A monoclonal antibody targeting hemagglutinin of avian influenza virus H5N1 and its application

Abstract

The present invention discloses a monoclonal antibody against the hemagglutinin of avian influenza virus H5N1 and its application. The monoclonal antibody can specifically bind to the hemagglutinin protein of avian influenza virus H5N1 and has good binding activity. It can be used for the early diagnosis and screening of diseases related to avian influenza virus infection. The present invention lays a foundation for the prevention and control of avian influenza virus infection and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and application thereof. Background Art

[0002] Avian influenza is a respiratory disease of birds caused by the influenza A virus, of which the H5N1 subtype is a particularly important, highly pathogenic variant. As a key subtype of avian influenza virus, H5N1 primarily causes disease in poultry, but it also has the ability to cross species and infect mammals. In humans, H5N1 infection occurs primarily as a sporadic event. Human infection with the H5N1 virus primarily occurs through contact with the excreta and secretions of infected sick or dead poultry, or through contact with environments and objects contaminated by these contaminants. Furthermore, infected secretions and excretions become airborne, forming aerosols that can enter the human body through various routes, including the respiratory tract, digestive tract, skin lesions, and conjunctiva. Patients infected with the H5N1 virus typically experience an acute onset and rapid progression. Although early symptoms are similar to those of common influenza, patients often develop severe lung disease as the disease progresses.

[0003] Given the rapid spread of H5N1, strengthening surveillance and developing rapid and accurate detection methods for the H5N1 avian influenza virus are crucial measures for preventing the spread of the virus. Traditional avian influenza virus detection methods (such as PCR and virus isolation) require specialized laboratories and a lengthy process, making them inadequate for rapid on-site diagnosis. Cross-reactivity between different subtypes (such as H5, H7, and H9) can also lead to misdiagnosis. Monoclonal antibodies (mAbs) can be designed against specific epitopes on the H5N1 hemagglutinin (HA) protein, enabling precise identification of H5N1 subtypes while avoiding cross-reactivity with other subtypes. This monoclonal antibody detection technology can enhance early detection of H5N1 avian influenza virus and reduce the risk of transmission. Therefore, developing a monoclonal antibody with strong binding activity to the H5N1 HA protein is of great practical significance. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a monoclonal antibody against the hemagglutinin of avian influenza virus H5N1 and its application. The present invention creatively discovered for the first time and confirmed through experiments that the monoclonal antibody (169-1 antibody) has good affinity and specificity for the hemagglutinin protein of avian influenza virus H5N1, providing a new idea and strategy for the research and development of detection reagents or detection products related to avian influenza virus H5N1, and has good application prospects.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides a monoclonal antibody against avian influenza virus H5N1 HA protein, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0007] The amino acid sequences of HCDR1-3 in the heavy chain variable region are shown in SEQ ID NOs: 1-3, respectively;

[0008] The amino acid sequences of LCDR1-3 in the light chain variable region are shown in SEQ ID NOs: 4-6, respectively.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13.

[0010] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14.

[0011] It is well known to those skilled in the art that, depending on the different CDR numbering schemes adopted, the corresponding HCDR1-3 and LCDR1-3 in the monoclonal antibody of the present invention have a variety of different amino acid sequences. As long as the HCDR1-3 and LCDR1-3 are defined based on the heavy chain variable region as shown in SEQ ID NO: 13 and the light chain variable region as shown in SEQ ID NO: 14 in the monoclonal antibody provided by the present invention, they are within the scope of protection of the present invention.

[0012] In the present invention, any CDR numbering scheme (existing CDR numbering scheme or new CDR numbering scheme to be generated in the future) is used to define HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO: 13 and LCDR1-3 in the light chain variable region as shown in SEQ ID NO: 14, respectively. The resulting HCDR1-3 and the amino acid sequence or nucleotide sequence of the monoclonal antibody corresponding to LCDR1-3 are all within the scope of protection of the present invention.

[0013] Illustratively, the CDR numbering scheme includes, but is not limited to, any one or a combination of any two or more (two or more) of the IMGT numbering scheme, the Kabat numbering scheme, the Chothia numbering scheme, the Martin (enhanced Chothia) numbering scheme, the AbM numbering scheme, the Aho numbering scheme, and the Contact numbering scheme.

[0014] In some embodiments, functional variants of the monoclonal antibodies provided herein as above are also included within the scope of protection of the present invention, and the functional variants refer to proteins having significant or significant sequence identity or similarity compared to the parent monoclonal antibody, and the functional variants retain the biological activity of the parent monoclonal antibody. Functional variants encompass, for example, the following variants of the monoclonal antibodies (parent antibodies) described herein above, which retain the ability to recognize the target antigen to a similar extent, to the same extent, or to a greater extent than the parent monoclonal antibody. With reference to the parent monoclonal antibody, the functional variants may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95% or more homology in amino acid sequence to the parent monoclonal antibody.

[0015] Furthermore, the functional variant may, for example, comprise the amino acid sequence of the parent monoclonal antibody with at least one conservative amino acid substitution. Alternatively or in addition, the functional variant may comprise the amino acid sequence of the parent monoclonal antibody with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is improved compared to the parent monoclonal antibody.

[0016] Furthermore, conservative amino acid substitutions are known in the art and include those in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. Exemplarily, the conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Leu, Met, Phe, Pro, Val, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., His, Lys, Arg, etc.), substitutions of an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Ser, Thr, Gln, Tyr, etc.), substitutions of an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr, and Val), and substitutions of an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., Phe, His, Trp, and Tyr).

[0017] The second aspect of the present invention provides a bispecific antibody, which comprises the monoclonal antibody described in the first aspect of the present invention and a second antibody that specifically binds to another antigen.

[0018] In some embodiments, the bispecific antibody comprises binding specificities for two different antigens (one antigen being the avian influenza virus H5N1 HA protein described herein, and the other antigen being an antigen other than the avian influenza virus H5N1 HA protein). In other embodiments, the bispecific antibody comprises two different binding specificities for the same antigen (e.g., the avian influenza virus H5N1 HA protein) (e.g., having different binding affinities and / or specific epitopes for the same antigen).

[0019] The third aspect of the present invention provides a nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.

[0020] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody described in the first aspect of the present invention is shown as SEQ ID NO: 15, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody described in the first aspect of the present invention is shown as SEQ ID NO: 16.

[0021] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the monoclonal antibody provided in the first aspect of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 80% or greater homology with the nucleotide sequence corresponding to the monoclonal antibody of the present invention, as long as they encode the monoclonal antibody of the first aspect of the present invention, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, and are also encompassed within the scope of protection of the present invention.

[0022] The fourth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0023] In some embodiments, the recombinant vector is an expression vector or a cloning vector. Any vector capable of delivering nucleic acid can be applied to the present invention. Exemplarily, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, a baculoviral vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, or any combination thereof.

[0024] The fifth aspect of the present invention provides a recombinant host cell, which comprises the recombinant vector described in the fourth aspect of the present invention.

[0025] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell. For example, suitable host cells for the present invention include, but are not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, HEK293 cells, COS cells, HeLa cells, NSO cells, BHK cells, or human cells.

[0026] A sixth aspect of the present invention provides any of the following products:

[0027] (1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly coupling the monoclonal antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention to a detectable label;

[0028] (2) A detection reagent comprising the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, and / or the antibody conjugate described therein;

[0029] (3) A detection product comprising the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate and / or the detection reagent.

[0030] In some embodiments, the detectable label includes, but is not limited to, a radionuclide, a chemiluminescent agent, a bioluminescent agent, a contrast agent, a fluorescent agent, a paramagnetic ion, an enzyme, and a photosensitive diagnostic agent.

[0031] In some embodiments, the detection product is a kit. Exemplarily, the kit includes but is not limited to: a chemiluminescence kit, a colloidal gold immunoassay kit, an enzyme-linked immunosorbent assay kit, a radioimmunoassay kit, a fluorescence immunoassay kit or a microfluidic chip.

[0032] A seventh aspect of the present invention provides any of the following methods:

[0033] (1) A method for preparing the monoclonal antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention, the method comprising: culturing the recombinant host cell of the fifth aspect of the present invention, and isolating the monoclonal antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention from the culture;

[0034] (2) A method for detecting avian influenza virus H5N1 HA protein for non-diagnostic and non-therapeutic purposes, the method comprising: contacting a sample to be tested with the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, a detection reagent and / or a detection product, and detecting the formation of an immune complex between the avian influenza virus H5N1 HA protein and the antibody;

[0035] (3) A method for preparing the recombinant host cell according to the fifth aspect of the present invention, the method comprising: introducing the recombinant vector according to the fourth aspect of the present invention into a host cell to obtain the recombinant host cell according to the fifth aspect of the present invention.

[0036] In some embodiments, the sample to be tested is selected from body fluids, excretions, and cells derived from a subject; for example, serum, plasma, whole blood, lymph, cerebrospinal fluid, tissue fluid, saliva, urine, lymphocytes, etc.

[0037] In the present invention, the subject refers to any animal, preferably a mammal, including but not limited to humans, higher primates, domestic and farm animals, as well as zoo animals, competitive animals or pets, such as horses, pigs, cows, dogs, cats and ferrets.

[0038] In addition, the present invention also provides a method for diagnosing or assisting in the diagnosis of avian influenza virus H5N1 infection, the method comprising: contacting the monoclonal antibody, bispecific antibody, antibody conjugate, detection reagent or detection product of the present invention as described above with a test sample derived from a subject, detecting whether the test sample derived from the subject contains the avian influenza virus H5N1 HA protein, and based on this, further determining whether the subject suffers from the avian influenza virus H5N1 infection or is at risk of suffering from the avian influenza virus H5N1 infection.

[0039] An eighth aspect of the present invention provides any of the following applications:

[0040] (1) Use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, and / or the recombinant host cell described in the fifth aspect of the present invention in the preparation of an antibody conjugate for detecting avian influenza virus H5N1 HA protein;

[0041] (2) Use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, and / or the antibody conjugate described in the sixth aspect of the present invention in the preparation of a detection reagent for detecting the avian influenza virus H5N1 HA protein;

[0042] (3) Use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate and / or detection reagent described in the sixth aspect of the present invention in the preparation of a detection product for detecting the avian influenza virus H5N1 HA protein;

[0043] (4) Use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate, detection reagent and / or detection product described in the sixth aspect of the present invention in detecting avian influenza virus H5N1 HA protein for non-diagnostic and non-therapeutic purposes;

[0044] (5) Use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate, detection reagent and / or detection product described in the sixth aspect of the present invention in the preparation of diagnostic products for diagnosing or assisting in the diagnosis of avian influenza virus H5N1 infection.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] The present invention discloses a novel monoclonal antibody against the hemagglutinin of the avian influenza virus H5N1. The monoclonal antibody can specifically bind to the hemagglutinin protein of the avian influenza virus H5N1 and has good binding activity. The antibody can be used for the early diagnosis and screening of diseases related to avian influenza virus infection. The present invention lays a foundation for the prevention and control of avian influenza virus infection and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the result of SDS-PAGE electrophoresis, in which, from left to right are protein molecular weight marker, reduced 169-1 antibody and non-reduced 169-1 antibody;

[0048] Figure 2 The figure shows the result that the 169-1 antibody has a high affinity for the H5N1 HA protein;

[0049] Figure 3 The graph shows the results of SPR detection of the affinity constant of 169-1 antibody and H5N1 HA;

[0050] Figure 4The figure shows the results corresponding to the high specificity of the 169-1 antibody to the H5N1 HA protein. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art. Unless otherwise specified, they can be obtained commercially. The experimental methods for which specific conditions are not specified in the present invention are typically tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0052] Example 1 Screening of antibodies targeting the hemagglutinin (HA) protein of avian influenza virus H5N1

[0053] 1. Experimental methods

[0054] After mice were immunized multiple times with H5N1 HA antigen (purchased from Sino Biological), the spleens of the mice were harvested to prepare single-cell suspensions, and antigen-specific B cells were obtained by flow cytometry sorting.

[0055] The obtained antigen-specific B cells are sent to Yiyan Technology for sequencing using the 10X technology platform to construct a single B cell library. During the library construction process, the immunoglobulin gene (gene encoding the antibody) in each B cell is sequenced, thereby obtaining a large amount of antibody gene sequence information.

[0056] 2. Experimental results

[0057] The 169-1 antibody targeting the avian influenza virus H5N1 HA protein was screened and obtained. The sequence information of the 169-1 antibody is as follows:

[0058] Heavy chain variable region CDR1 amino acid sequence: GGTFSRF (SEQ ID NO: 1);

[0059] Heavy chain variable region CDR2 amino acid sequence: IPILGR (SEQ ID NO: 2);

[0060] Heavy chain variable region CDR3 amino acid sequence: CARAFLTGHFDYW (SEQ ID NO: 3);

[0061] Light chain variable region CDR1 amino acid sequence: RSSTGAVTTSNYAN (SEQ ID NO: 4);

[0062] Light chain variable region CDR2 amino acid sequence: GTNNRAP (SEQ ID NO: 5);

[0063] Light chain variable region CDR3 amino acid sequence: CALWYSNHWVF (SEQ ID NO: 6);

[0064] Heavy chain variable region CDR1 nucleotide sequence: GGAGGCACCTTCAGCAGGTTT (SEQ ID NO: 7);

[0065] Heavy chain variable region CDR2 nucleotide sequence: ATCCCTATCCTTGGTAGA (SEQ ID NO: 8);

[0066] Heavy chain variable region CDR3 nucleotide sequence: TGTCGAGAGCTTTCTTAACTGGGCATTTTGACTACTGG (SEQ ID NO: 9);

[0067] Light chain variable region CDR1 nucleotide sequence: CGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAAC (SEQ ID NO: 10);

[0068] Light chain variable region CDR2 nucleotide sequence: GGTACCAACAACCGAGCTCCA (SEQ ID NO: 11);

[0069] Light chain variable region CDR3 nucleotide sequence: TGTGCTCTATGGTACAGCAACCATTGGGTGTTC (SEQ ID NO: 12);

[0070] Heavy chain variable region amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRFEISWVRQVPGQGLEWMGGIIPILGRTNYGQKFQGRVTITADKSTNTAYMELSSLRSEDTAVYYCARAFLTGHFDYWGQGTLVTVSS (SEQ ID NO: 13);

[0071] Light chain variable region amino acid sequence: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 14);

[0072] Heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGGTTTGAAATCAGCTGGGTGCGACAGGTCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCCTTGGTAGAACA AACTATGGACAGAAGTTTCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGCTTTCTAACTGGGCATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:15);

[0073] Light chain variable region nucleotide sequence: CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCA ACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAG (SEQ ID NO:16).

[0074] Example 2 Preparation of 169-1 Antibody Targeting Avian Influenza Virus H5N1 HA Protein

[0075] 1. Experimental methods

[0076] (1) The VH and VL DNA sequences of the 169-1 antibody obtained by sequencing in Example 1 were sent to Genewise for synthesis, and the synthesized sequences were installed in the laboratory expression vector by enzyme digestion and ligation.

[0077] (2) Transfect the expression vector into CHO mammalian cells for expression and collect the expression supernatant.

[0078] (3) Purify antibodies from CHO cell culture supernatant using Protein A on an AKTA protein purifier.

[0079] (4) Elution was performed with 3.5 mM citrate buffer and the pH was immediately adjusted to about 7-8 with 1 M Tris, pH 11.5, after collection was completed.

[0080] (5) After ultrafiltration and concentration using PBS (pH 7.2, 0.01 mol / L) in an ultrafiltration tube, sterilize by filtration using a 0.22 μm filter membrane.

[0081] (6) Use SDS-PAGE to detect the expression and purification of the antibody, and use the BCA method to detect the concentration of the purified antibody. Store at 4°C.

[0082] 2. Experimental results

[0083] The experimental results are as follows Figure 1 As shown, the results show that this example successfully prepared a monoclonal antibody (169-1 antibody) targeting the avian influenza virus H5N1 HA protein.

[0084] Example 3 ELISA detection of the binding of 169-1 antibody targeting avian influenza virus H5N1 HA protein to H5N1 HA

[0085] 1. Experimental methods

[0086] (1) Dilute the antigen to 0.25 mg / mL in sterile water for injection in a cell stand.

[0087] (2) Dilute the HA antigen to 1 μg / mL with coating solution, apply 100 μL / well of coating antigen, and incubate at 37°C for 2 h.

[0088] (3) Wash three times in a plate washer and pat dry on paper towels.

[0089] (4) Prepare 4% skim milk powder in PBS, 250 μL / well for blocking, incubate at 37°C for 1 h.

[0090] (5) Candidate 169-1 antibody: Dilute the candidate antibody with PBS to an initial concentration of 10 μg / mL.

[0091] (6) Perform a gradient dilution of 12 concentrations in a 96-deep-well plate, starting with a three-fold gradient dilution from 10 μg / mL. Add 250 μL of PBS to each well except the first column. Aspirate 125 μL of 10 μg / mL antibody from the first column and add it to the wells in the second column containing 250 μL of PBS, pipette and mix thoroughly, and dilute to the 12th well in sequence, completing a three-fold gradient dilution.

[0092] (7) Remove the plate washer from the incubator in step (4), wash the plate three times, and pat dry on paper towels.

[0093] (8) Use a 100 μL pipette to add each row of diluted antibodies at different concentrations to the blocked plate. Incubate at 37°C for 1 h.

[0094] (9) Wash three times in a plate washer and pat dry on paper towels.

[0095] (10) GAH-HRP was diluted 1:6000 and 100 μL / well was added to the plate and incubated at 37°C for 30 min.

[0096] (11) Wash three times in a plate washer and pat dry on paper towels.

[0097] (12) Prepare TMB working solution and add 100 μL / well to the plate after incubation with the primary antibody. After a clear color gradient is displayed, use 100 μL / well of 0.5 M sulfuric acid solution to stop the color development.

[0098] (13) The OD450 value was measured using a microplate reader within 5 min, the binding curve was drawn using Graphad Prism, and the EC50 value was calculated.

[0099] 2. Experimental results

[0100] The experimental results are as follows Figure 2 As shown, the results showed that the 169-1 antibody targeting the avian influenza virus H5N1 HA protein had a high affinity (nM level) for binding to the HA protein.

[0101] Example 4 SPR Detection of Affinity Constants of 169-1 Antibody Targeting H5N1 HA Protein of Avian Influenza Virus and H5N1 HA

[0102] 1. Experimental methods

[0103] The binding kinetics of the 169-1 antibody were measured using a Biacore T200 instrument (Cytiva) at 25°C using an HBS-EP mobile phase (BR100669, Cytiva). First, carboxyl groups on the CM5 sensor chip substrate were activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to generate active esters that react with amino groups. HA antigen was diluted to 1 μg / mL in 10 mM sodium acetate buffer, pH 5.0, and covalently coupled to an immobilization level of approximately 180 response units (RU). Unreacted active esters were subsequently blocked with ethanolamine.

[0104] Subsequently, multi-cycle kinetic analysis of HA binding to the 169-1 antibody was performed using a two-fold dilution range of 3.125–50 nM. The binding phase lasted 180 seconds, the dissociation phase lasted 300 seconds, and the flow rate was 30 μL / min, followed by regeneration. Kinetic parameters were calculated using a global kinetic fitting model (1:1 Langmuir) (Biacore T200 Evaluation Software 3.1, Cytiva).

[0105] 2. Experimental results

[0106] The experimental results are as follows Figure 3 As shown, the results showed that the affinity constant KD of the 169-1 antibody targeting the avian influenza virus H5N1 HA protein and the HA antigen was as high as 10-10 (KD (M) = 6.632E-10), which has a strong affinity.

[0107] Example 5 Detection of the specificity of the 169-1 antibody targeting the avian influenza virus H5N1 HA protein and H5N1 HA

[0108] 1. Experimental methods

[0109] The experimental method is the same as that in Example 3.

[0110] 2. Experimental results

[0111] The experimental results are as follows Figure 4 As shown, the results showed that the 169-1 antibody targeting the avian influenza virus H5N1 HA protein could not bind to the avian influenza virus H7N9 HA antigen, indicating that the 169-1 antibody targeting the avian influenza virus H5N1 HA protein screened and prepared by the present invention can specifically bind to the avian influenza virus H5N1 HA antigen.

[0112] The experimental results of the above examples show that the 169-1 antibody targeting the avian influenza virus H5N1 HA protein screened and prepared by the present invention can specifically bind to the avian influenza virus H5N1 HA antigen and has good specificity and affinity, and can be used in the detection of avian influenza virus H5N1 HA protein-related or auxiliary detection products.

Claims

1. A monoclonal antibody against avian influenza virus H5N1 HA protein, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of HCDR1-3 in the heavy chain variable region are shown in SEQ ID NOs: 1-3, respectively; The amino acid sequences of LCDR1-3 in the light chain variable region are shown in SEQ ID NOs: 4-6, respectively.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

13.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid molecule according to claim 4.

6. A recombinant host cell, characterized in that The recombinant host cell comprises the recombinant vector according to claim 5.

7. An antibody conjugate, characterized in that The antibody conjugate is a complex formed by directly or indirectly coupling the monoclonal antibody according to any one of claims 1 to 3 to a detectable label.

8. A detection reagent, characterized in that The detection reagent comprises the monoclonal antibody according to any one of claims 1 to 3 and / or the antibody conjugate according to claim 7.

9. A detection product, characterized in that: The detection product comprises the monoclonal antibody according to any one of claims 1 to 3, the antibody conjugate according to claim 7 and / or the detection reagent according to claim 8.

10. A method for preparing the monoclonal antibody according to any one of claims 1 to 3, characterized in that: The method comprises: culturing the recombinant host cell according to claim 6, and isolating the monoclonal antibody according to any one of claims 1 to 3 from the culture.

11. A method for detecting avian influenza virus H5N1 HA protein for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises: contacting a sample to be tested with the monoclonal antibody according to any one of claims 1 to 3, the antibody conjugate according to claim 7, the detection reagent according to claim 8, and / or the detection product according to claim 9, and detecting the formation of an immune complex between the avian influenza virus H5N1 HA protein and the antibody.

12. A method for preparing the recombinant host cell according to claim 6, characterized in that: The method comprises: introducing the recombinant vector according to claim 5 into a host cell to obtain the recombinant host cell according to claim 6.

13. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, and / or the recombinant host cell according to claim 6 in the preparation of an antibody conjugate for detecting avian influenza virus H5N1 HA protein.

14. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, and / or the antibody conjugate according to claim 7 in the preparation of a detection reagent for detecting avian influenza virus H5N1 HA protein.

15. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the antibody conjugate according to claim 7, and / or the detection reagent according to claim 8 in the preparation of a detection product for detecting avian influenza virus H5N1 HA protein.

16. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the antibody conjugate according to claim 7, the detection reagent according to claim 8, and / or the detection product according to claim 9 for detecting avian influenza virus H5N1 HA protein for non-diagnostic and non-therapeutic purposes.

17. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the antibody conjugate according to claim 7, the detection reagent according to claim 8, and / or the detection product according to claim 9 in the preparation of a diagnostic product for diagnosing or assisting in the diagnosis of avian influenza virus H5N1 infection.

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