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

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-influenza B virus antibody, 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, which includes human influenza virus, swine influenza virus, equine influenza virus, avian influenza virus, etc. Human influenza virus can be divided into three types: A (A), B (B), and C (C) based on the antigenicity of its nucleoprotein. It is the pathogen of influenza. Influenza virus can cause infection and disease in a variety of animals, including humans, poultry, pigs, horses, and bats. Among them, influenza A virus and influenza B virus are the main ones that can infect humans, mainly causing upper respiratory tract infections, but can also cause lower respiratory tract infections in children and adults, mainly pneumonia. Severe influenza in infants and young children is often accompanied by bronchitis and high fever.

[0003] Influenza B, caused by the influenza B virus, is characterized by an abrupt onset of illness, chills, and fever, with the temperature rising to a peak of 39-40°C or even higher within a few hours to 24 hours. It is accompanied by headaches, body aches, fatigue, and loss of appetite. Respiratory symptoms are mild, including dry throat, sore throat, dry cough, and possible diarrhea. Facial flushing, conjunctival hyperemia, pharyngeal congestion, and follicles on the soft palate can also occur. Treatment can include medications such as amantadine, an M2 ion blocker, and traditional Chinese medicine.

[0004] Influenza B viruses can produce numerous subtypes because their hemagglutinin (HA) and neuraminidase (NA) antigens are prone to mutation, allowing them to mutate at various amino acid sites. After each pandemic, these amino acid mutations create new strains of influenza virus. The human immune system generally lacks immunity to these mutated subtypes, making them susceptible to localized epidemics and, under unusual circumstances, widespread infection. Monitoring data from December 2017 to January 2018 show an increasing incidence of influenza B virus. Therefore, early and rapid screening for influenza viruses is crucial.

[0005] Influenza virus infection presents with atypical clinical symptoms, and clinical diagnosis relies primarily on laboratory testing. However, with over 200 respiratory viruses that can infect humans simultaneously, the sensitivity and specificity of detection reagents are crucial for clinical diagnosis. Selecting influenza virus screening technologies with short detection times and high detection rates is a key technical approach and prerequisite for ensuring rapid clinical diagnosis and targeted 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 relies on chicken embryo inoculation. Due to the complex operation and high technical requirements, it is rarely used in clinical practice. 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 has the advantages of high sensitivity and specificity, but the PCR method has high requirements for samples, experimental environment, and operators, and the amplification methodology is suitable for the detection of batch specimens. The report issuance time is long and it cannot well 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, helping the clinic to prescribe the right medicine as soon as possible to a great extent. 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 the 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] 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) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 1 to 3, and LCDR1 with amino acid sequences as shown in any one of SEQ ID NOs: 4, 9, 10, and 11, LCDR2 with amino acid sequences as shown in SEQ ID NO: 5, and LCDR3 with amino acid sequences 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: 12 to 13, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 29; 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: 30 to 31, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 32 to 47.

[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] To achieve the above object, according to a 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-mentioned antibody or functional fragment thereof or the above-mentioned 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, 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 objectives, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 1 The results of reducing SDS-PAGE of Anti-FluB 7G03mut1 to mut6 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, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 3, and LCDR1 having an amino acid sequence as shown in any one of SEQ ID NOs: 4, 9, 10, and 11, LCDR2 having shown in SEQ ID NO: 5, and LCDR3 having 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] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[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 the widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[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: 12 to 13, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 29. 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 of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0029] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following 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 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 having an amino acid sequence that is more than 80% identical to the sequence shown in b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in a). 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: 12 to 13, and a light chain framework region in a light chain variable region shown in any one of SEQ ID NOs: 14 to 29.

[0033] In an alternative 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 alternative embodiments, the constant region comprises 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 or λ type light chain constant region.

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

[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 shown as SEQ ID NO:7, and the light chain constant region (CL) sequence is shown as SEQ ID NO:8.

[0040] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 7 or 8).

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

[0043] 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.

[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: 30 to 31, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 32 to 47. 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 conjugated to the substance to be conjugated. Alternatively, the antibody is conjugated to the substance to be conjugated 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, radioisotopes, chemiluminescent reagents 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 falls within the scope of protection of the present invention.

[0052] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC) or its analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

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

[0054] In an alternative 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, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates 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 kit for detecting influenza B virus, wherein the reagent or kit comprises the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

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

[0066] In another aspect, the present invention provides a method for detecting influenza B virus, comprising: contacting the above-mentioned antibody or functional fragment thereof, antibody conjugate, or 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 that 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 conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it falls within the scope of protection of the present invention.

[0074] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

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

[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 person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

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

[0078] Example 1 Preparation of Anti-FluB 7G03 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 TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen.

[0080] 1 Construction of recombinant plasmid

[0081] (1) Antibody gene preparation

[0082] mRNA was extracted from a hybridoma cell line secreting monoclonal antibodies against influenza B virus. DNA products were obtained by RT-PCR. This product was subjected to an A-addition reaction using rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were transformed into DH5α competent cells, and colonies were grown. Heavy chain and light chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0083] (2) Sequence analysis of the variable region gene of the Anti-FluB 7G03 antibody

[0084] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 339 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 357 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

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

[0086] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed using the pcDNA3.4A expression vector, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI. It is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the above-mentioned pMD-18T, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protection bases at both ends, respectively. PCR amplification was used to amplify the 0.73kb light chain gene fragment and the 1.80kb heavy chain gene fragment.

[0087] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were 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] HEK293 cells were thawed in advance and subcultured to a 200 ml volume, reaching a cell density of 3–5 × 10⁶ cells / ml and a cell viability of >95%. Cells were washed by centrifugation and reconstituted with culture medium. The cell density was adjusted to 2.9 × 10⁶ cells / ml for use as a cell dilution. Plasmid DNA and transfection reagent dilutions were prepared separately using culture medium. The transfection reagent dilution was added to the plasmid DNA dilution, mixed, and allowed to stand at room temperature for 15 minutes. This mixture was slowly added to the cell dilution over 1 minute. After mixing, samples were collected and counted. Cell viability after transfection was recorded and observed. Cells were then incubated in a 35°C incubator at 120 rpm and 8% CO₂. After 13 days, samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electropherogram is shown in the figure. After reducing SDS-PAGE, two bands were shown, one with a Mr of 50 KD (heavy chain) and the other with a Mr of 28 KD (light chain).

[0090] Example 2 Activity Optimization

[0091] While the Anti-FluB 7G03 monoclonal antibody obtained in Example 1 possessed the ability to bind to influenza B virus antigens, its activity was suboptimal. Therefore, the applicants performed targeted mutagenesis on the antibody's light and heavy chain CDRs. Specifically, computer simulations were performed on the antibody variable region structure, the structure of the antigen-antibody variable region complex, key amino acid analysis, and mutation design. Bidirectional primers covering the mutation sites were designed and synthesized based on the mutagenesis strategy. Primers were synthesized at both ends of the target DNA, and a high-fidelity PCR reaction was performed. The PCR products were cloned into a vector, and the mutant antibodies were prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly enhanced activity were obtained and designated Anti-FluB 7G03mut1 to Anti-FluB 7G03mut6. The heavy and light chain amino acid sequences of each antibody are shown below.

[0092] Table 1 Antibody sequences

[0093] Sample name Heavy chain sequence number Light chain number Anti-FluB 7G03mut1 SEQ ID NO:30 SEQ ID NO:32 Anti-FluB 7G03mut2 SEQ ID NO:30 SEQ ID NO:35 Anti-FluB 7G03mut3 SEQ ID NO:30 SEQ ID NO:33 Anti-FluB 7G03mut4 SEQ ID NO:30 SEQ ID NO:34 Anti-FluB 7G03mut5 SEQ ID NO:30 SEQ ID NO:36 Anti-FluB 7G03mut6 SEQ ID NO:31 SEQ ID NO:35

[0094] Example 3 Activity and performance testing of antibodies

[0095] 1. Activity identification

[0096] Coat the microplates with 100 μL of goat anti-mouse IgG diluted in coating solution (main component NaHCO3) at 1 μg / ml, incubate at 4°C overnight. The next day, wash twice with washing solution (main component Na2HPO4+NaCl), pat dry. Add blocking solution (20% BSA+80% PBS), 120 μL per well, incubate at 37°C for 1 hour, pat dry. Add diluted purified antibody, 100 μL / well. 37°C for 60 min; shake off the liquid, pat dry, add 20% mouse negative blood to block, 120 μL per well, 37°C for 1 hour; shake off the liquid, pat dry, add diluted influenza B recombinant antigen (rFAN2-Ag, self-developed), 100 μL per well, 37°C for 40 min; wash five times with detergent, pat dry; add anti-influenza B monoclonal antibody HRP conjugate, 100 μL per well, 37°C for 30 min; add chromogenic buffer A (50 μL / well) and chromogenic buffer B (50 μL / well) for 10 min; add stop buffer (50 μL / well); read OD values ​​at 450 nm (reference 630 nm) on a microplate reader. Results are shown in the table below.

[0097] Table 2 Activity data

[0098]

[0099]

[0100] 2. Application of antibodies in colloidal gold detection

[0101] The above recombinant antibodies were used as labeled antibodies in combination with paired anti-influenza B monoclonal antibodies and 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 followed by "+" is slightly stronger than without color, 0.5-1C; the number followed by "-" is slightly lower than without color, 0.5-1C; "B" represents negative.

[0103] Table 3

[0104]

[0105] 3. Stability assessment

[0106] The above-mentioned antibodies were placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under the three test conditions for 21 days, and the activity did not show a downward trend with increasing test temperature, indicating that the above-mentioned antibodies are stable. The following table shows the OD results of the enzyme immunoassay activity assay after 21 days of antibody testing.

[0107] Table 4 Stability data

[0108]

[0109]

[0110] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0111] Some of the amino acid sequences involved in this application are as follows:

[0112] Sequence number Sequence fragments SEQ ID NO: 1 DYVIS SEQ ID NO:2 EIYPGSGRSYNNERFKD SEQ ID NO:3 DDARGGF SEQ ID NO:4 RSSQSLLYSNGNTYLH SEQ ID NO:5 KVSNRFS SEQ ID NO:6 YQSTHVP SEQ ID NO:9 RSSQSILYSNGNTYLH SEQ ID NO: 10 RSSQSLIYSNGNTYLH SEQ ID NO:11 RSSQSIIYSNGNTYLH

Claims

1. An anti-influenza B virus antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment includes: The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 1 to 3, and the amino acid sequences are LCDR1 as shown in any one of SEQ ID NOs: 4, 9, and 10, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO:

6.

2. An anti-influenza B virus antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment includes: The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 14; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 17; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 18; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

17.

3. An anti-influenza B virus antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment includes: A heavy chain variable region and a light chain variable region whose amino acid sequences are more than 80% identical to the heavy chain variable region and the light chain variable region described in claim 2, and include HCDR1 to HCDR3 and LCDR1 to LCDR3 described in claim 1.

4. The antibody or functional fragment thereof according to any one of claims 1 to 3, wherein: The antibody or functional fragment thereof further comprises a constant region.

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

6. The antibody or functional fragment thereof according to claim 5, 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.

7. The antibody or functional fragment thereof according to claim 4, 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.

8. The antibody or functional fragment thereof according to claim 7, characterized in that The species origin of the constant region is mouse.

9. The antibody or functional fragment thereof according to claim 5, characterized in that The heavy chain constant region sequence is as shown in SEQ ID NO: 7 or has at least 80% identity thereto, and the light chain constant region sequence is as shown in SEQ ID NO: 8 or has at least 80% identity thereto.

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

11. An anti-influenza B virus antibody, characterized in that The antibodies include: A heavy chain having an amino acid sequence as shown in SEQ ID NO: 30, and a light chain having an amino acid sequence as shown in SEQ ID NO: 32; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 30, and a light chain having an amino acid sequence as shown in SEQ ID NO: 35; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 30, and a light chain having an amino acid sequence as shown in SEQ ID NO: 33; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 30, and a light chain having an amino acid sequence as shown in SEQ ID NO: 34; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 30, and a light chain having an amino acid sequence as shown in SEQ ID NO: 36; or The heavy chain has an amino acid sequence as shown in SEQ ID NO:31, and the light chain has an amino acid sequence as shown in SEQ ID NO:

35.

12. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or functional fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11, wherein the antibody is conjugated to: biotin or a biotin derivative, a solid phase and / or 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 13, 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 functional fragment thereof according to any one of claims 1 to 10, the antibody according to claim 11, or the antibody conjugate according to any one of claims 12 to 14.

16. Use of the antibody or functional fragment thereof according to any one of claims 1 to 10, the antibody according to claim 11, or the antibody conjugate according to any one of claims 12 to 14 in the preparation of a reagent or kit for detecting influenza B virus, characterized in that: The method for detecting influenza B virus comprises: The antibody or functional fragment thereof according to any one of claims 1 to 10, the antibody according to claim 11, or the antibody conjugate according to any one of claims 12 to 14 is brought into contact 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 further comprises a second antibody that binds to the antibody or a functional 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 functional fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11.

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 functional fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11, characterized in that: It includes: Cultivate the cell of claim 21.

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