Antibodies, diagnostic kits, and preparation methods for influenza A virus

Abstract

This invention discloses an antibody against influenza A virus, a detection kit, and a preparation method, relating to the field of antibody technology. The antibody against influenza A virus disclosed in this invention includes a heavy chain complementarity-determining region (CDR) and a light chain CDR. This antibody exhibits good affinity for influenza A virus antigens, and its use in detecting influenza A virus demonstrates good sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an antibody against influenza A virus, a detection kit, and a preparation method thereof. Background Technology

[0002] Influenza virus (Flu), commonly known as influenza virus, is a representative species of the Orthomyxoviridae family. It includes human influenza virus, swine influenza virus, equine influenza virus, and avian influenza virus. Human influenza virus, based on the antigenicity of its nucleoprotein, can be divided into three types: A, B, and C. It is the pathogen of influenza. Influenza viruses can infect and cause illness in various animals, including humans, birds, pigs, horses, and bats. The main viruses that infect humans are influenza A and B viruses, primarily causing upper respiratory tract infections, but also 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 antigens are prone to mutation, and it 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 numerous global pandemics, with an annual peak. The severity of influenza A virus infection is related to individual immunity. Typical symptoms include chills, persistent high fever, and headache, accompanied by sore throat, cough, nasal congestion, and generally systemic symptoms such as body aches and fatigue. Literature reports a positive detection rate of 20-40% during epidemic periods, while the positive rate is 2-20% during non-epidemic seasons. The persistent prevalence of influenza A virus causes significant disruption and stress to people's health, lives, and public health systems, making it a major subject of epidemiological research.

[0004] Currently, the main detection methods for influenza A virus on the market include fluorescent PCR, immunoassay, and virus isolation and culture identification. Fluorescent PCR uses fluorescence signals to monitor the PCR process in real time during amplification, allowing for qualitative or quantitative detection; this method is the gold standard for pathogen detection. Immunoassay detects the target protein through the specific binding of 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 has good sensitivity and specificity, its short detection window allows for early diagnosis and treatment, reducing mortality and controlling outbreaks, making it a potential gold standard for diagnosis. However, this method requires highly skilled personnel with specialized training, and diagnostic testing must be performed in a qualified laboratory using specialized equipment. Therefore, it is not suitable for rapid diagnosis in clinical or epidemiological surveillance. Virus isolation, culture, and identification are time-consuming, have high environmental requirements, pose a significant risk of infection for operators, and have poor culture results, limiting their application in clinical diagnosis and epidemiological surveillance. Immunoassay reagents target antigens or antibodies in samples. They offer fast detection speeds and good accuracy, while requiring less expertise from laboratories and operators. They are widely applicable to primary screening in hospital laboratories and disease control system laboratories. They play a crucial 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 specific antibodies against the influenza A virus. However, currently available specific antibodies against influenza A virus have certain deficiencies in both specificity and sensitivity.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an antibody against influenza A virus, a detection kit, and a preparation method. The antibody has good affinity for influenza A virus antigen, and the detection of influenza A virus using this antibody has good sensitivity and specificity.

[0008] This invention is implemented as follows:

[0009] On one hand, the present invention provides an antibody or functional fragment thereof against influenza A virus, wherein the antibody or functional fragment thereof has the following complementary determining region:

[0010] CDR-VH1: GFS-X1-TA-X2-G-X3-H, where: X1 is I or L; X2 is F or Y; X3 is L, V or I;

[0011] CDR-VH2: X1-W-X2-GGSTNYN-X3-T-X4-MS, where: X1 is LL, LI, II or IL; X2 is G or A; X3 is P or A; X4 is L or Y;

[0012] CDR-VH3: A-X1-E-X2-TGRM, where: X1 is K or R; X2 is L, V or I;

[0013] CDR-VL1: KASQD-X1-N-X2-Y-X3-S, where: X1 is I or L; X2 is Q, R or N; X3 is I, V or L;

[0014] CDR-VL2: RANR-X1-X2-D, where: X1 is I or L; X2 is I, V or L;

[0015] CDR-VL3: X1-QY-X2-EFPY, where: X1 is I, V or L; X2 is N or D.

[0016] The antibody or functional fragment thereof against influenza A virus provided by the present invention has the above-mentioned complementarity-determining region structure. The above-mentioned complementarity-determining region structure enables the antibody or functional fragment thereof to specifically bind to influenza A virus antigen, and has good affinity for influenza A virus antigen. The detection of influenza A virus using the antibody or functional fragment thereof has good specificity and sensitivity.

[0017] In an optional implementation,

[0018] In CDR-VH1, X2 is F;

[0019] In CDR-VH2, X2 is A;

[0020] In CDR-VH3, X1 is R;

[0021] In CDR-VL1, X1 is I;

[0022] In CDR-VL2, X1 is L;

[0023] In CDR-VL3, X2 is D.

[0024] The inventors of this invention have discovered that when the mutation sites in each complementarity-determining region are the aforementioned amino acid residues, the antibody exhibits better affinity for influenza A virus.

[0025] In an optional implementation, in CDR-VH1, X1 is I.

[0026] In an optional implementation, in CDR-VH1, X1 is L.

[0027] In an optional implementation, in CDR-VH1, X3 is L.

[0028] In an optional implementation, in CDR-VH1, X3 is V.

[0029] In an optional implementation, in CDR-VH1, X3 is I.

[0030] In an optional implementation, in CDR-VH2, X1 is LL.

[0031] In an optional implementation, in CDR-VH2, X1 is LI.

[0032] In an optional implementation, in CDR-VH2, X1 is II.

[0033] In an optional implementation, X1 in CDR-VH2 is IL.

[0034] In an optional implementation, in CDR-VH2, X3 is P.

[0035] In an optional implementation, in CDR-VH2, X3 is A.

[0036] In an optional implementation, in CDR-VH2, X4 is L.

[0037] In an optional implementation, in CDR-VH2, X4 is Y.

[0038] In an optional implementation, in CDR-VH3, X2 is L.

[0039] In an optional implementation, in CDR-VH3, X2 is V.

[0040] In an optional implementation, in CDR-VH3, X2 is I.

[0041] In an optional implementation, in CDR-VL1, X2 is Q.

[0042] In an optional implementation, in CDR-VL1, X2 is R.

[0043] In an optional implementation, in CDR-VL1, X2 is N.

[0044] In an optional implementation, in CDR-VL1, X3 is I.

[0045] In an optional implementation, in CDR-VL1, X3 is V.

[0046] In an optional implementation, in CDR-VL1, X3 is L.

[0047] In an optional implementation, in CDR-VL2, X2 is I.

[0048] In an optional implementation, in CDR-VL2, X2 is V.

[0049] In an optional implementation, in CDR-VL2, X2 is L.

[0050] In an optional implementation, in CDR-VL3, X1 is I.

[0051] In an optional implementation, in CDR-VL3, X1 is V.

[0052] In an optional implementation, in CDR-VL3, X1 is L.

[0053] In an optional embodiment, the complementarity-determining regions of the antibody or its functional fragment are selected from any one of the following mutation combinations 1-62:

[0054]

[0055]

[0056] In an optional embodiment, the antibody or a functional fragment thereof is reacted with the influenza A virus at K... D ≤4.4×10 - 8 Affinity binding at mol / L.

[0057] In an optional implementation, K D ≤4×10 -8 mol / L, or K D ≤3×10 -8 mol / L, or K D ≤2×10 -8 mol / L, or K D ≤1×10 -8 mol / L, or K D ≤9×10 -9 mol / L, or K D ≤8×10 -9 mol / L, or K D ≤7×10 -9 mol / L, or K D ≤6×10 -9 mol / L, or KD ≤5×10 -9 mol / L, or K D ≤4×10 -9 mol / L, or K D ≤3×10 -9 mol / L, or K D ≤2×10 -9 mol / L.

[0058] In an optional implementation, 2.08 × 10 -9 mol / L≤K D ≤8.47×10 -9 mol / L.

[0059] K D The detection is performed in accordance with the method described in the embodiments of the present invention.

[0060] In an optional implementation,

[0061] In CDR-VH1, X2 is Y;

[0062] In CDR-VH2, X2 is G;

[0063] In CDR-VH3, X1 is K;

[0064] In CDR-VL1, X1 is L;

[0065] In CDR-VL2, X1 is I;

[0066] In CDR-VL3, X2 is N.

[0067] In an optional embodiment, the complementarity-determining regions of the antibody or its functional fragment are selected from any one of the following mutation combinations 63-70:

[0068]

[0069] In an optional embodiment, the antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:1-4, and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:5-8.

[0070] Typically, the variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0071] It should be noted that, in other embodiments, the amino acid sequences of each backbone region of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the corresponding backbone regions (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8) mentioned above.

[0072] In an optional implementation, the antibody further includes a constant region.

[0073] In an optional implementation, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0074] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0075] In an optional implementation, the constant region is derived from mice.

[0076] In an optional embodiment, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.

[0077] In an optional embodiment, the functional fragment is selected from any one of the antibody’s VHH, F(ab')2, Fab', Fab, Fv and scFv.

[0078] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0079] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0080] On the other hand, the present invention provides a reagent or kit for detecting influenza A virus, comprising an antibody or a functional fragment thereof as described in any of the preceding claims.

[0081] In an optional embodiment, the antibody or its functional fragment in the above reagent or kit is labeled with a detectable marker.

[0082] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0083] In optional embodiments, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0084] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0085] In optional 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), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0086] In optional embodiments, the enzymes that catalyze the color development of the substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0087] In optional embodiments, the radioactive isotope includes, but is not limited to, [other types of radioactive isotopes]. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I,188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0088] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0089] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0090] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0091] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0092] On the other hand, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its functional fragment.

[0093] On the other hand, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0094] On the other hand, the present invention provides recombinant cells containing the above-described carrier.

[0095] On the other hand, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing recombinant cells as described above, and separating and purifying the antibody or a functional fragment thereof from the culture product.

[0096] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in this invention, those skilled in the art will readily conceive of preparing the antibody or its functional fragment using genetic engineering or other techniques (chemical synthesis, hybridoma cells), such as isolating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention. Attached Figure Description

[0097] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0098] Figure 1 The results of the reducing SDS-PAGE of the anti-influenza A virus antibody in Example 1 are shown. Detailed Implementation

[0099] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0101] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0102] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0103] Example 1

[0104] In this embodiment, restriction endonucleases 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 Pharmaceuticals. Primer synthesis and gene sequencing were performed by Invitrogen.

[0105] 1. Construction of recombinant plasmids

[0106] (1) Antibody gene preparation

[0107] mRNA was extracted from a hybridoma cell line (4G2) that secretes antibodies against influenza A virus antigens. The DNA product was obtained by RT-PCR. The product was then inserted into the pMD-18T vector after being subjected to an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After the cells grew, four clones of the Heavy Chain and Light Chain genes were sent to a gene sequencing company for sequencing.

[0108] (2) Sequence analysis of antibody variable region gene

[0109] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 321 bp, belonging to the VkII gene family, and it had 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 348 bp, belonging to the VH1 gene family, and it had a 57 bp leader peptide sequence in front of it.

[0110] (3) Construction of recombinant antibody expression plasmid

[0111] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.73kb Light Chain gene fragment and the 1.43kb Heavy Chain gene fragment were amplified by PCR.

[0112] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0113] 2. Screening of stable cell lines

[0114] (1) Transient transfection of CHO cells with recombinant antibody expression plasmid to determine plasmid activity.

[0115] The plasmid was diluted to 400 ng / ml with ultrapure water and used to regulate CHO cells to 1.43 × 10⁻⁶. 7 Cells / ml were collected in centrifuge tubes, 100 μl of plasmid was mixed with 700 μl of cells, transferred to an electroporation cuvette, and electroporated. Samples were taken and counted on days 3, 5, and 7, and samples were collected and analyzed on day 7.

[0116] Coating buffer (mainly NaHCO3) was used to dilute goat anti-mouse IgG (1ug / ml) and coated onto microplates at 100μl per well, incubating overnight at 4°C. The next day, the cells were washed twice with washing buffer (mainly Na2HPO4 + NaCl) and blotted dry. Blocking buffer (20% BSA + 80% PBS) was added at 120μl per well, incubated at 37°C for 1 hour, and blotted dry. Diluted cell supernatant was added at 100μl per well, incubated at 37°C for 60 minutes. The liquid in the plate was discarded, the plate was blotted dry, and 20% mouse negative blood was added for blocking at 120μl per well, incubated at 37°C for 1 hour. h; Discard the liquid in the plate, blot dry, add diluted influenza A antigen, 100 μl per well, incubate at 37℃ for 40 min; wash 5 times with washing buffer, blot dry; add HRP-labeled influenza A monoclonal antibody, 100 μl per well, incubate at 37℃ for 30 min; add chromogenic solution A (50 μl / well), add chromogenic solution B (50 μl / well), incubate for 10 min; add stop solution, 50 μl / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader. The results showed that the OD of the reaction was still greater than 1.0 after 1000-fold dilution of cell supernatant, and the OD of the reaction in wells without cell supernatant was less than 0.1, indicating that the antibody produced after transient plasmid transfection is active against influenza A antigen.

[0117] (2) Linearization of recombinant antibody expression plasmid

[0118] Prepare the following reagents: Buffer 50 μl, DNA 100 μg / tube, PuvI enzyme 10 μl, and sterile water to a final volume of 500 μl. Incubate overnight at 37°C. Extract with an equal volume of phenol / chloroform / isoamyl alcohol (lower layer) in a 25:24:1 ratio, followed by extraction with chloroform (aqueous phase). Precipitate on ice with 0.1 volume (aqueous phase) of 3M sodium acetate and 2 volume of ethanol. Rinse the precipitate with 70% ethanol to remove organic solvents. After the ethanol has completely evaporated, reconstitute with an appropriate amount of sterile water. Finally, determine the concentration.

[0119] (3) Stable transfection with recombinant antibody expression plasmid, followed by pressure screening to select stable cell lines.

[0120] The plasmid was diluted to 400 ng / ml with ultrapure water and used to regulate CHO cells to 1.43 × 10⁻⁶. 7 Cells / ml were collected in centrifuge tubes, 100 μl of plasmid was mixed with 700 μl of cells, transferred to an electroporation cuvette, electroporated, and counted the cells the next day; cells were then cultured in 25 μmol / L MSX 96-well pressurized culture for about 25 days.

[0121] Observe the labeled clone wells containing cells under a microscope and record the degree of confluence; collect the culture supernatant and send it for testing; select cell lines with high antibody concentration and relative concentration to transfect 24-well cells, and transfect 6-well cells after about 3 days; after 3 days, preserve the culture in batch culture and adjust the cell density to 0.5 × 10⁻⁶ cells / well. 6 Cells / ml, 2.2ml batch culture, cell density 0.3×10⁶ 6 Cells / ml, 2ml for seeding; 6-well batch culture supernatant sent for testing after 7 days, cell lines with smaller antibody concentration and cell diameter were selected for TPP seeding and passage.

[0122] 3. Recombinant Antibody Production

[0123] (1) Cell expansion

[0124] After cell resuscitation, cells were initially cultured in 125ml shake flasks with an inoculation volume of 30ml in 100% Dynamis medium. The flasks were placed in a shaker at 120 rpm, 37°C, and 8% CO2. After 72 hours of culture, cells were propagated at a density of 500,000 cells / ml. The propagation volume was calculated based on production requirements, and the medium was 100% Dynamis. Propagation was then repeated every 72 hours. Once the cell count met production needs, the inoculation density was strictly controlled at approximately 500,000 cells / ml for production.

[0125] (2) Shake flask production and purification

[0126] Shake flask parameters: rotation speed 120 rpm, temperature 37℃, carbon dioxide 8%. Fed feeding: Starting after 72 hours of culture in the shake flask, feed was added daily. HyClone™ Cell Boost™ Feed 7a was used to feed 3% of the initial culture volume daily, and Feed 7b was used to feed 1 / 1000 of the initial culture volume daily, continuing until day 12 (feeding on day 12). Glucose was added at 3 g / L on day 6. Samples were collected on day 13. Affinity purification was performed using a protein A affinity chromatography column. 4 μg of purified antibody was subjected to reducing SDS-PAGE, with 4 μg of exogenous control antibody used as a control. Electrophoresis results are shown below. Figure 1 As shown, two bands are displayed after reducing SDS-PAGE: one with Mr 50 KD (heavy chain, SEQ ID NO.14) and the other with Mr 28 KD (light chain, SEQ ID NO.13).

[0127] Example 2

[0128] Antibody performance testing

[0129] (1) Example 1 Activity detection of antibodies and their mutants

[0130] The antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region is shown in SEQ ID NO:12. The amino acid sequences of the complementarity-determining regions on the heavy chain variable region are as follows:

[0131] CDR-VH1: GFSI(X1)-TAY(X2)-GL(X3)-H;

[0132] CDR-VH2: LI(X1)-WG(X2)-GGSTNYNA(X3)-TL(X4)-MS;

[0133] CDR-VH3: AK(X1)-EV(X2)-TGRM;

[0134] Its light chain variable region is shown in SEQ ID NO:11, wherein the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0135] CDR1-VL: KASQDL(X1)-NQ(X2)-YI(X3)-S;

[0136] CDR-VL2:RANRI(X1)-L(X2)-D;

[0137] CDR-VL3: I(X1)-QYN(X2)-EFPY.

[0138] Based on the anti-influenza A virus antibody (WT) of Example 1, mutations were made at sites related to antibody activity in the complementarity-determining region, where X1, X2, X3, and X4 are mutation sites. See Table 1 below.

[0139] Table 1 Mutation sites related to antibody activity

[0140]

[0141] Assay for antibody binding activity in Table 1:

[0142] Dilute goat anti-mouse IgG (1 μg / ml) with coating buffer (main component NaHCO3) and coat microplates with 100 μl per well, incubating overnight at 4°C. The next day, wash twice with washing buffer (main components Na2HPO4 + NaCl) and blot dry. Add blocking buffer (20% BSA + 80% PBS) at 120 μl per well, incubate at 37°C for 1 h, and blot dry. Add diluted purified antibody (Table 1) at 100 μl / well, incubate at 37°C for 60 min. Discard the liquid in the plate, blot dry, add 20% mouse negative blood for blocking at 120 μl per well, incubate at 37°C for 1 h. Discard the liquid in the plate, blot dry, add diluted influenza A antigen at 100 μl per well. Incubate at 37℃ for 40 min; wash 5 times with washing buffer and pat dry; add 100 μl of HRP-labeled influenza A monoclonal antibody (paired with purified antibody, obtained from Phytobio Biotechnology Co., Ltd.) per well, incubate at 37℃ for 30 min; add chromogenic solution A (50 μl / well, containing 2.1 g / L citric acid, 12.25 g / L citric acid, 0.07 g / L acetanilide, and 0.5 g / L urea peroxide), add chromogenic solution B (50 μl / well, containing 1.05 g / L citric acid, 0.186 g / L EDTA·2Na, 0.45 g / L TMB, and 0.2 ml / L concentrated HCl), incubate for 10 min; add stop solution (50 μl / well, containing 0.75 g / L EDTA·2Na and 10.2 ml / L concentrated H2SO4); read the OD value at 450 nm (630 nm for reference) on the microplate reader. The results are shown in Table 2 below.

[0143] Table 2 Activity data of WT antibodies and their mutants

[0144] Antibody concentration (ng / ml) 200 25 12.5 6.250 3.125 0 WT 2.089 1.339 0.972 0.466 0.121 0.073 Mutant 1 2.256 1.655 1.113 0.673 0.379 0.072 Mutant 2 2.129 1.645 1.085 0.64 0.339 0.082 Mutant 3 2.126 1.631 1.107 0.691 0.398 0.071 Mutant 4 2.114 1.62 1.148 0.688 0.301 0.077 Mutant 5 0.564 0.326 0.072 - - - Mutant 6 0.693 0.395 0.06 - - - Mutant 7 0.633 0.316 0.075 - - -

[0145] As can be seen from the results in Table 2, WT and mutant 1-mutant 4 antibodies have better binding activity. Among them, mutant 1 has the best binding activity, while mutant 5-mutant 7 antibodies have almost no binding activity.

[0146] (2) Affinity detection of antibodies and their mutants (a) Based on mutation 1, other sites were mutated. The sequences of each mutation are shown in Table 3 below.

[0147] Table 3 Mutation sites related to antibody affinity

[0148]

[0149]

[0150] Affinity Analysis

[0151] Using the AMC sensor, the purified antibody was diluted to 10 ug / ml with PBST, and the influenza A antigen was serially diluted with PBST to 20 ug / ml, 6.66 ug / ml, 2.22 ug / ml, 0.74 ug / ml, 0.24 ug / ml, 0.082 ug / ml, 0.027 ug / ml, and 0.0091 ug / ml.

[0152] Operating procedure: Equilibrate in Buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in Buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in Buffer 2 for 1200s, regenerate the sensor with 10mM pH 1.69 GLY solution and Buffer 3, and output data. K D This represents the equilibrium dissociation constant, i.e., the affinity. The results are shown in Table 4 below.

[0153] Table 4 Affinity Test Data

[0154]

[0155]

[0156]

[0157] As can be seen from the data in Table 3, mutation 1 and the antibodies obtained by mutations based on it (mutation 1-1 to mutation 1-61) all have good affinity; this indicates that the antibodies obtained by mutation 1 according to the mutation methods shown in Table 2 all have good affinity.

[0158] (b) Based on WT, other sites were mutated and the affinity of each mutant was tested. The sequences of each mutation are shown in Table 5 below, and the corresponding affinity data are shown in Table 6.

[0159] Table 5 Mutations performed using WT as the backbone

[0160]

[0161] Table 6 Affinity test results of WT antibodies and their mutants

[0162] K D (M) WT 4.40E-08 WT 1 2.93E-08 WT 2 2.77E-08 WT 3 3.17E-08 WT 4 3.65E-08 WT 5 2.87E-08 WT 6 3.73E-08 WT 7 3.25E-08

[0163] The data in Table 6 show that WT and its mutants (WT1-WT7) also have good affinity, indicating that antibodies obtained by mutation according to the mutation methods shown in Table 5 based on WT all have good affinity.

[0164] (3) Stability assessment of bare anti-electrode

[0165] The above-mentioned antibodies were placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of their state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three testing conditions after 21 days, and the activity did not decrease with increasing testing temperature, indicating that the above-mentioned antibodies were stable. Table 7 below shows the OD results of enzyme immunoassay for mutant 1 antibody after 21 days of testing.

[0166] Table 7

[0167] Sample concentration (ng / ml) 200 12.5 0 4°C, 21 day sample 2.199 1.156 0.053 -80°C, 21 day sample 2.211 1.124 0.051 37°C, 21 day sample 2.208 1.197 0.051

[0168] Example 3

[0169] Application of antibodies in colloidal gold detection

[0170] 1. Preparation of colloidal gold test strips

[0171] (1) Preparation of nitrocellulose membrane

[0172] Preparation of coating buffer: Use 0.01M pH 7.2 PBS buffer containing 6% methanol as the coating buffer. Filter through a 0.22μm membrane and store at 4℃. Shelf life is one week. The formula for 1000ml of 0.01M pH 7.2 PBS buffer containing 6% methanol is: NaCl 8g, KCl 0.2g, Na2HPO4·12H2O 2.9g, KH2PO4 0.2g, methanol 60ml, and double-distilled deionized water to a final volume of 1000ml.

[0173] Preparation of nitrocellulose membrane: Dilute the purified antibody to 1-5 mg / ml with coating buffer, adjust the machine, and draw the T line (detection line), approximately 5 mm from the gold-labeled pad. Dilute the goat anti-mouse IgG antibody to 1-5 mg / ml with coating buffer, adjust the machine, and draw the C line (control line), approximately 3 mm from the absorbent pad. The two lines should be 5-8 mm apart and uniform. Dry at 37°C and package for later use.

[0174] (2) Preparation of colloidal gold and gold-labeled monoclonal antibodies

[0175] (a) Solution preparation

[0176] ① Preparation of chloroauric acid: Dissolve chloroauric acid in double-distilled deionized water to prepare a 1% solution. Store at 4℃ for four months. Formula for 1000ml 1% chloroauric acid solution: 10g chloroauric acid: double-distilled deionized water to a final volume of 1000ml.

[0177] ② Preparation of trisodium citrate: Dissolve sodium citrate in double-distilled deionized water to prepare a 1% solution, filter through a 0.22μm membrane, store at 4 degrees Celsius for later use, and dilute to 1000ml before expiration.

[0178] ③ Preparation of 0.1M potassium carbonate: Prepare with double-distilled deionized water, filter through a 0.22μm membrane, store at 4℃ for use, shelf life is four months. Formula for 1000ml 0.1M potassium carbonate solution: 13.8g potassium carbonate; double-distilled deionized water to a final volume of 1000ml.

[0179] ④ Preparation of 2% PEG-20000: Prepare with double-distilled deionized water, filter through a 0.22μm membrane, store at 4℃ for later use, shelf life is four months. Formula for 1000ml 2% PEG-20000 solution: 20g PEG-20000; double-distilled deionized water to a final volume of 1000ml.

[0180] ⑤ Preparation of Label Washing Preservative Solution: 2% bovine serum albumin (BSA), 0.05% sodium azide (NaN3), 0.01M pH 7.2 PBS solution, filtered through a 0.22μm membrane, stored at 4℃, shelf life four months. 1000ml Label Washing Preservative Solution Formula: 20g BSA, 0.5g NaN3, 0.01M pH 7.2 PBS solution, brought to a final volume of 1000ml.

[0181] (b) Preparation of colloidal gold.

[0182] Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water, bring to a boil on an electric stove, and add 2 ml of 1% trisodium citrate per 100 ml of 0.01% chloroauric acid. Continue boiling until the liquid turns bright red, then stop heating. After cooling to room temperature, replenish the lost water. The prepared colloidal gold should be pure, clear, and free of precipitates and floating matter, and has a shelf life of one week.

[0183] (c) Preparation of colloidal gold-labeled antibodies.

[0184] Adjust the pH of the colloidal gold to 8.2 using 0.1M potassium carbonate. Add labeled influenza A antibody (available from Feipeng Biotechnology) at concentrations of 8–10 μg antibody / ml of colloidal gold. Mix with a magnetic stirrer for 30 min. Add BSA to a final concentration of 1% while stirring and let stand for 1 hour. Centrifuge at 13000 rpm and 4°C for 30 min. Discard the supernatant. Wash the precipitate twice with labeled wash and preservation buffer. Resuspend the precipitate in one-tenth of the initial colloidal gold volume of labeled wash and preservation buffer. Store at 4°C for one week.

[0185] (3) Preparation of gold-labeled pads

[0186] (a) Preparation of sealing solution.

[0187] Contains 2% BSA, 0.1% Triton X-100, 0.05% NaN3, and 0.01M pH 7.2 PBS solution. Filter through a 0.22μm membrane and store at 4℃. Shelf life is four months. Blocking solution formulation: 20g BSA, 0.5g NaN3, 1ml Triton X-100, and 0.01M pH 7.2 PBS solution, adjusted to 1000ml.

[0188] (b) Preparation of gold-labeled pads

[0189] The gold-labeled pads were immersed in blocking solution for 30 minutes and then dried at 37°C. The prepared gold-labeled antibodies were then evenly spread on the gold-labeled pads, with 20 square centimeters per milliliter of solution. The pads were then freeze-dried, encapsulated, and stored at 4°C for later use.

[0190] (4) Preparation of the sample pad for the test strip

[0191] (a) Preparation of sealing solution.

[0192] Contains 2% BSA, 0.1% TrtionX-100, 0.05% NaN3, and 0.01M pH7.2 PBS solution. Filter through a 0.22μm membrane and store at 4°C. Shelf life is four months. Blocking solution formulation: 20g BSA, 0.5g NaN3, 1ml TrtionX-100, and 0.01M pH7.2 PBS solution, adjusted to 1000ml.

[0193] (b) Preparation of sample pad.

[0194] After immersing the sample pad in the sealing solution for 30 minutes, dry it at 37°C, seal it, and store it at 4°C for later use.

[0195] (5) Assembly of test strips

[0196] The absorbent pad (purchased from Millipore), nitrocellulose membrane, gold label pad, and sample pad are placed on a non-absorbent support sheet and cut into 3mm wide strips. Ten strips are packaged together, desiccant is added, and they are vacuum sealed to obtain the colloidal gold test strip for detecting influenza A virus.

[0197] 2. Application of Antibodies in Colloidal Gold Detection

[0198] The assembled test strips were used to detect the presence of influenza A virus antigen in the test material, thereby determining the effectiveness of the antibodies obtained in the aforementioned examples for detecting influenza A virus antigen. A double-antibody sandwich method was used to detect the presence of influenza A virus antigen in the test material. During detection, the influenza A virus antigen first binds to colloidal gold-labeled influenza A antibody to form an influenza A antigen-colloidal gold-labeled influenza A antibody complex. Due to capillary action, the influenza A antigen-colloidal gold-labeled influenza A antibody complex migrates forward along the nitrocellulose membrane. Upon reaching the test line, the influenza A antigen-colloidal gold-labeled influenza A antibody complex binds to the influenza A antibody obtained in the examples, forming an influenza A antibody-influenza A antigen-colloidal gold-labeled influenza A antibody complex, which is then enriched on the test line, forming a red precipitate line. The influenza A antigen-colloidal gold-labeled influenza A antibody complex that does not bind to the influenza A antibody on the test line passes through the test line and is captured by goat anti-mouse IgG antibody, enriching on the control line, forming a red precipitate line. A positive result is indicated when both the test line and the control line show red precipitate lines. If the sample does not contain influenza A virus antigen, when the colloidal gold-labeled influenza A antibody that has not bound to influenza A virus antigen reaches the test line, it will not form a complex of influenza A antibody-influenza A antigen-colloidal gold labeling-influenza A antibody. The colloidal gold-labeled influenza A antibody complex that has not bound to influenza A antigen passes through the test line and only accumulates on the quality control line to form a red precipitation line. At this time, it is judged as a negative result.

[0199] The results are shown in Table 8 below.

[0200] Table 8

[0201]

[0202]

[0203] Note: The color development of gold-labeled products is represented by a "C" followed by a number. The smaller the number after the "C," the stronger the color development and the higher the activity; the larger the number after the "C," the weaker the color development and the lower the activity. A "+" after the number indicates slightly stronger color development (0.5-1C) than no number, and a "-" after the number indicates slightly weaker color development (0.5-1C) than no number. "B" indicates no activity.

[0204] As shown in Table 8, the antibodies provided in the embodiments of the present invention exhibit excellent detection activity when used on the gold standard platform for double antibody sandwich assay.

[0205] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Dongguan Pengzhi Biotechnology Co., Ltd. <120> Antibodies, diagnostic kits, and preparation methods for influenza A virus <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> PRT <213> Artificial sequence <400> 1 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Phe Leu Trp 1 5 10 15 Arg Val Thr Leu Thr Cys 20 <210> 2 <211> 15 <212> PRT <213> Artificial sequence <400> 2 Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Leu Ser 1 5 10 15 <210> 3 <211> 32 <212> PRT <213> Artificial sequence <400> 3 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser 1 5 10 15 Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys 20 25 30 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <400> 4 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <210> 5 <211> 25 <212> PRT <213> Artificial sequence <400> 5 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 6 <211> 14 <212> PRT <213> Artificial sequence <400> 6 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly 1 5 10 <210> 7 <211> 30 <212> PRT <213> Artificial sequence <400> 7 Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Glu 1 5 10 15 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys 20 25 30 <210> 8 <211> 13 <212> PRT <213> Artificial sequence <400> 8 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 9 <211> 106 <212> PRT <213> Artificial Sequence <400> 9 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 1 5 10 15 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 20 25 30 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 35 40 45 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 65 70 75 80 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 85 90 95 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 10 <211> 324 <212> PRT <213> Artificial Sequence <400> 10 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115 120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 225 230 235 240 Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val 245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro Gly Lys <210> 11 <211> 107 <212> PRT <213> artificial sequence <400> 11 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Phe Leu Trp 1 5 10 15 Arg Val Thr Leu Thr Cys Lys Ala Ser Gln Asp Leu Asn Gln Tyr Ile 20 25 30 Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Leu Ser 35 40 45 Arg Ala Asn Arg Ile Leu Asp Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu 65 70 75 80 Asp Met Gly Ile Tyr Tyr Cys Ile Gln Tyr Asn Glu Phe Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 12 <211> 116 <212> PRT <213> artificial sequence <400> 12 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Thr Ala Tyr 20 25 30 Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ala Thr Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Glu Val Thr Gly Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 13 <211> 213 <212> PRT <213> Artificial Sequence <400> 13 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Phe Leu Trp 1 5 10 15 Arg Val Thr Leu Thr Cys Lys Ala Ser Gln Asp Leu Asn Gln Tyr Ile 20 25 30 Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Leu Ser 35 40 45 Arg Ala Asn Arg Ile Leu Asp Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu 65 70 75 80 Asp Met Gly Ile Tyr Tyr Cys Ile Gln Tyr Asn Glu Phe Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asparagine Arginine Asparagine Glutamic acid Cysteine 210 <210> 14 <211> 440 <212> PRT <213> Artificial Sequence <400> 14 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Thr Ala Tyr 20 25 30 Gly Leu Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ala Thr Leu Met 35 40 45 Gly Leu Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ala Thr Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Glu Val Thr Gly Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala 115 120 125 Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu 130 135 140 Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly 145 150 155 160 Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp 165 170 175 Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro 180 185 190 Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 195 200 205 Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile 210 215 220 Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val 245 250 255 Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val 260 265 270 Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala 305 310 315 320 Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro 325 330 335 Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala 340 345 350 Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu 355 360 365 Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr 370 375 380 Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr 385 390 395 400 Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe 405 410 415 Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys 420 425 430 Ser Leu Ser His Ser Pro Gly Lys 435 440

Claims

1. An antibody or a functional fragment thereof against influenza A virus, characterized in that, The antibody or its functional fragment includes the following complementarity-determining regions: CDR-VH1: GFS-X1-TA-X2-G-X3-H, where: X2 is F; CDR-VH2: X1-W-X2-GGSTNYN-X3-T-X4-MS, where: X2 is A; CDR-VH3: A-X1-E-X2-TGRM, where: X1 is R; CDR-VL1: KASQD-X1-N-X2-Y-X3-S, where: X1 is I; CDR-VL2: RANR-X1-X2-D, where: X1 is L; CDR-VL3: X1-QY-X2-EFPY, where: X2 is D; Each complementarity-determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 1-62: 。 2. The antibody against influenza A virus or its functional fragment according to claim 1, characterized in that, The antibody or its functional fragment reacts with the influenza A virus antigen at K D ≤8.47×10 -9 Affinity binding at mol / L.

3. The antibody against influenza A virus or its functional fragment according to claim 1, characterized in that, The antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:1-4, and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:5-8.

4. The antibody against influenza A virus or its functional fragment according to claim 1, characterized in that, The antibody also contains a constant region.

5. The antibody against influenza A virus or its functional fragment according to claim 4, characterized in that, The constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

6. The antibody against influenza A virus or its functional fragment according to claim 4, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

7. The antibody against influenza A virus according to claim 6, characterized in that, The species source of the constant region is dairy cows.

8. The antibody against influenza A virus according to claim 6, characterized in that, The species source of the constant region is turkey or fighting cock.

9. The antibody against influenza A virus according to claim 6, or a functional fragment thereof, characterized in that, The species source of the constant region is mice.

10. The antibody against influenza A virus according to claim 9, characterized in that, The light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:

10.

11. The antibody against influenza A virus according to claim 1, or a functional fragment thereof, characterized in that, The functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

12. A reagent or kit for detecting influenza A virus, characterized in that, It includes the antibody or its functional fragment as described in any one of claims 1-11.

13. The reagent or kit according to claim 12, characterized in that, The antibody or its functional fragment is labeled with a detectable marker.

14. The reagent or kit according to claim 13, characterized in that, The detectable markers are selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

15. The reagent or kit according to claim 14, characterized in that, The fluorescent dyes are selected from fluorescein dyes, rhodamine dyes, Cy series dyes, Alexa series dyes, and protein dyes.

16. The reagent or kit according to claim 14, characterized in that, The enzyme that catalyzes the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

17. The reagent or kit according to claim 14, characterized in that, The radioactive isotope is selected from 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

18. The reagent or kit according to claim 14, characterized in that, The chemiluminescent reagent is selected from luminol, luciferin, fluorescein from crustaceans, ruthenium bipyridine, acridine ester, dioxane, rofenol, and peroxate.

19. The reagent or kit according to claim 14, characterized in that, The nanoparticle marker is selected from at least one of nanoparticles and colloids, and the nanoparticles are selected from at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

20. The reagent or kit according to claim 19, characterized in that, The colloid is selected from colloidal selenium, colloidal metal, disperse dye, dye-labeled microspheres, and dye-labeled latex.

21. The reagent or kit according to claim 20, characterized in that, The colloidal metal is selected from colloidal gold and colloidal silver.

22. A carrier, characterized in that, It comprises a nucleic acid molecule encoding an antibody or a functional fragment thereof as described in any one of claims 1-11.

23. A recombinant cell, characterized in that, It contains the carrier as described in claim 22.

24. A method for preparing an antibody or a functional fragment thereof as described in any one of claims 1-11, characterized in that, It includes: The recombinant cells of claim 23 are cultured, and the antibody or its functional fragment is isolated and purified from the culture product.

Images