WO2023224618A1

WO2023224618A1 - Recombinant antibodies, kits comprising the same, and uses thereof in diagnosing influenza virus

Info

Publication number: WO2023224618A1
Application number: PCT/US2022/029893
Authority: WO
Inventors: An-Suei Yang; Chung-Ming Yu; Chao-Ping Tung; Hung-Ju Hsu; Hung-Pin Peng; Chi-Yung Chen; Yu-Chuan Huang; Pei-Hsun Tsai; Szu-Yu Lin; Fei-Hung HUNG
Original assignee: Academia Sinica; Liu, Fu-Tong
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2023-11-23

Abstract

Disclosed herein are recombinant antibodies or the fragments thereof for detecting influenza A virus (IAV) or influenza B virus (IBV). Also disclosed herein are kits comprising the recombinant antibodies, and methods of making a diagnosis of the infection of IAV or IBV by using the recombinant antibody or kit.

Description

RECOMBINANT ANTIBODIES, KITS COMPRISING THE SAME, AND USES

THEREOF IN DIAGNOSING INFLUENZA VIRUS

BACKGROUND OF THE INVENTION

[0001] 1. FIELD OF THE INVENTION

[0002] The present disclosure in general relates to the field of disease diagnosis. More particularly, the present disclosure relates to recombinant antibodies specific to influenza A virus (IAV) or influenza B virus (IBV), and uses thereof in diagnosing IAV or IBV infection.

[0003] 2. DESCRIPTION OF RELATED ART

[0004] Influenza, commonly known as “flu”, is an infectious disease caused by an influenza virus. The symptoms of influenza include fever, runny or stuffy nose, sore throat, muscle and joint aches, headache, cough, fatigue, vomiting and diarrhea. The symptoms typically begin two days after exposure to the influenza virus, and last for several days to weeks. According to the reports of Centers for Disease Control and Prevention (CDC), influenza has resulted in 9-45 million illnesses, 140,000-810,000 hospitalizations and 12,000-61,000 deaths annually since 2010.

[0005] There are two main types of influenza virus, type A and type B, which are routinely spread in people (as human influenza viruses), and are responsible for seasonal flu epidemics every year. Influenza virus type A (also known as influenza A virus, IAV) may further be divided into subtypes based on the surface protein hemagglutinin (HA) and neuraminidase (NA). There are 18 different HA subtypes (from Hl to Hl 8) and 11 different NA subtypes (from N1 to Nil). Current subtypes of IAV circulating in the human population include H1N1 and H3N2. Rapid detection of these emerging influenza virus strains is a critical measure responding to the threats imposed by the influenza pandemic outbreaks and seasonal influenza epidemics on human society and economy. Rapid influenza diagnostic tests (RIDTs) for the nucleoprotein (NP) of influenza virus are frequently used to enable healthcare professionals to make immediate and effective treatment decisions and prevent unnecessary prescriptions of antibiotics and antiviral medications. Lateral flow immunoassay (LFIA)-based tests for IAV and IBV have been widely available as RIDTs, but the sensitive of these tests are nevertheless in the range of 40% to 70%, partly due to the difficulty to cover increasingly diverse influenza strains.

[0006] In view of the forging, there exists in the related art a need for an agent and/or a method for detecting IAV or IBV so as to establish a diagnostic platform for infection prevention and/or treatment purposes. SUMMARY

[0007] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0008] As embodied and broadly described herein, one aspect of the disclosure is directed to a recombinant antibody or the fragment thereof. According to embodiments of the present disclosure, the recombinant antibody or the antibody fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain, in which the VL domain comprises a first light chain complementarity determining region (CDR-L1), a second light chain CDR (CDR-L2) and a third light chain CDR (CDR-L3); and the VH domain comprises a first heavy chain CDR (CDR-H1), a second heavy chain CDR (CDR-H2) and a third heavy chain CDR (CDR-H3).

[0009] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to influenza A virus (IAV), in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 1-3, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 4-6. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 42, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 43. In some working examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 42 and 43.

[0010] According to certain embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IAV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 7-9, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 10-12. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 44, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 45. In some working examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 44 and 45.

[0011] According to certain embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IAV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 13, 2 and 14, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 15-17. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 46, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 47. In certain examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 46 and 47.

[0012] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IAV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 18-20, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 21-23. According to certain preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 48, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 49. In certain examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 48 and 49.

[0013] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IAV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 24-26, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 27-29. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 50, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 51. In certain working examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 50 and 51.

[0014] According to certain embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IAV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 30-32, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 33-35. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 52, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 53. In certain working examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 52 and 53. [0015] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IAV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 36-38, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 39-41. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 54, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 55. In some specific examples, the VL and VH domains of the anti-IAV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 54 and 55

[0016] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to influenza B virus (IBV), in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 56-58, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 59-61. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 91, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 92. In some working examples, the VL and VH domains of the anti-IBV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 91 and 92.

[0017] According to certain embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IBV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 62-64, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 65-67. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 93, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 94. In some working examples, the VL and VH domains of the anti-IBV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 93 and 94.

[0018] According to certain embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IBV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 68-70, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 71-73. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 95, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 96. In certain examples, the VL and VH domains of the anti-IBV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 95 and 96.

[0019] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IBV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 74-76, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 77, 10 and 78. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 97, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 98. In certain examples, the VL and VH domains of the anti-IBV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 97 and 98.

[0020] According to some embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IBV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 79-81, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 82-84. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 99, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 100. In certain working examples, the VL and VH domains of the anti-IBV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 99 and 100.

[0021] According to certain embodiments of the present disclosure, the recombinant antibody or antibody fragment is specific to IBV, in which the CDR-L1, CDR-L2 and CDR-L3 respectively have the amino acid sequences of SEQ ID NOs: 85-87, and the CDR-H1, CDR-H2 and CDR-H3 respectively have the amino acid sequences of SEQ ID NOs: 88-90. According to some preferred embodiments, the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 101, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 102. In certain working examples, the VL and VH domains of the anti-IBV recombinant antibody or antibody fragment respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 101 and 102.

[0022] Another aspect of the present disclosure is directed to a kit for detecting the presence of IAV or IBV in a biological sample. The kit comprises a first recombinant antibody, a second recombinant antibody, and a container containing the first and second recombinant antibodies. According to some embodiments, the first and second recombinant antibodies of the kit for detecting IAV are independently selected from the anti-IAV recombinant antibodies as described above.

[0023] In one exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 1-3 and 4-6, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 18-20 and 21-23. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 42 and 43, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 48 and 49.

[0024] In another exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 7-9 and 10-12, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 13, 2, 14, and 15-17. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 44 and 45, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 46 and 47.

[0025] In still another exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 24-26 and 27-29, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 36-38 and 39-41. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 50 and 51, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 54 and 55.

[0026] In further another exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 24-26 and 27-29, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 30-32 and 33-35. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 50 and 51, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 52 and 53.

[0027] According to certain embodiments, the first and second recombinant antibodies of the kit for detecting IBV are independently selected from the anti-IBV recombinant antibodies as described above.

[0028] In one exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 74-76, and 77, 10, 78, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 79-81 and 82-84. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 97 and 98, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 99 and 100.

[0029] In another exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 62-64 and 65-67, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 85-87 and 88-90. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 93 and 94, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 101 and 102.

[0030] In still another exemplary embodiment, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 56-58 and 59-61, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences of SEQ ID NOs: 68-70 and 71-73. Preferably, the VL and VH domains of the first recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 91 and 92, and the VL and VH domains of the second recombinant antibody respectively comprise amino acid sequences 100% identical to SEQ ID NOs: 95 and 96.

[0031] According to certain embodiments, one of the first and second recombinant antibodies serves as a capture antibody, and the other of the first and second recombinant antibodies serves as a detection antibody for use in a detection technique, e.g., an enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and western blotting (WB) assay.

[0032] Also provided herein is a method of making a diagnosis of whether a subject is infected by IAV or IBV via a biological sample isolated from the subject. The method comprises the steps of, detecting the presence or absence of the nucleoprotein of IAV or IBV (i.e., detecting the presence or absence of NPA or NPB) in the biological sample by use of the antibody fragment, the recombinant antibody or the kit of the present disclosure, wherein when the nucleoprotein is present in the biological sample, then diagnosing that the subject is infected by the IAV or IBV.

[0033] Based on the result, a skilled artisan or a clinical practitioner may administer to a subject in need thereof (e.g., a subject infected by IAV or IBV) an appropriate treatment in time. Specifically, in the case when the nucleoprotein is present in the biological sample of a subject, then an effective amount of an anti-viral treatment (e.g., oseltamivir, zanamivir, peramivir, baloxavir marboxil, amantadine, rimantadine, interferon-alpha (IFN-a), interferon-beta (IFN- ), or a combination thereof) is administered to the subject so as to alleviate and/or ameliorate the symptoms associated with IAV or IBV infection.

[0034] The subject is a mammal; preferably, a human.

[0035] Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:

[0037] Figs. 1A and IB are the results of western blotting assay according to Example 1 of the present disclosure, in which NPAs (i.e., the nucleoproteins derived from IAV, including NPA1 to NPA8) and NPBs (i.e., the nucleoproteins derived from IBV, NPB1 and NPB2) were detected by specified antibodies.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0039] I. Definition

[0040] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Also, unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more.

[0041] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0042] The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific or multivalent antibodies (e.g., bi-specific antibodies), and antibody fragments so long as they exhibit the desired biological activity. The term “antibody fragment” or “the fragment of an antibody” refers to a portion of a full-length antibody, generally the antigen binding or variable domain (i.e., VL and VH domains) of a full-length antibody. Examples of the antibody fragment include fragment antigen-binding (Fab), Fab’, F(ab’)2, single-chain variable fragment (scFv), diabody, linear antibody, single-chain antibody molecule, and multi-specific antibody formed from antibody fragments.

[0043] The term “complementarity determining region (CDR)” used herein refers to the hypervariable region of an antibody molecule that forms a surface complementary to the 3-dimensional surface of a bound antigen. Proceeding from N-terminus to C-terminus, each of the antibody heavy and light chains comprises three CDRs (i.e., CDR-1, CDR-2, and CDR-3). A HLA-DR antigen-binding site, therefore, includes a total of six CDRs that comprise three CDRs from the variable domain of a heavy chain (i.e., CDR-H1, CDR-H2, and CDR-H3), and three CDRs from the variable domain of a light chain (i.e., CDR-L1, CDR-L2, and CDR-L3). The amino acid residues of CDRs are in close contact with bound antigen, wherein the closest antigen contact is usually associated with the heavy chain CDR3. [0044] As used herein, the term “variable domain” or “variable region” of an antibody refers to the amino-terminal regions of heavy or light chain of the antibody. These regions are generally the most variable parts of an antibody and contain the antigen-binding sites. The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies, and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprises four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0045] “Percentage (%) sequence identity” with respect to any amino acid sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two amino acid sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI). The percentage sequence identity of a given sequence A to a subject sequence B (which can alternatively be phrased as a given sequence A that has a certain % sequence identity to a given sequence B) is calculated by the formula as follows:

where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in the subject sequence B.

[0046] As discussed herein, minor variations in the amino acid sequences of antibodies are contemplated as being encompassed by the presently disclosed and claimed inventive concept(s), providing that the variations in the amino acid sequence maintain at least 85% sequence identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity. Antibodies of the present disclosure may be modified specifically to alter a feature of the peptide unrelated to its physiological activity. For example, certain amino acids can be changed and/or deleted without affecting the physiological activity of the antibody in this study (i.e., the ability of binding to coronavirus). In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the peptide derivative. Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxyl-termini of fragments or analogs occur near boundaries of functional regions.

[0047] The term “subject” refers to a mammal including the human species that can be subjected to the diagnostic methods of the present invention. The term “subject” is intended to refer to both the male and female gender unless one gender is specifically indicated.

[0048] II. Description of the Invention

[0049] (II-l) Methods for selecting an antibody fragment specific to IAV or IBV

[0050] The first aspect of the present disclosure is directed to a method for selecting an antibody fragment specific to IAV or IBV. According to embodiments of the present disclosure, the method comprises the steps of,

(a) providing a phage-displayed single-chain variable fragment (scFv) library that comprises a plurality of phage-displayed scFvs, wherein the heavy chain variable (VH) domain of each phage-displayed scFvs has a binding affinity to protein A, and the light chain variable (VL) domain of each phage-displayed scFvs has a binding affinity to protein L;

(b) exposing the phage-displayed scFv library of the step (a) to at least one nucleoprotein of the IAV or IBV;

(c) selecting, from the phage-displayed scFv library of the step (b), a plurality of phages that respectively express scFvs exhibiting binding affinity to the nucleoprotein;

(d) respectively enabling the plurality of phages selected in the step (c) to express a plurality of soluble scFvs;

(e) exposing the plurality of soluble scFvs of the step (d) to the nucleoprotein;

(f) determining the respective binding affinity of the plurality of soluble scFvs to the nucleoprotein in the step (e); and

(g) based on the results determined in the step (f), selecting one soluble scFv that exhibits superior binding affinity to the nucleoprotein over the other soluble scFvs of the plurality of soluble scFvs as the antibody fragment.

[0051] The present method is useful in selecting an antibody fragment exhibiting a binding affinity and/or specificity to LAV or IBV, and accordingly, providing a potential means to detect various subtypes of influenza virus with diverse but highly similar antigens, for example, H1N1, H1N2, H2N2, H3N2, H5N1, H5N2, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N7.

[0052] In the step (a), a phage-displayed scFv library is provided. According to the embodiments of the present disclosure, the framework of the phage-displayed scFv library is based on the human IGKV1-NL1*O1/IGHV3-23*O4 germline sequence, and the complementarity determining region (CDR, including CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) thereof are diversified by PCR reaction using desired primers. After the selection of protein A and protein L, the phage-displayed scFv library (hereinafter as “GH2 library”) is produced, in which each of the plurality of phage-displayed scFvs has a VH domain capable of binding to protein A, and a VL domain capable of binding to protein L. This phage-displayed scFv library can be constructed using the method described in the co-pending PCT applications, PCT/US2016/19128 and PCT/US2018/56627. The entirety of the application is incorporated herein by reference. [0053] In the step (b), the GH2 library is exposed to NPA (i.e., a nucleoprotein of IAV) or NPB (i.e., a nucleoprotein of IBV). According to some embodiments, the NPA comprises the amino acid sequence of any of SEQ ID NOs: 103-110, respectively designated as “NPA1” to “NPA8”. In some working examples, the GH2 library is exposed to a mixture of NPA1-NPA8 (1:1:1:1:1:1:1:1). According to certain embodiments, the NPB comprises the amino acid sequence of SEQ ID NO: 111 or 112, respectively designated as “NPB1” and “NPB2”. In some working examples, the GH2 library is exposed to a mixture of NPB 1 and NPB2 (1:1). According to some working examples, the nucleoprotein of IAV or IBV is immobilized on a matrix (such as an agarose resin or polyacrylamide) and then mixed with the present GH2 library.

[0054] In the step (c), a plurality of phages i.e., a first plurality of phages) respectively expressing scFvs that exhibit binding affinity to the nucleoprotein of IAV or IBV are selected from the GH2 library. Specifically, the product of the step (b) is subjected to an elution buffer, which generally is an acidic solution (such as glycine solution, pH 2.2), so as to disrupt the binding between the nucleoprotein and phage-display scFv. By this way, the plurality of phages that respectively express scFvs exhibiting binding affinity to the nucleoprotein are collected.

[0055] Optionally, the step (c) is carried out under an acidic condition. Specifically, the product of the step (b) may be subjected to an acidic treatment (for example, a washing buffer having a pH value ranging between 5-7, such as pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7; preferably, a washing buffer having a pH value of 5.0) followed by the afore-mentioned elution step to collect the plurality of phages.

[0056] Optionally, for the purpose of enriching the population of scFvs with binding specificity to the nucleoprotein, the plurality of phages selected in the step (c) is subjected to the nucleoprotein in the presence of one or more scrambled nucleoproteins (as an optional step (c)-l), wherein each scrambled nucleoprotein comprises an amino acid sequence different from the amino acid sequence of the nucleoprotein. For example, in the case when the nucleoprotein comprises the amino acid sequence of SEQ ID NO: 103 (i.e., NPA1), then the scrambled nucleoprotein comprises the amino acid sequence of any of SEQ ID NOs: 104-112 (i.e., any of NPA2 to NPA8, NPB1 and NPB2); preferably, the scrambled nucleoprotein comprises the amino acid sequence of any of SEQ ID NOs: 104-110 (i.e., any of NPA2 to NPA8). Alternatively, when the nucleoprotein comprises the amino acid sequence of SEQ ID NO: 106 (i.e., NPA4) then the scrambled nucleoprotein comprises the amino acid sequence of any of SEQ ID NOs: 103-105 and 107-112 (i.e., any of NPA1 to NPA3, NPA5 to NPA8, NPB1 and NPB2); preferably, the scrambled nucleoprotein comprises the amino acid sequence of any of SEQ ID NOs: 103-105 and 107-110 (i.e., any of NPA1 to NPA3 and NPA5 to NPA8). Still alternatively, when the nucleoprotein comprises the amino acid sequence of SEQ ID NO: 110 (z.e., NPA8) then the scrambled nucleoprotein comprises the amino acid sequence of any of SEQ ID NOs: 103-109, 111 and 112 (i.e., any ofNPAl to NPA7, NPB1 and NPB2); preferably, the scrambled nucleoprotein comprises the amino acid sequence of any of SEQ ID NOs: 103-109 (i.e., any ofNPAl to NPA7). According to one embodiment, the nucleoprotein of IAV or IBV is immobilized on a matrix (such as an agarose resin or polyacrylamide), and then mixed with the present GH2 library in the presence of one or more scrambled nucleoproteins.

[0057] Still optionally, a plurality of phages i.e., a second plurality of phages) respectively expressing scFvs that exhibit binding specificity to the nucleoprotein in the presence of scrambled nucleoprotein(s) are selected from the first plurality of phages (as an optional step (c)-2). In a manner similar to the step (c), the product of the optional step (c)-l is subjected to an elution buffer, for example, an acidic solution (e.g., glycine solution, pH 2.2), so as to disrupt the binding between the nucleoprotein and phage-display scFv. By this way, the second plurality of phages that respectively express scFvs exhibiting binding specificity to the nucleoprotein are collected.

[0058] Next, in the step (d), the plurality of phages selected in the step (c) (i.e., the first plurality of phages) or selected in the optional step (c)-2 (i.e., the second plurality of phages) are subjected to conditions that enable them to produce a plurality of soluble scFvs. This step can be carried out by using methods known to any person having ordinary skill in the art. According to certain embodiments of the present disclosure, the expression of VH and VL domains may be driven by a lactose operon (lac operon); as known by one skilled artisan, the lac operon would be induced by isopropyl-thio-p-D-galactoside (IPTG), which then drives the expression of the down-stream genes (i.e., genes encoding the VH and VL domains). The produced scFv are then secreted into the supernatant of culture medium and could be collected therefrom.

[0059] In the step (e), the soluble scFvs produced in the step (d) are respectively mixed with the nucleoprotein of IAV or IBV so as to form the protein-scFv complexes.

[0060] Then, in the step (I), the level of the protein-scFv complexes formed in the step (e) is determined by a method known to a person having ordinary skill in the art for analyzing the binding affinity of two molecules (e.g., the binding affinity of an antibody to an antigen); for example, enzyme-linked immunosorbent assay (ELISA), western blotting (WB) assay, flow cytometry, surface plasmon resonance (SPR), or lateral flow immunoassay (LFIA). In general, the level of the protein-scFv complexes is proportional to the binding affinity of the scFv to the nucleoprotein. According to one working example, the level of the protein-scFv complex (i.e., the binding affinity of the soluble scFv to the nucleoprotein) is determined by ELISA.

[0061] Finally, in the step (g), the antibody fragment is selected based on the binding affinity determined in the step (f). More specifically, the soluble scFv that exhibits superior affinity to the nucleoprotein over the other soluble scFvs of the plurality of soluble scFvs is selected as the antibody fragment.

[0062] According to some embodiments of the present disclosure, the nucleoprotein for selecting the antibody fragment is derived from IAV i.e., NPA), in which 7 antibody fragments, respectively designated as “NPA-008 scFv”, “NPA-020 scFv”, “NPA-045 scFv”, “NPA-058 scFv”, “NPA-064 scFv”, “NPA-065 scFv” and “NPA-066 scFv”, are selected from different rounds of selection.

[0063] According to certain embodiments of the present disclosure, the nucleoprotein for selecting the antibody fragment is derived from IBV (i.e., NPB), in which 6 antibody fragments, respectively designated as “NPB-010 scFv”, “NPB-020 scFv”, “NPB-034 scFv”, NPB-042 scFv”, “NPB-046 scFv” and “NPB-055 scFv”, are selected from different rounds of selection.

[0064] (H-2) Methods for producing recombinant antibodies

[0065] The antibody fragment selected from section (II-l) of the present disclosure, e.g., any of the anti-NPA antibody fragments and the anti-NPB antibody fragments, is useful in the preparation of a recombinant antibody, which structurally comprises a VL domain, a light chain constant (CL) domain, a VH domain and a heavy chain constant (CH) domain. The method of using the selected antibody fragment to produce a recombinant antibody comprises the steps of,

(1) providing a phage that expresses the selected antibody fragment;

(2) extracting a phagemid DNA corresponding to the phage of the step (1);

(3) respectively amplifying a first nucleic acid sequence that encodes a VH domain, and a second nucleic acid sequence that encodes a VL domain by PCR using the phagemid DNA of the step (2) as a template;

(4) inserting the first and second nucleic acid sequences into an expression vector that comprises a third and a fourth nucleic acid sequences, wherein the third nucleic acid sequence encodes the CH domain of an immunoglobulin, and the fourth nucleic acid sequence encodes the CL domain of the immunoglobulin; and

(5) transfecting a host cell with the expression vector of the step (4) that comprises the first, second, third, and fourth nucleic acid sequences so as to produce the recombinant antibody. [0066] In the present method, the phage that expresses the selected antibody fragment is used as a starting material for the preparation of a recombinant antibody (i.e., the step (1)).

[0067] Then, the phagemid DNA corresponding to the antibody fragment-expressing phage is extracted as described in the step (2). Depending on intended purposes, the phagemid may be extracted by lysing the phage; alternatively, the phagemid may be obtained from a bacterial clone (i.e., the phagemid-containing bacterial clone). The extraction of phage DNA from the phage or bacterial clone could be achieved via any conventional DNA extraction technique; for example, the phenol/chloroform assay, and detergent (e.g., sodium dodecyl sulfate, TWEEN®-20, NP-40, and TRITON® X-100)/acetic acid assay.

[0068] In the step (3), the thus extracted phagemid DNA then serves as a template to respectively amplify the first nucleic acid sequence that encodes the CDR-H1, CDR-H2, and CDR-H3 by PCR using specific primers (forward primer: SEQ ID NO: 113; reverse primer: SEQ ID NO: 114), and the second nucleic acid sequence that encodes the CDR-L1, CDR-L2, and CDR-L3 by PCR using specific primers (forward primer: SEQ ID NO: 115; reverse primer: SEQ ID NO: 116).

[0069] In the step (4), the amplified first and second nucleic acid sequences are inserted into an expression vector, which comprises a third nucleic acid sequence encoding the constant domains of the heavy chain of an immunoglobulin, and a fourth nucleic acid sequence encoding the constant domains of the light chain of the immunoglobulin. As could be appreciated, the immunoglobulin can be any of IgG, IgA, IgD, IgE, and IgM. In one preferred embodiment of the present disclosure, the immunoglobulin is IgG. Specifically, the first and second nucleic acid sequences are first linked by a linker, which is amplified from plgG vector by PCR. According to the embodiment of the present disclosure, the linker comprises in sequence: the CL domain, a bovine growth hormone (BGH) polyadenylation (polyA) signal, a human CMV promoter, and a signal peptide of IgG heavy chain. For the presences of the complementary sequences between the 3 ’-end of second nucleic acid sequence and the 5 ’-end of linker, and the complementray sequences between the 3 ’-end of the linker and the 5 ’-end of the first nucleic acid sequence, the second nucleic acid sequence, the linker and the first nucleic acid sequence can be assembled in sequence via overlap extension polymerase chain reaction (OE-PCR). The assembled product is then inserted into the expression vector plgG by use of the restriction enzymes. Structurally, the constructed expression vector comprises in sequence: a first human CMV promoter, a signal peptide of IgG light chain, the second nucleic acid sequence, the CL domain, a first BGH-polyA signal, a second human CMV promoter, a signal peptide of IgG heavy chain, the first nucleic acid sequence, the CH domain, and a second BGH-polyA signal, in which the second nucleic acid sequence and the CL domain are driven by the first human CMV promoter so as to express the light chain of the recombinant antibody, and the first nucleic acid sequence and the CH domain are driven by the second human CMV promoter to express the heavy chain of the recombinant antibody.

[0070] In the step (5), the expression vector constructed in step (4) is transfected into a host cell so as to produce the present recombinant antibody. The commonly used host cell is a mammalian cell, such as a HEK293 cell. The transfection can be performed by any method familiar by one skilled artisan, including chemical-based method (e.g., calcium phosphate, liposome, and cationic polymer), non-chemical method (e.g., electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, and hydrodynamic delivery), particle-based method (e.g. gene gun, magnetofection, and impalefection), and viral method (e.g., adenoviral vector, sindbis viral vector, and lentiviral vector). The thus-produced recombinant antibody is secreted into the supernatant of the culture medium, and can be purified therefrom by any purification method familiar by any skilled person; for example, the purification can be achieved by affinity binding with protein A or protein G.

[0071] According to some embodiments of the present disclosure, 7 recombinant antibodies are produced from the anti-NPA antibody fragment of section (II- 7) of the present disclosure, and are designated as “NPA-008 IgG”, “NPA-020 IgG”, “NPA-045 IgG”, “NPA-058 IgG”, “NPA-064 IgG”, “NPA-065 IgG” and “NPA-066 IgG”, respectively.

[0072] According to certain of the present disclosure, 6 recombinant antibodies are respectively produced from the anti-NPB antibody fragment of section (II-I) of the present disclosure, and are designated as “NPB-010 IgG”, “NPB-020 IgG”, “NPB-034 IgG”, NPB-042 IgG”, “NPB-046 IgG” and “NPB-055 IgG”, respectively.

[0073] (II-I) Recombinant antibodies or the fragment thereof

[0074] According to certain embodiments, each of the antibody fragments selected from section (II-I) of the present disclosure (including NPA-008, NPA-020, NPA-045, NPA-058, NPA-064, NPA-065, NPA-066, NPB-010, NPB-020, NPB-034, NPB-042, NPB-046 and NPB-055 scFvs) and the recombinant antibodies prepared form section (II-2) of the present disclosure (including NPA-008, NPA-020, NPA-045, NPA-058, NPA-064, NPA-065, NPA-066, NPB-010, NPB-020, NPB-034, NPB-042, NPB-046 and NPB-055 IgGs) in structure comprises a VL domain and a VH domain, in which the VL domain comprises CDR-L1, CDR-L2 and CDR-L3, and the VH domain comprises CDR-H1, CDR-H2 and CDR-H3.

[0075] (A) Anti-NPA scFvs and IgGs [0076] According to some embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-008 scFv or NPA-008 IgG respectively have the amino acid sequences of SEQ ID NOs: 1-3 (i.e., respectively having the amino acid sequences 100% identical to SEQ ID NOs: 1-3), and the CDR-H1, CDR-H2 and CDR-H3 of NPA-008 scFv or NPA-008 IgG respectively have the amino acid sequences of SEQ ID NOs: 4-6 (i.e., respectively having the amino acid sequences 100% identical to SEQ ID NOs: 4-6). According to some embodiments, the VL domain of NPA-008 scFv or NPA-008 IgG comprises an amino acid sequence at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 42; and the VH domain of NPA-008 scFv or NPA-008 IgG comprises an amino acid sequence at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 43. As would be appreciated, the sequence (e.g., the framework sequence) of the VL and VH domains may vary (e.g., being substituted by conserved or non-conserved amino acid residues) without affecting the binding affinity and/or specificity of the present antibody. Preferably, the sequence(s) of the VL and VH domains is/are conservatively substituted by one or more suitable amino acid(s) with similar properties; for example, the substitution of leucine (an nonpolar amino acid residue) by isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (another nonpolar amino acid residue); the substitution of aspartate (an acidic amino acid residue) by glutamate (another acidic amino acid residue); or the substitution of lysine (an basic amino acid residue) by arginine or histidine (another basic amino acid residue). According to some preferred embodiments, the VL and VH domains of NPA-008 scFv or NPA-008 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 42 and 43. More preferably, the VL and VH domains of NPA-008 scFv or NPA-008 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 42 and 43. In one working example of the present disclosure, the VL domain of NPA-008 scFv or NPA-008 IgG has the amino acid sequence of SEQ ID NO: 42 (i.e., having an amino acid sequence 100% identical to SEQ ID NO: 42), and the VH domain of NPA-008 scFv or NPA-008 IgG has the amino acid sequence of SEQ ID NO: 43 (i.e., having an amino acid sequence 100% identical to SEQ ID NO: 43).

[0077] According to some embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-020 scFv or NPA-020 IgG respectively have the amino acid sequences of SEQ ID NOs: 7-9, and the CDR-H1, CDR-H2 and CDR-H3 of NPA-020 scFv or NPA-020 IgG respectively have the amino acid sequences of SEQ ID NOs: 10-12. According to some embodiments, the VL domain of NPA-020 scFv or NPA-020 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 44; and the VH domain of NPA-020 scFv or NPA-020 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 45. According to some preferred embodiments, the VL and VH domains of NPA-020 scFv or NPA-020 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 44 and 45. More preferably, the VL and VH domains of NPA-020 scFv or NPA-020 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 44 and 45. In one working example of the present disclosure, the VL domain of NPA-020 scFv or NPA-020 IgG has the amino acid sequence of SEQ ID NO: 44, and the VH domain of NPA-020 scFv or NPA-020 IgG has the amino acid sequence of SEQ ID NO: 45.

[0078] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-045 scFv or NPA-045 IgG respectively have the amino acid sequences of SEQ ID NOs: 13, 2 and 14, and the CDR-H1, CDR-H2 and CDR-H3 of NPA-045 scFv or NPA-045 IgG respectively have the amino acid sequences of SEQ ID NOs: 15-17. According to some embodiments, the VL domain of NPA-045 scFv or NPA-045 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 46; and the VH domain of NPA-045 scFv or NPA-045 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 47. According to certain preferred embodiments, the VL and VH domains of NPA-045 scFv or NPA-045 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 46 and 47. More preferably, the VL and VH domains of NPA-045 scFv or NPA-045 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 46 and 47. In one working example of the present disclosure, the VL domain of NPA-045 scFv or NPA-045 IgG has the amino acid sequence of SEQ ID NO: 46, and the VH domain of NPA-045 scFv or NPA-045 IgG has the amino acid sequence of SEQ ID NO: 47.

[0079] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-058 scFv or NPA-058 IgG respectively have the amino acid sequences of SEQ ID NOs: 18-20, and the CDR-H1, CDR-H2 and CDR-H3 of NPA-058 scFv or NPA-058 IgG respectively have the amino acid sequences of SEQ ID NOs: 21-23. According to some embodiments, the VL domain of NPA-058 scFv or NPA-058 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 48; and the VH domain of NPA-058 scFv or NPA-058 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 49. According to certain preferred embodiments, the VL and VH domains of NPA-058 scFv or NPA-058 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 48 and 49. More preferably, the VL and VH domains of NPA-058 scFv or NPA-058 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 48 and 49. In one working example of the present disclosure, the VL domain of NPA-058 scFv or NPA-058 IgG has the amino acid sequence of SEQ ID NO: 48, and the VH domain of NPA-058 scFv or NPA-058 IgG has the amino acid sequence of SEQ ID NO: 49.

[0080] According to some embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-064 scFv or NPA-064 IgG respectively have the amino acid sequences of SEQ ID NOs: 24-26, and the CDR-H1, CDR-H2 and CDR-H3 of NPA-064 scFv or NPA-064 IgG respectively have the amino acid sequences of SEQ ID NOs: 27-29. According to some embodiments, the VL domain of NPA-064 scFv or NPA-064 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 50; and the VH domain of NPA-064 scFv or NPA-064 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 51. According to some preferred embodiments, the VL and VH domains of NPA-064 scFv or NPA-064 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 50 and 51. More preferably, the VL and VH domains of NPA-064 scFv or NPA-064 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 50 and 51. In one working example of the present disclosure, the VL domain of NPA-064 scFv or NPA-064 IgG has the amino acid sequence of SEQ ID NO: 50, and the VH domain of NPA-064 scFv or NPA-064 IgG has the amino acid sequence of SEQ ID NO: 51.

[0081] According to alternative embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-065 scFv or NPA-065 IgG respectively have the amino acid sequences of SEQ ID NOs: 30-32, and the CDR-H1, CDR-H2 and CDR-H3 of NPA-065 scFv or NPA-065 IgG respectively have the amino acid sequences of SEQ ID NOs: 33-35. According to some embodiments, the VL domain of NPA-065 scFv or NPA-065 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 52; and the VH domain of NPA-065 scFv or NPA-065 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 53. According to certain preferred embodiments, the VL and VH domains of NPA-065 scFv or NPA-065 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 52 and 53. More preferably, the VL and VH domains of NPA-065 scFv or NPA-065 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 52 and 53. In one working example of the present disclosure, the VL domain of NPA-065 scFv or NPA-065 IgG has the amino acid sequence of SEQ ID NO: 52, and the VH domain of NPA-065 scFv or NPA-065 IgG has the amino acid sequence of SEQ ID NO: 53.

[0082] According to alternative embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPA-066 scFv or NPA-066 IgG respectively have the amino acid sequences of SEQ ID NOs: 36-38, and the CDR-H1, CDR-H2 and CDR-H3 of NPA-066 scFv or NPA-066 IgG respectively have the amino acid sequences of SEQ ID NOs: 39-41. According to some embodiments, the VL domain of NPA-066 scFv or NPA-066 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 54; and the VH domain of NPA-066 scFv or NPA-066 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 55. According to certain preferred embodiments, the VL and VH domains of NPA-066 scFv or NPA-066 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 54 and 55. More preferably, the VL and VH domains of NPA-066 scFv or NPA-066 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 54 and 55. In one working example of the present disclosure, the VL domain of NPA-066 scFv or NPA-066 IgG has the amino acid sequence of SEQ ID NO: 54, and the VH domain of NPA-066 scFv or NPA-066 IgG has the amino acid sequence of SEQ ID NO: 55.

[0083] (B) Anti-NPB scFvs and IgGs

[0084] According to some embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPB-010 scFv or NPB-010 IgG respectively have the amino acid sequences of SEQ ID NOs: 56-58, and the CDR-H1, CDR-H2 and CDR-H3 of NPB-010 scFv or NPB-010 IgG respectively have the amino acid sequences of SEQ ID NOs: 59-61. According to some embodiments, the VL domain of NPB-010 scFv or NPB-010 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 91; and the VH domain of NPB-010 scFv or NPB-010 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 92. According to some preferred embodiments, the VL and VH domains of NPB-010 scFv or NPB-010 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 91 and 92. More preferably, the VL and VH domains of NPB-010 scFv or NPB-010 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 91 and 92. In one working example of the present disclosure, the VL domain of NPB-010 scFv or NPB-010 IgG has the amino acid sequence of SEQ ID NO: 91, and the VH domain of NPB-010 scFv or NPB-010 IgG has the amino acid sequence of SEQ ID NO: 92.

[0085] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPB-020 scFv or NPB-020 IgG respectively have the amino acid sequences of SEQ ID NOs: 62-64, and the CDR-H1, CDR-H2 and CDR-H3 of NPB-020 scFv or NPB-020 IgG respectively have the amino acid sequences of SEQ ID NOs: 65-67. According to some embodiments, the VL domain of NPB-020 scFv or NPB-020 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 93; and the VH domain of NPB-020 scFv or NPB-020 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 94. According to certain preferred embodiments, the VL and VH domains of NPB-020 scFv or NPB-020 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 93 and 94. More preferably, the VL and VH domains of NPB-020 scFv or NPB-020 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 93 and 94. In one working example of the present disclosure, the VL domain of NPB-020 scFv or NPB-020 IgG has the amino acid sequence of SEQ ID NO: 93, and the VH domain of NPB-020 scFv or NPB-020 IgG has the amino acid sequence of SEQ ID NO: 94.

[0086] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPB-034 scFv or NPB-034 IgG respectively have the amino acid sequences of SEQ ID NOs: 68-70, and the CDR-H1, CDR-H2 and CDR-H3 of NPB-034 scFv or NPB-034 IgG respectively have the amino acid sequences of SEQ ID NOs: 71-73. According to some embodiments, the VL domain of NPB-034 scFv or NPB-034 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 95; and the VH domain of NPB-034 scFv or NPB-034 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 96. According to some preferred embodiments, the VL and VH domains of NPB-034 scFv or NPB-034 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 95 and 96. More preferably, the VL and VH domains of NPB-034 scFv or NPB-034 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 95 and 96. In one working example of the present disclosure, the VL domain of NPB-034 scFv or NPB-034 IgG has the amino acid sequence of SEQ ID NO: 95, and the VH domain of NPB-034 scFv or NPB-034 IgG has the amino acid sequence of SEQ ID NO: 96.

[0087] According to some embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPB-042 scFv or NPB-042 IgG respectively have the amino acid sequences of SEQ ID NOs: 74-76, and the CDR-H1, CDR-H2 and CDR-H3 of NPB-042 scFv or NPB-042 IgG respectively have the amino acid sequences of SEQ ID NOs: 77, 10 and 78. According to some embodiments, the VL domain of NPB-042 scFv or NPB-042 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 97; and the VH domain of NPB-042 scFv or NPB-042 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 98. According to certain preferred embodiments, the VL and VH domains of NPB-042 scFv or NPB-042 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 97 and 98. More preferably, the VL and VH domains of NPB-042 scFv or NPB-042 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 97 and 98. In one working example of the present disclosure, the VL domain of NPB-042 scFv or NPB-042 IgG has the amino acid sequence of SEQ ID NO: 97, and the VH domain of NPB-042 scFv or NPB-042 IgG has the amino acid sequence of SEQ ID NO: 98. [0088] According to alternative embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPB-046 scFv or NPB-046 IgG respectively have the amino acid sequences of SEQ ID NOs: 79-81, and the CDR-H1, CDR-H2 and CDR-H3 of NPB-046 scFv or NPB-046 IgG respectively have the amino acid sequences of SEQ ID NOs: 82-84. According to some embodiments, the VL domain of NPB-046 scFv or NPB-046 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 99; and the VH domain of NPB-046 scFv or NPB-046 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 100. According to some preferred embodiments, the VL and VH domains of NPB-046 scFv or NPB-046 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 99 and 100. More preferably, the VL and VH domains of NPB-046 scFv or NPB-046 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 99 and 100. In one working example of the present disclosure, the VL domain of NPB-046 scFv or NPB-046 IgG has the amino acid sequence of SEQ ID NO: 99, and the VH domain of NPB-046 scFv or NPB-046 IgG has the amino acid sequence of SEQ ID NO: 100.

[0089] According to alternative embodiments, the CDR-L1, CDR-L2 and CDR-L3 of NPB-055 scFv or NPB-055 IgG respectively have the amino acid sequences of SEQ ID NOs: 85-87, and the CDR-H1, CDR-H2 and CDR-H3 of NPB-055 scFv or NPB-055 IgG respectively have the amino acid sequences of SEQ ID NOs: 88-90. According to some embodiments, the VL domain of NPB-055 scFv or NPB-055 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 101; and the VH domain of NPB-055 scFv or NPB-055 IgG comprises an amino acid sequence at least 85% identical to SEQ ID NO: 102. According to certain preferred embodiments, the VL and VH domains of NPB-055 scFv or NPB-055 IgG respectively comprise amino acid sequences at least 90% identical to SEQ ID NOs: 101 and 102. More preferably, the VL and VH domains of NPB-055 scFv or NPB-055 IgG respectively comprise amino acid sequences at least 95% identical to SEQ ID NOs: 101 and 102. In one working example of the present disclosure, the VL domain of NPB-055 scFv or NPB-055 IgG has the amino acid sequence of SEQ ID NO: 101, and the VH domain of NPB-055 scFv or NPB-055 IgG has the amino acid sequence of SEQ ID NO: 102.

[0090] (H-4) Uses of antibody fragments and recombinant antibodies in diagnosing IAV or

IBV infection

[0091] According to some examples of the present disclosure, each of the present anti-NPA antibody fragments and recombinant antibodies is useful in detecting IAV, and each of the present anti-NPB antibody fragments and recombinant antibodies is useful in detecting IBV. Accordingly, these antibody fragments and recombinant antibodies may serve as detecting agents for diagnosing IAV or IBV infection.

[0092] It is therefore another aspect of the present disclosure to provide a kit for the detection of IAV or IBV infection in a subject (i.e., in a human subject). The kit includes, at least, a first antibody, a second antibody, and a container containing the first and second antibodies. According to some embodiments, the first and second antibodies of the kit for detecting LAV infection are independently selected from the anti-NPA antibody fragments (including NPA-008, NPA-020, NPA-045, NPA-058, NPA-064, NPA-065 and NPA-066 scFvs) and the anti-NPA recombinant antibodies (including NPA-008, NPA-020, NPA-045, NPA-058, NPA-064, NPA-065 and NPA-066 IgGs) as described in sections (II-l) to (II-3) of the present disclosure. According to certain embodiments, the first and second antibodies of the kit for detecting IBV infection are independently selected from the anti-NPB antibody fragments (including NPB-010, NPB-020, NPB-034, NPB-042, NPB-046 and NPB-055 scFvs) and the anti-NPB recombinant antibodies (including NPB-010, NPB-020, NPB-034, NPB-042, NPB-046 and NPB-055 IgGs) as described in sections (II-l) to (II-3) of the present disclosure. The present kit is useful in detecting the IAV or IBV infection in a biological sample via any detection technique known to a skilled artisan, such as ELISA, LFIA, SPR, western blotting assay, and flow cytometry. According to some working examples, the first and second antibody fragments/recombinant antibodies of the present kit respectively serve as a capture antibody and a detection antibody for use in ELISA, western blotting assay or LFIA.

[0093] According to some embodiment, the kit is designed to detect IAV. In one example, the kit designated as “D08C58” comprises NPA-058 IgG as the first antibody, and NPA-008 IgG as the second antibody. In another example, the kit designated as “D45C20” comprises NPA-020 IgG as the first antibody, and NPA-045 IgG as the second antibody. In another example, the kit designated as “D64C66” comprises NPA-066 IgG as the first antibody, and NPA-064 IgG as the second antibody. In still another example, the kit designated as “D64C65” comprises NPA-065 IgG as the first antibody, and NPA-064 IgG as the second antibody. In further another example, the kit designated as “D65C64” comprises NPA-064 IgG as the first antibody, and NPA-065 IgG as the second antibody.

[0094] According to some embodiment, the kit is designed to detect IBV. In one example, the kit designated as “D46C42” comprises NPB-042 IgG as the first antibody, and NPB-046 IgG as the second antibody. In another example, the kit designated as “D55C20” comprises NPB-020 IgG as the first antibody, and NPB-055 IgG as the second antibody. In still another example, the kit designated as “D34C10” comprises NPB-010 IgG as the first antibody, and NPB-034 IgG as the second antibody.

[0095] Optionally, the kit may further comprise a legend indicating how to use the antibody fragment or the recombinant antibody for detecting IAV or IBV infection.

[0096] Also included herein is a method of making a diagnosis of whether a subject is infected by IAV or IBV via a biological sample isolated from the subject. The method comprises detecting the presence or absence of a nucleoprotein of IAV or IBV (i.e., detecting the presence or absence of NPA or NPB) in the biological sample by use of the antibody fragment, the recombinant antibody, or the kit of the present disclosure, wherein when the nucleoprotein is present in the biological sample, then diagnosing that the subject is infected by the IAV or IBV. Non-limiting examples of the NPA detectable by the present method include, NPA1 (SEQ ID NO: 103), NPA2 (SEQ ID NO: 104), NPA3 (SEQ ID NO: 105), NPA4 (SEQ ID NO: 106), NPA5 (SEQ ID NO: 107), NPA6 (SEQ ID NO: 108), NPA7 (SEQ ID NO: 109), NPA8 (SEQ ID NO: 110), and a variant or derivative thereof (e.g., a NPA mutant having an amino acid sequence at least 85% identical to any of SEQ ID NOs: 103-110). Examples of the NPB detectable by the present method include, but are not limited to, NPB1 (SEQ ID NO: 111), NPB2 (SEQ ID NO: 112), and a variant or derivative thereof (e.g., a NPB mutant having an amino acid sequence at least 85% identical to SEQ ID NO: 111 or 112).

[0097] Basically, the biological sample is a sample obtained from the respiratory tract of the subject; preferably, the upper respiratory tract of the subject. Non-limiting examples of the biological sample suitable to be used in the present method include, a mucosa tissue, a fluid, or a secretion (e.g., sputum) isolated from the oral cavity, nasal cavity, trachea, bronchus, or lung of the subject.

[0098] Based on the diagnostic result, a skilled artisan or a clinical practitioner may administer to a subject need thereof (e.g., a subject suffering from IAV or IBV infection) a suitable treatment (such as, an anti-viral treatment) thereby ameliorating and/or alleviating the symptom(s) associated with the IAV or IBV infection. Examples of the anti-viral treatment suitable to be used in the present method include, but are not limited to, oseltamivir, zanamivir, peramivir, baloxavir marboxil, amantadine, rimantadine, interferon-alpha (IFN-a), interferon-beta (IFN- ), and a combination thereof.

[0099] The subject that can be subjected to the diagnosis and/or treatment methods of the present invention is a mammal, such as a human, a mouse, a rat, a monkey, a sheep, a goat, a cat, a dog, a horse, or a chimpanzee. Preferably, the subject is a human. [0100] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

EXAMPLE

[0101] Materials and Methods

[0102] Preparation of recombinant nucleoproteins oflAV and IB V (NPA and NPB)

[0103] Eight representative NPAs i.e., NPA1 to NPA8; SEQ ID NOs: 103-110) and two representative NPBs (i.e., NPB1 and NPB2; SEQ ID NOs: 111 and 112) were prepared in this study. Specifically, the coding regions of NPA/NPB genes were optimized for E. coli expression and cloned into expression vector pET15b linearized with Nde I and Xho I restriction enzymes; the recombinant NPA/NPB protein contained a Hise-tag and a thrombin cleavage sequence upstream to the NPA/NPB sequence. These NPA/NPB constructs were overexpressed in BL21 (DE3) cell with 0.5 mM isopropyl -D-1 -thiogalactopyranoside (IPTG) induction at 16°C. The NPA/NPB recombinant protein expressed in E. coli was purified using Ni²⁺ charged chelating sepharose column (for Hise-tag binding), heparin column (for RNA-free NP binding), and size exclusive separation with buffer containing 40 mM Tris, pH 7.5, 600 mM NaCl. To obtain the NPA/NPB protein free of RNA, RNaseA (20 pg/ml) was applied to cell lysis of E. coli, followed by the purification procedures. Purified NPA/NPB proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0104] Cell lines

[0105] 293T (ATCC CRL-3216) was cultured in DMEM medium supplemented with 10% fetal bovine serum, penicillin-streptomycin (lOOx). Suspension Expi293F™ cells were cultured in Expi293™ Expression Medium at 37°C with shaking 110 rpm in 8% CO2 incubator.

[0106] Characterization of the IgGls derived from the selection and screening procedure with phage-displayed synthetic scFv libraries

[0107] The construction and characterization of the phage-displayed synthetic scFv libraries followed the same procedure, without modification, as described in the co-pending PCT applications, PCT/US2016/19128 and PCT/US2018/56627. The experimental procedures for panning the phage display libraries, selecting and screening of phage-displayed scFv binders, characterizing the scFvs binding to the cognate antigens and Protein A/L with ELISA, reformatting scFvs into IgGls, and expressing and purifying IgGls have been described in the co-pending PCT applications, PCT/US2016/19128 and PCT/US2018/56627.

[0108] Detection ofNPA/NPB with immunoblotting

[0109] The recombinant NPA/NPB (250 ng) were resolved on an 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to a polyvinylidene difluoride nylon (PVDF) membrane, and probed with specified IgG antibodies (1 pg/mL). The specific bands were detected by HRP-conjugated antibody (1 mg/ml, 1:2,500 dilution), and revealed by chemiluminescent substrate and chemiluminescent imaging system.

[0110] EC 50 for antibody-antigen interaction

[0111] The IgG ECso was determined by the titrations of IgG antibody on immobilized recombinant NPA/NPB with ELISA. Specifically, the NPA/NPB (0.5 pg per well) diluted in PBS buffer (pH 7.4) was coated on 96-well immunoplates, and then blocked with 5% skim milk in PBST for 1 hour. In the meantime, two-fold serial dilutions of the antibody (stock concentration: 0.5 pg/mL) in PBST with 5% milk were performed, and 11 different concentrations of the antibody were generated. After blocking, 100 pL diluted antibody samples were added to each well, and incubated for 1 hour under gentle shaking. The wells were washed three times with 300 pL PBST, and then reacted with 100 pL of l:5,000-diluted HRP-conjugated goat anti-human IgG antibody in PBST with 5% milk for 1 hour incubation. The wells were washed three times with 300 pL PBST buffer and twice with 300 pL PBS, developed for 5 minutes with TMB substrate, quenched with 1.0 M HC1 and read spectrophotometrically at 450 nm.

[0112] Detection of NPA/NPB with sandwich ELISA

[0113] Capture antibody (1 pg per well) diluted in PBS buffer (pH7.4) was coated on 96-well immunoplates overnight. The plates were blocked with 5% skim milk in PBST for 1 hour. Then, two-fold serial dilutions of NPA/NPB (stock concentration: 1 pg/mL) were added to the plates (100 pL per well), and incubated at room temperature for 1 hour under gentle shaking. The wells were washed three times with 300 pL PBST, and then reacted with 100 pL HRP-conjugated detection antibody (1:2,000 dilution) in 5% milk/PBST for 1 hour incubation with gently shaking at room temperature. The wells were washed three times with 300 pL PBST buffer and twice with 300 pL PBS, developed for 5 minutes with TMB substrate, quenched with 1.0 M HC1 and read spectrophotometrically at 450 nm.

[0114] Preparation of colloidal gold-conjugated AL2C and IgGs [0115] 50 pl of 0.2 M K2CO3 (pH 11.5) was mixed with 10 ml colloidal gold solution (pH 5-6) to adjust pH (final pH 8), and then added 500 pg of IgG or 167 pg of AL2C to the colloidal gold solution to react 40 minutes at room temperature. The reaction was stopped by adding 1 ml of blocking buffer (10% bovine serum albumin (B SA) in 20 mM sodium borate, pH 9.3) for 15 minutes at room temperature, followed by centrifugation (15,000 xg, 30 minutes, 4°C). The supernatant was discarded, and the pellet was completely resuspended in 10 ml wash buffer (1% BSA in 20 mM sodium borate, pH 9.3), followed by centrifugation (15,000 xg, 30 minutes, 4°C). The washing procedure was repeated twice, and the pellet was resuspended in 1 ml of 1% BSA in 20 mM sodium borate (pH 9.3) for the procedure preparing the conjugate pad.

[0116] Assembly of the LFIA strips

[0117] 1 pg of the capture antibody, antigen or AL2C in PBS buffer were stripped on nitrocellulose membrane per cm with lateral flow dispenser driven by syringe infusion pump. All other procedures for the preparation of the nitrocellulose membrane with immobilized antigen or capture antibody, the preparation of the conjugate pad and the sample pad, and the preparation of the LFIA strip assembly were followed the protocol previously reported.

[0118] Example 1 Characterization of recombinant antibodies

[0119] The NPA and NPB were synthesized for selecting a panel of anti-NPA or anti-NPB antibodies in accordance with the methodology described in the co-pending PCT applications, PCT/US2016/19128 and PCT/US2018/56627.

[0120] 7 anti-NPA scFvs and 6 anti-NPB scFvs were selected from the phage-displayed scFv libraries, and reformatted into IgG Is. The thus-obtained anti-NPA IgGs were respectively designated as “NPA-008 IgG”, “NPA-020 IgG”, “NPA-045 IgG”, “NPA-058 IgG”, “NPA-064 IgG”, “NPA-065 IgG” and “NPA-066 IgG”; and the thus-obtained anti-NPB IgGs were respectively designated as “NPB-010 IgG”, “NPB-020 IgG”, “NPB-034 IgG”, NPB-042 IgG”, “NPB-046 IgG” and “NPB-055 IgG”. The VL and VH sequences of the anti-NPA IgGs and anti-NPB IgGs were summarized in Tables 1 and 2. The ELISA EC50 of these IgG Is against the NPA and NPB were respectively summarized in Tables 3 and 4. [0121] Table 1 VL and VH sequences of specified anti-NPA antibodies

SEQ

Name Domain Amino acid sequence ID

NO

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVGWGVAWYQQKPGKAP KLLISSPPFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDF 42 PITFGQGTKVEIKR NPA-008 VH EVQLVESGGGLVQPGGSLRLSCAASGFTIDGYGIHWVRQAPGKGLE WVAGIGPSWGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA 43

VYYCARFFGSSGYMDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCSGSSSNIGFSGVYWYQQKPGKAP KLLISGPTWLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAAYSY 44 NNNGITFGQGTKVEIKR

VH EVQLVESGGGLVQPGGSLRLSCAASGFTIGGWGIHWVRQAPGKGLE WVAGIWPYWGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA 45 VYYCARFYSYYSNMDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVNNDVAWYQQKPGKAP KLLISSPPFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYGS 46 PVTFFGQGTKVEIKR

VH EVQLVESGGGLVQPGGSLRLSCAASGFTINSYYIHWVRQAPGKGLEW VASIWPYGGSTFYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY 47 YCARSWSSSYWDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVGGFVAWYQQKPGKAP KLLISGSRFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSN 48

SPLTFGQGTKVEIKR

VH EVQLVESGGGLVQPGGSLRLSCAASGFTISSYFIHWVRQAPGKGLEW VASIWPYGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV 49

YYCARSYFGSYWD YWGQGTL VTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVDDDVAWYQQKPGKAP KLLISSARGLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYN 50 SPMTFGQGTKVEIKR NPA-064 VH EVQLVESGGGLVQPGGSLRLSCAASGFTINDYGIHWVRQAPGKGLE WVAGIWPSGGSTF YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA 51

VYYC ARFHSYSGNMD YWGQGTL VTVS SAS AAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVGFGVAWYQQKPGKAP KLLISFPGSLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSF 52 PITFGQGTKVEIKR

* The CDR sequences were marked in boldface, including three CDRs (i.e., CDR-L1, CDR-L2 and CDR-L3, from N-terminus to C-terminus, in sequence) in the VL domain, and three CDRs (i.e., CDR-H1, CDR-H2 and CDR-H3, from N-terminus to C-terminus, in sequence) in the VH domain. [0122] Table 2 VL and VH sequences of specified anti-NPB antibodies

SEQ

Name Domain Amino acid sequence ID

NO

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT 91

PPTFGQGTKVEIKR NPB-010 VH EVQLVESGGGLVQPGGSLRLSCAASGFTIGGFGIHWVRQAPGKGLE WVAGIGPFWGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA 92 VYYCARFDSHSSYSYGDSLMDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVWGGVAWYQQKPGKAP KLLIFGTGWLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYY 93 NYPVTFFGQGTKVEIKR

VH EVQLVESGGGLVQPGGSLRLSCAASGFTISDSSIHWVRQAPGKGLEW VAGIWPSWGYTFYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV 94 YYCARFHSYSGSGVMDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVGDSVAWYQQKPGKAP KLLIYSTSWLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYS 95 YPITFGQGTKVEIKR

VH EVQLVESGGGLVQPGGSLRLSCAASGFTINNSGIHWVRQAPGKGLEW VAWIGPYWGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV 96 YYCAREYSFWSFDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVGSSVAWYQQKPGKAPK LLIYGTSSLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYNF 97

PVTFGQGTKVEIKR VH EVQLVESGGGLVQPGGSLRLSCAASGFTIGGWGIHWVRQAPGKGLE

WVAGIGPFWGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA 98 VYYCARFDGYSSYSSSHGLMDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCLLNSDGWTSLNWYQQKPGKAP KLLIYWTSFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSND 99 FPYTFGQGTKVEIKR NPB-046 VH EVQLVESGGGLVQPGGSLRLSCTFSHWSMHWVWVRQAPGKGLEWV ADISPNFGHTHYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY 100 CARGIWLDYWGQGTLVTVSSASAAA

VL MADIQMTQSPSSLSASVGDRVTITCRASQDVYFGVAWYQQKPGKAP KLLISYSASLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSF 101 PLTFGQGTKVEIKR NPB-055 VH EVQLVESGGGLVQPGGSLRLSCAASGFTIDNSGIHWVRQAPGKGLEW VAGIWPSWGSTS YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV 102

YYCARGSYWSDYWGQGTLVTVSSASAAA

* The CDR sequences were marked in boldface, including three CDRs (i.e., CDR-L1, CDR-L2 and CDR-L3, from N-terminus to C-terminus, in sequence) in the VL domain, and three CDRs (i.e., CDR-H1, CDR-H2 and CDR-H3, from N-terminus to C-terminus, in sequence) in the VH domain. [0123] Table 3 Binding affinities (ECso) of specified IgGl against the NPA

[0124] Table 4 Binding affinities (EC50) of specified IgGl against the NPB

[0125] The binding affinities of the present IgGs to NPA or NPB were further examined by sandwich ELISA. According to the analytic results, each of the antibody pair D08C58, D45C20, D64C66, D64C65 and D65C64 was useful in detecting at least three NPAs (Table 5), and each of the antibody pair D46C42, D55C20 and D34C10 was useful in detecting NPBs (Table 6). These data demonstrated that the present antibodies may serve as the detection and capture antibodies for detecting IAV or IBV.

[0126] Table 5 Binding affinities (ECso) of specified antibody pairs against NPAs in sandwich ELISA

Antibody pair D08C58 comprises NPA-058 IgG as the capture antibody and NPA-008 IgG as the detection antibody.

Antibody pair D45C20 comprises NPA-020 IgG as the capture antibody and NPA-045 IgG as the detection antibody.

Antibody pair D64C66 comprises NPA-066 IgG as the capture antibody and NPA-064 IgG as the detection antibody.

Antibody pair D64C65 comprises NPA-065 IgG as the capture antibody and NPA-064 IgG as the detection antibody.

Antibody pair D65C64 comprises NPA-064 IgG as the capture antibody and NPA-065 IgG as the detection antibody.

[0127] Table 6 Binding affinities (EC50) of specified antibody pairs against NPBs in sandwich ELISA

Antibody pair D46C42 comprises NPB-042 IgG as the capture antibody and NPB-046 IgG as the detection antibody.

Antibody pair D55C20 comprises NPB-020 IgG as the capture antibody and NPB-055 IgG as the detection antibody.

Antibody pair D34C10 comprises NPB-010 IgG as the capture antibody and NPB-034 IgG as the detection antibody

[0128] In addition to ELISA, the present antibodies may also serve as detecting antibodies for IAV or IBV in western blotting assay. According to the data of Fig. 1A, NPA-008, NPA-020, NPA-045, NPA-058, NPA-064, NPA-065 and NPA-066 IgGs, 7 representative anti-NPA antibodies, were capable of specifically recognizing one or more NPAs (i.e., NPA1 to NPA8) without binding to NPBs (i.e., NPB1 and NPB2). The data of Fig. IB indicated that each of NPB-010, NPB-020, NPB-042, NPB-046, NPB-055 and NPB-034 recognized the conformational epitope of NPAs (i.e., NPA1 to NPA8) and NPBs (i.e., NPB1 and NPB2), but not the linear epitope of these NPAs and NPBs.

[0129] Example ! Establishing LFIA devices for detecting IAV or IBV

[0130] For the purpose of constructing LFIA devices, 7 representative anti-NPA antibodies and 6 representative anti-NPB antibodies were conjugated to colloidal gold in accordance with the procedures described in “Materials and Methods” of the present disclosure. The data of Tables 7 and 8 depicted that the present antibodies may efficiently conjugate to colloidal gold.

[0131] Table 7 SB buffer exchange efficiency of anti-NPA IgGs prior to gold conjugation

No. IgG Name Input (mg) Output Vol. (ml) Cone, (mg/ml) Output (mg) Recovery (%)

1 NPA-008 2.00 0.35 5.62 1.97 98.35

2 NPA-020 2.00 0.29 6.64 1.93 96.28

3 NPA-045 2.00 0.31 5.63 1.75 87.27

4 NPA-058 2.00 0.44 3.86 1.70 84.92

5 NPA-064 2.00 0.44 4.38 1.93 96.36

6 NPA-065 2.00 0.33 5.56 1.83 91.74

7 NPA-066 2.00 0.38 5.13 1.95 97.47

[0132] Table 8 SB buffer exchange efficiency of anti-NPB IgGs prior to gold conjugation

No. IgG Name Input (mg) Output Vol. (ml) Cone, (mg/ml) Output (mg) Recovery (%)

1 NPB-010 2.00 0.4 5.16 2.06 103.20

2 NPB-020 2.00 0.45 5.59 2.52 125.78

3 NPB-034 2.00 0.48 4.29 2.06 102.96

4 NPB-042 2.00 0.45 4.8 2.16 108.00

5 NPB-046 2.00 0.45 4.31 1.94 96.98

6 NPB-055 2.00 0.44 4.84 2.13 106.48

[0133] Five prototypes of LFIA device for detecting IAV were established in this example, in which D08C58 LFIA prototype was constructed with NPA-058 IgG as the capture antibody and NPA-008 IgG as the colloidal gold-conjugated detection antibody; D45C20 LFIA prototype was constructed with NPA-020 IgG as the capture antibody and NPA-045 IgG as the colloidal gold-conjugated detection antibody; D64C66 LFIA prototype was constructed with NPA-066 IgG as the capture antibody and NPA-064 IgG as the colloidal gold-conjugated detection antibody; D64C65 LFIA prototype was constructed with NPA-065 IgG as the capture antibody and NPA-064 IgG as the colloidal gold-conjugated detection antibody; and D65C64 LFIA prototype was constructed with NPA-064 IgG as the capture antibody and NPA-065 IgG as the colloidal gold-conjugated detection antibody. The detection limit of each LFIA prototype was determined with two sample preparations containing NPA (i.e., any of NPA1-NPA6 and NPA8). The first sample preparation contained Expi 293 cell-expressed NPA, and the second sample preparation contained E. coZz-expressed NPA. The detection limits for both sample preparations are in the range of 2-62.5 ng/test (data not shown).

[0134] Further, three prototypes of LFIA device for detecting IBV were established in this example, in which D46C42 LFIA prototype was constructed with NPB-042 IgG as the capture antibody and NPB-046 IgG as the colloidal gold-conjugated detection antibody; D55C20 LFIA prototype was constructed with NPB-020 IgG as the capture antibody and NPB-055 IgG as the colloidal gold-conjugated detection antibody; and D34C10 LFIA prototype was constructed with NPB-010 IgG as the capture antibody and NPB-034 IgG as the colloidal gold-conjugated detection antibody. The detection limit of each LFIA prototype was determined with two sample preparations containing NPB1. The first sample preparation contained Expi 293 cell-expressed NPB, and the second sample preparation contained E.

NPB. The detection limits for both sample preparations are in the range of 1.0-3.9 ng/test (data not shown).

[0135] In summary, the antibodies selected from the GH synthetic antibody libraries were demonstrated to be capable of binding to the nucleoprotein of IAV or IBV with high affinities and specificities. The present antibodies (including 7 anti-NPA IgGs and 6 anti-NPB IgGs) derived from the GH antibody libraries without further affinity maturation were used in sandwich ELISA and LFIA to detect the corresponding NPA or NPB with detection limit of 1-2 ng/test. Accordingly, the present antibodies may serve as potential antibodies for detecting IAV or IBV thereby making a diagnosis of LAV or IBV infection.

[0136] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

Claims

WHAT IS CLAIMED IS:

1. A recombinant antibody or the fragment thereof, comprising a variable light chain (VL) domain and a variable heavy chain (VH) domain, wherein the VL domain comprises the amino acid sequences of SEQ ID NOs: 1-3, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 4-6; the VL domain comprises the amino acid sequences of SEQ ID NOs: 7-9, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 10-12; the VL domain comprises the amino acid sequences of SEQ ID NOs: 13, 2 and 14, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 15-17; the VL domain comprises the amino acid sequences of SEQ ID NOs: 18-20, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 21-23; the VL domain comprises the amino acid sequences of SEQ ID NOs: 24-26, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 27-29; the VL domain comprises the amino acid sequences of SEQ ID NOs: 30-32, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 33-35; or the VL domain comprises the amino acid sequences of SEQ ID NOs: 36-38, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 39-41.

2. The recombinant antibody of claim 1, wherein the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 42, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 43; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 44, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 45; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 46, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 47; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 48, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 49; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 50, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 51; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 52, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 53; or the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 54, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 55.

3. The recombinant antibody of claim 2, wherein the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 42, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 43; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 44, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 45; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 46, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 47; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 48, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 49; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 50, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 51; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 52, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 53; or the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 54, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 55.

4. A kit for detecting influenza A virus (IAV), comprising a first recombinant antibody and a second recombinant antibody, independently according to any of claim 1; and a container containing the first and second recombinant antibodies.

5. The kit of claim 4, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 1-3, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 4-6; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 18-20, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 21-23.

6. The kit of claim 5, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 42, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 43; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 48, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 49.

7. The kit of claim 4, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 7-9, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 10-12; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 13, 2 and 14, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 15-17.

8. The kit of claim 7, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 44, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 45; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 46, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 47.

9. The kit of claim 4, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 24-26, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 27-29; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 36-38, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 39-41.

10. The kit of claim 9, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 50, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 51; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 54, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 55.

11. The kit of claim 4, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 24-26, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 27-29; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 30-32, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 33-35.

12. The kit of claim 11, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 50, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 51; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 52, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 53.

13. A method of determining whether a subject is infected by IAV via a biological sample isolated from the subject, comprising detecting the presence or absence of a nucleoprotein of the IAV in the biological sample by use of the recombinant antibody of claim 1 or the kit of claim 4, wherein when the nucleoprotein is present in the biological sample, then diagnosing that the subject is infected by the IAV.

14. A recombinant antibody or the fragment thereof, comprising a variable light chain (VL) domain and a variable heavy chain (VH) domain, whereinthe VL domain comprises the amino acid sequences of SEQ ID NOs: 56-58, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 59-61; the VL domain comprises the amino acid sequences of SEQ ID NOs: 62-64, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 65-67; the VL domain comprises the amino acid sequences of SEQ ID NOs: 68-70, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 71-73; the VL domain comprises the amino acid sequences of SEQ ID NOs: 74-76, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 77, 10 and 78; the VL domain comprises the amino acid sequences of SEQ ID NOs: 79-81, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 82-84; or the VL domain comprises the amino acid sequences of SEQ ID NOs: 85-87, and the VH domain comprises the amino acid sequences of SEQ ID NOs: 88-90.

15. The recombinant antibody of claim 14, wherein the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 91, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 92; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 93, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 94; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 95, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 96; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 97, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 98; the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 99, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 100; or the VL domain comprises an amino acid sequence at least 85% identical to SEQ ID NO:

101, and the VH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO:

102.

16. The recombinant antibody of claim 15, wherein the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 91, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 92; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 93, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 94; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 95, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 96; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 97, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 98; the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 99, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 100; or the VL domain comprises an amino acid sequence 100% identical to SEQ ID NO: 101, and the VH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 102.

17. A kit for detecting influenza B virus (IBV), comprising a first recombinant antibody and a second recombinant antibody, independently according to any of claim 14; and a container containing the first and second recombinant antibodies.

18. The kit of claim 17, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 74-76, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 77, 10 and 78; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 79-81, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 82-84.

19. The kit of claim 18, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 97, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 98; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 99, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 100.

20. The kit of claim 17, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 62-64, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 65-67; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 85-87, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 88-90.

21. The kit of claim 20, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 93, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 94; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 101, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 102.

22. The kit of claim 17, wherein the VL domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 56-58, and the VH domain of the first recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 59-61; and the VL domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 68-70, and the VH domain of the second recombinant antibody comprises the amino acid sequences of SEQ ID NOs: 71-73.

23. The kit of claim 22, wherein the VL domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 91, and the VH domain of the first recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 92; and the VL domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 95, and the VH domain of the second recombinant antibody comprises an amino acid sequence 100% identical to SEQ ID NO: 96.

24. A method of determining whether a subject is infected by IBV via a biological sample isolated from the subject, comprising detecting the presence or absence of a nucleoprotein of the IBV in the biological sample by use of the recombinant antibody of claim 14 or the kit of claim 17, wherein when the nucleoprotein is present in the biological sample, then diagnosing that the subject is infected by the IBV.