An antibody against avian influenza virus and application thereof

Abstract

This invention discloses an antibody against avian influenza virus and its applications, relating to the field of antibodies. The anti-avian influenza virus antibody disclosed in this invention includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. This antibody provides an important source of raw materials for the detection of avian influenza virus and exhibits good activity, detection sensitivity, and specificity.

Description

[0001] Cross-reference to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202411633035.9, filed on November 14, 2024, entitled "An antibody against avian influenza virus and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to an antibody against avian influenza virus and its application. Background Technology

[0004] Influenza viruses, as pathogens widely found in nature, pose a serious threat to the health of humans and many animals. Among them, influenza A viruses, due to their antigenic variability and high infectivity, have become a focus of global public health attention. Influenza A viruses can be further subdivided into several subtypes, such as H1N1, H3N2, H5N1, and H7N9. These subtypes differ in antigenic structure, but all can cause severe respiratory infections, even leading to pneumonia and death.

[0005] Avian influenza virus (H5N1), a highly pathogenic influenza virus, poses a significant threat to humans and poultry. In recent years, outbreaks of the H5N1 subtype of avian influenza virus have raised widespread concern about its transmissibility and potential public health impact. Its potential recombination and mutation capabilities make it a potential source of future influenza pandemics. Therefore, developing detection and identification technologies for the H5N1 virus is of paramount importance.

[0006] The H5N1 virus has a complex structure, primarily composed of two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). The HA protein is the virus's main surface antigen, playing a crucial role in the virus's invasion of host cells. The HA protein acts like a "key" in the virus's hand, binding to receptors on the surface of host cells through specific receptor binding sites, thereby unlocking and invading the host cell. This unique binding mechanism makes the HA protein an ideal target for developing antibody detection technologies.

[0007] Immunoassay based on the HA protein can not only detect the presence of the H5N1 virus, but also provide crucial information for virus control and vaccine development by identifying different viral variants. Traditional detection methods, such as virus isolation and PCR amplification, while highly sensitive, are complex, time-consuming, and require specialized laboratory equipment and personnel. In contrast, immunoassay is simple to operate, rapid, and accurate, making it more suitable for large-scale screening and on-site testing. Immunoassay is primarily based on the principle of antigen-antibody binding. Currently, there are relatively few antibody products on the market targeting avian influenza viruses, and their performance varies.

[0008] Based on the above background, the present invention aims to provide a high-performance monoclonal antibody against avian influenza virus and its preparation method, so as to play an important role in the field of rapid diagnosis of avian influenza virus. Summary of the Invention

[0009] This application provides an antibody against avian influenza virus, which provides an important source of raw materials for the detection of avian influenza virus. It has good activity and sensitivity, and can effectively detect different subtypes of H5N1 antigen and strains. It binds only to H5N1 antigen and does not bind to other subtypes of avian influenza antigen, thus exhibiting good detection specificity.

[0010] To achieve the above objective, according to a first aspect of the present invention, an antibody against avian influenza 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 of SEQ ID NO:31 to SEQ ID NO:35 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:36 to SEQ ID NO:40.

[0011] To achieve the above objectives, according to two aspects of the present invention, an antibody against avian influenza virus is provided, wherein the complementarity-determining region of the aforementioned antibody includes any one of (a) to (e):

[0012] (a) HCDR1 with amino acid sequences as shown in SEQ ID NO:1 (SYAMG), HCDR2 with amino acid sequences as shown in SEQ ID NO:2 (IINTAGSAYYASWAKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:3 (GAHSIDYTYFDI), and LCDR1 with amino acid sequences as shown in SEQ ID NO:4 (QASQSISSYLA), LCDR2 with amino acid sequences as shown in SEQ ID NO:5 (QASKLAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:6 (QSYYGTSGTASYNA);

[0013] (b) HCDR1 with amino acid sequences as shown in SEQ ID NO:7 (SNAMG), HCDR2 with amino acid sequences as shown in SEQ ID NO:8 (TITTSGTTYYASWAKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:9 (PYIGSSWGYYFNI), and LCDR1 with amino acid sequences as shown in SEQ ID NO:10 (QASENIYSGLA), LCDR2 with amino acid sequences as shown in SEQ ID NO:11 (SASTLAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:12 (LYGDYTISSAFA);

[0014] (c) HCDR1 with amino acid sequences as shown in SEQ ID NO:13 (GSWMN), HCDR2 with amino acid sequences as shown in SEQ ID NO:14 (RTYPGDGDSKYNGIFKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:15 (GRIPYYFDS), and LCDR1 with amino acid sequences as shown in SEQ ID NO:16 (RASESVDNYGNSFMN), LCDR2 with amino acid sequences as shown in SEQ ID NO:17 (LASNLEA), and LCDR3 with amino acid sequences as shown in SEQ ID NO:18 (QQNNEDPWT);

[0015] (d) HCDR1 as shown in SEQ ID NO:19 (DSWIS), HCDR2 as shown in SEQ ID NO:20 (RIFPGDGDSKYSGKFKG), HCDR3 as shown in SEQ ID NO:21 (GVLPWYFDV), and LCDR1 as shown in SEQ ID NO:22 (RASESVDNYGNSFMH), LCDR2 as shown in SEQ ID NO:23 (RASNLES), and LCDR3 as shown in SEQ ID NO:24 (QQSNEDPFT); and

[0016] (e) HCDR1 with amino acid sequences as shown in SEQ ID NO:25 (SYNFH), HCDR2 with amino acid sequences as shown in SEQ ID NO:26 (CIYPGNGGTNYSQKFRG), HCDR3 with amino acid sequences as shown in SEQ ID NO:27 (SYGTSYVGAMDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:28 (RASESVEYSGISLLQ), LCDR2 with amino acid sequences as shown in SEQ ID NO:29 (AASNVES), and LCDR3 with amino acid sequences as shown in SEQ ID NO:30 (QQSRKVPST).

[0017] To achieve the above objectives, according to a third aspect of the present invention, an antibody against avian influenza 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:31 to SEQ ID NO:35, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:36 to SEQ ID NO:40.

[0018] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody against avian influenza virus is provided, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as any one of SEQ ID NO:41, 42, 43, 44, 45, 56, and the amino acid sequence of the light chain is shown as SEQ ID NO:46 to SEQ ID NO:50.

[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 or antibody conjugate described above.

[0021] To achieve the above objectives, according to a seventh aspect of the present invention, the use of the above-described antibody and antibody-drug conjugate in the preparation of products for detecting avian influenza 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 avian influenza 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:31 to SEQ ID NO:35 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:36 to SEQ ID NO:40.

[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 is still able to 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 of antibodies 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 recognition unit of an antibody. Antigen-binding fragments of antibodies typically have the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand from the description of this invention that the antigen-binding fragment of an antibody 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 fragment of the antibody.

[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 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.

[0028] 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.

[0029] 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). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of 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.

[0030] Table 1: CDR Definition 1

[0031] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0032] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain 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., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0033] 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.

[0034] 3If neither H35A nor H35B exists, then HCDR1 ends at bit 35; if only H35A exists, then HCDR1 ends at bit 35A; if both H35A and H35B exist, then HCDR1 ends at bit 35B.

[0035] 4 If neither H35A nor H35B exists, then HCDR1 ends at bit 32; if only H35A exists, then HCDR1 ends at bit 33; if both H35A and H35B exist, then HCDR1 ends at bit 34.

[0036] 5 If neither H35A nor H35B exists, then HCDR1 ends at bit 33; if only H35A exists, then HCDR1 ends at bit 34; if both H35A and H35B exist, then HCDR1 ends at bit 35.

[0037] 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.

[0038] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0039] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0040] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0041] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0042] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0043] 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.

[0044] Secondly, embodiments of the present invention provide an antibody against avian influenza virus, wherein the complementarity-determining region of the aforementioned antibody includes any one of (a) to (e):

[0045] (a) HCDR1 with amino acid sequences as shown in SEQ ID NO:1 (SYAMG), HCDR2 with amino acid sequences as shown in SEQ ID NO:2 (IINTAGSAYYASWAKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:3 (GAHSIDYTYFDI), and LCDR1 with amino acid sequences as shown in SEQ ID NO:4 (QASQSISSYLA), LCDR2 with amino acid sequences as shown in SEQ ID NO:5 (QASKLAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:6 (QSYYGTSGTASYNA);

[0046] (b) HCDR1 with amino acid sequences as shown in SEQ ID NO:7 (SNAMG), HCDR2 with amino acid sequences as shown in SEQ ID NO:8 (TITTSGTTYYASWAKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:9 (PYIGSSWGYYFNI), and LCDR1 with amino acid sequences as shown in SEQ ID NO:10 (QASENIYSGLA), LCDR2 with amino acid sequences as shown in SEQ ID NO:11 (SASTLAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:12 (LYGDYTISSAFA);

[0047] (c) HCDR1 with amino acid sequences as shown in SEQ ID NO:13 (GSWMN), HCDR2 with amino acid sequences as shown in SEQ ID NO:14 (RTYPGDGDSKYNGIFKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:15 (GRIPYYFDS), and LCDR1 with amino acid sequences as shown in SEQ ID NO:16 (RASESVDNYGNSFMN), LCDR2 with amino acid sequences as shown in SEQ ID NO:17 (LASNLEA), and LCDR3 with amino acid sequences as shown in SEQ ID NO:18 (QQNNEDPWT);

[0048] (d) HCDR1 as shown in SEQ ID NO:19 (DSWIS), HCDR2 as shown in SEQ ID NO:20 (RIFPGDGDSKYSGKFKG), HCDR3 as shown in SEQ ID NO:21 (GVLPWYFDV), and LCDR1 as shown in SEQ ID NO:22 (RASESVDNYGNSFMH), LCDR2 as shown in SEQ ID NO:23 (RASNLES), and LCDR3 as shown in SEQ ID NO:24 (QQSNEDPFT); and

[0049] (e) HCDR1 with amino acid sequences as shown in SEQ ID NO:25 (SYNFH), HCDR2 with amino acid sequences as shown in SEQ ID NO:26 (CIYPGNGGTNYSQKFRG), HCDR3 with amino acid sequences as shown in SEQ ID NO:27 (SYGTSYVGAMDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:28 (RASESVEYSGISLLQ), LCDR2 with amino acid sequences as shown in SEQ ID NO:29 (AASNVES), and LCDR3 with amino acid sequences as shown in SEQ ID NO:30 (QQSRKVPST).

[0050] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0051] In optional embodiments, the antibodies described in the first and second aspects further include a framework region.

[0052] In an optional embodiment, the antibody described in the first and second aspects further comprises the framework regions shown by HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0053] In an optional embodiment, the heavy chain variable region and the light chain variable region of the antibody described in the first aspect and the second aspect are selected from any one of (a') to (e'):

[0054] (a') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:31, the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:36, and including the complementarity-determining region shown in (a) of the second aspect;

[0055] (b') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:32, the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:37, and including the complementarity-determining region shown in (b) of the second aspect;

[0056] (c') The heavy chain variable region has at least 80% identity with the amino acid sequence of SEQ ID NO:33, and the light chain variable region has at least 80% identity with the amino acid sequence of SEQ ID NO:38, and includes the complementarity-determining region shown in (c) of the second aspect;

[0057] (d') The heavy chain variable region having at least 80% identity with SEQ ID NO:34, and the light chain variable region having at least 80% identity with SEQ ID NO:39, and including the complementarity-determining region shown in (d) of the second aspect; and

[0058] (e') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:35, the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:40, and including the complementarity-determining region shown in (e) of the second aspect.

[0059] 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 complementarity-determining region (CDR). 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.

[0060] 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.

[0061] 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.

[0062] In other embodiments, the amino acid sequences of each frame region of the antibody against avian influenza 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.

[0063] Thirdly, embodiments of the present invention provide an antibody against avian influenza 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:31 to SEQ ID NO:35, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:36 to SEQ ID NO:40.

[0064] In an optional implementation, the combination of the above-mentioned heavy chain variable region and light chain variable region is selected from any combination of (A) to (E):

[0065] (A) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:31, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:36;

[0066] (B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:37;

[0067] (C) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:38;

[0068] (D) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:39; and

[0069] (E) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:35, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:40.

[0070] In optional embodiments, the antibodies described in the first, second, or third aspects above further include a constant region.

[0071] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and a light chain constant region.

[0072] 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.

[0073] 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, and / or CH4 of IgM.

[0074] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0075] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0076] 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.

[0077] In an optional implementation, the species source of the aforementioned constant region is rabbit.

[0078] In an optional implementation, the species source of the aforementioned constant region is a mouse.

[0079] 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:housemouse(Mus musculus)IGHC,IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. theinternational ImMunoGenetics information 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.

[0080] In an optional implementation, the aforementioned constant region is selected from any one of (F) to (I):

[0081] (F) The amino acid sequence CH as shown in SEQ ID NO:51; and the amino acid sequence CL as shown in SEQ ID NO:52; or an amino acid sequence having at least 80% identity with each of the constant regions;

[0082] (G) The amino acid sequence CH as shown in SEQ ID NO:53; and the amino acid sequence CL as shown in SEQ ID NO:55; or an amino acid sequence having at least 80% identity with each of the constant regions;

[0083] (H) amino acid sequences CH as shown in SEQ ID NO:54; and amino acid sequences CL as shown in SEQ ID NO:55; or amino acid sequences having at least 80% identity with each of the constant regions; and

[0084] (I) The amino acid sequence CH as shown in SEQ ID NO:57; and the amino acid sequence CL as shown in SEQ ID NO:52; or an amino acid sequence having at least 80% identity with each of the constant regions.

[0085] 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.

[0086] In an optional embodiment, the antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0087] Fourthly, the present invention provides an antibody against avian influenza virus, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the aforementioned heavy chain is shown in any one of SEQ ID NO:41, 42, 43, 44, 45, 56, and the amino acid sequence of the light chain is shown in any one of SEQ ID NO:46 to SEQ ID NO:50.

[0088] In an optional implementation, the combination of the heavy chain and the light chain described above is selected from any combination of (A') to (F'):

[0089] (A') The heavy chain with an amino acid sequence as shown in SEQ ID NO:41, and the light chain with an amino acid sequence as shown in SEQ ID NO:46;

[0090] (B') The heavy chain with the amino acid sequence shown in SEQ ID NO:42, and the light chain with the amino acid sequence shown in SEQ ID NO:47;

[0091] (C') The heavy chain with an amino acid sequence as shown in SEQ ID NO:43, and the light chain with an amino acid sequence as shown in SEQ ID NO:48;

[0092] (D') The heavy chain with an amino acid sequence as shown in SEQ ID NO:44, and the light chain with an amino acid sequence as shown in SEQ ID NO:49;

[0093] (E') The heavy chain with the amino acid sequence shown in SEQ ID NO:45, and the light chain with the amino acid sequence shown in SEQ ID NO:50; and

[0094] (F') The heavy chain with an amino acid sequence as shown in SEQ ID NO:56, and the light chain with an amino acid sequence as shown in SEQ ID NO:46.

[0095] Fifthly, the present invention provides an antibody conjugate, wherein the aforementioned antibody conjugate includes the antibody described above.

[0096] In an optional embodiment, the antibody conjugate includes biotin or a biotin derivative.

[0097] In an optional embodiment, the antibody conjugate may further include a marker or purification tag.

[0098] 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.

[0099] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0100] 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.

[0101] 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).

[0102] 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.

[0103] 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, 99mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 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.

[0104] 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.

[0105] 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.

[0106] In optional embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0107] In optional embodiments, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0108] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated with the antibody.

[0109] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0110] 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.

[0111] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or antibody conjugate described above.

[0112] As previously stated, the antibodies in some embodiments or examples of this invention can effectively bind to avian influenza viruses. Therefore, reagents or kits containing the aforementioned avian influenza virus antibodies can effectively perform qualitative or quantitative detection of avian influenza viruses. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of avian influenza viruses and their 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 avian influenza viruses; therefore, reagents or kits containing the aforementioned antibodies have higher detection sensitivity or specificity.

[0113] In a seventh aspect, the present invention provides a method for detecting avian influenza virus, comprising: a) contacting the aforementioned antibody, antibody-conjugate, reagent, or kit with avian influenza 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.

[0114] In an optional embodiment, the immune complex further includes a second antibody, which binds to the antibody.

[0115] In an optional embodiment, the immune complex further includes a second antibody that binds to the avian influenza virus.

[0116] Eighthly, the present invention provides the use of the above-mentioned anti-avian influenza virus antibodies and antibody-drug conjugates in the preparation of products for detecting avian influenza virus.

[0117] It should be noted that the products of this invention include, but are not limited to, reagents, kits, test strips, or reagent plates.

[0118] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0119] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0120] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0121] In a twelfth aspect, the present invention provides a method for preparing antibodies against avian influenza virus, comprising: culturing cells as described above.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] Based on the amino acid sequence of the anti-avian influenza 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-avian influenza 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-avian influenza virus antibody of this invention, it falls within the protection scope of this invention.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0130] Example 1: Antibody Discovery of Monoclonal Antibodies

[0131] Method 1:

[0132] 1. Animal immunization

[0133] H5N1 HA protein (from Phypeng Biotechnology) was mixed with an equal volume of Freund's complete adjuvant to obtain an oily emulsion. This emulsion was injected subcutaneously at multiple sites into BALB / c mice at a dose of 0.2 ml per mouse. Fourteen days later, mice were immunized intraperitoneally with the same antigen and adjuvant. Immunization continued until the fourth injection, and tail blood was collected for titer testing. If the titer met the fusion requirements, a booster immunization was administered intraperitoneally three days before fusion. The same dose of antigen was mixed with an equal volume of 0.9% sodium chloride injection.

[0134] 2. Preparation of hybridoma cell lines

[0135] On the third day after booster immunization of mice, spleens were removed under sterile conditions. Mouse tumor cells and immune spleen cells were mixed at a ratio of 1:10, fused, and cultured. The culture medium was changed twice on the sixth day. On the seventh day after fusion, the cell supernatant was collected for antibody detection to screen for hybridoma cell lines secreting specific antibodies. Three lines were identified and named 12F8, 10B6, and 6H4.

[0136] 3. Hybridoma cell antibody gene sequencing

[0137] RNA was extracted from 12F8, 10B6, and 6H4 hybridoma cells and reverse transcribed into cDNA. Then, antibody gene fragments were amplified by PCR. Next, the antibody gene fragments were 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.

[0138] The anti-H5N1 rabbit monoclonal antibody sequence obtained by the above method was expressed via eukaryotic recombination.

[0139] Method 2:

[0140] 1. Immunized animals

[0141] An emulsion of H5N1 HA protein prepared with incomplete Freund's adjuvant was administered subcutaneously to stimulate an immune response in 4-6 week old New Zealand white rabbits. Serum was collected before and after immunization at days 0, 14, 28, 42, and 69 for serum titer testing. Rabbits with acceptable titers were selected, and their spleens were surgically removed to prepare spleen cell suspensions. Fresh individual spleen cells were then isolated and cultured overnight in B cell culture medium.

[0142] 2. Specific single B cell acquisition

[0143] 1) Prepare a fresh cell suspension by diluting spleen cells with dissolved oxygen in PBS containing 2-3% fetal bovine serum and 1 mM EDTA.

[0144] 2) Individual B cells were sorted for H5N1HA antigen specificity using a Sony MA900 flow cytometer (Sonybiotechnology, Japan) and placed in each well of a 96-well plate.

[0145] 3) Add primary B cells with H5N1 HA specificity to B cell culture medium and then culture them at 37°C and 5.5% CO2 for 7-10 days.

[0146] 4) At the end of the primary B cell culture, the B cell culture supernatant was screened and identified by ELISA. Finally, two B cell positive clones with superior detection performance against H5N1 were selected and named 8J13 and 10D13, respectively.

[0147] 3. B-cell antibody gene sequencing:

[0148] RNA was extracted from 8J13 and 10D13 positive clones 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.

[0149] The anti-H5N1 rabbit monoclonal antibody sequence obtained by the above method was expressed via eukaryotic recombination.

[0150] Example 2: Preparation of Monoclonal Antibodies

[0151] 1. Construction of recombinant antibody expression plasmid

[0152] pcDNA TM 3.4 The vector is a constructed eukaryotic expression vector for the recombinant antibody. 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 variable region genes of the above-mentioned antibody 12F8, 10B6, 6H4, 8J13, and 10D13, gene-specific primers for the light chain variable region and heavy chain variable region of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The light chain gene fragment and the heavy chain gene fragment were amplified by PCR amplification.

[0153] 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.

[0154] 2. Recombinant antibody production

[0155] 2.1 Recombinant Cell Antibody Expression

[0156] HEK293 cells were revived early and passaged to a 200 mL 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 3.3 × 10⁻⁶ cells / mL. 6 Cells / mL were used as cell dilution buffers. 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. The cells were then incubated in a 35°C incubator at 120 rpm with 8% CO2. After 13 days, the cells were centrifuged and the supernatant was analyzed by ELISA.

[0157] 2.1.1 Indirect ELISA supernatant detection

[0158] 1) Coating: H5N1 antigen (from Feipeng Biotechnology) was coated at a concentration of 0.5 μg / mL and incubated overnight at 4°C.

[0159] 2) Wash twice with PBST, pat dry, block with 120 μL / well of 20% bovine serum, incubate at 37°C for 1 h, and pat dry.

[0160] 3) Dilute the supernatant of 8J13 and 10D13 cells with 20% bovine serum at 500-fold, 2000-fold, 2000-fold, and 10000-fold, respectively, and add 100 μL / well to the ELISA plate in step 2); dilute the supernatant of 12F8, 10B6, and 6H4 cells with 20% bovine serum, serially diluting from 240-fold to 15360-fold, and add 100 μL / well to the ELISA plate in step 2); incubate at 37°C for 30 min.

[0161] 4) Wash 5 times with PBST, pat dry, add goat anti-rabbit IgG-HRP (1% casein diluted 5000 times) to the ELISA plates corresponding to 8J13 and 10D13, add goat anti-mouse IgM-HRP (1% casein diluted 5000 times) to the ELISA plates corresponding to 12F8, 10B6, and 6H4, and incubate at 37℃ for 30 min.

[0162] 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 results using a microplate reader. The detection results for 8J13 and 10D13 are shown in Table 3, and the detection results for 12F8, 10B6, and 6H4 are shown in Table 4. The results show that antibodies 8J13, 10D13, 12F8, 10B6, and 6H4 can all effectively bind to the H5N1 antigen.

[0163] Table 3: Cell supernatant binding activity data by indirect ELISA - 1

[0164] Dilution factor Original Double 500 times 2000 times 10000 times 10D13 2.142 2.155 2.226 1.786 8J13 2.026 2.169 2.134 2.008

[0165] Table 4: Cell supernatant binding activity data by indirect ELISA - 2

[0166] Dilution factor 240 480 960 1920 3840 7680 15360 12F8 1.991 1.953 1.642 1.229 0.732 0.365 0.256 10B6 2.178 1.864 1.134 0.632 0.362 0.2 0.107 6H4 2.017 2.034 1.771 1.225 0.71 0.411 0.225

[0167] 2.2 Antibody supernatant purification

[0168] 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 10D13 Rmb, 8J13 Rmb, 12F8 Rmb, 10B6 Rmb, and 6H4 Rmb. The heavy chain amino acid sequences and light chain amino acid sequences of each antibody are shown in Table 5.

[0169] Table 5: Antibody Sequences

[0170]

[0171]

[0172] Example 3: Antibody Performance Detection

[0173] 1. Antibody labeling process

[0174] (1) Adjust the pH of the label: Take 10 mL of 40,000 colloidal gold, centrifuge, add 150 μL of 0.2 M K2CO3, and stir to mix for 2 min;

[0175] (2) Antibody conjugation: Add 200 μg of antibody 6H4 Rmb, 8J13 Rmb and 10D13 Rmb respectively, and stir for 15 min.

[0176] (3) Sealing: Add 10% (by mass volume) BSA and stir for 15 min;

[0177] (4) Centrifugation and storage: 10000 rpm / 7 min / room temperature, remove supernatant, resuspend in gold standard solution to 1 mL, and store at 4℃ for later use;

[0178] 2. Wrapped

[0179] (1) Assemble the nitrocellulose membrane and colloidal gold PVC base plate for later use;

[0180] (2) Dilute antibodies 12F8 Rmb, 10B6 Rmb, and 8J13 Rmb to 1.0-2.0 mg / mL respectively, and use a gold sputtering membrane spectrometer to draw lines evenly on the NC membrane. Then place the membrane in a 50℃ incubator for drying for at least 4 hours. Assemble the membrane, cut the strips, and add samples for detection.

[0181] 3. Testing

[0182] (1) Sample:

[0183] Sample 1: H5N1 recombinant antigen at different concentrations;

[0184] Sample 2: H5N1 strains from different sources (from NIBSC);

[0185] Sample 3: Different subtypes of avian influenza virus;

[0186] (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 label the activity of the product.

[0187] The smaller the number, the stronger the color development and the higher the activity; the higher the number, the weaker the color development and the lower the activity; a "+" after the number indicates slightly stronger color development, a "-" after the number indicates slightly weaker color development, and "B" represents negative.

[0188] (3) Test results

[0189] The results of antibody pairing detection are shown in Table 6. The results show that (1) the pairing of antibodies 10D13 Rmb, 8J13 Rmb, 12F8 Rmb, 10B6 Rmb and 6H4 Rmb can effectively detect H5N1, and they only bind to H5N1 antigen and do not bind to other subtype avian influenza antigens, so the detection specificity is good.

[0190] Table 6: Detection Results of Colloidal Gold Platform

[0191]

[0192] Example 4: Construction, preparation, and performance testing of IgG / IgM hybrid recombinant antibodies

[0193] 1. Construction and preparation of IgG / IgM hybrid recombinant antibodies

[0194] The constant region of the 8J13 Rmb antibody was modified to construct an IgG / IgM hybrid recombinant antibody.

[0195] 1.1 Construction of expression plasmid for IgG / IgM hybrid recombinant antibody

[0196] The heavy chain variable region encoding 8J13 Rmb was spliced ​​together with the sequence encoding the IgM constant region via overlap extension PCR. The polynucleotides obtained by PCR amplification were then ligated into the 3.4A expression vector after double digestion with HindIII / EcoRI. Since the polymeric properties of IgM depend on the heavy chain, the light chain expression plasmid of 8J13 Rmb was used.

[0197] 1.2. Preparation of IgG / IgM hybrid recombinant antibody expression

[0198] The heavy and light chain expression plasmids of the IgG / IgM hybrid recombinant antibody were diluted 1:1 and mixed with CHO cells. After electroporation, the cells were cultured under MSX pressure for approximately 25 days. Cell viability was recorded and observed. Cell lines with high antibody concentrations were selected for expansion culture and placed in a 37°C incubator at 120 rpm with 8% CO2. After 13 days, the cells were centrifuged to obtain the IgG / IgM hybrid recombinant antibody cell line, which was named 8J13A. The supernatant of 8J13A cells was diluted 100-fold, 200-fold, and 400-fold with 20% bovine serum, and ELISA was performed using the same method as in 2.1.1. The results are shown in Table 7, indicating that 8J13A can effectively bind to the H5N1 antigen.

[0199] Table 7: Cell supernatant binding activity data by indirect ELISA - 3

[0200] Dilution factor Original Double 100 times 200 times 400 times 8J13A 2.155 1.907 1.315 0.756

[0201] 2. Recombinant antibody expression

[0202] Using two packing materials, captoL (Cytiva) and CHT (Bio-Ray), the supernatant of the IgG / IgM hybrid recombinant antibody expression was purified in two steps to obtain the recombinant antibody, named 8J13A Rmb. The heavy chain amino acids of 8J13A Rmb are shown in SEQ ID NO:56, and the light chain amino acids are shown in SEQ ID NO:46.

[0203] 3. Antibody performance testing

[0204] The paired reagent composed of antibody 8J13A Rmb was used to detect different samples. The detection method was the same as in Example 3. The samples included H5N1 recombinant antigens of different subtypes, H5N1 strains from different sources (from NIBSC), and avian influenza viruses of different subtypes. The detection results are shown in Table 8. The results show that the paired reagent composed of 8J13A Rmb can detect different H5N1 recombinant antigens and different H5N1 strains more effectively. Moreover, it only binds to H5N1 antigen and does not bind to other subtypes of avian influenza antigens, showing good detection specificity.

[0205] Table 8: Detection Results of Colloidal Gold Platform - 2

[0206]

[0207]

[0208] The partial amino acid sequences involved in this application are shown in Table 9:

[0209] Table 9: Amino Acid Sequences

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] 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 avian influenza virus, characterized in that, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibody are, in order: The amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:31 and the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:36; The amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:33 and the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:38; or The amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:34 and the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:39; The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody against avian influenza virus, characterized in that, The antibody's HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include any one of (a) to (c): (a) HCDR1 with amino acid sequences as shown in SEQ ID NO:1 (SYAMG), HCDR2 with amino acid sequences as shown in SEQ ID NO:2 (IINTAGSAYYASWAKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:3 (GAHSIDYTYFDI), and LCDR1 with amino acid sequences as shown in SEQ ID NO:4 (QASQSISSYLA), LCDR2 with amino acid sequences as shown in SEQ ID NO:5 (QASKLAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:6 (QSYYGTSGTASYNA); (b) HCDR1 with amino acid sequences as shown in SEQ ID NO:13 (GSWMN), HCDR2 with amino acid sequences as shown in SEQ ID NO:14 (RTYPGDGDSKYNGIFKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:15 (GRIPYYFDS), and LCDR1 with amino acid sequences as shown in SEQ ID NO:16 (RASESVDNYGNSFMN), LCDR2 with amino acid sequences as shown in SEQ ID NO:17 (LASNLEA), and LCDR3 with amino acid sequences as shown in SEQ ID NO:18 (QQNNEDPWT); and (c) HCDR1 with amino acid sequences as shown in SEQ ID NO:19 (DSWIS), HCDR2 with amino acid sequences as shown in SEQ ID NO:20 (RIFPGDGDSKYSGKFKG), HCDR3 with amino acid sequences as shown in SEQ ID NO:21 (GVLPWYFDV), and LCDR1 with amino acid sequences as shown in SEQ ID NO:22 (RASESVDNYGNSFMH), LCDR2 with amino acid sequences as shown in SEQ ID NO:23 (RASNLES), and LCDR3 with amino acid sequences as shown in SEQ ID NO:24 (QQSNEDPFT).

3. The antibody according to any one of claims 1 to 2, characterized in that, The antibody also includes a framework region; Optionally, the heavy chain variable region and the light chain variable region of the antibody are selected from any one of (a') to (c'): (a') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:31, and the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:36, and including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in claim 2(a); (b') The heavy chain variable region having at least 80% identity with SEQ ID NO:33, and the light chain variable region having at least 80% identity with SEQ ID NO:38, and including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in claim 2 (b); and (c') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:34, and the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:39, and including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in claim 2 (c).

4. An antibody against avian influenza virus, comprising a heavy chain variable region and a light chain variable region, characterized in that, The combination of the heavy chain variable region and the light chain variable region is selected from any combination of (A) to (C): (A) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:36; (B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:38; and (C) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:

39.

5. The antibody according to any one of claims 1 to 4, characterized in that, The antibody also contains a constant region.

6. The antibody according to claim 5, characterized in that, The constant region includes the heavy chain constant region and the light chain constant region.

7. The antibody according to claim 6, characterized in that, The heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, and IgD.

8. The antibody according to claim 6, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

9. The antibody according to claim 6, characterized in that, The light chain constant region includes light chain constant regions selected from κ-type or λ-type.

10. The antibody according to claim 5, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

11. The antibody according to claim 5, characterized in that, The species origin of the constant region is rabbit.

12. The antibody according to claim 5, characterized in that, The species origin of the constant region is mice.

13. The antibody according to claim 5, characterized in that, The constant region is selected from any one of (F) to (I): (F) The amino acid sequence CH as shown in SEQ ID NO:51; and the amino acid sequence CL as shown in SEQ ID NO:52; or an amino acid sequence having at least 80% identity with each of the constant regions; (G) The amino acid sequence CH as shown in SEQ ID NO:53; and the amino acid sequence CL as shown in SEQ ID NO:55; or an amino acid sequence having at least 80% identity with each of the constant regions; (H) The amino acid sequence CH as shown in SEQ ID NO:54; and the amino acid sequence CL as shown in SEQ ID NO:55; or an amino acid sequence having at least 80% identity with each of the constant regions; and (I) The amino acid sequence CH as shown in SEQ ID NO:57; and the amino acid sequence CL as shown in SEQ ID NO:52; or an amino acid sequence having at least 80% identity with each of the constant regions.

14. An antibody against avian influenza virus, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:41, and the amino acid sequence of the light chain is shown in SEQ ID NO:

46. The amino acid sequence of the heavy chain is shown in SEQ ID NO:43, and the amino acid sequence of the light chain is shown in SEQ ID NO:48; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:44, and the amino acid sequence of the light chain is shown in SEQ ID NO:

49.

15. An antibody conjugate, characterized in that, The antibody conjugate includes the antibody according to any one of claims 1 to 14, and the antibody conjugate includes biotin, a label, a purification tag, or a solid-phase carrier conjugated to the antibody.

16. The antibody conjugate according to claim 15, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

17. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 14 or the antibody conjugate as described in any one of claims 15 and 16.

18. Use of the antibody according to any one of claims 1 to 14, or the antibody conjugate according to any one of claims 15 and 16, in the preparation of products for detecting avian influenza virus.

19. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1 to 14.

20. A carrier, characterized in that, The vector contains the nucleic acid molecule encoding the claim 19.

21. A cell, characterized in that, The cell contains the nucleic acid molecule of claim 19 or the vector of claim 20.

22. A method for preparing the antibody according to any one of claims 1 to 14, characterized in that, The method comprises culturing the cells of claim 21.

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