Anti-influenza A virus antibody and application thereof

Abstract

The invention discloses an anti-influenza A virus antibody and application thereof, and relates to the field of antibodies. The anti-influenza A virus antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of influenza A virus, and has good specificity and sensitivity.

Description

[0001] Cross-referencing:

[0002] This application claims priority to Chinese Patent Application No. 202411135479.X, filed on August 19, 2024, entitled "An antibody against influenza A virus and its application", the entire contents of which are incorporated herein by reference.

[0003] This application claims priority to Chinese Patent Application No. 202411570925.X, filed on November 5, 2024, entitled "An antibody against influenza A virus and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to the field of antibody technology, and more specifically, to an antibody against influenza A virus and its application. Background Technology

[0005] Influenza A virus (IAV) is an important zoonotic pathogen belonging to the Orthomyxoviridae family. It is an enveloped, segmented, single-stranded, negative-sense RNA virus. Historically, it has caused several influenza pandemics and sporadic avian influenza infections in humans, seriously threatening public health and socio-economic development. The influenza virus has two important envelope proteins on its surface—hemagglutinin (HA) and neuraminidase (NA). Different subtypes of influenza viruses bind to receptors primarily through the HA protein, and HA binding to receptors exhibits species specificity.

[0006] Influenza A viruses are further divided into several subtypes based on the differences in their H and N antigens. H has 18 subtypes (H1–H18), and N has 11 subtypes (N1–N11). Of these, H1N1, H2N2, and H3N2 primarily infect humans, while many other subtypes have various avian and animal hosts in their natural habitat. Influenza A viruses are most prone to mutation; their surface antigens frequently undergo minor variations known as "antigenic drift," which allows the virus to evade recognition by the human immune system.

[0007] Influenza viruses are primarily transmitted through droplets, with patients and asymptomatic carriers being the main sources of infection. Influenza typically presents with an acute onset, accompanied by symptoms such as fever, headache, muscle pain, and general malaise. Severe cases can lead to various complications and even death. Prevention and control of influenza require a comprehensive approach, including vaccination, antiviral treatment, and daily protective measures.

[0008] Currently, detection techniques for influenza A virus mainly include fluorescent PCR, immunoassay, and virus isolation and culture identification. Among these, fluorescent PCR, with its high sensitivity and specificity, has become the gold standard for pathogen detection; however, it requires highly skilled personnel and must be performed in a specialized laboratory, limiting its application in clinical and epidemiological surveillance. While virus isolation and culture identification allows direct observation of virus growth, it has a long testing cycle, stringent environmental requirements, a high risk of operator infection, and its culture results are affected by various factors, thus limiting its clinical application.

[0009] Immunoassay, with its rapid, accurate, and convenient characteristics, has shown broad application prospects in the detection of influenza A virus. Immunoassay is based on the principle of specific antigen-antibody binding, diagnosing viral infection by detecting antigens or antibodies in a sample. Currently, various immunoassay-based influenza A virus detection reagents are available on the market, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. These reagents are widely used in hospital laboratories and disease control system laboratories for primary screening, playing an important role in the initial detection, epidemic control, and treatment guidance of influenza A.

[0010] However, despite the availability of various immunodiagnostic reagents on the market, specific antibodies against the influenza A virus remain a crucial step in the research and development process. The quality of the specific antibody directly affects the sensitivity and specificity of the test reagent, and consequently, the accuracy of the test results. Therefore, developing an antibody that can specifically recognize the influenza A virus and efficiently bind to its antigen is of great significance for improving the performance of influenza A virus detection reagents. Summary of the Invention

[0011] This application provides an antibody against influenza A virus, which provides an important source of raw materials for the detection of influenza A virus and has good specificity and sensitivity.

[0012] To achieve the above objective, according to a first aspect of the present invention, an antibody against influenza A virus is provided, the antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:17, 35, 53 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:18, 37, 55.

[0013] To achieve the above objective, according to a second aspect of the present invention, an antibody against influenza A virus is provided, wherein the complementarity-determining region of the aforementioned antibody includes any one of (a) to (c):

[0014] (a) The amino acid sequence of HCDR1 is RYGVT (SEQ ID NO:1); the amino acid sequence of HCDR2 is YIDLVFGSTYYASWVND (SEQ ID NO:2); the amino acid sequence of HCDR3 is ETDYNTGFFGI (SEQ ID NO:3); the amino acid sequence of LCDR1 is QASEDIEKYLA (SEQ ID NO:4); the amino acid sequence of LCDR2 is DASDLAS (SEQ ID NO:5); and the amino acid sequence of LCDR3 is QNAYYPSNNHIA (SEQ ID NO:6).

[0015] (b) The amino acid sequence of HCDR1 is SYNMG (SEQ ID NO:21); the amino acid sequence of HCDR2 is VINSYRITYYASWAKG (SEQ ID NO:22); the amino acid sequence of HCDR3 is ETVYFYLDL (SEQ ID NO:23); the amino acid sequence of LCDR1 is QGSQSISSSYLS (SEQ ID NO:24); the amino acid sequence of LCDR2 is KASTLAS (SEQ ID NO:25); the amino acid sequence of LCDR3 is LYEDYIMSNNKA (SEQ ID NO:26); and

[0016] (c) The amino acid sequence of HCDR1 is SFAVG (SEQ ID NO:39); the amino acid sequence of HCDR2 is IISGIGSVYYASWAKG (SEQ ID NO:40); the amino acid sequence of HCDR3 is AIPSYTGGYNL (SEQ ID NO:41); the amino acid sequence of LCDR1 is QASQSISIYLA (SEQ ID NO:42); the amino acid sequence of LCDR2 is DASTLAS (SEQ ID NO:43); and the amino acid sequence of LCDR3 is LQTYRSSAVDYA (SEQ ID NO:44).

[0017] To achieve the above objectives, according to a third aspect of the present invention, an antibody against influenza A virus is provided, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown in any one of SEQ ID NO:17, 35, and 53, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:18, 37, and 55.

[0018] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody against influenza A virus is provided, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:19, 36, and 54, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:20, 38, and 56.

[0019] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibodies described above.

[0020] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the antibody, antibody conjugate, or antibody pair described above.

[0021] To achieve the above objectives, according to a seventh aspect of the present invention, the use of the above-described antibody, antibody-drug conjugate, antibody pair, reagent or kit in the preparation of products for detecting influenza A virus is provided.

[0022] To achieve the above objectives, the present invention also provides a nucleic acid molecule, a vector, a cell, and a method for preparing the above-mentioned antibody. Detailed Implementation

[0023] In a first aspect, embodiments of the present invention provide an antibody against influenza A virus, the aforementioned antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:17, 35, 53 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:18, 37, 55.

[0024] In an optional implementation, the complementary determination region of the aforementioned variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0025] In this invention, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment of an antibody is a substance containing an antibody CDR that lacks some amino acids present in the full-length chain but can still specifically bind to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and the smallest antibody recognition unit. The antigen-binding fragments of the aforementioned antibodies can bind to the same antigen as the parent antibody. Those skilled in the art will readily understand from the description of this invention that the aforementioned antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the antigen-binding fragments of the aforementioned antibodies.

[0026] Antigen-binding fragments of antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by automated peptide synthesizers, such as those sold by Applied BioSystems.

[0027] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0028] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0029] In this document, the term "and / or" includes both "and" and "or".

[0030] In this invention, the subtypes of the term "type A influenza virus" include, but are not limited to, H1N1, H1N2, H2N2, H3N2, H5N1, and H7N9.

[0031] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of antibodies.

[0032] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0033] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, “Kabat definition” refers to the definition system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For “Chothia definition,” see Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes but will still overlap at least partially with the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0034] Table 1: CDR Definition 1

[0035] CDR Kabat AbM2 IMGT Chothia <![CDATA[Contact 6 ]]> HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> H30--H35 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 H47--H58 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 H93--H101 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 L30--L36 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 L46--L55 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97 L89--L96

[0036] 1In Table 1, except for the Contact definition system where the CDR is based on the Chothia numbering system, the CDRs defined in other definition systems are based on the Kabat numbering system (see below). Amino acid numbers on the heavy chain are represented by "H + number," and amino acid numbers on the light chain are represented by "L + number." Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia numbering" as used herein refers to the numbering system described by Al-Lazikani et al., (1997) JMB 273, 927-948.

[0037] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0038] 3 If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0039] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0040] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0041] 6 The CDR numbering shown in Table 1 under the Contact definition system is based on the Chothia numbering system, and the Contact definition system is only applicable to the Chothia or Martin numbering system.

[0042] According to embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0043] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0044] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0045] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0046] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0047] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0048] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0049] Secondly, embodiments of the present invention provide an antibody against influenza A virus, wherein the complementarity-determining region of the aforementioned antibody includes any one of (a) to (c):

[0050] (a) The amino acid sequence of HCDR1 is RYGVT (SEQ ID NO:1); the amino acid sequence of HCDR2 is YIDLVFGSTYYASWVND (SEQ ID NO:2); the amino acid sequence of HCDR3 is ETDYNTGFFGI (SEQ ID NO:3); the amino acid sequence of LCDR1 is QASEDIEKYLA (SEQ ID NO:4); the amino acid sequence of LCDR2 is DASDLAS (SEQ ID NO:5); and the amino acid sequence of LCDR3 is QNAYYPSNNHIA (SEQ ID NO:6).

[0051] (b) The amino acid sequence of HCDR1 is SYNMG (SEQ ID NO:21); the amino acid sequence of HCDR2 is VINSYRITYYASWAKG (SEQ ID NO:22); the amino acid sequence of HCDR3 is ETVYFYLDL (SEQ ID NO:23); the amino acid sequence of LCDR1 is QGSQSISSSYLS (SEQ ID NO:24); the amino acid sequence of LCDR2 is KASTLAS (SEQ ID NO:25); the amino acid sequence of LCDR3 is LYEDYIMSNNKA (SEQ ID NO:26); and

[0052] (c) The amino acid sequence of HCDR1 is SFAVG (SEQ ID NO:39); the amino acid sequence of HCDR2 is IISGIGSVYYASWAKG (SEQ ID NO:40); the amino acid sequence of HCDR3 is AIPSYTGGYNL (SEQ ID NO:41); the amino acid sequence of LCDR1 is QASQSISIYLA (SEQ ID NO:42); the amino acid sequence of LCDR2 is DASTLAS (SEQ ID NO:43); and the amino acid sequence of LCDR3 is LQTYRSSAVDYA (SEQ ID NO:44).

[0053] According to an embodiment of the present invention, the above-mentioned HCDRs and LCDRs are defined by the Kabat system.

[0054] In optional embodiments, the antibodies described in the first and second aspects further include a framework region.

[0055] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0056] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In optional embodiments, the antibodies described in the first and second aspects further comprise HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0058] In an optional implementation, HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from any one of (a') to (c'):

[0059] (a') The amino acid sequences are as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, namely HFR1, HFR2, HFR3, and HFR4; and the amino acid sequences are as shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, namely LFR1, LFR2, LFR3, and LFR4; or the amino acid sequences that have at least 80% identity with the aforementioned frame region sequences;

[0060] (b') The amino acid sequences are as shown in SEQ ID NO:27 to EQ ID NO:30, namely HFR1, HFR2, HFR3, and HFR4; and the amino acid sequences are as shown in SEQ ID NO:31 to SEQ ID NO:34, namely LFR1, LFR2, LFR3, and LFR4; or the amino acid sequences having at least 80% identity with the sequences of each of the frame regions.

[0061] (c') The amino acid sequences are as shown in SEQ ID NO:45 to EQ ID NO:48, namely HFR1, HFR2, HFR3, and HFR4; and the amino acid sequences are as shown in SEQ ID NO:49 to SEQ ID NO:52, namely LFR1, LFR2, LFR3, and LFR4; or the amino acid sequences having at least 80% identity with the sequences of each of the frame regions.

[0062] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0063] In other embodiments, the amino acid sequences of each frame region of the antibody against influenza A virus provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions described above.

[0064] Thirdly, embodiments of the present invention provide an antibody against influenza A virus, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown in any one of SEQ ID NO:17, 35, and 53, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:18, 37, and 55.

[0065] In an optional embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from any combination of the following:

[0066] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:17 SEQ ID NO:18 2 SEQ ID NO:35 SEQ ID NO:37 3 SEQ ID NO:53 SEQ ID NO:55

[0067] In optional embodiments, the antibodies described in the first, second, or third aspects above further include a constant region.

[0068] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and a light chain constant region.

[0069] In an optional embodiment, the aforementioned heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0070] In an optional embodiment, the aforementioned heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM, and / or the tail peptide of IgM.

[0071] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant region to perform its biological function. The heavy chain constant region, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0072] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved towards the IgM CH1 region) or backward by several amino acid residues (i.e., moved towards the IgM CH3 region) to obtain a sequence of the corresponding segment that is longer or shorter than the reference sequence.

[0073] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline, thus allowing for stretching and bending, and typically contains at least one proline (P). Hinge regions are usually dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0074] In this paper, the term "tail peptide" refers to a short peptide sequence of about a dozen amino acid residues at the end of the CH3 or CH4 region of an antibody. Tail peptides from different species are well known.

[0075] In this document, the term "IgM tail peptide" refers to a short peptide sequence at the end of the CH4 region of an IgM antibody, located at the C-terminus of the CH4 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. The IgM tail peptide contains cysteine ​​residues that participate in polymer formation and can also bind to the J chain to further stabilize the multimeric structure.

[0076] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0077] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0078] In an optional implementation, the species source of the aforementioned constant region is cattle, horses, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0079] In an optional implementation, the species source of the aforementioned constant region is rabbit.

[0080] In this paper, the partitioning of the variable and constant regions is based on the IMGT partitioning method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGHC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGLC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region delineated by IMGT or the N-terminus of the constant region. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0081] In an optional embodiment, the antibody includes the following constant regions: CH with an amino acid sequence as shown in SEQ ID NO:15; and CL with an amino acid sequence as shown in SEQ ID NO:16; or an amino acid sequence having at least 80% identity with the aforementioned constant regions.

[0082] In other embodiments, the above-described constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the constant region (SEQ ID NO: 15 or 16).

[0083] In an optional embodiment, the antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0084] Fourthly, the present invention provides an antibody against influenza A virus, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO:19, 36, and 54, and the amino acid sequence of the light chain is shown in any one of SEQ ID NO:20, 38, and 56.

[0085] In an optional embodiment, the heavy chain and light chain amino acid sequences of the above-mentioned antibody are selected from any combination of the following:

[0086] combination Heavy chain Light chain 1 SEQ ID NO:19 SEQ ID NO:20 2 SEQ ID NO:36 SEQ ID NO:38 3 SEQ ID NO:54 SEQ ID NO:56

[0087] Fifthly, the present invention provides an antibody conjugate, wherein the aforementioned antibody conjugate includes the antibody described above.

[0088] In an optional embodiment, the antibody conjugate includes biotin or a biotin derivative.

[0089] In an optional embodiment, the antibody conjugate may further include a marker or purification tag.

[0090] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0091] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0092] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0093] In optional embodiments, the fluorescent dyes mentioned above include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0094] In optional embodiments, the enzymes mentioned above include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0095] In optional embodiments, the aforementioned radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0096] In optional embodiments, the chemiluminescent reagents mentioned above include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0097] In optional embodiments, the above-mentioned nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0098] In optional embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0099] In optional embodiments, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0100] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated with the antibody.

[0101] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0102] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

[0103] In this study, the conjugation of antibodies to conjugates does not affect the original properties of the antibodies.

[0104] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or antibody conjugate described above.

[0105] As previously stated, the antibodies in some embodiments or examples of this invention can effectively bind to influenza A virus. Therefore, reagents or kits containing the aforementioned influenza A virus antibodies can effectively perform qualitative or quantitative detection of influenza A virus. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of influenza A virus and its antibodies, such as immunoblotting and immunoprecipitation. As previously stated, the antibodies in some embodiments or examples of this invention have higher binding activity and specificity with influenza A virus; therefore, reagents or kits containing the aforementioned antibodies have higher detection sensitivity or specificity.

[0106] In a seventh aspect, the present invention provides a method for detecting influenza A virus, comprising: a) contacting the aforementioned antibody, antibody-conjugate, reagent, or kit with influenza A virus in a sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the aforementioned immune complex, the presence of which indicates the presence of the aforementioned antigen in the test sample.

[0107] In an optional embodiment, the immune complex further includes a second antibody, which binds to the antibody.

[0108] In an optional embodiment, the immune complex further includes a second antibody, which is in combination with influenza A virus.

[0109] Eighthly, the present invention provides the use of the above-mentioned anti-influenza A virus antibodies and antibody-drug conjugates in the preparation of products for detecting influenza A virus.

[0110] It should be noted that the products of this invention include, but are not limited to, reagents, kits, test strips, or reagent plates.

[0111] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0112] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0113] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0114] In a twelfth aspect, the present invention provides a method for preparing antibodies against influenza A virus, comprising: culturing cells as described above.

[0115] In this invention, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0116] In this invention, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include plasmids, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors.

[0117] In this invention, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be introduced, or have already been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." When the recombinant cell contains a vector, the vector can be introduced into mammalian cells to construct recombinant cells, which are then used to express the antibodies or antigen-binding fragments provided by this invention. The corresponding antibodies can be obtained by culturing the recombinant cells. Suitable mammalian cells include CHO cells, etc.

[0118] Based on the amino acid sequence of the anti-influenza A virus antibody disclosed in this invention, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, recombinant expression) to prepare the anti-influenza A virus antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing any of the antibodies described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-influenza A virus antibody of this invention, it falls within the protection scope of this invention.

[0119] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0121] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0122] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0123] Example 1: Antibody Discovery of Monoclonal Antibodies

[0124] 1. Animal immunization

[0125] An emulsion of influenza A virus N protein prepared with incomplete Freund's adjuvant was injected subcutaneously into 4-6 week old New Zealand white rabbits to stimulate an immune response. Serum was collected before and after immunization on days 0, 14, 28, 42 and 69 for titer testing. The spleen of rabbit #2, which met the titer requirements, was surgically removed to prepare a spleen cell suspension.

[0126] 2. B cell preparation

[0127] 2.1 Fresh single spleen cells were isolated and cultured overnight in B cell culture medium prepared in our laboratory.

[0128] 2.2 Fresh single-cell suspensions were prepared by diluting spleen cells with dissolved oxygen in PBS containing 2-3% fetal bovine serum and 1 mM EDTA.

[0129] 2.3 Individual B cells were sorted for influenza A NP antigen specificity using a Sony MA900 flow cytometer (Sonybiotechnology, Japan) and placed in each well of a 96-well plate.

[0130] 2.4 Primary B cells with influenza A NP antigen specificity were added to B cell culture medium and then cultured at 37°C and 5.5% CO2 for 7-10 days.

[0131] 2.5 At the end of the primary B cell culture, the B cell culture supernatant was screened and identified by ELISA. Three B cell positive clones with superior anti-influenza A virus N protein were screened and named Anti-FluA-9C15, Anti-FluA-15G2, and Anti-FluA-7B10.

[0132] 2.6 B-cell antibody gene sequencing:

[0133] RNA was extracted from Anti-FluA-9C15, Anti-FluA-15G2, and Anti-FluA-7B10 and reverse transcribed into cDNA. Then, the antibody gene fragment was amplified by PCR. Next, the antibody gene fragment was inserted into a sequencing T vector (purchased from Takara). Finally, the antibody gene was sequenced to obtain the gene sequence of the antibody variable region.

[0134] Example 2: Preparation of Monoclonal Antibodies

[0135] 1. Construction of recombinant antibody expression plasmid

[0136] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region gene, gene-specific primers for the light chain variable region and heavy chain variable region of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The light chain and heavy chain gene fragments of the antibody were amplified by PCR amplification.

[0137] The heavy chain and light chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the heavy chain gene and light chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of the heavy chain and light chain, respectively.

[0138] 2. Recombinant antibody production

[0139] 2.1 Recombinant Expression

[0140] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 3.0 × 10⁶ cells / ml. 6 Cells were washed at a concentration of [number] cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. Cells were then incubated at 35°C (120 rpm) with 8% CO2 for 13 days. After 13 days, the cells were centrifuged, and the cell supernatant was analyzed by ELISA. The experimental steps are as follows:

[0141] 1) Coating: Influenza A virus antigen PK2-FAN3 (from Feipeng Biotechnology) was coated at a concentration of 2ug / ml and incubated overnight at 4℃.

[0142] 2) Wash twice with PBST, blot dry, block with 120 μL / well of 20% bovine serum, incubate at 37°C for 1 h, and blot dry.

[0143] 3) Dilute the cell supernatant with 20% bovine serum 50 times and 100 times respectively, add 100 μL / well to the ELISA plate in step 2), and incubate at 37°C for 30 min.

[0144] 4) Wash 5 times with PBST, pat dry, add 100ul / well of goat anti-rabbit IgG-HRP (1% casein diluted 5000 times), and incubate at 37℃ for 30min.

[0145] 5) Wash 5 times with PBST, pat dry, add 50 μL each of solution A and solution B, incubate in the dark for 10 min, add 50 μL of stop solution, and read the values ​​using a microplate reader. The results are shown in Table 2. The results show that the supernatant of the recombinant antibodies expressing Anti-FluA-9C15, Anti-FluA-15G2, and Anti-FluA-7B10 has good binding activity.

[0146] Table 2: Cell supernatant binding activity data

[0147] Dilution factor Original Double 50 times 100 times Anti-FluA-9C15 supernatant 2.118 2.22 0.946 Anti-FluA-15G2 Supercleaner 2.18 1.967 1.536 Anti-FluA-7B10 Superfine Cleaner 2.166 2.062 1.095

[0148] 2.2 Antibody supernatant purification

[0149] The supernatant of the recombinant expressed antibody was centrifuged and purified using a Protein A affinity chromatography column to obtain purified antibodies. The obtained antibodies were named Anti-FluA-9C15Rmb, Anti-FluA-15G2Rmb, and Anti-FluA-7B10Rmb. The heavy chain amino acid sequence of antibody Anti-FluA-9C15Rmb is shown in SEQ ID NO:19, and the light chain amino acid sequence is shown in SEQ ID NO:20; the heavy chain variable region amino acid sequence of antibody Anti-FluA-15G2Rmb is shown in SEQ ID NO:36, and the light chain variable region amino acid sequence is shown in SEQ ID NO:38; the heavy chain variable region amino acid sequence of antibody Anti-FluA-7B10Rmb is shown in SEQ ID NO:54, and the light chain variable region amino acid sequence is shown in SEQ ID NO:56.

[0150] Example 3: Detection performance of antibodies on a colloidal gold platform

[0151] 1. Antibody labeling process

[0152] (1) Adjust the pH of the label: Take 10 ml of 40,000 colloidal gold, centrifuge with 150 μl of 0.2 M K2CO3, and stir to mix for 2 min;

[0153] (2) Antibody conjugation: Add 200ug of Anti-FluA-A, Anti-FluA-15G2Rmb, Anti-FluA-7B10Rmb and Anti-FluA-B (from Feipeng Biotechnology) respectively, and stir for 15min.

[0154] (3) Sealing: Add 10% (by mass volume) BSA and stir for 15 min;

[0155] (4) Centrifugation and storage: 10000 rpm / 7 min / room temperature, remove supernatant, resuspend in gold standard reconstitution solution to 1 ml, and store at 4℃ for later use;

[0156] 2. Wrapped

[0157] (1) Assemble the nitrocellulose membrane and colloidal gold PVC base plate for later use;

[0158] (2) Dilute Anti-FluA-9C15Rmb, Anti-FluA-B, and Anti-FluA-C (from Feipeng Biotechnology) to 1.0-2.0 mg / ml, and use a gold sputtering membrane spectrometer to draw lines evenly on the NC membrane. Then, place the membrane in a 50°C incubator for drying for at least 4 hours. Assemble the membrane, cut the strips, and add samples for detection.

[0159] The paired antibody tests are as follows:

[0160] Group Group 1 Group 2 Group 3 Group 4 Group 5 Wrapped Anti-FluA-9C15Rmb Anti-FluA-B Anti-FluA-C Anti-FluA-C Anti-FluA-C mark Anti-FluA-A Anti-FluA-A Anti-FluA-15G2Rmb Anti-FluA-7B10Rmb Anti-FluA-B

[0161] 3. Testing

[0162] (1) Sample:

[0163] Sample 1: Quality control samples: H3N2 recombinant antigen, H1N1 recombinant antigen, H5N1 recombinant antigen, H7N9 recombinant antigen, H1N2 recombinant antigen, and Cetura antigen.

[0164] Sample 2: Clinical samples positive for influenza A virus and influenza B virus

[0165] Sample 3: Clinical sample negative for influenza A virus

[0166] Sample 4: Influenza B strain 87 / 774 diluted 10-fold and 100-fold respectively.

[0167] (2) Detection method: Colloidal gold detection. The intensity of the test line is observed visually to determine the result. The intensity of the displayed band color indicates the activity of antigen-antibody binding in the sample. The color of the T-line developed by the colloidal gold test strip is compared with the standard color chart. The closest color is selected, and the corresponding color number is used to indicate the activity of the product. The smaller the number, the stronger the color development and the higher the activity; the larger the number, the weaker the color development and the lower the activity. A "+" after the number indicates slightly stronger, a "-" after the number indicates slightly weaker, and "B" represents negative.

[0168] 4. Test Results

[0169] (1) The test results of quality control products and positive clinical samples are shown in Tables 3 and 4. The results show that the activity and sensitivity of the test reagent composed of Anti-FluA-9C15, Anti-FluA-15G2Rmb and Anti-FluA-7B10Rmb are significantly better than those of the control antibody for different quality control products and positive clinical samples.

[0170] Table 3: Test Results of Quality Control Samples and Positive Clinical Samples - 1

[0171]

[0172] Table 4: Test Results of Quality Control Samples and Positive Clinical Samples - 2

[0173]

[0174]

[0175] (2) The detection results of paired antibodies of Group 1 and Group 2 against influenza B strain 87 / 774 10 are shown in Table 5. The detection results of paired antibodies of Group 3, Group 4 and Group 5 against positive clinical samples of influenza B virus are shown in Table 6. The results show that the detection reagent composed of antibody Anti-FluA-9C15 has no cross-reaction with influenza B strains of different concentrations and only binds to influenza A virus, with better specificity than the control antibody. The detection reagent composed of antibodies Anti-FluA-15G2Rmb and Anti-FluA-7B10Rmb has no cross-reaction with positive clinical samples of influenza B virus and only binds to influenza A virus, with good specificity.

[0176] Table 5: Detection results of influenza B strain

[0177] sample Group 1 Group 2 Influenza B strain 87 / 774, 10-fold dilution B 3+ Influenza B strain 87 / 774, 100-fold dilution B 4+

[0178] Table 6: Detection results of clinical samples positive for influenza B virus

[0179] sample Group 3 Group 4 Group 5 B Yang 1# B B B B Yang 2# B B B B Yang 3# B B B B Yang 4# B B B B Yang 5# B B B B Yang 6# B B B B Yang 7# B B B B Yang 8# B B B

[0180] (3) The paired antibodies of Group 1 and Group 2 were tested against 31 clinical samples of negative influenza A. All results were negative and no false positives were found. The specificity of the test reagent composed of Anti-FluA-9C15 was 100%.

[0181] The partial amino acid sequences involved in this application are shown in Table 7:

[0182] Table 7: Amino Acid Sequence List

[0183]

[0184]

[0185]

[0186]

[0187] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against influenza A virus, characterized in that, the antibody comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 35, 53 and three complementarity determining regions of a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 18, 37, 55; Optionally, the complementarity determining regions of the variable region are defined by any one of Kabat, Chothia, IMGT, AbM or Contact system or a combination of multiple systems.

2. An antibody against influenza A virus, characterized in that, The complementarity determining regions of the antibody comprise any one of (a)-(c): (a) the amino acid sequence of HCDR1 is RYGVT (SEQ ID NO: 1); the amino acid sequence of HCDR2 is YIDLVFGSTYYASWVND (SEQ ID NO: 2); the amino acid sequence of HCDR3 is ETDYNTGFFGI (SEQ ID NO: 3); the amino acid sequence of LCDR1 is QASEDIEKYLA (SEQ ID NO: 4); the amino acid sequence of LCDR2 is DASDLAS (SEQ ID NO: 5); and the amino acid sequence of LCDR3 is QNAYYPSNNHIA (SEQ ID NO: 6); (b) the amino acid sequence of HCDR1 is SYNMG (SEQ ID NO: 21); the amino acid sequence of HCDR2 is VINSYRITYYASWAKG (SEQ ID NO: 22); the amino acid sequence of HCDR3 is ETVYFYLDL (SEQ ID NO: 23); the amino acid sequence of LCDR1 is QGSQSISSSYLS (SEQ ID NO: 24); the amino acid sequence of LCDR2 is KASTLAS (SEQ ID NO: 25); and the amino acid sequence of LCDR3 is LYEDYIMSNNKA (SEQ ID NO: 26); and (c) the amino acid sequence of HCDR1 is SFAVG (SEQ ID NO: 39); the amino acid sequence of HCDR2 is IISGIGSVYYASWAKG (SEQ ID NO: 40); the amino acid sequence of HCDR3 is AIPSYTGGYNL (SEQ ID NO: 41); the amino acid sequence of LCDR1 is QASQSISIYLA (SEQ ID NO: 42); the amino acid sequence of LCDR2 is DASTLAS (SEQ ID NO: 43); and the amino acid sequence of LCDR3 is LQTYRSSAVDYA (SEQ ID NO: 44).

3. The antibody according to any one of claims 1 to 2, characterized in that, The antibody further comprises framework regions as set forth in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are selected from any one of (a')-(c'): (a') the amino acid sequence of HFR1 is RIKS (SEQ ID NO: 7); the amino acid sequence of HFR2 is VVYSGG (SEQ ID NO: 8); the amino acid sequence of HFR3 is RIKD (SEQ ID NO: 9); the amino acid sequence of HFR4 is RLD (SEQ ID NO: 10); the amino acid sequence of LFR1 is KASQD (SEQ ID NO: 11); the amino acid sequence of LFR2 is YTSRLHS (SEQ ID NO: 12); the amino acid sequence of LFR3 is QVYTLP (SEQ ID NO: 13); and the amino acid sequence of LFR4 is QLYDYTDPK (SEQ ID NO: 14); (a') HFR1, HFR2, HFR3, HFR4 of the amino acid sequences set forth, in order, in SEQ ID NO: 7 to SEQ ID NO: 10; and LFR1, LFR2, LFR3, LFR4 of the amino acid sequences set forth, in order, in SEQ ID NO: 11 to SEQ ID NO: 14; or amino acid sequences having at least 80% identity to each of the framework region sequences; (b') HFR1, HFR2, HFR3, HFR4 of the amino acid sequences set forth, in order, in SEQ ID NO: 27 to SEQ ID NO: 30; and LFR1, LFR2, LFR3, LFR4 of the amino acid sequences set forth, in order, in SEQ ID NO: 31 to SEQ ID NO: 34; or amino acid sequences having at least 80% identity to each of the framework region sequences; (c') HFR1, HFR2, HFR3, HFR4 of the amino acid sequences set forth, in order, in SEQ ID NO: 45 to SEQ ID NO: 48; and LFR1, LFR2, LFR3, LFR4 of the amino acid sequences set forth, in order, in SEQ ID NO: 49 to SEQ ID NO: 52; or amino acid sequences having at least 80% identity to each of the framework region sequences.

4. An anti-influenza A virus antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, the heavy chain variable region amino acid sequence is set forth in any one of SEQ ID NO: 17, 35, 53; and the light chain variable region amino acid sequence is set forth in any one of SEQ ID NO: 18, 37, 55.

5. The antibody according to any one of claims 1 to 4, characterized in that, the antibody further comprises a constant region; optionally, the constant region comprises a heavy chain constant region and a light chain constant region; optionally, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments; optionally, the heavy chain constant region comprises CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM and / or a tail peptide of IgM; optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; optionally, the species origin of the constant region is rabbit; optionally, the antibody comprises the following constant regions: CH of the amino acid sequence set forth in SEQ ID NO: 15; and CL of the amino acid sequence set forth in SEQ ID NO: 16; or amino acid sequences having at least 80% identity to each of the constant regions.

6. An anti-influenza A virus antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 1, and the light chain comprises SEQ ID NO:

2. the amino acid sequence of the heavy chain is set forth in any one of SEQ ID NO: 19, 36, 54; and the amino acid sequence of the light chain is set forth in any one of SEQ ID NO: 20, 38, 56.

7. An antibody conjugate, characterized in that, the antibody conjugate comprises the antibody of any one of claims 1 to 6; optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; optionally, the antibody conjugate further comprises a label or a purification tag conjugated to the antibody. Optionally, the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label. Optionally, the antibody conjugate further comprises a solid support coupled to the antibody.

8. A reagent or kit characterized in that, The reagent or kit comprises the antibody of any one of claims 1 to 6, the antibody conjugate of claim 7.

9. Use of the antibody of any one of claims 1 to 6, the antibody conjugate of claim 7, or the reagent or kit of claim 8 in the manufacture of a product for detecting influenza A virus; Optionally, the use comprises: a) contacting the antibody of any one of claims 1 to 6, the antibody conjugate of claim 7, the reagent or kit of claim 8 with influenza A virus in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immunocomplex further comprises a second antibody, the second antibody binding to the antibody; Optionally, the immunocomplex further comprises a second antibody, the second antibody binding to influenza A virus.

10. A nucleic acid molecule encoding the antibody of any one of claims 1 to 6, a vector comprising the nucleic acid molecule encoding the antibody of any one of claims 1 to 6, a cell comprising the nucleic acid molecule or the vector, or a method of making the antibody of any one of claims 1 to 6, the method comprising culturing the cell.

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