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

Abstract

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

Description

Technical Field

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

[0002] Influenza virus (Flu), also known as influenza virus for short, is a representative species of the Orthomyxoviridae family, including human influenza virus, swine influenza virus, equine influenza virus, avian influenza virus, etc. Human influenza virus can be divided into three types: A, B, and C according to the antigenicity of its nucleoprotein, and is the pathogen of influenza. Influenza virus can cause infection and disease in many animals such as humans, poultry, pigs, horses, and bats. Among them, influenza A virus and influenza B virus can infect humans, mainly causing 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 B is a flu caused by influenza B virus, characterized by an acute onset, chills, fever, and a temperature that reaches a peak of 39-40°C or even higher within a few hours to 24 hours. It is accompanied by headache, body aches, fatigue, and loss of appetite. Respiratory symptoms are mild, with dry throat, sore throat, dry cough, and possible diarrhea. Facial flushing, conjunctival hyperemia, pharyngeal hyperemia, and follicles on the soft palate. It can be treated with drugs such as amantadine as an M2 ion blocker, or with traditional Chinese medicine.

[0004] Influenza B virus can produce many subtypes, because the hemagglutinin (HA) and neuraminidase (NA) antigens of influenza virus are prone to change, and their constituent amino acid sites can mutate. After each pandemic of influenza virus, new influenza viruses are produced through the above-mentioned amino acid site mutations. The human immune system generally lacks resistance to the mutated subtypes, which can easily cause local epidemics and cause large-scale population infections under special circumstances. Monitoring data from the Centers for Disease Control and Prevention (CDC) in the United States and China from December 2017 to January 2018 showed that the incidence of influenza B virus was on the rise. Therefore, early and rapid screening of influenza viruses is particularly important.

[0005] The clinical symptoms of influenza virus infection are atypical, and clinical diagnosis mainly relies on laboratory testing. However, there are more than 200 respiratory viruses that can infect humans at the same time, so the sensitivity and specificity of the detection reagents are crucial for clinical diagnosis. Selecting influenza virus screening technology with short detection time and high detection rate is a technical path and prerequisite guarantee to ensure rapid clinical diagnosis and symptomatic treatment.

[0006] There are many laboratory detection methods for influenza viruses. Referring to the changes in the Ministry of Health's "Influenza Diagnostic Criteria", it can be seen that early detection relied on chicken embryo inoculation, which is rarely used in clinical practice due to its complex operation and high technical requirements. Modern detection technologies include antigen detection and nucleic acid PCR detection. The new technologies are rapid, sensitive and specific, providing great help for clinical diagnosis.

[0007] Fluorescence PCR amplification technology, the advantage of this method is that the sensitivity and specificity are both high, but the PCR method has high requirements for samples, test environment, and operators, and the amplification methodology is suitable for the detection of batch specimens. The report time is long and it cannot meet the requirements of clinical rapid diagnosis. The immune colloidal gold technology for detecting viral antigens can be used as a preferred method for rapid diagnosis of influenza B. It has a short detection time and can effectively assist clinical diagnosis, which greatly helps clinical symptomatic medication as soon as possible. It is necessary to strengthen the monitoring of influenza B virus and provide assistance for the rapid and accurate screening of influenza B virus infection. The focus is on optimizing the quality of rapid detection reagents, shortening the sample lysis time, reducing the sample detection concentration limit, and improving the sensitivity and specificity of reagents. Therefore, there is a strong demand in this field for antibodies that effectively and specifically bind to influenza B virus and detect it.

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

[0009] The present application provides an anti-influenza B virus antibody with enhanced activity to improve the detection of influenza B virus and provide an important source of raw materials for the detection of influenza B virus.

[0010] In order to achieve the above object, according to one aspect of the present invention, an anti-influenza B virus antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises:

[0011] a) an amino acid sequence of HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2 or 9, HCDR3 as shown in SEQ ID NO: 3, and an amino acid sequence of LCDR1 as shown in SEQ ID NO: 4 or 10, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; or

[0012] b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 12, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 13 to 20; or

[0013] c) a heavy chain variable region and a light chain variable region whose amino acid sequences are more than 80% identical to the sequences shown in b), and include HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequences shown in a); or

[0014] d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 21 to 22, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 23 to 30.

[0015] In order to achieve the above object, according to a second aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0016] In order to achieve the above object, according to the third aspect of the present invention, a reagent or kit for detecting influenza B virus is provided, wherein the reagent or kit comprises the above antibody or its functional fragment or the above antibody conjugate.

[0017] To achieve the above object, according to a fourth aspect of the present invention, a method for detecting influenza B virus is provided, comprising: contacting the above antibody or its functional fragment, conjugate or reagent or kit with influenza B virus in a sample to be detected to form an immune complex.

[0018] In order 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

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The results of reducing SDS-PAGE of Anti-FluB 5C11mut1 to mut4 are shown. DETAILED DESCRIPTION

[0021] The present invention provides an anti-influenza B virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0022] a) HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2 or 9, HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3, and LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4 or 10, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6. The above antibody has improved activity.

[0023] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0024] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0025] 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: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0026] In another aspect, the present invention provides an anti-influenza B virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0027] b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 12, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 13 to 20. The above antibody has improved activity.

[0028] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0029] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0030] In another aspect, the present invention provides an anti-influenza B virus antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0031] c) a heavy chain variable region and a light chain variable region whose amino acid sequence has more than 80% identity with the sequence shown in b), and includes HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in a). The above antibody has improved activity.

[0032] In an optional embodiment, the antibody or a functional fragment thereof comprises a heavy chain framework region in a heavy chain variable region shown in any one of SEQ ID NOs: 11 to 12, and a light chain framework region in a light chain variable region shown in any one of SEQ ID NOs: 13 to 20.

[0033] In an optional embodiment, the framework region amino acid sequence of the antibody or its functional fragment may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned framework region.

[0034] In an optional embodiment, any of the above antibodies or functional fragments thereof further comprises a constant region.

[0035] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0036] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0037] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0038] In an alternative embodiment, the species origin of the constant region is mouse.

[0039] In an optional 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.

[0040] 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-mentioned constant region (SEQ ID NO: 7 or 8).

[0041] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0042] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents 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 split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0043] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0044] In another aspect, the present invention provides an anti-influenza B virus antibody, comprising:

[0045] d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 21 to 22, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 23 to 30. The above antibody has improved activity.

[0046] In another aspect, the present invention provides an antibody conjugate, comprising the above-mentioned antibody, wherein the antibody is directly or indirectly covalently coupled to the object to be coupled, or the antibody is coupled to the object to be coupled by non-covalent adsorption.

[0047] In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to biotin or a biotin derivative.

[0048] In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to a label.

[0049] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0050] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0051] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0052] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0053] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0054] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0055] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0056] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0057] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0058] In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0059] In an optional embodiment, the colloidal metal is colloidal gold.

[0060] In an alternative embodiment, the antibody in the above antibody conjugate is coupled to a solid phase.

[0061] In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.

[0062] In an optional embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0063] In an alternative embodiment, the solid phase is a nitrocellulose membrane.

[0064] In another aspect, the present invention provides a reagent or a kit for detecting influenza B virus, wherein the reagent or the kit comprises the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.

[0065] In another aspect, the present invention provides use of the above-mentioned antibody or its functional fragment, antibody conjugate, or the above-mentioned reagent or kit in the detection of influenza B virus.

[0066] In another aspect, the present invention provides a method for detecting influenza B virus, comprising: contacting the above-mentioned antibody or its functional fragment, antibody conjugate or the above-mentioned reagent or kit with influenza B virus in a sample to be detected to form an immune complex.

[0067] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0068] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to influenza B virus.

[0069] In another aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0070] In another aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0071] In another aspect, the present invention provides a cell containing the above-mentioned vector.

[0072] In another aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cell as described above.

[0073] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0074] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0076] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0077] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0078] Example 1 Preparation of Anti-FluB 5C11 Monoclonal Antibody

[0079] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen.

[0080] 1 Construction of recombinant plasmid

[0081] (1) Antibody gene preparation

[0082] mRNA was extracted from the hybridoma cell line secreting monoclonal antibodies against influenza B virus, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0083] (2) Sequence analysis of the variable region gene of Anti-FluB 5C11 antibody

[0084] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. In the gene fragment amplified by the Light Chain, the VL gene sequence was 339 bp, and there was a 57 bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 354 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

[0085] (3) Construction of recombinant antibody expression plasmid

[0086] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced 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; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.73kb Light Chain gene fragment and the 1.80kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0087] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0088] 2. Sample preparation of recombinant antibodies

[0089] Resuscitate HEK293 cells in advance, subculture to 200ml system, make the cell density reach 3-5×106cells / ml, cell viability>95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 2.9×106cells / ml as cell diluent. Prepare plasmid DNA and transfection reagent diluent with culture medium respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15min; slowly add the mixture to cell diluent within 1min, mix well and count, record and observe the viability of cells after transfection, and place them in a 35℃ constant temperature incubator for culture, speed 120rmp, CO2 content 8%, and centrifuge after 13 days. The supernatant of centrifugation was affinity purified using proteinA affinity chromatography column. Take 6μg purified antibody for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands were shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0090] Example 2 Activity Optimization

[0091] Although the Anti-FluB 5C11 monoclonal antibody obtained in Example 1 has the ability to bind to influenza B virus antigens, the antibody activity is not ideal, so the applicant performs directed mutations on the light chain CDR and heavy chain CDR of the antibody. That is, the antibody variable region structure simulation, the antigen and antibody variable region complex structure simulation, the antibody key amino acid analysis and mutation design are performed using a computer, and bidirectional primers covering the mutation site are designed and synthesized according to the mutation scheme, and primers at both ends of the target DNA are synthesized, and a high-fidelity PCR reaction is performed. The PCR product is cloned into a vector, and then the mutant antibody is prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly improved antibody activity are obtained and named: Anti-FluB5C11mut1 to Anti-FluB 5C11mut7, and the heavy chain and light chain amino acid sequences of each antibody are as follows.

[0092] Table 1 Antibody sequences

[0093] Sample name Heavy chain number Light chain number Anti-FluB 5C11mut1 SEQ ID NO:21 SEQ ID NO:23 Anti-FluB 5C11mut2 SEQ ID NO:22 SEQ ID NO:23 Anti-FluB 5C11mut3 SEQ ID NO:21 SEQ ID NO:24 Anti-FluB 5C11mut4 SEQ ID NO:21 SEQ ID NO:25 Anti-FluB 5C11mut5 SEQ ID NO:21 SEQ ID NO:26 Anti-FluB 5C11mut6 SEQ ID NO:22 SEQ ID NO:26 Anti-FluB 5C11mut7 SEQ ID NO:22 SEQ ID NO:28 Anti-FluB 5C11mut8 SEQ ID NO:22 SEQ ID NO:30

[0094] Example 3 Antibody Activity and Performance Testing

[0095] 1. Activity identification

[0096] Coating solution (main component NaHCO3) diluted goat anti-mouse IgG 1ug / ml for microplate coating, 100uL per well, 4℃ overnight; the next day, wash twice with washing solution (main component Na2HPO4+Nacl), pat dry; add blocking solution (20% BSA+80% PBS), 120uL per well, 37℃, 1h, pat dry; add diluted purified antibody, 100uL / well, 37℃, 60min; shake off the liquid in the plate, pat dry, add 20% mouse negative blood for blocking, 120ul per well, 37℃, 1h; shake off the liquid in the plate , pat dry, add diluted influenza B recombinant antigen (rFAN2-Ag, self-developed), 100uL per well, 37℃, 40min; wash 5 times with detergent, pat dry; add anti-influenza B monoclonal antibody HRP marker, 100uL per well, 37℃, 30min; add colorimetric solution A (50uL / well), add colorimetric solution B (50uL / well), 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on the microplate reader. The results are shown in the table below.

[0097] Table 2 Activity data

[0098]

[0099]

[0100] 2. Performance Evaluation

[0101] The above recombinant antibodies were used as labeled antibodies and paired with anti-influenza B monoclonal antibodies to be applied to the colloidal gold platform for combined sample testing. Specific data are shown in the table below:

[0102] Note: The gold label colorimetric consists of C plus a number. The smaller the number after C, the stronger the color and the higher the activity; the higher the number after C, the weaker the color and the lower the activity. The number with "+" is slightly stronger than without color by 0.5-1C, and the number with "-" is slightly lower than without color by 0.5-1C. "B" represents negative.

[0103] Table 3

[0104]

[0105] 3. Stability assessment

[0106] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. The following table shows the OD results of the enzyme immunoassay activity test for 21 days of antibody test.

[0107] Table 4 Stability data

[0108] Sample concentration (ng / ml) 62.50 31.25 0 4℃, 21-day samples 1.534 1.027 0.021 -80℃, 21 days sample 1.522 1.066 0.022 37℃, 21 days sample 1.564 1.081 0.021

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0110] The partial amino acid sequences involved in this application are as follows:

[0111] Sequence number Sequence fragment SEQ ID NO:1 RYWIQ SEQ ID NO:2 EVL PGSGSTDYNEKFED SEQ ID NO:3 DYRYGAL SEQ ID NO:4 KSSQSL LKSSDQRNYLA SEQ ID NO:5 FASTRES SEQ ID NO:6 QQQHYSSP SEQ ID NO:9 EVI PGSGSTDYNEKFED SEQ ID NO:10 KSSQSI LKSSDQRNYLA

Claims

1. An anti-influenza B virus antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: a) an amino acid sequence of HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2 or 9, HCDR3 as shown in SEQ ID NO: 3, and an amino acid sequence of LCDR1 as shown in SEQ ID NO: 4 or 10, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; or b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 12, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 13 to 20; or c) a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region having more than 80% identity with the heavy chain variable region in the sequence shown in b), the light chain variable region having more than 80% identity with the light chain variable region in the sequence shown in b), and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence shown in a), and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence shown in a); or d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 21 to 22, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 23 to 30.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The constant region includes a heavy chain constant region and / or a light chain constant region.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

5. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

6. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The species origin of the constant region is mouse.

7. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The heavy chain constant region sequence is as shown in SEQ ID NO: 7 or has at least 80% identity thereto; the light chain constant region sequence is as shown in SEQ ID NO: 8 or has at least 80% identity thereto.

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

9. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The antibody conjugate according to claim 9, characterized in that The antibody was conjugated to biotin.

11. The antibody conjugate according to claim 9, characterized in that The antibody is coupled to a solid phase.

12. The antibody conjugate according to claim 9, characterized in that The antibody is conjugated to a label.

13. The antibody conjugate according to claim 12, characterized in that: The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

14. The antibody conjugate according to claim 12, characterized in that: The label is colloidal gold.

15. A reagent or kit for detecting influenza B virus, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the antibody conjugate according to any one of claims 9 to 14.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody conjugate according to any one of claims 9 to 14, or the reagent or kit according to claim 15 in the preparation of a product for detecting influenza B virus, characterized in that: include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody conjugate according to any one of claims 9 to 14, or the reagent or kit according to claim 15 is contacted with influenza B virus in a sample to be detected to form an immune complex.

17. The use according to claim 16, characterized in that The immune complex also includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

18. The use according to claim 16, characterized in that The immune complex also includes a second antibody that binds to the influenza B virus.

19. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

20. A carrier, characterized in that It contains the nucleic acid according to claim 19.

21. A cell, characterized in that It contains the nucleic acid according to claim 19 or the vector according to claim 20.

22. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that: It includes: Cultivate the cell of claim 21.

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