JP2007261988A - Specific detection of h5 subtype influenza virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody specifically recognizing H5 subtype influenza virus, a method for detecting the H5 subtype influenza virus and a kit therefor. <P>SOLUTION: The anti-HA5 monoclonal antibody capable of specifically recognizing the H5 subtype influenza virus in type A influenza viruses, the anti-NP monoclonal antibody capable of recognizing the type A influenza virus, the method for detecting the H5 subtype influenza virus using the antibody, and the kit therefor are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses, an anti-NP monoclonal antibody capable of recognizing influenza A virus, and antibodies thereof The present invention relates to a method for detecting H5 subtype influenza virus and a kit therefor.

  Influenza viruses are classified into type A, type B, and type C based on the difference in antigenicity between nucleoprotein (NP) and membrane protein (M). Among them, influenza A virus is further classified into a plurality of subtypes depending on the amino acid sequence or antigenicity of hemagglutinin (HA) and neuraminidase (NA).

  Avian influenza is called “bird flu” and is caused by an influenza A virus infection, and many do not show severe symptoms. However, some H5, H7, and H9 subtype influenza viruses show severe symptoms when infected with birds. These are called “highly pathogenic influenza viruses,” and there is concern about the spread of infection in humans.

At present, a method for diagnosing influenza that is performed in clinical practice detects all subtypes A1 to H15 (Non-Patent Documents 1 to 12). Therefore, if a human shows signs of influenza, it is impossible to diagnose whether it is due to a highly pathogenic avian influenza virus or a human influenza virus. Therefore, there is a need to develop a rapid type diagnosis method for influenza A viruses.
Hideyuki Ikematsu et al .: Journal of infectious diseases. 73: 1153-1158, 1999 Atsuo Goto et al .: Clinical and viruses. 28: 248-252, 2000 Masahiko Yamazaki et al .: Journal of infectious diseases. 73: 1064-1068, 1999 Keiko Mitamura et al .: Journal of infectious diseases. 73: 1069-1073, 1999 Toshimi Watanabe et al .: Journal of infectious diseases. 73: 1199-1204, 1999 Masahiko Yamazaki et al .: Journal of infectious diseases. 74: 1032-1037, 1999 Hideaki Shimizu et al .: Journal of infectious diseases. 74: 1038-1043, 1999 Keiko Mitamura et al .: Journal of infectious diseases. 74: 12-16, 2000 Kawakami Chiharu et al .: Journal of infectious diseases. 75: 792-799, 2001 Masahiko Yamazaki et al .: Journal of infectious diseases. 75: 1047-1053, 2001 Keiko Mitamura et al .: Influenza. 3: 105-109, 2002 Okuno Yoshinobu et al .: Medicine and pharmacy. 48: 895-904, 2002

  The problem to be solved by the present invention is to obtain an antibody that specifically recognizes H5 subtype influenza virus among influenza A viruses, and to provide a specific, rapid and simple detection method for H5 subtype influenza virus and a kit therefor Is to develop.

  As a result of intensive studies in view of the above circumstances, the present inventors recognize an anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among type A influenza viruses, and type A influenza virus. The present inventors have succeeded in producing a hybridoma producing an anti-NP monoclonal antibody capable of producing the present invention.

That is, the present invention
(1) an anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses,
(2) The antibody according to (1), produced by a hybridoma having an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center receipt number FERM AP-20821,
(3) an anti-NP monoclonal antibody capable of recognizing influenza A virus,
(4) The antibody according to (3), which is produced by a hybridoma having an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center receipt number FERM AP-20822,
(5) A method for detecting H5 subtype influenza virus in a sample, comprising using the antibody according to (1),
(6) The method according to (5), which is based on an immunochromatography method,
(7) The method according to (5), wherein the antibody according to (3) is used in combination.
(8) A kit for detecting H5 subtype influenza virus in a sample, comprising the antibody according to (1) as an essential component;
(9) The kit according to (8), wherein an immunochromatography method is used,
(10) The kit according to (8), further comprising the antibody according to (3),
Is to provide.

  According to the present invention, an anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses, an anti-NP monoclonal antibody capable of recognizing influenza A virus, and these A rapid and easy method for detecting an H5 subtype influenza virus using an antibody and a kit therefor are provided.

  In one aspect, the present invention relates to an anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses. There are at least 15 types of HA of influenza A virus, and influenza A viruses are classified into subtypes H1 to H15 based on the difference. Since the antibody of the present invention is directed against HA of H5 influenza virus, it can specifically recognize H5 influenza virus among influenza A viruses. The antibody of this aspect of the present invention, for example, immunizes a mammal such as a mouse with H5 subtype influenza virus, and fuses the spleen cells of the immunized animal with a myeloma cell-derived strain derived from the same animal to produce a hybridoma. Can be obtained by culturing. The antibody of this embodiment may be produced using other known methods such as a genetic recombination method or a chemical synthesis method.

One specific example of an antibody of this aspect of the invention is an anti-HA monoclonal antibody (named 1C10) produced by the hybridoma Mouse-Mouse hybridoma 1C10. The hybridoma was deposited with the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) (1st, 1st, 1st, 1st, 1st, 6th, Tsukuba, Ibaraki Prefecture) and received on February 24, 2006, with the receipt number FERM AP- 20821. This hybridoma is a fusion cell of a mouse myeloma cell-derived strain and mouse lymphocytes, and produces an antibody (1C10) that binds to HA of H5 subtype influenza virus. For example, 1C10 is a hybridoma Mouse-Mouse hybridoma 1C10 cultured in RPMI 1640 medium (SIGMA R6540) containing 10% fetal serum, 10 mM L-Glutamine, 0.25% NaHCO ₃ at 37 ° C., 5% CO ₂ . It can be produced by recovering and purifying the produced 1C10 by a known method.

  Anti-HA monoclonal antibodies of this aspect of the invention include fragments thereof, and modified and mutated antibodies such as chimeric and humanized antibodies thereof. These fragments, modified antibodies or mutant antibodies also have the same specificity for H5 subtype influenza virus as the original antibody. These can be produced by means and methods known to those skilled in the art.

  In another aspect, the present invention relates to an anti-NP monoclonal antibody capable of recognizing influenza A virus. Since the antibody of this aspect of the present invention specifically binds to NP of influenza A virus, it can recognize any of H1 to H15 subtypes of influenza A virus. The antibody of this aspect of the present invention, for example, immunizes a mammal such as a mouse with H5 subtype influenza virus, and fuses the spleen cells of the immunized animal with a myeloma cell-derived strain derived from the same animal to produce a hybridoma. Can be obtained by culturing. The antibody of this embodiment may be produced using other known methods such as a genetic recombination method or a chemical synthesis method.

One specific example of an antibody of this aspect of the invention is an anti-NP monoclonal antibody (named 4E3) produced by the hybridoma Mouse-Mouse hybridoma 4E3. The hybridoma was deposited with the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) (1st, 1st, 1st, 1st, 1st, 6th, Tsukuba, Ibaraki Prefecture) and received on February 24, 2006, with the receipt number FERM AP- 20822. This hybridoma is a fusion cell of a mouse myeloma cell-derived strain and mouse lymphocyte, and produces a monoclonal antibody (4E3) that binds to NP of H4-H15 subtype influenza virus. 4E3, for example, hybridoma Mouse-Mouse hybridoma 4E3 is cultured in RPMI 1640 medium (SIGMA R6540) containing 10% fetal serum, 10 mM L-Glutamine, 0.25% NaHCO ₃ at 37 ° C., 5% CO ₂ , The produced 4E3 can be produced by collecting and purifying by a known method.

  The anti-NP monoclonal antibodies of this aspect of the invention include fragments thereof, and modified and mutated antibodies such as chimeric and humanized antibodies thereof. These fragments, modified antibodies or mutant antibodies also have the same specificity for H5 subtype influenza virus as the original antibody. These can be produced by means and methods known to those skilled in the art.

  In another aspect, the present invention relates to a method for detecting an H5 subtype influenza virus in a sample, characterized by using the anti-HA antibody of the present invention. Two or more anti-HA antibodies may be used in the method of the present invention. By using two or more types of anti-HA antibodies, the sensitivity and specificity of the method of the present invention are increased and the accuracy is improved. The sample may be any sample that may contain H5 subtype influenza virus, such as throat swab, nasal swab, nasal aspirate, virus culture, feces, stool, various tissues・ Organs. By detecting the H5 subtype influenza virus in such a sample using the method of the present invention, whether the subject from which the sample is derived suffers from an infection caused by the H5 subtype influenza virus can be quickly, easily and reliably Can be diagnosed.

  In the detection method of the present invention, for example, the sample is incubated with the anti-HA antibody of the present invention to form a complex of the anti-HA antibody and the HA antigen in the sample, and then the complex is detected. , May be done. For example, the method of the present invention can be carried out using an immunochromatography method, an ELISA method, a Western blot method, an immunoblot method, or the like. The immunochromatography method is preferably used because it does not require special equipment / equipment and the like and can detect H5 subtype influenza virus quickly and easily at an actual medical site.

  The detection method of the present invention may be performed in combination with the anti-NP antibody of the present invention. Two or more anti-NP antibodies may be used in combination. By using an anti-NP antibody in combination, the sensitivity and specificity of the method of the present invention are further increased, and the accuracy is further improved. The antibody used in the method of the present invention may be labeled. Various labels are known and can be appropriately selected and used by those skilled in the art. Examples thereof include gold colloid, enzymes such as peroxidase, and particles such as gold particles. By attaching such a label, the method of the present invention can be performed efficiently.

  In still another aspect, the present invention relates to a kit for detecting H5 subtype influenza virus in a sample, comprising the anti-HA antibody of the present invention as an essential component. Two or more anti-HA antibodies may be included in the kit of the present invention. By including two or more types of anti-HA antibodies, the sensitivity, specificity, and accuracy of virus detection performed by the kit of the present invention can be improved.

  In the kit of the present invention, an immunochromatography method, an ELISA method, a Western blot method, an immunoblot method, or the like may be used. By using the immunochromatography method in the kit of the present invention, the virus can be detected more rapidly and easily.

  The kit of the present invention may further contain the anti-NP antibody of the present invention, and two or more anti-NP antibodies may be contained. By further including an anti-NP antibody, the sensitivity, specificity, and accuracy of virus detection performed by the kit of the present invention can be further improved. In addition to the anti-NP antibody of the present invention, the kit of the present invention contains, for example, other anti-HA antibody or anti-NP antibody, sampling means, containers for containing antibodies and reagents, labels, reaction containers, detection reagents and the like. May be included. In general, an instruction manual is attached to the kit. In the kit of the present invention, the antibody may be labeled. Since the H5 subtype influenza virus can be detected quickly and easily using the kit of the present invention, rapid diagnosis of infections caused by the H5 subtype influenza virus at clinical sites is possible. In addition, research and analysis of influenza viruses can be performed using the kit of the present invention.

  The present invention will be described in detail and specifically with reference to examples, but the examples are not intended to limit the present invention.

Preparation of monoclonal antibody-producing hybridoma that recognizes H5 subtype influenza virus Balb / c mice were immunized twice with H5N9 influenza virus. Three days later, the spleen of the immunized mouse was removed, and 1 × 10 ⁸ spleen cells were mixed with 3 × 10 ⁷ mouse myeloma cell-derived strain P3U1 cells. After centrifugation, the supernatant was aspirated and 1 ml of 50% polyethylene glycol solution was added at 37 ° C. over 1 minute. Next, 50 ml of RPMI 1640 medium was added over 10 minutes. After centrifugation, RMPI 1640 medium containing 10% fetal bovine serum was added and dispensed into 96-well microplates previously seeded with Balb / c mouse splenocytes. After culturing for about 10 days, the supernatant was screened for the presence or absence of antibodies by the enzyme antibody method using infected cells. The procedure of the enzyme antibody method is as follows. MDCK cells were incubated with H5 subtype influenza virus or H3 subtype influenza virus for 12 hours in a 96-well microplate to infect them. The infected MDCK cells were then fixed with ethanol and incubated with the hybridoma supernatant for 30 minutes at room temperature. After incubation, the plate was washed with PBS, and then reacted with peroxidase-labeled anti-mouse immunoglobulin antibody for 30 minutes. After the reaction, it was washed with PBS, and diaminobenzidine was added to visualize the anti-mouse immunoglobulin antibody. Antibody-producing hybridomas (Mouse-Mouse hybridoma 1C10 and Mouse-Mouse hybridoma 4E3) that did not react with H5 subtype influenza virus-infected cells and did not react with H3 subtype influenza virus-infected cells were obtained.

Identification of recognition site of monoclonal antibody that recognizes H5 subtype influenza virus Agglutination inhibition test between H5 subtype influenza virus and human blood cells and enzyme antibody method using influenza virus-infected MDCK cells shown in Table 1 (same as above) The recognition sites of monoclonal antibodies 1C10 and 4E3 produced by the resulting hybridoma were determined. The results are shown in Table 1 (inhibition test results are not shown). Since the 1C10 antibody has only reactivity with the H5 subtype influenza virus and inhibits the aggregation of the H5 subtype influenza virus and human blood cells, the 1C10 antibody can specifically recognize the H5 subtype influenza virus. It was confirmed that it was an anti-HA antibody. It was confirmed that the 4E3 antibody has reactivity with the H4-15 subtype influenza virus.

  In order to identify the recognition site of 4E3 antibody, an immunoprecipitation reaction method using Celluler Labeling and immunoprecipitation kit (Roche) was performed by the following procedure. MDCK cells were incubated with H3 or H5 influenza viruses for 24 hours. After incubation, the virus was recovered by centrifugation. The recovered virus was labeled with a luminescent substance (hereinafter also referred to as an antigen), and then an antibody was mixed to form a complex. Anti-mouse antibody binding beads were added to precipitate the complex. The precipitate was solubilized and electrophoresed. As a negative control for antigen, a culture supernatant (NC) derived from MDCK cells not infected with virus, as a positive control for anti-NP antibody, 7304 antibody known to bind to NP, and as a negative control for antibody, anti-NP B antibody (Anti-B), anti-A antibody (2 types; Anti-A) and F49 were used. The results are shown in FIG. When the H5 subtype influenza virus-derived antigen and 4E3 antibody were used, a band was confirmed at the position of the molecular weight of NP, and 4E3 antibody was identified as an anti-NP antibody.

＋は感染ＭＤＣＫ細胞と抗体が結合すること、−は結合しないことを示す。

+ Indicates that the infected MDCK cell and the antibody bind, and − indicates that the antibody does not bind.

Purification of Monoclonal Antibody Recognizing H5 Subtype Influenza Virus The resulting hybridomas Mouse-Mouse hybridoma 1C10 and Mouse-Mouse hybridoma 4E3 were prepared in RPMI 1640 medium containing 10% fetal serum, 10 mM L-Glutamine, 0.25% NaHCO ₃ ( In SIGMA R6504), the culture supernatant was recovered after culturing at 37 ° C. and 5% CO _{2 for} 5 days. The collected culture supernatant was purified using a protein G column (Pharmacia) to obtain anti-HA monoclonal antibody 1C10 and anti-NP monoclonal antibody 4E3.

Immunochromatographic method for detecting H5 subtype influenza virus by using together 1C10 and 4E3 antibodies. Materials and Methods 2 mg / ml 1C10 antibody was dropped and bound to a filter membrane for immunochromatography (Millipore). After drying, it was stored at 4 ° C. Gold colloid was bound to 30 μg / ml 4E3 antibody using a gold colloid kit (BBlnternational) and stored at −20 ° C. 25 μl of gold colloid-labeled 4E3 antibody diluted 10-fold with the reaction solution (0.5% BSA / 2% TritonX-100 / 0.1M Tris-HCl, pH 8.0) and 25 μl of the sample diluted with water were added to the microplate. Mix in wells. A 1C10 binding filter was erected in the prepared liquid and allowed to stand for 15 minutes. When a clear band was observed, it was determined as positive.

B. Detection of H5 subtype influenza virus Using H5N1, H5N2 and H5N3 influenza viruses, it was evaluated whether 1C10 antibody could detect H5 subtype influenza viruses other than H5N9 influenza virus. H5N9 influenza virus was used as a positive control. The results are shown in FIG. A clear band was observed in all of the H5N1, H5N2 and H5N3 influenza viruses as well as the positive control, and immunochromatography using the 1C10 and 4E3 antibodies was specific for the H5 subtype influenza virus in a short time of 15 minutes. It was confirmed that it could be detected.

C. Evaluation of sensitivity Using four serially diluted H5 subtype influenza virus (H5N1, H5N2, H5N3 and H5N9) solutions, the minimum detectable virus titer of each was determined, and the immunochromatographic method using 1C10 and 4E3 antibodies was used. Sensitivity was evaluated. Distilled water (DW) was used as a negative control. The results are shown in FIG. The minimum detectable virus titers of H5N1, H5N2, H5N3, and H5N9 are 5 × 10 ⁵ , 1 × 10 ⁷ , 3 × 10 ⁶ , 1 × 10 ⁴ FFU / ml (1 FFU (Focus Forming Unit) is 1 infectious particle, respectively. The immunochromatography method using 1C10 and 4E3 antibodies was found to have sufficient sensitivity.

D. Evaluation of specificity Influenza virus other than H5 subtype (virus titer 1 × 10 ⁷ FFU / ml or more), virus other than influenza virus (virus titer 1 × 10 ⁶ TCID 50 ml or more), and bacteria (candida albicans amount) Was sufficient for the experiment, and the others were 1 × 10 ⁶ CFU / ml or more) to evaluate the specificity of the immunochromatography method using 1C10 and 4E3 antibodies. H5N9 influenza virus was used as a positive control. The results are shown in Table 2 and Table 3, and FIG. Immunochromatographic results were negative for all viruses and bacteria other than influenza virus (Tables 2 and 3).

  INDUSTRIAL APPLICABILITY According to the present invention, anti-HA5 monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses, anti-NP monoclonal antibody capable of recognizing influenza A virus, and such antibody Since a method for detecting H5 subtype influenza virus and a kit therefor can be obtained, it is extremely useful in the field of type-by-type diagnosis of influenza A virus.

FIG. 1 shows the results of an immunoprecipitation reaction using H5 subtype influenza virus-derived antigen and 4E3 antibody. FIG. 2 shows the immunochromatographic results for H5N1, H5N2 and H5N3 influenza viruses. FIG. 3 shows the minimum detectable virus titer of H5N1, H5N2, H5N3 and H5N9 influenza viruses by immunochromatography. FIG. 4 shows the results of immunochromatography for H1N1, H2N2, H3N2, and influenza B virus.

Claims (10)

  An anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses.   The antibody according to claim 1, which is produced by a hybridoma having the National Institute of Advanced Industrial Science and Technology patent biological deposit center receipt number FERM AP-20821.   An anti-NP monoclonal antibody capable of recognizing influenza A virus.   The antibody according to claim 3, which is produced by a hybridoma having the National Institute of Advanced Industrial Science and Technology patent biological deposit center receipt number FERM AP-20822.   A method for detecting an H5 subtype influenza virus in a sample, wherein the antibody according to claim 1 is used.   The method according to claim 5, wherein the method is by immunochromatography.   The method according to claim 5, wherein the antibody according to claim 3 is used in combination.   A kit for detecting H5 subtype influenza virus in a sample, comprising the antibody according to claim 1 as an essential component.   The kit according to claim 8, wherein immunochromatography is used. The kit according to claim 8, further comprising the antibody according to claim 3.

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