KR101429194B1 - Antibody of specific binding to influenza viruses - Google Patents

Abstract

The present invention relates to a binding molecule specifically binding to a swine influenza virus. The binding molecule specific to the swine influenza virus of the present invention is useful for the prevention and treatment of a disease derived from a swine influenza virus, And can be used to diagnose a new influenza virus.

Description

Antibody specific binding to influenza viruses < RTI ID = 0.0 >

The present invention relates to an antibody that specifically binds to a swine influenza virus.

The causative virus of influenza is Orthomyxoviridae Family < / RTI > RNA (ssRNA) virus. Influenza viruses are usually divided into A, B, and C types. Influenza A virus infects not only humans but also birds and swine. Influenza C causes only mild illness in humans. Influenza A is subtyped again by surface antigens hemagglutanin (H) and neuramidase (N) antigens. H antigens are known to H1-H16, and N antigens are known to N1-N9. The gene for type A influenza is composed of 8 gene fragments, and for this reason, genetic recombination can occur.

Avian influenza viruses and swine influenza viruses do not infect humans well because of interspecies barriers. However, in rare cases infectious diseases exceed species barriers. In 1976, a case of infection with swine influenza (H1N1) occurred in New Jersey, USA. There were 13 infected patients and one of them died. In 1997 in Hong Kong, an infectious disease caused by avian influenza H5N1 occurred, and infection by the H5N1 influenza virus is still sporadic. By June 1, 2009, there were 432 cases of avian influenza in all over the world, of which 262 (60%) died.

Influenza A H1N1 has been identified as the causative microorganism of the new influenza A (H1N1) pdm09. However, unlike Influenza A (H1N1), which caused seasonal influenza, it became known that the causative virus of swine influenza [A (H1N1) pdm09] was caused by pigs and became known as S-OIV (swine influenza virus) ) The name changed to pdm09 virus. Molecular biology research has shown that the A (H1N1) pdm09 virus is a recombination of four influenza virus genes. The four influenza viruses are the North American swine influenza virus, the North American avian influenza virus, the human influenza virus and the Eurasian swine influenza virus, respectively.

Currently, the influenza virus diagnosis method is viral culture, and it is known that the sensitivity of the diagnosis is good when the culture is well matched (Reina, 1997). However, the virus culture method has a disadvantage in that it is costly in terms of experimental results, and it takes much time to deliver and confirm the virus to the laboratory.

As a commercialized rapid diagnosis reagent, there is a simultaneous detection kit of A-type circular and B-type rounds for human body, and a kit for detecting only A-type rounds in case of animals. However, the current commercialized influenza rapid diagnostic kit has a disadvantage that it can not distinguish the seasonal flu from the swine flu [A (H1N1) pdm09] virus which is currently popular. The current method for distinguishing between seasonal influenza and the new influenza A (H1N1) pdm09 virus is real-time polymerase chain reaction (RT-PCR) However, it has expensive equipment and high technical power, which makes it less convenient to use.

It is an object of the present invention to provide a binding molecule specifically binding to a swine influenza virus.

The present invention relates to an antibody that specifically binds to a swine influenza virus. According to the Kabat method, the light chain is L1; D; L2 is I; L3 is K or V; L < 4 > is L or M; L5 is T; L6 is Q; L7 is S or A; L8 is P or A; L9 is S, A or F; L10 is S; L11 is L or N; L12 is Y, A or P; L13 is V; L14 is S or T; L15 is L; L16 is G; L17 is E, Q or T; L18 is R or S; L19 is V or A; L20 is T or S; L21 is I; L22 is T or S; L23 is C; L24 is K or R; L25 is A or S; L26 is S; L27 is Q, E or K; L27A is X or S; L27B is X, V or L; L27C is X, D or L; L27D is X, N or Y; L27E is X or S; L28 is D, Y or N; L29 is I or G; L30 is Q or I; L31 is S or T; L32 is F or Y; L33 is M or L; L34 is N or S; L35 is W; L36 is F or Y; L37 is Q or L; L38 is Q; L39 is K; L40 is P; L41 is W or G; L42 is K or Q; L43 is P or S; L44 is P; L45 is K or Q; L46 is T or L; L47 is L; L48 is I; L49 is S or Y; L50 is Y, A or Q; L51 is A or M; L52 is T or S; L53 is S or N; L54 is Q or L; L55 is G or A; L56 is D or S; L57 is G; L58 is V; L59 is P; L60 is S, A or D; L61 is R; L62 is F; L63 is S; L64 is G; L65 is S; L66 is G; L67 is S; L68 is G; L69 is Q or T; L70 is E or D; L71 is Y or F; L72 is S or T; L73 is L; L74 is T, N or R; L75 is I; L76 is H or S; L77 is S, P or R; L78 is L or V; L79 is E; L80 is S, E or A; L81 is D or E; L82 is D; L83 is T or L; L84 is A or G; L85 is T, M or V; L86 is Y; L87 is F or Y; L88 is C; L89 is L, Q or A; L90 is Q; L91 is H, S or N; L92 is G, K or R; L93 is E; L94 is S, V or L; L95 is P or X; L96 is Y or W; L97 is T; L98 is F; L99 is G; L100 is G; L101 is G; L102 is T; L103 is K or T; L104 is V or L; L105 is E; L106 is I; L107 is K; L108 is R; And L109 are A; ego; H1 is either D or Q; H2 is V or I; H3 is Q; H4 is L; H5 is K, Q or V; H6 is E or Q; H7 is S; H8 is G; H9 is P; H10 is G or E; H11 is L; H12 is V or K; H13 is Q or K; H14 is P; H15 is S or G; H16 is Q or E; H17 is S or T; H18 is L or V; H19 is S or K; H20 is F or I; H21 is T or S; H22 is C; H23 is T or K; H24 is V or A; H25 is T or S; H26 is G; H27 is F or Y; H28 is S or T; H29 is I or L; H30 is T; H31 is S or K; H32 is D or Y; H33 is Y or G; H34 is V, A or M; H35 is H, W or N; H35A is N or X; H36 represents W; H37 is I or V; H38 is R or K; H39 is Q; H40 is S, F or A; H41 is P; H42 is G; H43 is N or K; H44 is G, K or D; H45 is L; H46 is E or K; H47 is W; H48 is L or M; H49 is A or G; H50 is V, Y or W; H51 is I; H52 is W, R or N; H52A is X or T; H53 is R, Y or N; H54 is G, S or T; H55 is G; H56 is S, T or E; H57 is T or P; H58 is D, T or T; H59 is Y; H60 is N or A; H61 is A, P or E; H62 is A, S or E; H63 is L or F; H64 is K or K; H65 is S or G; H66 is R; H67 is L, I or F; H68 is S or A; H69 is I or F; H70 is T or S; H71 is K, R or L; H72 is D or E; H73 is N, I or T; H74 is S; H75 is K or A; H76 is N or S; H77 is Q or T; H78 is V, F or A; H79 is F or Y; H80 is F or L; H81 is K or Q; H82 is M, L or I; H82A is N; H82B is S or N; H82C is V or L; H83 is Q, T or K; H84 is A, T or N; H85 is D or E; H86 is D; H87 is T; H88 is A; H89 is I or T; H90 is Y; H91 is Y or F; H92 is C; H93 is A; H94 is K or R; H95 is N or G; H96 is Y or A; H97 is G, Y or M; H98 is S, G or X; H99 is S, N or X; H100 is Y or X; H100A is N, V or X; H100B is Y, G or X; H100C is A, W or X; H100D is M, Y or X; H100E is F or X; H101 is D; H102 is V or Y; H103 represents W; H104 is G; H105 is A or Q; H106 is G; H107 is T; H108 is T or S; H109 is V; H110 is T; H111 is V; H112 is S; And H113 are S; : Wherein X is a Kabat sequence in which no amino acid is present.

In one embodiment of the present invention, the light chain is any one of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and in one embodiment, the heavy chain is SEQ ID NO: 8, SEQ ID NO: 12, and in one embodiment of the invention said antibody comprises SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; Or SEQ ID NO: 11 and SEQ ID NO: 12.

In one embodiment of the present invention, the antibody; And an antibody-drug conjugate comprising a novel influenza virus therapeutic agent.

In one embodiment of the invention, there is provided a vector comprising a gene encoding said antibody.

In one embodiment of the present invention, there is provided a cell line comprising the vector of claim 6, wherein the cell line is selected from the group consisting of F2N, HEK293, CHOK1, DG44, DXB11, CHO-S, BHK, Sp2 / 0, or NS.

In one embodiment of the present invention, there is provided a method for producing a binding molecule by transfecting the vector of claim 6 into a cell line. In one embodiment of the present invention, the cell line is F2N, HEK293, CHOK1, DG44, DXB11 , CHO-S, BHK, Sp2 / 0, or NS.

In one embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of swine influenza virus comprising the antibody, wherein the composition for preventing or treating is in the range of 0.001 to 100 mg / kg In one embodiment of the present invention, the composition for preventing or treating is for a mouse, a dog, a pig, a cattle, a horse, a rabbit, a llama or a pellet. In one embodiment of the present invention, The therapeutic composition may be in the form of an oral preparation, an external preparation, a pre-filled syringe solution, a suppository, a sterilized injection solution or a lyophilized form.

In one embodiment of the present invention, there is provided a method for providing information on the presence or absence of a pandemic influenza virus, which comprises reacting the antibody with a target sample.

In one embodiment of the present invention, there is provided a diagnostic kit for swine influenza virus comprising the antibody.

The transforming growth method used in the present invention can be carried out by a method known in the art, for example, but not limited to, by making a competent cell using CaCl ₂ buffer, and then introducing the expression vector into a host cell The transfection may be introduced into the host cell by transfection, transfection, transient transfection, microinjection, cell fusion, calcium phosphate precipitation, electroporation, or the like.

The novel influenza therapeutic agent used in the present invention may be, but is not limited to, Tamiflu or Relenza.

Since the antibody of the present invention specifically binds to the new influenza virus, it is useful as a method for diagnosing treatment and infection of the new influenza virus.

Hereinafter, the word of the present invention will be defined as follows.

Binding molecule " as used herein refers to an intact immunoglobulin comprising a monoclonal antibody, such as a chimeric, humanized or human monoclonal antibody, or an immunoglobulin that binds to an antigen, for example, Receptor or protein capable of binding to a variable domain or substrate comprising an immunoglobulin fragment competing with intact immunoglobulin for binding to influenza A virus monomer HA or trimer HA. Regardless of structure, the antigen-binding fragments bind to the same antigen recognized by the intact immunoglobulin. The antigen-binding fragment may comprise two or more contiguous amino acid sequences of the binding molecule, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, , At least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, A peptide or polypeptide comprising an amino acid sequence of at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues. "Antigen-binding fragments" specifically include Fab, F (ab '), F (ab') ₂ , Fv, dAb, Fd, complementarity determining region (CDR) fragments, single- Single chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind to a particular antigen to a polypeptide, and the like. The fragment may be produced synthetically or by enzymatic or chemical degradation of the complete immunoglobulin, or may be genetically engineered by recombinant DNA technology. Methods of generation are well known in the art.

As used herein, the term " pharmaceutically acceptable excipient " means an inert material combined with an active molecule, such as a drug, agent, or binding molecule to produce an acceptable or convenient dosage form. Pharmaceutically acceptable excipients are non-toxic or at least those excipients which are acceptable for their intended use in the recipient at the dosages and concentrations for which they are used and include other components of the formulation including drugs, Can be compatible.

As used herein, the term " therapeutically effective amount " refers to the amount of the binding molecule of the present invention effective for prevention or treatment before or after exposure of influenza A virus.

The composition containing the substance according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral liquid preparations such as suspensions, emulsions, syrups and aerosols, external preparations, pre-filled syringe solution, or lyophilized form. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The compounds of the present invention, particularly the binding molecules of the present invention, are particularly useful as diagnostic reagents in the detection of swine influenza virus.

Preferably, the compounds of the present invention used in the diagnostic composition are detectably labeled. Various methods that can be used to label biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. These methods are described in Tijssen, 'Practice and theory of enzyme immunoassays', Burden, RH and von Knippenburg (Eds), Volume 15 (1985), 'Basic methods in molecular biology'; Davis LG, Dibmer MD; Acad. Press, London (1987), or in the series 'Methods in Enzymology', Academic Press, Inc .; Battey Elsevier (1990), Mayer et al., (Eds.) Immunochemical methods in cell and molecular biology.

There are many other markings and marking methods known to those skilled in the art. Examples of marking classes that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.

Commonly used markers include fluorescent substances (e.g., fluorescein, rhodamine, Texas red, etc.), enzymes (such as horseradish peroxidase,? -Galactosidase, alkaline phosphatase), radioactive isotopes 32 P or 125 I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (such as dioxetane, luminol or acridinium). Methods of labeling enzymes or biotinyl groups such as covalent bonding, iodination, phosphorylation, biotinylation, and the like are well known in the art.

Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzyme reactions, and the like. Detection assays commonly used include radioisotope or non-radioisotope methods. Among them, they include Western blotting, overlay-analysis, RIA (Radioimmunoassay) and IRMA (Immune Radioimmunoassay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay) Assay).

The antibody according to the present invention can be used in the form of a drug-conjugated antibody-drug conjugate. When an antibody-drug conjugate (ADC), that is, an immunoconjugate, is used for local delivery of a drug, the drug moiety can be targeted to the infected cells. If the drug is administered without conjugation, This can lead to unacceptable levels of toxicity. By increasing the selectivity of polyclonal and monoclonal antibodies (mAbs) as well as drug-linking and drug-releasing properties, the maximum efficacy and minimal toxicity of the ADC can be improved.

Conventional means of attaching a drug moiety to an antibody, i. E., Through covalent bonds, generally result in a heterogeneous molecular mixture in which the drug moiety is attached to numerous sites on the antibody. For example, a cytotoxic drug can typically be conjugated to an antibody, often through numerous lysine residues of the antibody, to produce a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, such a heterogeneous mixture typically has an antibody distribution of 0 to about 8 or more attached to the drug moiety. In addition, each aliquot of the conjugate with a specific integer ratio drug moiety-to-antibody is a potentially heterogeneous mixture in which the drug moiety is attached to various sites on the antibody. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Reactivity with linker reagents and drug-linker intermediates is dependent on such factors as pH, concentration, salt concentration and cosolvent.

Hereinafter, the present invention will be described in detail. However, the following examples are intended to illustrate the contents of the present invention and are not intended to limit the scope of the invention. The documents cited in the present invention are incorporated herein by reference.

Various embodiments described herein are described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions, and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in any detail, in order not to unnecessarily obscure the present invention.

Example

Example  1. Virus Concentration and Purification

Inactivated swine influenza virus culture (A / Korea / 01/2009) was provided. The method of S. Kodiballi et al. (1993) was used to concentrate and purify antigens from the newly isolated influenza virus (A / Korea / 01/2009, A (H1N1) pdm09).

Specifically, the swine influenza virus cultured for about ²⁵ activity was inactivated with β-propiolactone and subjected to primary centrifugation at 4,000 × g for 30 minutes. The supernatant was then centrifuged at 70,000 × g for 3 hours (L8-70M, Beckman, USA). The centrifuged pellet was resuspended in sucrose (1/25 times the amount of sucrose), sucrose density gradient ultracentrifugation (100,000 xg, 90 min) And then the fraction showing hemagglutination activity was isolated. To this concentrated virus fraction, 5% (w / v) n-octyl-bD-gluco pyranoside was added and the virus was disrupted by treatment at 22 ° C for 2 hours . Thereafter, the pellet was centrifuged at 70,000 xg for 90 minutes, the pellet was dissolved in 0.1 M NaCl, and centrifuged again at 10,000 xg for 30 minutes to isolate the influenza virus.

Example  2. Monoclonal antibody production

2.1 Immunogen preparation

The immunogen was mixed with complete Freund's adjuvant (Sigma) and emulsified sufficiently. The mice were inoculated 3 times at 1 week intervals with 200 ㎍ each, and the tail vein injection was performed 3 times.

2.2 Serum collection, Potency  Measurement and cell fusion

A small amount of blood was collected from the tail of the immunized mouse (Balb / c), and the serum was separated, and the activity was measured by enzyme immunoassay. Antibody titers were adsorbed on an ELISA plate, and antiserum was diluted 10, 100, 1,000, and 10,000 times with 10 times dilution. Secondary reactions were performed using goat anti-mouse IgG-peroxidase (Goat anti-mouse IgG-peroxidase) and developed with TMB. Cell fusion was performed when the absorbance of the enzyme was about 3 times higher than the absorbance of normal mouse serum as a cutoff and the titer of the antiserum was more than 1,000 times and the titer was more than the cutoff (Table 1).

The splenic cells of the immunized mouse were isolated and hybridized with myeloma cells (SP2 / 0) to produce a monoclonal antibody. The hybridoma cell line was transfected with a hemagglutinin inhibition assay (Hemagglutinin Inhibition Assay) And several monocytic cells were established by limiting dilution method. Cells were mass-cultured and cells were inoculated into the peritoneal cavity of Balb / c mice to obtain ascites containing a large amount of antibody, and the IgG antibody was purified using protein G gel.

Dilution of antiserum dilution 10 times 100 times 1,000 times 10,000 times Normal mouse serum
Absorbance ( OD ₄₅₀ ) 0.091 0.095 0.110 0.096 Immune mouse serum
Absorbance ( OD ₄₅₀ ) 1.820 1.026 0.450 0.201

2.3 Mouse monoclonal swine influenza virus Hemiglutinin  Antibody screening

30 μl of PBS (phosphate buffered saline) was added to each well of the U-plated. 30 μl of the sample was mixed with the first well and diluted by 1/2 with the next well. After diluting 30 μl with the final well, And the control group did not contain any specimen. The virus was diluted to 2 ² HAU (hemagglutination unit) in various types of suntypes and 30 μl of each sample was added to the well. In the control, 2 ² HA virus was added to the first well and diluted 3 times with 1/2 Respectively.

Thereafter, the cells were allowed to stand at room temperature for 30 minutes, and 30 쨉 l of 1% chicken erythrocytes were dispensed into each well, and red blood cells were similarly dispensed into the control group. The cells were allowed to stand at room temperature again for 40 minutes, and cell lines in which erythrocyte sedimentation occurred were selected as compared with the control group.

2.4 Mouse Monoclonal  Antibody purification

A large number of cell strains selected in Example 2.3 were cultured and inoculated into peritoneal cavity of Balb / c mice to obtain a plurality of antibodies containing a large amount of antibody, and then the plurality was centrifuged at 2500 rpm for 10 minutes and the precipitate was removed. The plurality was diluted with the above-mentioned 4-fold volumes of binding buffer and allowed to stand at room temperature for 1 hour.

The diluted solution was centrifuged at 12000 rpm at 4 캜 for 5 minutes (High speed centrifugation) and the precipitate was removed and the protein G gel was washed with 1.5M to 3M NaSCN in OD ₂₈₀ Lt; RTI ID = 0.0 > 0. < / RTI > The washed protein G gel was allowed to equilibrate with binding buffer. The prepared specimen was loaded and washed with binding buffer until the OD ₂₈₀ value of the washing solution was less than 0.05. Elution was carried out by elution buffer (2 times repeated), and after standing for 10 minutes, OD ₂₈₀ value of each fraction was measured.

A The measured OD ₂₈₀ value of 0.9 or more tubes only PD-10 in PBS (pH7.2) 0.1% NaN ₃ after dialysis, filtering the dialyzed antibody as a filter having a pore size of 0.2 ㎛, by measuring the OD ₂₈₀ value of the concentration .

Example  3. Swine influenza virus [A ( H1N1 ) pdm09 ] Binding characteristics of specific antibodies

In order to confirm whether the monoclonal antibodies (mAbI310, mAbI360 and mAbI430) obtained through Example 2 specifically bind to A (H1N1) pdm09, hemagglutination inhibition activity was assayed by HAI (Hemagglutinin Inhibition) assay Respectively. The experiment was performed in the same manner as conventionally known, wherein ^a -pdmH1N1 H1N1 and ^b is Ferret antisera obtained from NIID (National Institute of Infectious Diseases in Japnan) for each of the A / Korea / 01/2009 and A (H1N1) in pdm09 ( ferret antiserum) control sheep antiserum. On the other hand, anti-IFN-B ^c and anti-IFN-B ^d are the influenza B reference antiserum against B / brisbane / 60/2008 and B / Florida / 4/2006, respectively.

As a result, as shown in Table 2 below, mAbI319 antibody against other influenza subtypes showed no binding activity to H1N1, H3N2, B (Brisbane) and B (Florida) except for H1N1pdm, And it was found.

virus Antibody or anti-serum mAb
I310 mAb
D360 mAb
D430 anti-pDM H1N1 ^a anti-PDM H1N1 ^b anti-H1N1 anti-H3N2 anti-IFN-B ^c anti-IFN-B ^d H1N1pdm 64 256 32 1280 2560 - - - - H1N1 - - - - - 640 - - - H3N2 - - - - - - 2560 - - B (Brisbane) - - - - - - 40 2560 80 B
(Florida) - - - - - - 80 40 2560

Example  4. Monoclonal antibody CDR

The DNA sequence of the Fab region of the monoclonal antibodies prepared in Example 2 was confirmed and the results are shown in Table 3 below.

Light chain Heavy-span Fab (Fd) mAbI310 SEQ ID NO: 1 SEQ ID NO: 2 mAbI360 SEQ ID NO: 3 SEQ ID NO: 4 mAbI430 SEQ ID NO: 5 SEQ ID NO: 6

In addition, the proteins to be translated from SEQ ID NO: 1 to SEQ ID NO: 6 are SEQ ID NO: 7 to SEQ ID NO: 12, respectively.

<110> Republic of Korea <120> Antibody specific binding to influenza viruses <130> IPDB46125 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 413 <212> DNA <213> Mouse <400> 1 atgagggccc ctgctcagtt ttttgggttc ttgttgctct ggtttccagg tatcagatgt 60 gacatcaaga tgacccagtc tccatcctcc atgtatgtat cgctgggaga gagagtcact 120 atcacttgca aggcgagtca ggacattcaa agctatttaa gctggtacca gcagaaacca 180 tggaaatctc ctaagaccct gatctcttat gcaacaagct tggcagatgg ggtcccatca 240 agattcagtg gcagtggatc tgggcaagaa tattctctaa ccatcagcag cctggagtct 300 gacgatacag caacttattt ctgtctacag catggggaga gcccgtacac gttcggaggg 360 gggaccaagg tggaaataaa acgggctgat gctgcaccaa ctgtatccat ctt 413 <210> 2 <211> 435 <212> DNA <213> Mouse <400> 2 atggctgtct tagggctgtt cttctgcctg gtgacattcc caagctgtgt cctgtcccag 60 gtgcagctga agcagtcagg acctggccta gtgcagccct cacagagcct gtccataacc 120 tgcacagtct ctggtttctc attaactagc tatggtgtac actgggttcg ccagtctcca 180 ggaaagggtc tggagtggct gggagtgata tggagaggtg gaagcacaga ctacaatgca 240 gctttcatgt ccagactgag catcaccaag gacaactcca agagccaagt tttctttaaa 300 atgaacagtc tgcaagctga tgacactgcc atatactact gtgccaaaaa ttacggtagt 360 agctacaact atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctcagcc 420 aaaacaacac cccca 435 <210> 3 <211> 420 <212> DNA <213> Mouse <400> 3 atggagacag acacactcct gctatgggtc ctgcttctct gggttccagg ttccacaggt 60 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 120 atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc 180 caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc 240 ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 300 cctttggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 360 acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420                                                                          420 <210> 4 <211> 438 <212> DNA <213> Mouse <400> 4 atgagagtgc tgattctttt gtggctgttc acagcctttc ctggtatcct gtctgatgta 60 cagcttcagg agtcgggacc tggcctggtg aaaccttctc agtctctgtc cttcacctgc 120 actgtcactg gctactcaat caccagtgac tatgcctgga actggatccg gcagtttcca 180 ggaaacaaac tggagtggat ggcctacata aggtacagtg gtaccaccac ctacaaccca 240 tctctcaaaa gtcgaatctc tatcactcga gacatatcca agaaccagtt cttcctgcag 300 ttgaattctg tgactactga ggacacagcc acatattact gtgcaagagg ggcgtatggt 360 aattacgtcg gctggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 420 gctacaacaa cagcccca 438 <210> 5 <211> 420 <212> DNA <213> Mouse <400> 5 atgatgttct cggctcagtt cctgaggctg cttgtgctct ggatccctgg atccactgca 60 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 120 atctcctgca ggtctagtaa gagtctcctg tatagtaatg gcatcactta tttgtcttgg 180 tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 240 tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 300 agcagagtgg aggctgagga tttgggtgtt tattactgtg ctcaaaatcg agaactgtgg 360 acgttcggtg gaggcaccac gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420                                                                          420 <210> 6 <211> 417 <212> DNA <213> Mouse <400> 6 atggcttggg tgtggaactt gctattcctg atggcagctg cccaaagtat ccaagcacag 60 atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc 120 tgcaaggctt ctgggtatac cctcacaaaa tacggaatga actgggtgaa gcaggctcca 180 ggaaaggatt taaagtggat gggctggata aacaccaaca ctggagagcc aacatatgct 240 gaagagttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300 cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag aggcgctatg 360 gactactggg gtcaaggaac ctcagtcacc gtctcctcag ctacaacaac agcccca 417 <210> 7 <211> 137 <212> PRT <213> Mouse <400> 7 Met Arg Ala Pro Ala Gln Phe Phe Gly Phe Leu Leu Leu Trp Phe Pro   1 5 10 15 Gly Ile Arg Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr              20 25 30 Val Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp          35 40 45 Ile Gln Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro      50 55 60 Lys Thr Leu Ile Ser Tyr Ala Thr Ser Leu Ala Asp Gly Val Ser Ser  65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Gln Glu Tyr Ser Leu Thr Ile Ser                  85 90 95 Ser Leu Glu Ser Asp Asp Thr Ala Thr Tyr Phe Cys Leu Gln His Gly             100 105 110 Glu Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Ala Asp Ala Ala Pro Thr Val Ser Ile     130 135 <210> 8 <211> 145 <212> PRT <213> Mouse <400> 8 Met Ala Val Leu Gly Leu Phe Phe Cys Leu Val Thr Phe Pro Ser Cys   1 5 10 15 Val Leu Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln              20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu          35 40 45 Thr Ser Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu      50 55 60 Glu Trp Leu Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala  65 70 75 80 Ala Phe Met Ser Arg Leu Ser Ile Thr Lys Asp Ser Ser Ser Ser Ser Gln                  85 90 95 Val Phe Phe Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr             100 105 110 Tyr Cys Ala Lys Asn Tyr Gly Ser Ser Tyr Asn Tyr Ala Met Asp Tyr         115 120 125 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro     130 135 140 Pro 145 <210> 9 <211> 140 <212> PRT <213> Mouse <400> 9 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala              20 25 30 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser          35 40 45 Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro      50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser  65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser                  85 90 95 Leu Asn Ile His Pro Leu Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys             100 105 110 Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu         115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser     130 135 140 <210> 10 <211> 146 <212> PRT <213> Mouse <400> 10 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile   1 5 10 15 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro              20 25 30 Ser Gln Ser Leu Ser Phe Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr          35 40 45 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu      50 55 60 Glu Trp Met Ala Tyr Ile Arg Tyr Ser Gly Thr Thr Thr Tyr Asn Pro  65 70 75 80 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Ile Ser Lys Asn Gln                  85 90 95 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr             100 105 110 Tyr Cys Ala Arg Gly Ala Tyr Gly Asn Tyr Val Gly Trp Tyr Phe Asp         115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Thr Thr Thr     130 135 140 Ala Pro 145 <210> 11 <211> 140 <212> PRT <213> Mouse <400> 11 Met Met Phe Ser Ala Gln Phe Leu Arg Leu Leu Val Leu Trp Ile Pro   1 5 10 15 Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro              20 25 30 Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Ser Ser Ser          35 40 45 Leu Leu Tyr Ser Asn Gly Ile Thr Tyr Leu Ser Trp Tyr Leu Gln Lys      50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala  65 70 75 80 Ser Gly Val Pro Asp Arg Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Thr Asp Phe                  85 90 95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr             100 105 110 Cys Ala Gln Asn Arg Glu Leu Trp Thr Phe Gly Gly Gly Thr Thr Leu         115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser     130 135 140 <210> 12 <211> 139 <212> PRT <213> Mouse <400> 12 Met Ala Trp Val Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser   1 5 10 15 Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys              20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Leu          35 40 45 Thr Lys Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Asp Leu      50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala  65 70 75 80 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser                  85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr             100 105 110 Tyr Phe Cys Ala Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser         115 120 125 Val Thr Val Ser Ser Ala Thr Thr Ala Pro     130 135

Claims (16)

A monoclonal antibody comprising a light chain consisting of the amino acid sequence of SEQ ID NO: 7 and an amino acid sequence of SEQ ID NO: 8, which specifically binds to A / Korea / 01/2009 virus. 9. A monoclonal antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO: 9 and an amino acid sequence of SEQ ID NO: 10, which specifically binds to A / Korea / 01/2009 virus. 12. A monoclonal antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO: 11 and an amino acid sequence of SEQ ID NO: 12, which specifically binds to A / Korea / 01/2009 virus. A vector comprising a gene encoding a monoclonal antibody according to any one of claims 1 to 3. 4. A cell line comprising the vector according to claim 4. 6. The method of claim 5,
Wherein the cell line is F2N, HEK293, CHOK1, DG44, DXB11, CHO-S, BHK, Sp2 / 0 or NS. A method for producing a monoclonal antibody by transfecting a vector according to claim 4 into a cell line. 8. The method of claim 7, wherein the cell line is F2N, HEK293, CHOK1, DG44, DXB11, CHO-S, BHK, Sp2 / 0 or NS. A method for providing information on the presence or absence of an A / Korea / 01/2009 virus comprising reacting a monoclonal antibody according to any one of claims 1 to 3 with a target sample. A diagnostic kit for A / Korea / 01/2009 virus comprising a monoclonal antibody according to any one of claims 1 to 3. delete delete delete delete delete delete