Anti-influenza A virus antibodies, reagents and kits for detecting influenza A virus

Abstract

The present invention discloses an anti-influenza A virus antibody, a reagent for detecting influenza A virus, and a kit, relating to the field of antibody technology. The anti-influenza A virus antibody disclosed herein comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important source of raw materials for the detection of influenza A virus and has improved affinity and activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-influenza A virus antibody, a reagent and a kit for detecting influenza A virus. Background Art

[0002] Influenza virus (Flu), also known as influenza virus, is a representative species of the Orthomyxoviridae family, including human influenza virus, swine influenza virus, equine influenza virus, and avian influenza virus. Human influenza virus can be divided into three types: A (A), B (B), and C (C) based on the antigenicity of its nucleoprotein, and 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, the main influenza viruses that can infect humans are influenza A virus and influenza B virus, which mainly cause 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.

[0003] Influenza A virus (Flu-A), first isolated in 1933, is prone to antigenic variation and can be further divided into subtypes, including H1N1, H3N2, H5N1, and H7N9 (H represents the hemagglutinin of the influenza virus, and N represents the neuraminidase of the influenza virus). It has repeatedly caused global pandemics, with annual peaks. The severity of influenza A infection is related to individual immunity. Typical symptoms include chills, persistent high fever, and headache, often accompanied by sore throat, cough, nasal congestion, and generally, 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 spread 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.

[0004] 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, its detection window is relatively short, buying time for early diagnosis and treatment, reducing mortality, and controlling the epidemic, making it the gold standard for diagnosis. However, this method requires high standards for personnel, requiring specialized training and diagnostic testing in qualified laboratories using specialized equipment. 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.

[0005] 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 to the influenza A virus.

[0006] Therefore, there is a strong need in the art for antibodies that can effectively and specifically bind to and detect influenza A virus (Flu-A).

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The present application provides an anti-influenza A virus antibody with improved affinity and / or activity to improve the detection of influenza A virus and provide an important source of raw materials for the detection of influenza A virus.

[0009] To achieve the above object, according to one aspect of the present invention, an anti-influenza A virus antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises:

[0010] a) HCDR1, HCDR2, HCDR3 with amino acid sequences as shown in SEQ ID NOs: 1 to 3, and LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 4 to 6; or

[0011] b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 9 to 12, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 13; or

[0012] 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); or

[0013] d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17, and a light chain having an amino acid sequence as shown in SEQ ID NO: 18.

[0014] In order to achieve the above object, according to a second aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0015] To achieve the above object, according to a third aspect of the present invention, a reagent or kit for detecting influenza A virus is provided, wherein the reagent or kit comprises the above-mentioned antibody or functional fragment thereof or the above-mentioned antibody conjugate.

[0016] To achieve the above object, according to a fourth aspect of the present invention, a method for detecting influenza A virus is provided, comprising: contacting the above antibody or its functional fragment, conjugate, reagent or kit with influenza A virus in a sample to be detected to form an immune complex.

[0017] In order to achieve the above objectives, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The results of reducing SDS-PAGE of Anti-FluA 5C17mut1 to mut4 are shown. DETAILED DESCRIPTION

[0020] The present invention provides an anti-influenza A virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0021] a) HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 3, and LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 4 to 6. The above antibodies have improved affinity and / or activity.

[0022] 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.

[0023] 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.

[0024] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in this field include: Kabat numbering scheme, IMGT numbering scheme, 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 a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark CDR regions, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0025] In another aspect, the present invention provides an anti-influenza A virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0026] b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 9 to 12, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 13. The above antibody has improved affinity and / or activity.

[0027] 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.

[0028] 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.

[0029] In another aspect, the present invention provides an anti-influenza A virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0030] c) a heavy chain variable region and a light chain variable region having an amino acid sequence greater than 80% identical to the sequence shown in b) above, and including HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in a) above. The above antibody has improved affinity and / or activity.

[0031] In an optional embodiment, the antibody or a functional fragment thereof comprises a heavy chain framework region in the heavy chain variable region shown in any one of SEQ ID NOs: 9 to 12, and a light chain framework region in the light chain variable region shown in SEQ ID NO: 13.

[0032] 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.

[0033] In an optional embodiment, the antibody or its functional fragment has a KD of less than 9.87×10 -09 The affinity of M binds to influenza A virus.

[0034] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -09 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12The affinity of M binds to influenza A virus.

[0035] In an optional embodiment, the antibody or its functional fragment has a KD≤9.90×10 -09 The affinity of M binds to influenza A virus.

[0036] In an optional embodiment, any of the above antibodies or functional fragments thereof further comprises a constant region.

[0037] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0038] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0039] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0040] In an alternative embodiment, the species origin of the constant region is mouse.

[0041] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:7, and the light chain constant region (CL) sequence is shown as SEQ ID NO:8.

[0042] It should be noted that, in other embodiments, the constant region sequence 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 constant region (SEQ ID NO: 7 or 8).

[0043] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0044] 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.

[0045] 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.

[0046] In another aspect, the present invention provides an anti-influenza A virus antibody comprising

[0047] d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17, and a light chain having an amino acid sequence as shown in SEQ ID NO: 18. The above-mentioned antibody has improved affinity and / or activity.

[0048] In another aspect, the present invention provides an antibody conjugate comprising the above-mentioned antibody, wherein the antibody is directly or indirectly covalently conjugated to the substance to be conjugated. Alternatively, the antibody is conjugated to the substance to be conjugated by non-covalent adsorption.

[0049] In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to biotin or a biotin derivative.

[0050] In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to a label.

[0051] 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.

[0052] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0053] 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.

[0054] 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.).

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0060] In an alternative embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0061] In an optional embodiment, the colloidal metal is colloidal gold.

[0062] In an alternative embodiment, the antibody in the above antibody conjugate is coupled to a solid phase.

[0063] In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.

[0064] 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.

[0065] In an alternative embodiment, the solid phase is a nitrocellulose membrane.

[0066] In another aspect, the present invention provides a reagent or kit for detecting influenza A virus, wherein the reagent or kit comprises the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

[0067] In another aspect, the present invention provides use of the above-mentioned antibody or functional fragment thereof, antibody conjugate, or the above-mentioned reagent or kit in the detection of influenza A virus.

[0068] In another aspect, the present invention provides a method for detecting influenza A virus, comprising: contacting the above-mentioned antibody or functional fragment thereof, antibody conjugate, or reagent or kit with influenza A virus in a sample to be detected to form an immune complex.

[0069] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0070] In an alternative embodiment, the immune complex further comprises a second antibody that binds to influenza A virus.

[0071] In another aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0072] In another aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0073] In another aspect, the present invention provides a cell containing the above-mentioned vector.

[0074] In another aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cell as described above.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0080] Example 1 Preparation of Anti-FluA 5C17 Monoclonal Antibody

[0081] 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 Invitrogen.

[0082] 1 Construction of recombinant plasmid

[0083] (1) Antibody gene preparation

[0084] mRNA was extracted from a hybridoma cell line secreting monoclonal antibodies against influenza A virus. DNA products were obtained by RT-PCR. This product was subjected to an A-addition reaction using rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0085] (2) Sequence analysis of the variable region gene of the Anti-FluA 5C17 antibody

[0086] 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 357 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

[0087] (3) Construction of recombinant antibody expression plasmid

[0088] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.73kb Light Chain gene fragment and a 1.43kb Heavy Chain gene fragment.

[0089] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. 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.

[0090] 2. Sample preparation of recombinant antibodies

[0091] HEK293 cells were thawed in advance and subcultured to a 200 ml volume, reaching a cell density of 3–5 × 10⁶ cells / ml and a cell viability of >95%. Cells were washed by centrifugation and reconstituted with culture medium. The cell density was adjusted to 2.9 × 10⁶ cells / ml for use as a cell dilution. Plasmid DNA and transfection reagent dilutions were prepared separately using culture medium. The transfection reagent dilution was added to the plasmid DNA dilution, mixed, and allowed to stand at room temperature for 15 minutes. This mixture was slowly added to the cell dilution over 1 minute. After mixing, samples were collected and counted. Cell viability after transfection was recorded and observed. Cells were then incubated in a 35°C incubator at 120 rpm and 8% CO₂. After 13 days, samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electropherogram is shown in the figure. 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).

[0092] Example 2 Affinity and activity optimization

[0093] Although the Anti-FluA 5C17 monoclonal antibody obtained in Example 1 had the ability to bind to influenza A virus antigens, its affinity and antibody activity were less than ideal. Therefore, the applicants performed targeted mutagenesis on the light and heavy chain CDRs of the antibody. Specifically, computer simulations of the antibody variable region structure, the structure of the complex interacting with the antigen and antibody variable region, key amino acid analysis of the antibody, and mutation design were performed. Bidirectional primers covering the mutation sites were designed and synthesized according to the mutation scheme. 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 the mutant antibodies were 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 5C17mut1 to Anti-FluA5C17mut4. The heavy and light chain amino acid sequences of each antibody are as follows.

[0094] Table 1 Antibody sequences

[0095] Sample name Heavy chain sequence number Light chain number Anti-FluA 5C17mut1 SEQ ID NO:14 SEQ ID NO: 18 Anti-FluA 5C17mut2 SEQ ID NO: 17 SEQ ID NO: 18 Anti-FluA 5C17mut3 SEQ ID NO:15 SEQ ID NO: 18 Anti-FluA 5C17mut4 SEQ ID NO:16 SEQ ID NO: 18

[0096] Example 3 Affinity Analysis and Activity Identification

[0097] 1. Affinity Analysis

[0098] Using the AMC sensor, the purified antibody was diluted to 10 μg / ml with PBST, and the human influenza A recombinant antigen (rFAN1-Ag, self-developed) was gradiently diluted with PBST.

[0099] Operation process: 60 s equilibration in buffer 1 (PBST), 300 s antibody immobilization in antibody solution, 180 s incubation in buffer 2 (PBST), 420 s binding in antigen solution, 1200 s dissociation in buffer 2, sensor regeneration with 10 mM pH 1.69 GLY solution and buffer 3 (PBST), and data output.

[0100] (KD represents the equilibrium dissociation constant, i.e. affinity; kon represents the association rate; kdis represents the dissociation rate. The main components of PBST are Na2HPO4+NaCl+TW-20).

[0101] Table 2 Affinity data

[0102]

[0103]

[0104] 2. Activity Identification

[0105] The rFAN1-Ag recombinant antigen (self-developed) was diluted to 2ug / ml with coating solution (main component NaHCO3), 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+Nacl) and patted dry; blocking solution (20% BSA+80% PBS) was added, 120uL per well, incubated at 37°C for 1h, and patted dry; diluted purified antibody and control antibody were added, 100uL / well, incubated at 37°C for 30min; the cells were washed 5 times with washing solution and patted dry; goat anti-mouse IgG-HRP was added, 100uL per well, incubated at 37°C for 30min; the cells were washed 5 times with washing solution and patted dry; colorimetric solution A (50uL / well) and colorimetric solution B (50uL / well) were added for 10min; stop solution was added, 50uL / well; OD value was read at 450nm (reference 630nm) on a microplate reader.

[0106] Table 3 Activity data

[0107] Sample concentration (ng / ml) 156.25 78.13 39.06 19.53 9.77 0.00 comparison 1.497 0.923 0.551 0.302 0.161 0.035 Anti-FluA 5C17mut1 1.849 1.458 1.034 0.596 0.370 0.038 Anti-FluA 5C17mut2 1.717 1.204 0.763 0.401 0.224 0.037 Anti-FluA 5C17mut3 1.782 1.279 0.823 0.432 0.268 0.036 Anti-FluA 5C17mut4 1.832 1.312 0.981 0.498 0.277 0.039

[0108] 3. Performance Evaluation

[0109] The above recombinant antibodies were used as labeled antibodies in combination with paired anti-influenza A monoclonal antibodies and applied to the colloidal gold platform for combined sample testing. Specific data are shown in the table below:

[0110] Note: The gold label colorimetric consists of C plus a number. The smaller the number after C, the stronger the color and the higher the activity; the higher the number after C, the weaker the color and the lower the activity. The number followed by "+" is slightly stronger than without color, 0.5-1C; the number followed by "-" is slightly lower than without color, 0.5-1C; "B" represents negative.

[0111] Table 4

[0112]

[0113]

[0114] 3. Stability assessment

[0115] 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 the three test conditions for 21 days, and the activity did not show a downward trend with increasing test temperature, indicating that the above-mentioned antibodies are stable. The table below shows the OD results of the enzyme immunoassay activity assay after 21 days of antibody testing.

[0116] Table 3 Stability data

[0117] Sample concentration (ng / ml) 78.13 39.06 0 4℃, 21-day samples 1.412 0.987 0.008 -80℃, 21-day sample 1.408 0.992 0.007 37℃, 21-day samples 1.411 0.978 0.008

[0118] 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.

[0119] Some of the amino acid sequences involved in this application are as follows:

[0120] Sequence number Sequence fragments SEQ ID NO: 1 GYYMH SEQ ID NO:2 RINPYDGATTYNQNFKD SEQ ID NO:3 YYGSSDY SEQ ID NO:4 KASQDIYRYLS SEQ ID NO:5 RANRLVD SEQ ID NO:6 LQYNEFP

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 with amino acid sequences as shown in SEQ ID NOs: 1 to 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 4 to 6.

2. An anti-influenza A virus antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 9 to 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

13.

3. 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 variable region and a light chain variable region whose amino acid sequences are more than 80% identical to the sequences of the heavy chain variable region and the light chain variable region described in claim 2, and comprises HCDR1 to HCDR3 and LCDR1 to LCDR3 described in claim 1.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment thereof has a KD of <9.87×10 -09 The affinity of M for binding to influenza A virus.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof further comprises a constant region.

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

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

8. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

9. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that The species origin of the constant region is mouse.

10. The antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain constant region sequence is shown in SEQ ID NO: 7 or has at least 80% identity thereto, and the light chain constant region sequence is shown in SEQ ID NO: 8 or has at least 80% identity thereto.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

12. An anti-influenza A virus antibody, characterized in that The antibodies include: A heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17, and a light chain having an amino acid sequence as shown in SEQ ID NO:

18.

13. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the antibody according to claim 12, wherein the antibody is conjugated to: biotin or a biotin derivative, a solid phase or a label.

14. The antibody conjugate according to claim 13, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

15. The antibody conjugate according to claim 13, characterized in that The label is colloidal gold.

16. A reagent or kit for detecting influenza A virus, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the antibody according to claim 12, or the antibody conjugate according to any one of claims 13 to 15.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the antibody according to claim 12, or the antibody conjugate according to any one of claims 13 to 15 in the preparation of a reagent or kit for detecting influenza A virus, characterized in that: The detection of influenza A virus comprises: contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the antibody according to claim 12, or the antibody conjugate according to any one of claims 13 to 15 with the influenza A virus in the sample to be detected to form an immune complex.

18. The use according to claim 17, characterized in that The immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

19. The use according to claim 17, characterized in that The immune complex also includes a second antibody that binds to the influenza A virus.

20. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the antibody according to claim 12.

21. A carrier, characterized in that It contains the nucleic acid according to claim 20.

22. A cell, characterized in that It contains the nucleic acid according to claim 20 or the vector according to claim 21.

23. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the antibody according to claim 12, characterized in that: It includes: Cultivate the cell of claim 22.

Images