Anti-influenza A virus antibody, reagent and kit for detecting influenza A virus

Abstract

The present invention discloses an anti-influenza A virus antibody, a reagent and a kit for detecting influenza A virus, which relate to the technical field of antibodies. The anti-influenza A virus antibody disclosed by the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. This antibody provides an important raw material source for the detection of influenza A virus and has improved affinity and activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, 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 the representative species of the Orthomyxoviridae family, including human influenza virus, swine influenza virus, equine influenza virus, avian influenza virus, etc. Among them, human influenza virus can be divided into three types: A (H1N1), B (H3N2), and C (H5N1) according to the antigenicity of its nucleoprotein, and it is the pathogen of influenza. Influenza virus can cause infection and disease in various animals such as humans, birds, pigs, horses, and bats. Among them, the main influenza viruses that can infect humans are influenza A virus and influenza A virus, which mainly cause upper respiratory tract infections, and 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) was successfully isolated in 1933. Its antigen is prone to mutation and can be further divided into subtypes such as H1N1, H3N2, H5N1, and H7N9 (H represents the hemagglutinin of the influenza virus, and N represents the neuraminidase of the influenza virus). It has caused several worldwide pandemics, with a peak every year. The degree of influenza A virus infection and illness is related to personal immunity. The typical symptoms are chills, persistent high fever, and headache, accompanied by sore throat, cough, and nasal congestion. Generally, there are systemic symptoms such as body aches and fatigue. Literature reports that the positive detection rate during the epidemic period is 20-40%, while the positive rate in the non-epidemic season is 2-20%. The continuous prevalence of influenza A virus will bring great interference and pressure to people's health, life, and the social public epidemic prevention system. It has become one of the main research objects in epidemiology.

[0004] Currently, the main detection methods for influenza A virus on the market are fluorescence PCR method, immunoassay, and virus isolation and culture identification. The fluorescence PCR method is to detect the PCR process in real time through fluorescence signals during PCR amplification for qualitative or quantitative detection of the target. This method is the gold standard for pathogen detection; the immunoassay detects the target protein through specific binding of antigen and antibody; common methods for virus isolation and culture include chicken embryo inoculation, animal inoculation, tissue (cell) culture, etc., and then the results are observed and analyzed. Although the fluorescence PCR method has good sensitivity and specificity, its detection window period is short, which can gain time for early diagnosis, early treatment, reducing mortality and controlling the epidemic situation of the disease, and can be used as the gold standard for diagnosis. However, this method has high requirements for the detection personnel, who need to undergo professional skill training, and the diagnostic detection can only be carried out in a professional laboratory with qualified instruments and equipment. Therefore, this method is not suitable for rapid diagnosis in the front line of clinical or epidemiological monitoring. Virus isolation and culture identification takes a long time, has high environmental requirements, a high risk of infection for the operator, and poor culture effects, and its application in clinical diagnosis and epidemiological monitoring is very limited. The immunoassay detection reagent targets antigens or antibodies in the sample, has a relatively fast detection speed and good accuracy, and has relatively low requirements for the laboratory and operators. It is generally applicable to grass-roots screening in hospital clinical laboratories, CDC system laboratories, etc., and plays a very important role in the initial detection of influenza A, successfully controlling outbreaks and guiding treatment in hospitals and communities.

[0005] Currently, the main immunoassay diagnostic reagent products for influenza A on the market are enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography, such as the influenza A virus antigen detection reagent (colloidal gold method) of Guangzhou Wondfo and the influenza A ELISA kit of R&D. In all of the above diagnostic reagent products, specific antibodies against influenza A virus are required.

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

[0007] In view of this, the present invention is specifically 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 raw material source for the detection of influenza A virus.

[0009] To achieve the above object, according to one aspect of the present invention, there is provided an anti-influenza A virus antibody or its functional fragment, and the antibody or its functional fragment includes:

[0010] a) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1 to 3, and LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4 or 9, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5 or 10, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6 or 11; or

[0011] b) A heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 12 to 13, and a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 14 to 21; or

[0012] c) A heavy chain variable region and a light chain variable region with an amino acid sequence having more than 80% identity to the sequences shown in b), and including HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequences shown in a); or

[0013] d) A heavy chain with an amino acid sequence as shown in any one of SEQ ID NO: 22 to 23, and a light chain with an amino acid sequence as shown in any one of SEQ ID NO: 24 to 31.

[0014] To achieve the above object, according to the second aspect of the present invention, an antibody conjugate is provided, and the antibody conjugate includes the above antibody or its functional fragment.

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

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

[0017] To achieve the above object, the present invention also provides a vector, a cell, and a method for preparing the above antibody or its functional fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1Results of the reducing SDS-PAGE of Anti-FluA 9G2mut1 to mut4. Detailed implementation mode

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

[0021] a) HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO: 1 to 3, and LCDR1 with an amino acid sequence shown in SEQ ID NO: 4 or 9, LCDR2 with an amino acid sequence shown in SEQ ID NO: 5 or 10, and LCDR3 with an amino acid sequence shown in SEQ ID NO: 6 or 11. The above antibody has 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, as long as they exhibit the required biological activity.

[0023] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the main amino acid residues that play a role in the binding of an antibody or an antigen-binding fragment to the antigen or epitope it recognizes. In the specific implementation mode of the present invention, the CDRs refer to the highly variable 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 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. in 1997 for all protein sequences of the immunoglobulin superfamily. 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 the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label the CDR region, but the CDR regions labeled by other methods also fall within the protection scope of the present invention.

[0025] On the other hand, the present invention provides an anti-influenza A virus antibody or a functional fragment thereof, and the antibody or the functional fragment thereof includes:

[0026] b) A heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 12 to 13, and a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 14 to 21. The above-mentioned 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, which refers to the regions other than the CDRs in the heavy chain variable region and the light chain variable region of the antibody; wherein, the heavy chain framework region can be further divided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further divided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0028] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1 - HCDR1 - HFR2 - HCDR2 - HFR3 - HCDR3 - HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1 - LCDR1 - LFR2 - LCDR2 - LFR3 - LCDR3 - LFR4.

[0029] On the other hand, the present invention provides an antibody against influenza A virus or a functional fragment thereof, and the antibody or its functional fragment includes:

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

[0031] In an alternative embodiment, the antibody or its functional fragment includes the heavy chain framework region in the heavy chain variable region shown in any one of SEQ ID NO: 12 to 13, and the light chain framework region in the light chain variable region shown in any one of SEQ ID NO: 14 to 21.

[0032] In an alternative embodiment, the amino acid sequence of the framework region of the antibody or its functional fragment may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above-mentioned framework region.

[0033] In an alternative embodiment, the antibody or its functional fragment binds to influenza A virus with an affinity of KD < 9.92×10 -09 M.

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

[0035] In an alternative embodiment, the antibody or its functional fragment binds to influenza A virus with an affinity of KD ≤ 9.89×10 -09 M.

[0036] In an alternative embodiment, any one of the above antibodies or its functional fragment further comprises a constant region.

[0037] In an alternative embodiment, 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 regions 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 regions.

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

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

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

[0042] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant region (SEQ ID NO:7 or 8).

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

[0044] Functional fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It will be readily understood by those skilled in the art from the content described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the structure of the intact antibody disclosed in the present invention, those skilled in the art can readily obtain the above functional fragments.

[0045] Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0046] On the other hand, the present invention provides an antibody against influenza A virus, comprising

[0047] d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NO: 22 to 23, and a light chain having an amino acid sequence as shown in any one of SEQ ID NO: 24 to 31. The above antibody has improved affinity and / or activity.

[0048] On the other hand, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above antibody. Among them, the antibody is directly or indirectly covalently conjugated to the conjugate to be conjugated. Alternatively, the antibody is conjugated to the conjugate to be conjugated in a non-covalent adsorption manner.

[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 alternative embodiment, the label refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics.

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

[0053] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0054] In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (e.g., including but not limited to rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (e.g., including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (e.g., including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0055] In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0056] In alternative embodiments, the radioisotopes 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 alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives.

[0058] In alternative embodiments, 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 colloids include, but are 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 alternative embodiment, the colloidal metal is colloidal gold.

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

[0063] In an alternative embodiment, the solid phase is selected from microspheres, plates, and membranes.

[0064] In an alternative embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

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

[0066] On the other hand, the present invention provides a reagent or kit for detecting influenza A virus, and the reagent or kit includes the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

[0067] On the other hand, the present invention provides the use of the above-mentioned antibody or its functional fragment, antibody conjugate, or the above-mentioned reagent or kit in the detection of influenza A virus.

[0068] On the other hand, the present invention provides a method for detecting influenza A virus, including: contacting the above-mentioned antibody or its functional fragment, antibody conjugate, or the above-mentioned reagent or kit with influenza A virus in a sample to be detected to form an immune complex.

[0069] In an alternative embodiment, the immune complex further includes a second antibody that binds to the antibody or its functional fragment.

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

[0071] On the other hand, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its functional fragment.

[0072] On the other hand, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0073] On the other hand, the present invention provides a cell containing the above-mentioned vector.

[0074] On the other hand, the present invention provides a method for preparing an antibody or its functional fragment, which includes: culturing the cell as described above.

[0075] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in the present invention, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (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 recombinant cells capable of recombinantly expressing the antibody or its functional fragment described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the antibody or its functional fragment of the present invention, it falls within the protection scope of the present invention.

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0078] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0079] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0080] Example 1 Preparation of Anti-FluA 9G2 Monoclonal Antibody

[0081] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen.

[0082] 1 Construction of recombinant plasmid

[0083] (1) Preparation of antibody gene

[0084] mRNA was extracted from the hybridoma cell line secreting monoclonal antibody against influenza A virus. DNA products were obtained by RT-PCR method. After adding A reaction to the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew out, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to the gene sequencing company for sequencing.

[0085] (2) Sequence analysis of the variable region genes of Anti-FluA 9G2 antibody

[0086] The gene sequences obtained from the above sequencing were analyzed in the Kabat antibody database, and the VNTI 11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 321 bp, and there was a 57 bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 360 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

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

[0088] pcDNA TM 3.4 The vector pcDNA 3.4 was the constructed eukaryotic expression vector for recombinant antibody. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protective bases at both ends. A 0.73 kb Light Chain gene fragment and a 1.45 kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0089] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0090] 2 Sample preparation of recombinant antibody

[0091] Resuscitate HEK293 cells in advance, passage and culture them in a 200 ml system until the cell density reaches 3 - 5×106 cells / ml and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×106 cells / ml as the cell dilution solution. Prepare the plasmid DNA and transfection reagent dilution solutions with the medium respectively. Add the transfection reagent dilution solution to the plasmid DNA dilution solution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution solution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in an incubator at 35°C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0092] Example 2 Affinity and activity optimization

[0093] Although the Anti-FluA 9G2 monoclonal antibody obtained in Example 1 has the ability to bind to the influenza A virus antigen, its affinity and antibody activity are not ideal enough. Therefore, the applicant carried out site-directed mutagenesis on the light chain CDR and heavy chain CDR of this antibody. That is, use a computer to simulate the structure of the antibody variable region, the structure of the antigen-antibody variable region interaction complex, analyze and design mutations of key amino acids of the antibody, design and synthesize bidirectional primers covering the mutation sites according to the mutation scheme, synthesize primers at both ends of the target DNA, carry out a high-fidelity PCR reaction, clone the PCR product into the vector, and then prepare the mutant antibody 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 9G2mut1 to Anti-FluA 9G2mut7. The heavy chain and light chain amino acid sequences of each antibody are as follows.

[0094] Table 1 Antibody sequences

[0095] Sample Name Heavy Chain Serial Number Light Chain Serial Number Anti-FluA 9G2mut1 SEQ ID NO:22 SEQ ID NO:24 Anti-FluA 9G2mut2 SEQ ID NO:23 SEQ ID NO:24 Anti-FluA 9G2mut3 SEQ ID NO:22 SEQ ID NO:25 Anti-FluA 9G2mut4 SEQ ID NO:22 SEQ ID NO:26 Anti-FluA 9G2mut5 SEQ ID NO:22 SEQ ID NO:27 Anti-FluA 9G2mut6 SEQ ID NO:23 SEQ ID NO:25 Anti-FluA 9G2mut7 SEQ ID NO:23 SEQ ID NO:30

[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 recombinant human influenza A antigen (rFAN1-Ag, self-developed) was serially diluted with PBST.

[0099] Operation process: Equilibrate in Buffer 1 (PBST) for 60 s, immobilize the antibody in the antibody solution for 300 s, incubate in Buffer 2 (PBST) for 180 s, bind in the antigen solution for 420 s, dissociate in Buffer 2 for 1200 s, regenerate the sensor with 10 mM pH 1.69 GLY solution and Buffer 3 (PBST), and output the data.

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

[0101] Table 2 Affinity Data

[0102] Sample Name KD(M) kon(1 / Ms) kdis(1 / s) Control 9.92E-09 9.13E+04 9.06E-04 Anti-FluA 9G2mut1 7.83E-10 1.40E+05 1.10E-04 Anti-FluA 9G2mut2 9.87E-10 8.56E+04 8.45E-05 Anti-FluA 9G2mut3 8.13E-10 1.26E+05 1.02E-04 Anti-FluA 9G2mut4 1.59E-10 6.89E+05 1.10E-04 Anti-FluA 9G2mut5 8.34E-10 1.45E+05 1.21E-04 Anti-FluA 9G2mut6 9.89E-10 7.42E+04 7.32E-05 Anti-FluA 9G2mut7 9.72E-10 8.24E+04 8.01E-05

[0103] 2. Activity Identification

[0104] Dilute the rFAN1-Ag recombinant antigen (self-developed) to 3 μg / ml with the coating solution (main component NaHCO3), 100 μL per well, overnight at 4°C; the next day, wash 2 times with the washing solution (main components Na2HPO4 + NaCl), pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0105] Table 3 Activity Data

[0106] Sample Concentration (ng / ml) 125.00 62.50 31.25 15.63 7.81 0.00 Control 1.602 0.994 0.512 0.266 0.144 0.025 Anti-FluA 9G2mut1 1.899 1.662 1.073 0.641 0.266 0.026 Anti-FluA 9G2mut2 1.831 1.641 0.954 0.522 0.267 0.029 Anti-FluA 9G2mut3 1.839 1.607 0.935 0.520 0.292 0.024 Anti-FluA 9G2mut4 1.807 1.666 0.909 0.516 0.212 0.019 Anti-FluA 9G2mut5 1.843 1.612 0.947 0.511 0.274 0.024 Anti-FluA 9G2mut6 1.872 1.654 0.933 0.597 0.211 0.026 Anti-FluA 9G2mut7 1.795 1.592 0.901 0.546 0.209 0.026

[0107] 3. Performance Evaluation

[0108] The above recombinant antibody was used as a coating antibody in combination with a pairable anti-influenza A monoclonal antibody for combined sample testing on a colloidal gold platform. The specific data are shown in the following table:

[0109] Note: The gold-labeled color development is composed of C plus a number. The smaller the number after C, the stronger the color development and the higher the activity; the higher the number after C, the weaker the color development and the lower the activity; the number with a "+" after it is slightly stronger than without by 0.5 - 1C, and the number with a "-" after it is slightly weaker than without by 0.5 - 1C. "B" represents negative.

[0110] Table 4

[0111]

[0112] 3. Stability assessment

[0113] The above antibody was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Samples were taken at 7 days, 14 days, and 21 days for status observation, and the 21-day samples were subjected to activity detection. The results showed that there were no obvious protein status changes after the antibody was placed for 21 days under the three assessment conditions, and the activity did not show a downward trend with the increase in the assessment temperature, indicating that the above antibody is stable. The following table shows the OD results of the enzyme immunoassay activity detection of the antibody after 21 days of assessment.

[0114] Table 5 Stability data

[0115] Sample Concentration (ng / ml) 62.50 15.63 0 Sample at 4°C for 21 days 1.689 0.658 0.017 Sample at -80°C for 21 days 1.672 0.648 0.017 Sample at 37°C for 21 days 1.668 0.607 0.016

[0116] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0117] Some of the amino acid sequences involved in this application are shown as follows:

[0118] Sequence Number Sequence Fragment SEQ ID NO:1 GYTMN SEQ ID NO:2 LINPYNGGTNYNQKFQG SEQ ID NO:3 TGGYDYVS SEQ ID NO:4 KASDHINSWLA SEQ ID NO:5 GAIRLEN SEQ ID NO:6 QQYWKIP SEQ ID NO:9 KASDHLNSWLA SEQ ID NO:10 GALRLEN SEQ ID NO:11 QQYWKLP

Claims

1. An anti-influenza A virus antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment comprises: HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO: 1 to 3 in sequence, and LCDR1, LCDR2, and LCDR3 with amino acid sequences shown in SEQ ID NO: 4 or 9, SEQ ID NO: 5 or 10, and SEQ ID NO: 6 or 11 in sequence, respectively.

2. An anti-influenza A virus antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment comprises: a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 12, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 14; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 14; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 12, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 15; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 12, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 16; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 12, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 17; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 15; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region with an amino acid sequence shown in SEQ ID NO:

20.

3. An anti-influenza A virus antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment comprises: a heavy chain variable region and a light chain variable region with amino acid sequences having more than 80% identity with the sequences of the heavy chain variable region and the light chain variable region described in claim 2, and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 described in claim 1.

4. The antibody or its functional fragment according to any one of claims 1 to 3, characterized in that, The antibody or its functional fragment binds to influenza A virus with an affinity of KD < 9.92×10 -09 M.

5. The antibody or its functional fragment according to any one of claims 1 to 3, characterized in that, The antibody or its functional fragment further comprises a constant region.

6. The antibody or functional fragment thereof according to claim 5, wherein The constant region comprises a heavy chain constant region and / or a light chain constant region.

7. The antibody or functional fragment thereof according to claim 6, wherein The heavy chain constant region is selected from the heavy chain constant regions 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 regions.

8. The antibody or functional fragment thereof according to claim 5, wherein The species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

9. The antibody or functional fragment thereof according to claim 8, wherein The species origin of the constant region is mouse.

10. The antibody or functional 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 therewith, and the light chain constant region sequence is shown in SEQ ID NO: 8 or has at least 80% identity therewith.

11. The antibody or its functional fragment according to any one of claims 1 to 3, characterized in that, The functional 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 antibody comprises: a heavy chain with an amino acid sequence shown in SEQ ID NO: 22, and a light chain with an amino acid sequence shown in SEQ ID NO: 24; or A heavy chain having an amino acid sequence as shown in SEQ ID NO:23, and a light chain having an amino acid sequence as shown in SEQ ID NO:24; or A heavy chain having an amino acid sequence as shown in SEQ ID NO:22, and a light chain having an amino acid sequence as shown in SEQ ID NO:25; or A heavy chain having an amino acid sequence as shown in SEQ ID NO:22, and a light chain having an amino acid sequence as shown in SEQ ID NO:26; or A heavy chain having an amino acid sequence as shown in SEQ ID NO:23, and a light chain having an amino acid sequence as shown in SEQ ID NO:25; or A heavy chain having an amino acid sequence as shown in SEQ ID NO:23, and a light chain having an amino acid sequence as shown in SEQ ID NO:

30.

13. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1 to 11 or a functional fragment thereof or the antibody according to claim 12.

14. The antibody conjugate according to claim 13, wherein, The antibody is conjugated to biotin or a biotin derivative.

15. The antibody conjugate according to claim 13, wherein The antibody is conjugated to a solid phase.

16. The antibody conjugate according to claim 13, wherein The antibody is conjugated to a label.

17. The antibody conjugate according to claim 16, wherein, The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

18. The antibody conjugate according to claim 17, wherein The label is colloidal gold.

19. A reagent or kit for detecting influenza A virus, characterized in that, The reagent or kit comprises the antibody according to any one of claims 1 to 11 or a functional fragment thereof or the antibody according to claim 12 or the antibody conjugate according to any one of claims 13 to 18.

20. Use of the antibody or its functional fragment according to any one of claims 1 to 11, or the antibody according to claim 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 reagent or kit for detecting influenza A virus, characterized in that, A method for detecting influenza A virus comprises: Contacting the antibody according to any one of claims 1 to 11 or a functional fragment thereof, the antibody according to claim 12, the antibody conjugate according to any one of claims 13 to 18, or the reagent or kit according to claim 19 with influenza A virus in a sample to be detected to form an immune complex.

21. The application according to claim 20, wherein The immune complex further comprises a second antibody that binds to the antibody or a functional fragment thereof.

22. The application according to claim 20, wherein, The immune complex further comprises a second antibody that binds to influenza A virus.

23. A nucleic acid, characterized in that, It encodes the antibody according to any one of claims 1 to 11 or a functional fragment thereof or the antibody according to claim 12.

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

25. A cell, characterized in that, It contains the nucleic acid according to claim 23 or the vector according to claim 24.

26. A method for preparing the antibody or its functional fragment according to any one of claims 1 to 11 or the antibody according to claim 12, characterized in that, It comprises: Culturing the cell according to claim 25.

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