Anti-influenza A virus antibodies or functional fragments thereof, reagents and kits for detecting influenza A virus

Abstract

The present invention discloses anti-influenza A virus antibodies or functional fragments thereof, as well as reagents and kits for detecting influenza A virus, relating to the field of antibodies. The anti-influenza A virus antibodies disclosed in the present invention include heavy chain complementary determining regions and light chain complementary determining regions. The antibodies provide an important source of raw materials for the detection of influenza A virus and have good affinity and activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to application number 202211010996.5, filed on August 23, 2022, entitled “Anti-influenza A virus antibodies or functional fragments thereof, reagents and kits for detecting influenza A virus,” the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present invention relates to the field of antibody technology, and in particular to anti-influenza A virus antibodies or functional fragments thereof, and reagents and kits for detecting influenza A virus. Background Art

[0004] Influenza virus (Flu), also known as influenza virus for short, is a representative species of the Orthomyxoviridae family, which includes human influenza virus, swine influenza virus, equine influenza virus, avian influenza virus, etc. Human influenza virus can be divided into three types: A (A), B (B), and C (C) based on the antigenicity of its nucleoprotein. It is the pathogen of influenza. Influenza virus can cause infection and disease in a variety of animals, including humans, poultry, pigs, horses, and bats. Among them, influenza A virus and influenza B virus are the main ones that can infect humans, mainly causing upper respiratory tract infections, but can also cause lower respiratory tract infections in children and adults, mainly pneumonia. Severe influenza in infants and young children is often accompanied by bronchitis and high fever.

[0005] Influenza A virus (Flu-A), first isolated in 1933, is prone to antigenic variation and can be further divided into subtypes: H1N1, H3N2, H5N1, and H7N9 (H represents the hemagglutinin of the influenza virus, and N represents the neuraminidase of the influenza virus). Infection with influenza A virus has an annual peak. The severity of infection is related to individual immunity. Typical symptoms include chills, persistent high fever, and headache, often accompanied by sore throat, cough, nasal congestion, and often systemic symptoms such as body aches and fatigue. Literature reports that the positive detection rate during epidemic periods is 20-40%, while the positive rate during non-epidemic seasons is 2-20%. The persistent prevalence of influenza A virus can significantly disrupt and strain public health, daily life, and public health systems, making it a major focus of epidemiological research.

[0006] Currently, the main methods for influenza A virus detection on the market include fluorescent PCR, immunoassays, and virus isolation, culture, and identification. Fluorescent PCR uses fluorescent signals to monitor the PCR process in real time during the PCR amplification process for qualitative or quantitative detection purposes. This method is the gold standard for pathogen detection. Immunoassays detect the target protein through specific binding between antigens and antibodies. Common methods for virus isolation and culture include chicken embryo inoculation, animal inoculation, and tissue (cell) culture, followed by observation and analysis of the results. While fluorescent PCR offers good sensitivity and specificity, it has a short detection window, which allows for early diagnosis, treatment, and mortality reduction, making it the gold standard for diagnosis. However, this method requires high standards for personnel, requiring specialized skills training. Diagnostic testing requires specialized equipment and equipment in qualified laboratories. Therefore, it is not suitable for rapid diagnosis in clinical settings or epidemiological surveillance. Virus isolation, culture, and identification testing are time-consuming, require strict environmental requirements, pose a high risk of infection to the operator, and have poor culture results, limiting their application in clinical diagnosis and epidemiological surveillance. Immunoassay detection reagents target antigens or antibodies in samples. They have a fast detection speed and good accuracy. At the same time, they have low requirements for laboratories and operators. They are generally suitable for grassroots screening such as hospital laboratories and disease control system laboratories. They play a very important role in the initial detection of influenza A, the successful control of outbreaks in hospitals and communities, and the guidance of treatment.

[0007] Currently, the main immunodiagnostic reagents for influenza A on the market include enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography, such as Guangzhou Wondfo's influenza A virus antigen detection reagent (colloidal gold method) and R&D's influenza A ELISA kit. All of these diagnostic reagents require antibodies specific for influenza A. Therefore, there is a strong demand in the field for antibodies that effectively and specifically bind to and detect influenza A (Flu-A).

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of the present invention is to provide anti-influenza A virus antibodies or functional fragments thereof, and reagents and kits for detecting influenza A virus.

[0010] The present invention is achieved in that:

[0011] In a first aspect, an embodiment of the present invention provides an anti-influenza A virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises three complementarity determining regions having a heavy chain variable region of any one of the amino acid sequences of SEQ ID NOs: 7, 8, 9, and 10 and three complementarity determining regions having a light chain variable region of any one of the amino acid sequences of SEQ ID NOs: 11, 12, 13, and 14.

[0012] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises any one of (a) to (c): (a) HCDR1 to 3 and LCDR1 to 3, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1 to 3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID Nos: 4 to 6, respectively; (b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 10 and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 14; (c) a heavy chain variable region and a light chain variable region having an amino acid sequence that is more than 80% identical to the sequence shown in (b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a).

[0013] In a third aspect, an embodiment of the present invention provides an antibody conjugate, which includes the antibody or a functional fragment thereof as described in the above embodiments.

[0014] In a fourth aspect, an embodiment of the present invention provides a reagent or kit, which includes the antibody or its functional fragment as described in the above embodiments or the antibody conjugate as described in the above embodiments.

[0015] In a fifth aspect, an embodiment of the present invention provides a method for detecting influenza A virus, comprising: mixing the antibody or its functional fragment as described in the above embodiment with a sample to be detected, allowing the antibody or its functional fragment to contact the influenza A virus in the sample to be detected to form an immune complex.

[0016] In a sixth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or a functional fragment thereof described in the aforementioned embodiment.

[0017] In a seventh aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

[0018] In an eighth aspect, an embodiment of the present invention provides a cell containing the isolated nucleic acid described in the preceding embodiment or the vector described in the preceding embodiment.

[0019] In a ninth aspect, an embodiment of the present invention provides a method for preparing the antibody or functional fragment thereof described in the preceding embodiment, comprising: culturing the cells described in the preceding embodiment.

[0020] In a tenth aspect, embodiments of the present invention provide use of the above-mentioned antibodies or functional fragments thereof, antibody conjugates, reagents or kits in detecting influenza A virus antigens or preparing products for detecting influenza A virus antigens.

[0021] The present invention has the following beneficial effects:

[0022] The anti-influenza A virus antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region. The antibody provides an important source of raw materials for the detection of influenza A virus and has excellent or improved affinity and activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The results of reducing SDS-PAGE of Anti-FluA 2F13 are shown. DETAILED DESCRIPTION

[0025] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0026] The present invention provides anti-influenza A virus antibodies or functional fragments thereof, reagents and kits for detecting influenza A virus, which have excellent activity, affinity, specificity or sensitivity.

[0027] In some embodiments, the present invention provides anti-influenza A virus antibodies or functional fragments thereof, reagents and kits for detecting influenza A virus, which have improved at least one property including activity, affinity, specificity or sensitivity.

[0028] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0029] On the one hand, an embodiment of the present invention provides an anti-influenza A virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises three complementarity determining regions having a heavy chain variable region of any one of the amino acid sequences of SEQ ID NOs: 7, 8, 9, and 10 and three complementarity determining regions having a light chain variable region of any one of the amino acid sequences of SEQ ID NOs: 11, 12, 13, and 14.

[0030] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or its functional fragment described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or its functional fragment described in the first aspect.

[0031] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 7; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 11.

[0032] On the other hand, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0033] (a) The amino acid sequences of HCDR1 to 3 and LCDR1 to 3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1 to 3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID Nos: 4 to 6, respectively.

[0034] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0035] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0036] In the present invention, the heavy chain complementary determining region is represented by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be the widely used standard for numbering antibody residues. The embodiments of the present invention use the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0037] Methods for defining CDRs are well known in the art, including the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200200201154, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196: 901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0038] Table 1: CDR Definition 1

[0039]

[0040]

[0041] 1The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0042] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0043] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0044] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0045] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0046] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

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

[0048] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0049] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0050] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0051] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0052] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0053] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0054]

[0055]

[0056] On the other hand, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0057] (a) The amino acid sequences of HCDR1-3 and LCDR1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1-3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID Nos: 4-6, respectively;

[0058] The HCDR1, HCDR2 and HCDR3 are, respectively, the amino acid sequences of positions 31 to 35 (SEQ ID No: 1), 50 to 65 (SEQ ID No: 2), and 95 to 98 (SEQ ID No: 3) of the heavy chain variable region according to Kabat numbering; the LCDR1, LCDR2 and LCDR3 include or are, respectively, the amino acid sequences of positions 24 to 34 (SEQ ID No: 4), 50 to 56 (SEQ ID No: 5), and 89 to 95 (SEQ ID No: 6) of the light chain variable region according to Kabat numbering.

[0059] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0060] (b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 10 and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 14.

[0061] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0062] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0063] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0064] (c) A heavy chain variable region and a light chain variable region whose amino acid sequences are more than 80% identical to the sequences shown in (b), and include HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequences shown in (a).

[0065] In an optional embodiment, the antibody or a functional fragment thereof comprises a heavy chain framework region in a heavy chain variable region shown in any one of SEQ ID NOs: 7 to 10, and a light chain framework region in a light chain variable region shown in any one of SEQ ID NOs: 11 to 14.

[0066] In an optional embodiment, the framework region amino acid sequence of the antibody or its functional fragment may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned framework region.

[0067] In an alternative embodiment, the antibody or functional fragment thereof further comprises a constant region.

[0068] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0069] In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and the light chain constant region is selected from the κ or λ type light chain constant region.

[0070] In an optional embodiment, the species origin of the constant region is any one of cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose and human.

[0071] In an alternative embodiment, the species origin of the constant region is mouse.

[0072] In an alternative embodiment, the heavy chain constant region sequence is as shown in SEQ ID NO: 23 or has at least 80% identity thereto.

[0073] In an alternative embodiment, the light chain constant region sequence is as shown in SEQ ID NO: 24 or has at least 80% identity thereto.

[0074] Specifically, the constant region sequence can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 23 or 24).

[0075] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0076] The functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they are derived. Based on the disclosure herein, those skilled in the art will readily appreciate that the functional fragments of the above-mentioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the above-mentioned functional fragments.

[0077] Functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0078] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises: a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 15 to 18, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 19 to 22.

[0079] In an optional embodiment, the antibody or its functional fragment described in the above embodiment has a KD of less than 2.02×10 - 11 The affinity of M for binding to influenza A virus.

[0080] In an optional embodiment, the antibody or its functional fragment described in the above embodiment has a KD≤10 -11 M or KD≤10 -12 The affinity of M binds to influenza A virus.

[0081] In an optional embodiment, the antibody or functional fragment thereof described in the above embodiment has a KD ≤ 2.27×10 - 13 The affinity of M binds to influenza A virus.

[0082] On the other hand, an embodiment of the present invention further provides an antibody conjugate, which includes the antibody or a functional fragment thereof as described in the above embodiment.

[0083] In an optional embodiment, the antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or a functional fragment thereof.

[0084] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof.

[0085] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0086] In an optional embodiment, the label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0087] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0088] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0089] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0090] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0091] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0092] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0093] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0094] In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0095] In an optional embodiment, the colloidal metal is colloidal gold.

[0096] In an optional embodiment, the antibody conjugate further comprises a solid phase support coupled to the antibody or its functional fragment. In the antibody conjugate, the antibody is coupled to the solid phase support.

[0097] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0098] In an optional embodiment, the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0099] In an optional embodiment, the solid phase carrier is a nitrocellulose membrane.

[0100] On the other hand, an embodiment of the present invention further provides a reagent or kit, which includes the antibody or its functional fragment as described in any of the foregoing embodiments or the antibody conjugate as described in any of the foregoing embodiments.

[0101] On the other hand, an embodiment of the present invention further provides a method for detecting influenza A virus antigens, comprising:

[0102] The antibody or functional fragment thereof as described in any of the preceding embodiments, the antibody conjugate as described in any of the preceding embodiments, or the reagent or kit as described in any of the preceding embodiments is contacted with a sample to be detected to form an immune complex.

[0103] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0104] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to influenza A virus.

[0105] On the other hand, embodiments of the present invention further provide use of the antibody or functional fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in detecting influenza A virus antigens or preparing products for detecting influenza A virus antigens.

[0106] On the other hand, an embodiment of the present invention further provides the use of the antibody or functional fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in the preparation of a product having at least one of the following uses, wherein the uses include: diagnosing or assisting in the diagnosis of diseases related to influenza A virus infection, and predicting or assisting in the prediction of the prognosis and efficacy of diseases related to influenza A virus infection.

[0107] In an optional embodiment, the related diseases caused by influenza A virus infection include at least one of viral pneumonia, secondary bacterial pneumonia, myocarditis and pericarditis.

[0108] In an alternative embodiment, the product comprises a reagent or a kit.

[0109] On the other hand, an embodiment of the present invention further provides an isolated nucleic acid encoding the antibody or a functional fragment thereof described in any of the aforementioned embodiments.

[0110] On the other hand, an embodiment of the present invention further provides a vector comprising the isolated nucleic acid described in any of the aforementioned embodiments.

[0111] On the other hand, an embodiment of the present invention further provides a cell comprising the isolated nucleic acid described in any of the foregoing embodiments or the vector described in any of the foregoing embodiments.

[0112] On the other hand, an embodiment of the present invention further provides a method for preparing the antibody or functional fragment thereof as described in any of the aforementioned embodiments, comprising: culturing the cell as described in any of the aforementioned embodiments.

[0113] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it falls within the scope of protection of the present invention.

[0114] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0116] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0117] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0118] Example 1 Preparation of Anti-FluA 2F13 Monoclonal Antibody

[0119] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by a gene sequencing company.

[0120] 1. Expression Plasmid Construction

[0121] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by a gene sequencing company.

[0122] 1.1. Preparation of Anti-FluA 2F13 Antibody Gene

[0123] mRNA was extracted from a hybridoma cell line secreting the Anti-FluA 2F13 monoclonal antibody. DNA was obtained by RT-PCR and inserted into the pMD-18T vector. The DNA was then transformed into DH5α competent cells. After colonies were grown, four positive clones each for the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0124] 1.2. Sequence Analysis of the Variable Region Gene of Anti-FluA 2F13 Antibody

[0125] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 321 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 342 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

[0126] 1.3 Construction of recombinant antibody expression plasmid

[0127] pcDNA TM 3.4 Vector is a constructed recombinant antibody eukaryotic expression vector, which has been modified to introduce multiple cloning restriction enzyme sites and is subsequently referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the above-mentioned pMD-18T, VL and VH gene-specific primers of the Anti-FluA2F13 antibody were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The 0.73KB Light Chain gene fragment and the 1.40kb Heavy Chain gene fragment were amplified by PCR.

[0128] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0129] 2. Recombinant Antibody Sample Preparation

[0130] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, resolubilize with culture medium, and adjust the cell density to 2.9×10 6 cells / ml, as cell diluent. Use culture medium to prepare plasmid DNA and transfection reagent diluent respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a speed of 120 rpm and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The supernatant of the centrifuge was affinity purified using a protein A affinity chromatography column. Take 6 μg of purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is as shown below. Figure 1 After reducing SDS-PAGE, two bands were shown, one with a Mr of 50 KD (heavy chain) and the other with a Mr of 28 KD (light chain).

[0131] Example 2 Affinity and activity optimization

[0132] Although the Anti-FluA 2F13 monoclonal antibody obtained in Example 1 has the ability to bind to influenza A virus, its affinity and antibody activity are not ideal. Therefore, the applicant conducted targeted mutagenesis on the variable region of the antibody. That is, a computer was used to simulate the structure of the antibody variable region, the structure of the complex of the antigen and the antibody variable region, and the key amino acids of the antibody were analyzed and designed for mutations. According to the mutation scheme, bidirectional primers covering the mutation sites were designed and synthesized. Primers at both ends of the target DNA were synthesized, and a high-fidelity PCR reaction was performed. The PCR product was cloned into a vector, and then the mutant antibody was prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained and named Anti-FluA-2F13RMb1 to Anti-FluA-2F13RMb5. The amino acid sequences of their heavy and light chains are as follows:

[0133] Table 1 Antibody sequences

[0134] Sample name Heavy chain sequence number Light chain number Anti-FluA-2F13RMb1 SEQ ID NO:15 SEQ ID NO: 19 Anti-FluA-2F13RMb2 SEQ ID NO:16 SEQ ID NO: 19 Anti-FluA-2F13RMb3 SEQ ID NO: 17 SEQ ID NO: 19 Anti-FluA-2F13RMb4 SEQ ID NO:15 SEQ ID NO:20 Anti-FluA-2F13RMb5 SEQ ID NO:15 SEQ ID NO:21

[0135] Example 3 Affinity Analysis

[0136] Purified antibodies were diluted in advance, and influenza A recombinant antigen (obtained from Feipeng Bio) was serially diluted. The binding and dissociation curves of the antigen and antibody were measured on a Biacore 8K+ instrument using a CM5 chip pre-coupled with goat anti-mouse IgG. The instrument automatically fitted the affinity constant, association rate, and dissociation rate (KD represents the equilibrium dissociation constant, i.e., affinity constant; ka represents the association rate; and kd represents the dissociation rate).

[0137] Table 2 Affinity analysis data

[0138] Sample name <![CDATA[K D (M)]]> ka kd Control Antibody 2.02E-11 1.07E+05 2.17E-06 Anti-FluA-2F13RMb1 1.96E-13 1.18E+06 2.31E-07 Anti-FluA-2F13RMb2 1.10E-13 2.11E+06 2.32E-07 Anti-FluA-2F13RMb3 2.27E-13 1.37E+06 3.11E-07 Anti-FluA-2F13RMb4 1.63E-13 1.95E+06 3.17E-07 Anti-FluA-2F13RMb5 1.72E-13 1.87E+06 3.22E-07

[0139] Example 4 Activity Identification

[0140] Dilute influenza A antigen (obtained from Feipeng Bio) to 3ug / ml with coating solution (main component NaHCO3), add 100uL to each well, and incubate at 4℃ overnight; the next day, wash twice with washing solution (main component Na2HPO4+Nacl) and pat dry; add blocking solution (20% BSA+80% PBS), 120uL to each well, incubate at 37℃ for 1h, and pat dry; add diluted purified antibody and control antibody, 100uL / well, incubate at 37℃ for 30min; wash 5 times with washing solution and pat dry; add goat anti-mouse IgG-HRP, 100uL to each well, incubate at 37℃ for 30min; wash 5 times with washing solution and pat dry; add colorimetric solution A (50uL / well) and colorimetric solution B (50uL / well) for 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on microplate reader. The same method was used to detect that the above antibodies had no cross-reactivity with other viruses (such as Coxsackievirus A16).

[0141] Table 3 Activity data

[0142] Concentration (ng / ml) 250 125 62.5 31.25 15.63 0 Control Antibody 1.312 0.947 0.616 0.359 0.183 0.013 Anti-FluA-2F13RMb1 1.612 1.335 0.921 0.617 0.345 0.016 Anti-FluA-2F13RMb2 1.674 1.342 0.972 0.641 0.394 0.018 Anti-FluA-2F13RMb3 1.634 1.316 0.931 0.644 0.338 0.012 Anti-FluA-2F13RMb4 1.689 1.378 0.936 0.631 0.373 0.014 Anti-FluA-2F13RMb5 1.679 1.305 0.952 0.624 0.360 0.014

[0143] Example 5 Antibody Stability Assessment

[0144] The above-mentioned antibodies were placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under all three test conditions for 21 days, and the activity did not decrease with increasing test temperature, indicating that the expressed antibodies were stable. The table below shows the OD results of the enzyme immunoassay activity assay after 21 days of testing.

[0145] Table 4 Stability data

[0146]

[0147]

[0148] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0149] Some of the amino acid sequences involved in this application are shown in Table 5 below:

[0150]

[0151]

[0152]

Claims

1. An anti-influenza A virus antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are sequentially identical to the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as set forth in SEQ ID NO: 7; and the LCDR1, LCDR2, and LCDR3 are sequentially identical to the LCDR1, LCDR2, and LCDR3 of the light chain variable region as set forth in SEQ ID NO: 11; or The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are sequentially identical to the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in SEQ ID NO: 9; the LCDR1, LCDR2, and LCDR3 are sequentially identical to the light chain variable region LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NO: 11; or The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are identical to the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in SEQ ID NO: 7, respectively; the LCDR1, LCDR2, and LCDR3 are identical to the LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 12, respectively; The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined by any one of the systems of Kabat, Chothia, IMGT, Lesk, AbM or Contact.

2. An anti-influenza A virus antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof includes any one of (a) to (b): (a) The amino acid sequences of HCDR1-3 and LCDR1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1-3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 4-6, respectively; (b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 10 and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 14.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that The antibody or antigen-binding fragment thereof is expressed as K D <2.02×10 -11 The affinity of M binds to influenza A virus.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein The antibody or antigen-binding fragment thereof further comprises a constant region.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that The constant region includes a heavy chain constant region and / or a light chain constant region.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that The species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

8. The antibody or antigen-binding fragment thereof according to claim 4, wherein The species origin of the constant region is mouse.

9. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The heavy chain constant region sequence is as shown in SEQ ID NO: 23 or has at least 80% identity thereto.

10. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The light chain constant region sequence is as shown in SEQ ID NO: 24 or has at least 80% identity thereto.

11. An anti-influenza A virus antibody or antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 15 to 18, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 19 to 22.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or 5 to 11, wherein: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

13. An antibody conjugate, characterized in that It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. The antibody conjugate according to claim 13, characterized in that The antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or antigen-binding fragment thereof.

15. The antibody conjugate according to claim 13, characterized in that The antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

16. The antibody conjugate according to claim 13, characterized in that The antibody conjugate further includes a label coupled to the antibody or antigen-binding fragment thereof.

17. The antibody conjugate according to claim 16, characterized in that The label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

18. The antibody conjugate according to claim 16, characterized in that The label is colloidal gold.

19. A reagent or kit, characterized in that: It includes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the antibody conjugate according to any one of claims 13 to 18.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the antibody conjugate according to any one of claims 13 to 18, or the reagent or kit according to claim 19 in the preparation of a product for detecting influenza A virus antigens, characterized in that: It includes: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the antibody conjugate according to any one of claims 13 to 18, or the reagent or kit according to claim 19 is contacted with a sample to be detected to form an immune complex.

21. The use according to claim 20, characterized in that The immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

22. The use according to claim 20, characterized in that The immune complex further includes a second antibody that binds to an influenza A virus antigen.

23. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

24. A carrier, characterized in that The vector contains the nucleic acid according to claim 23.

25. A cell, characterized in that The cell contains the nucleic acid of claim 23 or the vector of claim 24.

26. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, characterized in that: The method comprises culturing the cell of claim 25.

27. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the antibody conjugate according to any one of claims 13 to 18, or the reagent or kit according to claim 19 in the preparation of a product for detecting influenza A virus antigens.

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