N100 + 0.15 M NaCl + 2% Sucrose + 0.5% BSA + 0.1% PC300, pH 8) (0 ng / mL), did not show color development, indicating that the limit of detection for H9-HA recombinant protein on the test strip is 1 ng / mL.

[0051] 9. Monoclonal antibody binding activity assay Based on the screening of potential paired antibodies using a double-antibody sandwich ELISA, the selected paired monoclonal antibodies and other murine unrelated monoclonal antibodies were serially diluted (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 H9-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 diagram, "Ctrl" represents the negative control, influenza A virus NP mouse monoclonal antibody 1B5 (inventor's commercially available product, M100014). Monoclonal antibodies 5E2 and 1F3 showed significant positive reactions with the H9 protein at a concentration of 10 ng / mL, indicating that the monoclonal antibodies possess strong binding activity.

[0052] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs that specifically recognize the H9-HA protein. This combined antibody pair can efficiently recognize the H9-HA recombinant protein, exhibiting good specificity and sensitivity. This invention applies the monoclonal antibody pair 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 H9-HA recombinant protein and shows no cross-reactivity with other proteins.

[0053] 10. Gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasyMiniKit 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 then 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.

[0054] membrane-scraped monoclonal antibody 5E2 sequence Light chain variable region nucleotide sequence: The nucleotide sequence encoding the variable region of the 5E2 light chain of the monoclonal antibody is shown in SEQ ID NO.18: CAAATTGTTCTTCCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTGTGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATC CACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGCAAGCAATCTTATGATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTACGGTG.

[0055] Light chain variable region amino acid sequence: The amino acid sequence of the variable region of the light chain of the monoclonal antibody 5E2 is shown in SEQ ID NO.14; QIVLSQSPSSLAVSAGEKVTVSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYDLYTFGGGTKLELKRTV.

[0056] Light chain CDR area annotation: The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 5E2 is shown in SEQ ID NO.4: CDR-L1: KSSQSLLNSRTRKNYLA; The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 5E2 is shown in SEQ ID NO. 5: CDR-L2: WASTRES; The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 5E2 is shown in SEQ ID NO. 6: CDR-L3: KQSYDLYT.

[0057] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.17: GAGTTCCAGCTGCAGCAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAAGTCTCCTGTGCAGTCTCTGGATTCACTTTCAGTACTTATGTCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCGACTATCCA GACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTACAAGACACGGGTCCCTTTATGATGTGTACTGGGAACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0058] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.13; EFQLQQSGGDLVKPGGSLKVSCAVSGFTFSTYVMSWVRQTPEKRLEWVATISSGGSYTDYPDSVKGRFTISRDNAKNTLYLQMTSLRSEDTAMYYCTRHGSLYDGTGNFDVWGAGTTVTVSS.

[0059] Heavy chain CDR region annotation: The amino acid sequence of CDR-H1 of the monoclonal antibody 5E2 is shown in SEQ ID NO.1: CDR-H1: TYVMS; The amino acid sequence of CDR-H2 of the monoclonal antibody 5E2 is shown in SEQ ID NO.2: CDR-H2: TISSGGSYTDYPDSVKG; The amino acid sequence of CDR-H3 of the monoclonal antibody 5E2 is shown in SEQ ID NO.3: CDR-H3: HGSLYDGTGNFDV.

[0060] Gold monoclonal antibody 1F3 sequence Light chain variable region nucleotide sequence: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1F3 is shown in SEQ ID NO.20: GACATCCTGATGACCCAATCTCCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGTAGGGCAAGTCAGGACCTTAACAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAAGTCCTGATCTACTACACATCAAGATTACGT TCAGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAAGAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAGGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACATTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0061] Light chain variable region amino acid sequence: The amino acid sequence of the variable region of the light chain of the monoclonal antibody 1F3 is shown in SEQ ID NO.16: DILMTQSPSSLSASLGDRVTISCRASQDLNNYLNWYQQKPDGTVKVLIYYTSRLRSGVPSRFSGSGSGRDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIKRTV.

[0062] Light chain CDR area annotation: The light chain variable region of monoclonal antibody 1F3 includes the amino acid sequence of CDR-L1 as shown in SEQ ID NO.10: CDR-L1: RASQDLNNYLN; The light chain variable region of monoclonal antibody 1F3 includes the amino acid sequence of CDR-L2 as shown in SEQ ID NO.11: CDR-L2: YTSRLRS; The light chain variable region of monoclonal antibody 1F3 includes the amino acid sequence of CDR-L3 as shown in SEQ ID NO.12: CDR-L3: QQGNTLPYT.

[0063] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.19: GAGGTGCAGCTGGTGGAGTCTGGACCTGGCCTGGTGAAACCTTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCGCCTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCTGGAAACAAACTGGAGTGGATGGCCTACATGAGTTACAGAGGTACCACTAACTAC AACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACCACTGAGGACACAGCCACATATTACTGTGCAGGAGAGATTTACTACGGTAGTAGAGGATTTGTTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0064] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.15; EVQLVESGPGLVKPSQSLSLTCTVTGASITSDYAWNWIRQFPGNKLEWMAYMSYRGTTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAGEIYYGSRGFVYWGQGTLVTVSA.

[0065] Heavy chain CDR region annotation: The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 1F3 is shown in SEQ ID NO.7: CDR-H1: SDYAWN; The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 1F3 is shown in SEQ ID NO. 8: CDR-H2: YMSYRGTTNYNPSLKS; The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 1F3 is shown in SEQ ID NO. 9: CDR-H3: EIYYGSRGFVY.

Claims

1. A monoclonal antibody combination against the hemagglutinin protein of avian influenza virus H9 subtype, characterized in that, The monoclonal antibody combination includes monoclonal antibody 5E2 and monoclonal antibody 1F3. The heavy chain variable region of the monoclonal antibody 5E2 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 5E2 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 1F3 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 1F3 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 avian influenza virus H9 subtype hemagglutinin protein according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody 5E2 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 5E2 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 1F3 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 1F3 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 avian influenza virus H9 subtype hemagglutinin protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.

14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination against avian influenza virus H9 subtype hemagglutinin protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5E2 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination against avian influenza virus H9 subtype hemagglutinin protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for recognizing the hemagglutinin protein of avian influenza virus H9.

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

8. The application according to claim 7, characterized in that, The colloidal gold test strip uses monoclonal antibody 5E2 as the capture antibody and monoclonal antibody 1F3 as the labeling antibody.

9. The application 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 application 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 5E2, the control line is coated with goat anti-mouse IgG, and the gold-labeled conjugate pad is coated with monoclonal antibody 1F3.

Images