WO2011087092A1

WO2011087092A1 - Anti-influenza antibody and influenza detection device

Info

Publication number: WO2011087092A1
Application number: PCT/JP2011/050558
Authority: WO
Inventors: 和良生田; 正大佐々木; 律子纐纈; アナリワドゥ; 里佳水池
Original assignee: 国立大学法人大阪大学
Priority date: 2010-01-15
Filing date: 2011-01-14
Publication date: 2011-07-21
Also published as: JP2013060367A

Abstract

Disclosed are: an antibody which enables the easy detection of an influenza type-A virus H1N1 subtype (2009); a test device which enables the easy detection of an influenza type-A virus H1N1 subtype (2009); and a method for detecting the H1N1 subtype (2009). Specifically disclosed is a monoclonal antibody produced using, as an antigen, a virus of A/Osaka/168/2009(pdm) strain and/or A/Suita/1/2009(pdm) strain, which is an influenza type-A virus H1N1 subtype (2009), specifically using particles of the virus. More specifically, the monoclonal antibody is produced from a hybridoma specified as accession No. FERM ABP-11329 or FERM ABP-11330 in accordance with the international deposition under the Budapest treaty.

Description

Anti-influenza antibodies and influenza detection devices

The present invention relates to a test device that can easily detect an anti-influenza A virus H1N1 subtype (2009) antibody and an influenza A virus H1N1 subtype (2009).

Influenza viruses belong to the Orthomyxoviridae family and are classified into three genera, type A, type B, and type C, and are referred to as influenza A virus, influenza B virus, and influenza C virus, respectively. In general, influenza viruses often refer to types A and B in particular. The difference between A-type, B-type and C-type is based on the antigenicity difference between M1 protein and NP protein among the proteins constituting the virus particle. In addition, even with the same type A and B, due to the difference in antigenicity of hemagglutinin (hereinafter also referred to simply as “HA”) and neuraminidase (NA), which are molecules on the surface of the envelope, For example, influenza A virus is classified into subtypes such as H1N1, H2N2, and H3N2. Since human influenza A virus periodically mutates HA and NA, the reactivity of conventional simple test kits using antibodies targeting the antigenicity of mutant viruses decreases. Or vaccinations corresponding to previous subtypes cannot be expected.

HA of influenza A virus is composed of regions with different structures, the head region and the stem region, which contain a receptor binding site for the virus to bind to the target cell. Involved in the hemagglutination activity of HA, the stem region contains a fusion peptide necessary for membrane fusion between the viral envelope and the endosomal membrane of the cell, and is involved in the fusion activity (Non-patent Document 1). Most anti-HA antibodies that recognize H1N1 and H3N2 subtypes of influenza A virus recognize the globular region of HA. However, this region is the most prone to antigenic mutations, and these antibodies do not react in common with human influenza A virus subtypes and lose their recognition as the viral HA antigen changes. There are many cases to do.

Since each subtype of influenza A virus has high variability in RNA genome, new strains are frequently generated. After the recognition of the epidemic in Mexico in April 2009, the influenza that is said to have spread worldwide is type A H1N1 subtype influenza, new influenza, swine influenza, pandemic influenza A (H1N1), swine flu, H1N1 It is called flu, A / H1N1 pdm. It is said that the virus that was prevalent among pigs was directly transmitted from pigs to humans on farms and then spread among humans. Hereinafter, in the present specification, the new influenza virus is referred to as “influenza A virus H1N1 subtype (2009)” and may be simply referred to as “H1N1 subtype (2009)”. H1N1 subtype (2009) is distinct from seasonal A Soviet influenza (influenza A virus H1N1 subtype) and A Hong Kong influenza (influenza A virus H3N2 subtype) Used.

Kits for easily detecting influenza A virus antigen or B virus antigen by immunochromatography are already commercially available and widely used (for example, manufactured by Becton Dickinson). However, as a method for detecting the above-mentioned H1N1 subtype (2009), only a gene amplification method such as PCR has been established, and a simple immunological detection kit such as immunochromatography has been put into practical use. It has not been. Therefore, for the detection of the H1N1 subtype (2009), if it is determined that the influenza A virus antigen is positive by the simple test method, a test by the gene amplification method is further performed as a secondary test. The situation was, the inspection was complicated, and it took time. Therefore, development of a simple inspection method that can easily detect the H1N1 subtype (2009) has been desired.

An object of the present invention is to provide an antibody that can easily detect influenza A virus H1N1 subtype (2009). Furthermore, it is an object to provide an inspection device that can easily detect the H1N1 subtype (2009), and further to provide a detection method of the H1N1 subtype (2009).

In order to solve the above problems, the present inventors have conducted extensive research, and as a result, influenza A virus H1N1 subtype (2009) A / Osaka / 168/2009 (pdm) strain or A / Suita / 1 It is based on a monoclonal antibody prepared using the virus of / 2009 (pdm) strain as an antigen, specifically, the virus particles as an antigen.

That is, this invention consists of the following.
1. Anti-influenza A virus H1N1 subtype (2009) -specific monoclonal antibody using influenza A virus H1N1 subtype (2009) as an antigen.
2. 2. The monoclonal antibody according to item 1, which is produced from a hybridoma specified by international deposit receipt number FERM ABP-11329 (domestic deposit number FERM P-21892).
3. 2. The monoclonal antibody according to item 1, which is produced from a hybridoma specified by international deposit receipt number FERM ABP-11330 (domestic deposit number FERM P-21893).
4). 4. The anti-influenza A virus H1N1 subtype according to any one of the preceding items 1 to 3, which is capable of antigen-antibody reaction against the hemagglutinin region or nucleoprotein of influenza A virus H1N1 subtype (2009) ) Monoclonal antibody.
5. A hybridoma for producing a monoclonal antibody identified by international deposit receipt number FERM ABP-11329 (domestic deposit number FERM P-21892).
6). A hybridoma for producing a monoclonal antibody identified by international deposit receipt number FERM ABP-11330 (domestic deposit number FERM P-21893).
7). A device for detecting influenza A virus H1N1 subtype (2009), comprising at least one monoclonal antibody according to any one of items 1 to 4.
8). The device for detecting influenza A virus H1N1 subtype (2009) according to item 7, wherein the device according to item 7 is a carrier for immunochromatography.
9. An influenza A virus H1N1 subtype (2009) detection kit comprising the influenza A virus H1N1 subtype (2009) detection device according to item 7 or 8.
10. Method for detecting influenza A virus H1N1 subtype (2009) comprising the following steps:
1) a step of contacting a specimen collected from a subject with at least one monoclonal antibody according to any one of 1 to 4 above;
2) a step of reacting an antigen in the sample with an anti-influenza A virus H1N1 subtype (2009) monoclonal antibody by an antigen-antibody reaction;
3) A step of detecting an antigen-antibody reaction product.

The H1N1 subtype (2009) can be easily detected by a monoclonal antibody that specifically reacts with the anti-influenza A virus H1N1 subtype (2009) of the present invention. Conventionally, detection of the H1N1 subtype (2009) had to be performed by a nucleic acid amplification method such as PCR by amplifying a gene specific for the H1N1 subtype (2009), whereas the present invention By using this antibody, an immunological test can be easily performed. In addition, by including antibodies specific for other types, such as seasonal influenza A virus H1N1 subtype and influenza A virus H3N2 subtype, in the measurement system, It can be distinguished and detected.

For example, existing seasonal influenza and so-called new influenza that prevailed in 2009 can be distinguished and easily detected.

It is a photograph figure which shows the result of the IF (fluorescence antibody method) test with respect to various influenza virus stocks about a monoclonal antibody (N-SW2-6, N-SW4-6, NC1-10, NC1-12). It is a photograph figure which shows the result of the IF (fluorescence antibody method) test with respect to various influenza virus stocks about a monoclonal antibody (N-SW1-1, N-SW4-5, N-SW5-1, N-SW5-6). . It is a photograph figure which shows the result of IF (fluorescence antibody method) test with respect to various influenza virus strains about the monoclonal antibody (N-SW5-7, N-SW5-8).

The anti-influenza A virus H1N1 subtype (2009) -specific monoclonal antibody of the present invention is a monoclonal antibody prepared using the influenza A virus H1N1 subtype (2009) as an antigen, and is simply referred to as “anti-H1N1 subtype ( 2009) Monoclonal antibody ". The anti-H1N1 subtype (2009) monoclonal antibody of the present invention is derived from mammals, and examples thereof include mouse type, rat type, hamster type, rabbit type, goat type, and horse type. The antibody is not limited to IgG, but may be IgM.

As a method for producing the anti-H1N1 subtype (2009) monoclonal antibody of the present invention, a method known per se or any method developed in the future can be adopted. A monoclonal antibody and a hybridoma producing the monoclonal antibody can be prepared specifically by the method described below by fusing spleen cells derived from an immunized animal with various myeloma cells.

The antigen for producing the anti-H1N1 subtype (2009) monoclonal antibody of the present invention is not particularly limited as long as it is an antigen derived from influenza A virus H1N1 subtype (2009). Particularly preferably, the influenza A virus H1N1 subtype (2009), A / Osaka / 168/2009 (pdm) strain and / or A / Suita / 1/2009 (pdm) strain virus is used as an antigen. it can. As the viral antigen, for example, a virus inactivated by a method known per se can be used, for example, an influenza virus vaccine strain inoculated into an embryonated chicken egg and a influenza HA antigen prepared by a method known per se is used. be able to. The mammal is immunized by administration of the antigen, preferably with an adjuvant.

In the production of the monoclonal antibody of the present invention, an antigen solution obtained by dissolving or suspending the antigen in an appropriate buffer such as a phosphate buffer (PBS) can be used. The antigen solution is usually prepared to a concentration containing about 50 to 500 μg / mL of the antigen substance. Examples of animals immunized with the antigen include mice, rats, hamsters, horses, goats, and rabbits.

At this time, in order to enhance the responsiveness of the immunized animal to the antigen, the antigen solution can be mixed with an adjuvant and administered. Adjuvants that can be used here are Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Boredetella pertussis vaccine), Muramyl Examples include dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof, but combinations using FCA at the time of primary immunization and FIA or Ribi adjuvant at the time of booster immunization are particularly preferable.

The immunization method can appropriately change the injection site, schedule, etc. depending on the type of antigen to be used and the presence / absence of adjuvant mixing. For example, when a mouse is used as the immunized animal, an adjuvant mixed antigen solution 0.05 to 1 is used. Inject intraperitoneally, subcutaneously, intramuscularly (in the tail) vein with mL (antigen substance 10-200 μg), and perform additional immunization 1 to 4 times every 4 to 21 days from the first immunization, and further about 1 to 4 weeks A final immunization is performed later. The antigen solution can be administered without using an adjuvant by increasing the amount of antigen and intraperitoneal injection. The antibody titer is examined by collecting blood about 5 to 10 days after the boost. The antibody titer can be measured by a conventional method according to the antibody titer assay described below. Approximately 3 to 5 days after the final immunization, spleen cells are separated from the immunized animal to obtain antibody-producing cells.

The anti-H1N1 subtype (2009) monoclonal antibody of the present invention can be prepared, for example, according to the method of Kohler and Milstein (Kohler and Milstein, Nature 256, 495-497, 1975). As myeloma cells, those derived from mice, rats, humans, etc. are used, such as mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2 / O-Ag14, P3X63-Ag8 / 653, PAI, etc. Examples are established myeloma cells. Some myeloma cells produce an immunoglobulin light chain, and if this is used as a fusion target, the immunoglobulin heavy chain produced by the antibody-producing cell and this light chain may bind randomly. In particular, it is preferable to use myeloma cells that do not produce an immunoglobulin light chain, such as P3X63-Ag8 · 653, SP2 / O-Ag14, and PAI. The antibody-producing cells and the myeloma cells are preferably derived from the same species, particularly the same strain. The myeloma cells may be stored according to a method known per se. For example, those subcultured in a general medium supplemented with fetal calf serum (FCS) are stored by freezing. For cell fusion, cells in the logarithmic growth phase are preferably used.

Examples of methods for producing hybridomas by fusing antibody-producing cells and myeloma cells include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device. For example, in the case of the PEG method, spleen cells and myeloma cells are placed in an appropriate medium or buffer containing about 30 to 60% PEG (average molecular weight 1,000 to 6,000) in an amount of 1 to 10: 1, preferably 5 to 10: 1. The mixture may be suspended at a temperature of about 25 to 37 ° C. and pH 6 to 8 for about 30 seconds to 3 minutes. After completion of the reaction, the cells are washed, removed from the PEG solution, resuspended in the medium, seeded in a microtiter plate, and the culture is continued.

Cells after the fusion operation are cultured in a selective medium to select hybridomas. The selection medium is a medium in which the parent cell line can be killed and only the fused cells can grow, and usually a HAT (hypoxanthine, aminopterin, thymidine) medium is used. Selection of hybridoma is usually carried out by exchanging a part of the medium, preferably about half of the medium, with the selective medium 1 to 7 days after the fusion operation, and further culturing while repeating the same medium exchange every 2 or 3 days. The well where the hybridoma colony is growing is confirmed by microscopic observation.

The anti-H1N1 subtype (2009) monoclonal antibody of the present invention can be prepared more specifically by the following method. Influenza virus antigen is intraperitoneally administered 2-3 times to 4-week-old BALB / c mice, and spleens are removed 3 to 4 days after the final immunization to obtain antibody-producing cells. Hybridomas can be easily and efficiently produced by cell fusion of the antibody-producing cells and PAI cells that are mouse myeloma cells by the PEG method.

In order to know whether the growing hybridoma is producing the desired antibody, the culture supernatant is collected and an antibody titer assay may be performed by a method known per se. Specifically, antibody-producing cells having an activity to bind to influenza virus-derived proteins can be obtained by using influenza-infected cells as antigens, by IC (immunocytochemistry), IF (immunofluorescence), IHC (immunohistochemistry) sputum staining, etc. Can be sorted.

Furthermore, a single clone producing the monoclonal antibody of the present invention can be isolated by a limiting dilution method, a soft agar method, a method using a fluorescence excitation cell sorter, or the like. For example, in the case of the limiting dilution method, a hybridoma clone that produces the target antibody can be isolated by serially diluting a hybridoma colony with a medium so as to be about 1 cell / well. The obtained antibody-producing hybridoma clone is frozen in the presence of about 10% dimethyl sulfoxide (DMSO) or glycerin, or a cryoprotectant such as a commercially available cell banker ^TM , and stored at -70 to -196 ° C. It can be stored for half a year to semi-permanently. The cells are used after being rapidly thawed in a constant temperature bath at around 37 ° C. It is desirable to use after thoroughly washing so that the cytotoxicity of the cryoprotectant does not remain.

The method for obtaining a monoclonal antibody from a hybridoma can be appropriately selected depending on the required amount and the properties of the hybridoma. Examples thereof include a method of obtaining from the mouse ascites transplanted with the hybridoma and a method of obtaining from the culture supernatant by cell culture. If it is a hybridoma capable of growing in the mouse abdominal cavity, a high concentration monoclonal antibody of several mg / mL can be obtained from ascites. Hybridomas that cannot grow in vivo can be obtained from the culture supernatant of the cell culture. Obtaining monoclonal antibodies by cell culture has the advantage that antibody production is lower than that obtained in vivo, but there is little contamination with immunoglobulins and other contaminants contained in the mouse abdominal cavity, and purification is easy. .

When obtaining a monoclonal antibody from the abdominal cavity of a mouse transplanted with a hybridoma, for example, the abdominal cavity of a BALB / c mouse that has been previously administered with an immunosuppressive substance such as pristane (2, 6, 10, 14-tetramethylpentadecane). The hybridoma (about 10 ⁶ or more) is transplanted into it, and the ascites collected after about 1 to 3 weeks is collected. In the case of heterologous hybridomas (for example, mice and rats), it is preferable to use nude mice or radiation-treated mice.

On the other hand, when the antibody is obtained from the cell culture supernatant, for example, in addition to the stationary culture method used for cell maintenance, the hybridoma is cultured by using a culture method such as a high-density culture method or a spinner flask culture method. A culture supernatant containing is obtained. The serum contained in the culture solution contains other contaminants such as antibodies and albumin, and antibody purification is often complicated, so it is desirable to reduce the addition to the culture solution. More preferably, the hybridoma is acclimated to a serum-free medium by a conventional method and cultured using the serum-free medium. Antibody purification is facilitated by culturing in a serum-free medium.

Purification of monoclonal antibody from ascites or culture supernatant can be performed by a method known per se. For example, as a method for purifying immunoglobulins, a fractionation method by salting out using ammonium sulfate or sodium sulfate, a polyethylene glycol (PEG) fractionation method, an ethanol fractionation method, a DEAE ion exchange chromatography method, a gel filtration method, etc. It is easily achieved by applying. Further, when the monoclonal antibody is IgG, it can be easily purified by affinity chromatography using a protein A or protein G binding carrier.

The anti-H1N1 subtype (2009) monoclonal antibody of the present invention is specifically an influenza A virus H1N1 subtype (2009), A / Osaka / 168/2009 (pdm) strain and / or A / Suita It is possible to produce a virus of / 1/2009 (pdm) strain as an antigen. Such a monoclonal antibody is, for example, a monoclonal antibody (N-SW2-6) or Mouse-Mouse produced from Mouse-Mouse hybridoma N-SW2-6 (hereinafter referred to as “hybridoma N-SW2-6”). and monoclonal antibody (N-SW4-6) produced from hybridoma N-SW4-6 (hereinafter referred to as “hybridoma N-SW4-6”). Each of the above hybridomas was established on January 15, 2010 in the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (IPOD) (6th Tsukuba Center 1-1-1 Tsukuba City, Ibaraki Prefecture 305-8566). An application for deposit was made, and hybridoma N-SW2-6 was deposited under the accession number FERM P-21892 and hybridoma N-SW4-6 under the accession number FERM P-21893. Later, on January 11, 2011, an application for transfer to an international deposit under the Budapest Treaty was also filed at IPOD, and international deposit receipt numbers FERM ABP-11329 and FERM ABP-11330 were assigned to these hybridomas, respectively. In addition to the monoclonal antibodies described above, Mouse-Mouse hybridomas prepared by the above-described methods (N-SW1-1, N-SW4-5, N-SW5-1, N-SW5-6, N-SW, which are hybridomas, respectively) Monoclonal antibodies produced from SW5-7, N-SW5-8), such as N-SW1-1, N-SW4-5, N-SW5-1, N-SW5-6, N-SW5-7, N -SW5-8 can be acquired.

The present invention extends to each anti-influenza A virus H1N1 subtype (2009) specific monoclonal antibody produced from the hybridomas identified by international deposit receipt numbers FERM ABP-11329 and FERM ABP-11330, and these monoclonal clones. It extends to hybridomas for antibody production.

The present invention further extends to a device for detecting influenza A virus H1N1 subtype (2009) comprising the above monoclonal antibody. Examples of such a device include a device for immunoassay containing at least one of the above monoclonal antibodies, and examples thereof include a carrier for immunochromatography, a carrier for immunodiffusion measurement, a carrier for ELISA, and the like. Preferably, for example, a carrier for immunochromatography or a carrier for immunodiffusion measurement. The immunoassay device includes at least the anti-influenza A virus H1N1 subtype (2009) monoclonal antibody of the present invention, and further includes a seasonal anti-influenza A virus H1N1 subtype-specific antibody and an anti-influenza A virus H3N2 Subtype-specific antibodies can also be included. By comparing the reactivity with these antibodies, it is possible to determine the type of influenza virus that may be mixed in the subject's sample in a single test. Furthermore, the present invention extends to an influenza A virus H1N1 subtype (2009) detection kit including a device for detecting influenza A virus H1N1 subtype (2009).

The present invention also extends to a method for detecting influenza A virus H1N1 subtype (2009) comprising the following steps.
1) A step of contacting a sample collected from a subject with the anti-H1N1 subtype (2009) monoclonal antibody of the present invention;
2) antigen-antibody reaction of the antigen in the specimen and the anti-H1N1 subtype (2009) monoclonal antibody described above;
3) A step of detecting an antigen-antibody reaction product.

The detection of the antigen-antibody reaction product in the detection method of the influenza A virus H1N1 subtype (2009) is not particularly limited as long as it is a method capable of detecting the antigen-antibody reaction, but for example, IC (immunocytochemistry), IF In addition to staining methods such as (immunofluorescence) and IHC (immunohistochemistry) sputum, immunochromatography, immunodiffusion measurement, ELISA, and the like can be mentioned.

In order to help understanding of the present invention, the present invention will be specifically described with reference to the following examples, but it is needless to say that the present invention is not limited to the examples.

Example 1 Production of Anti-H1N1 Subtype (2009) Monoclonal Antibody In this example, a monoclonal antibody was produced using hybridomas identified by international deposit receipt numbers FERM ABP-11329 and FERM ABP-11330. Things will be described.

1) Virus antigen Influenza A virus H1N1 subtype (2009) was isolated from the A / Osaka / 168/2009 (pdm) strain distributed by the Osaka Prefectural Public Health Research Institute, or the National University Corporation Osaka University Using A / Suita / 1/2009 (pdm) strain, each was infected with MDCK cells (canine renal epithelial cell strain) and cultured at 37 ° C. for 2 to 3 days in the presence of trypsin, and then the culture supernatant was collected. Each influenza virus culture supernatant was concentrated by ultracentrifugation at 25,000 rpm, and virus particles inactivated with formalin were used as virus antigens.

2) Immunization method using a viral antigen A 4-week-old BALB / c mouse is immunized by intraperitoneally administering the viral antigen prepared by the above method three times. Specifically, 50-500 mg / animal of viral antigen is first mixed with Freund's complete adjuvant (FCA), the second is mixed with Freund's incomplete adjuvant (FIA), and the third is administered intraperitoneally without adjuvant. went. The second and subsequent immunizations were carried out about every 2 weeks from the first immunization, and the spleen was removed about 3 to 4 days after the third immunization and used for the production of hybridomas.

3) Preparation of hybridoma The spleen derived from the immunized mouse was crushed and washed with serum-free DMEM before cell fusion to obtain a spleen cell as a hybridoma preparation cell. PAI cells, which are IL-6-independent mouse myeloma cells, were used as partner cells for hybridoma production. PAI cells were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS) for 2 days and then washed with serum-free DMEM before cell fusion.

Next, the spleen cells and PAI cells obtained above were mixed at a cell number ratio of 1: 5 to 1:10 and centrifuged to remove the supernatant. After sufficiently loosening the precipitated cell mass, 0.6 mL of a 50% PEG1500-PBS solution was slowly added over 1 minute while stirring, and then 10 mL of serum-free DMEM was added slowly over 2 minutes. Further, 10 mL of DMEM supplemented with 15% FCS was added to complete the cell fusion. Next, the supernatant was removed after centrifugation and washed with 20 mL of serum-free DMEM. Finally, loosen the cells gently and add HAT medium {15% FCS-added DMEM with medium additives for mouse hybridoma such as HAT (hypoxanthine, aminopterin, thymidine) and BM condimed H1 ^TM (Roche) } Was added, and the cells were gently suspended using a measuring pipette.

4) Cloning of hybridoma The cell suspension of 3) above is dispensed into five 96-well microplates for culture and cultured at 37 ° C. for 7 to 10 days in an incubator containing 5% CO ₂ . During this period, half of the HAT medium was changed at intervals of 3 to 4 days. Subsequently, a portion of the culture supernatant was taken and screened for hybridomas.

The IF (immunofluorescent antibody) method was used for screening. MDCK cells were seeded in a 96-well culture microplate and cultured overnight at 37 ° C. in an incubator containing 5% CO ₂ . After washing the cells with PBS (−), various influenza virus diluted solutions appropriately diluted with serum-free MEM were added, and the cells were cultured at 37 ° C. for 6 to 10 hours. By adding 4% formalin-added PBS solution to fix cells and inactivate influenza virus, by permeabilizing infected cells with 1% Triton-X (polyoxyethylene-p-isooctylphenol) -added PBS, A plate for IF (immunofluorescence) was prepared. After the IF plate was washed with PBS (−), 50 μL of the hybridoma culture supernatant stock solution was added to each well and allowed to react at room temperature for 30 minutes to 1 hour. After washing with PBS (−), 40 μL of FITC-labeled anti-mouse antibody was added to each well and reacted at room temperature for 30 minutes to 1 hour to prepare a FITC-labeled immune complex. After washing with PBS (-), anti-influenza mouse antibodies were detected in the culture supernatant with a fluorescence microscope.

Next, the cells contained in each well of the culture microplate in which the cells in which the antibody production was confirmed were growing were taken out, the limiting dilution method was performed three times, and the target cells were cloned by the same method. Of the cloned hybridomas, hybridoma N-SW2-6 was obtained by immunizing mice with an antigen derived from the A / Osaka / 168/2009 (pdm) strain three times. Hybridoma N-SW4 -6 shows that among the three immunizations, the first immunization was performed using an antigen derived from the A / Osaka / 168/2009 (pdm) strain, and the second and third immunizations were performed using A / Suita / 1/2009 ( pdm) was obtained by immunization with an antigen derived from a strain.

5) Purification of antibody Each hybridoma was reduced in the FCS content in the culture solution from 10% to 2%, and finally cultured in a serum-free culture solution. 100 ml of the culture supernatant of each hybridoma cultured in serum-free medium for 3-7 days is centrifuged at 2,000 rpm for 10 minutes, and the resulting supernatant is filtered through a 0.45 μm filter to remove solid components. Was purified with 1 mL of a 6% agarose gel (HiTrap Protein G HP ^™ , manufactured by GE Healthcare). The monoclonal antibody produced by the hybridoma N-SW2-6 was designated N-SW2-6, and the monoclonal antibody produced by the hybridoma N-SW4-6 was designated N-SW4-6. These hybridomas were applied for domestic deposit on January 15th 2010 at the Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology, and for hybridoma N-SW2-6, accession number FERM P-21892, and Hybridoma N-SW4-6 was accepted under the accession number FERM P-21893. Later, on January 11, 2011, an application for transfer to an international deposit under the Budapest Treaty was also filed at IPOD, and international deposit receipt numbers FERM ABP-11329 and FERM ABP-11330 were assigned to these hybridomas, respectively.

In addition to the above, hybridomas produced by the above-mentioned method and cloned are N-SW1-1, N-SW4-5, N-SW5-1, N-SW5-6, N-SW5-7 and N-SW4-7. Here, N-SW1-1 was obtained by immunizing a mouse twice with an antigen derived from the A / Osaka / 168/2009 (pdm) strain, and N-SW4-5, N-SW5-1 , N-SW5-6, N-SW5-7 and N-SW4-7 are prepared by using an antigen derived from A / Osaka / 168/2009 (pdm) strain as the first immunization among the three immunizations. The first and third immunizations were obtained by immunization using an antigen derived from the A / Suita / 1/2009 (pdm) strain.

(Comparative Example 1) Production of anti-H1N1 subtype (seasonal influenza virus) monoclonal antibody As an H1N1 subtype (seasonal influenza virus), A / New Caledonia / 20/1999 vaccine strain is used as an antigen, and monoclonal antibody-producing hybridoma Was made. The influenza antigen was prepared by the following method. The above influenza virus vaccine strain was inoculated into embryonated chicken eggs, cultured at 33-35 ° C. for 2 days, and then allowed to stand at 4 ° C. overnight to collect the infected allantoic fluid. Subsequently, it concentrated by the ultrafiltration method etc., and the virus particle was refine | purified by the sucrose density gradient centrifugation method. That is, ultracentrifugation was performed at a rotational speed of 35,000 rpm in a sucrose density gradient of 0 to 60%, and fractions with a sucrose density of around 40% were collected. After this concentrated virus fraction was treated with ether, formalin was added and further purified by sucrose density gradient centrifugation to obtain influenza HA antigen.

The mice were immunized three times with the same method as in Example 1 using the influenza HA antigen prepared by the method described above to prepare hybridomas. Two hybridomas were cloned, and each of the hybridomas NC1-10 and NC1-12 was cloned. I got it.

(Experimental Example 1) Properties of each hybridoma culture supernatant The antigen-antibody reactivity of each hybridoma culture supernatant prepared in Example 1 and Comparative Example 2 against various influenza viruses was used for screening at the time of hybridoma cloning. Performed according to IF method. That is, MDCK cells are seeded on a 96-well microplate for culture, cultured overnight at 37 ° C. in an incubator containing 5% CO ₂ , washed with PBS (−), and then added with no serum. Various influenza virus diluted solutions shown in the remarks in Table 1 that were appropriately diluted with MEM were added, followed by culturing at 37 ° C. for 6 to 10 hours. A 4% formalin-added PBS solution was added to fix cells and inactivate influenza virus, and the infected cells were permeabilized with 1% Triton-X-added PBS to prepare IF plates. After the IF plate was washed with PBS (−), 50 μL of the hybridoma culture supernatant stock solution was added to each well and allowed to react at room temperature for 30 minutes to 1 hour. After washing with PBS (−), 40 μL of FITC-labeled anti-mouse antibody was added to each well and reacted at room temperature for 30 minutes to 1 hour to prepare a FITC-labeled immune complex. After washing with PBS (-), the presence or absence of antigen-antibody reaction was examined with a fluorescence microscope.

The results of FITC immunostaining are shown in FIG. 1-3. As a result of observing the staining pattern of the culture supernatant of each hybridoma against cells containing each virus strain shown in the remarks of Table 1 below, the culture supernatant of hybridoma N-SW2-6 reacts with HA and is similarly a hybridoma. Each culture supernatant of N-SW4-6, N-SW1-1, N-SW4-5, N-SW5-1, N-SW5-6, N-SW5-7 and N-SW4-7 reacts with NP It was thought that it was doing. Moreover, it was considered that each culture supernatant of NC1-10 and NC1-12, which are hybridomas of Comparative Examples, reacted with HA. The reactivity in the IF method is shown in Table 1. As a result, each of the culture supernatants of the hybridomas NC1-10 and NC1-12 of the comparative example reacts with various types of seasonal influenza of the influenza A virus H1N1 subtype, but the two types of viruses of the H1N1 subtype (2009) It did not react. Meanwhile, hybridomas N-SW2-6, N-SW4-6, N-SW1-1, N-SW4-5, N-SW5-1, N-SW5-6, N-SW5-7 and N-SW4 -7 culture supernatants did not respond or weakly responded to seasonal influenza of H1N1 subtype, whereas two cultures of H1N1 subtype (2009) Reacted.

(Experimental example 2) Properties of culture supernatant of each hybridoma Each hybridoma N-SW2-6, N-SW4-6, N-SW4-5, N-SW5-6, N-SW5-7 prepared in Example 1 The antigen-antibody reactivity of these culture supernatants to various influenza viruses was performed according to the IF method used for screening at the time of hybridoma cloning. That is, antigen-antibody reactivity was confirmed by the same method as in Experimental Example 1 except that the influenza virus strains used in the IF method were the strains shown in Table 2.

The reactivity in the IF method is shown in Table 2. As a result, each of the culture supernatants of hybridomas N-SW4-6, N-SW4-5, N-SW5-6 and N-SW5-7 was subcultured of H3N8, H4N5, H5N1, H10N7, H11N6, H12N5 and H14N5. It was confirmed to react to either type of virus. Unlike seasonal influenza, these viruses and H1N1 subtype (2009) virus are common in that they are viruses derived from animals other than humans. Monoclonal antibodies produced from each of the above hybridomas are non-human antibodies. It was suggested that the antibody recognizes a consensus sequence epitope of animal-derived virus NP. On the other hand, the culture supernatant of hybridoma N-SW2-6 does not react with the various viruses shown in Table 2. For example, monoclonal antibody (N-SW2-6) and monoclonal antibody (N-SW4-6) Suggested different epitopes for influenza virus.

As described in detail above, the influenza A virus H1N1 subtype (2009) can be easily detected with the anti-H1N1 subtype (2009) monoclonal antibody of the present invention. Conventionally, detection of the influenza A virus H1N1 subtype (2009) had to be performed by a nucleic acid amplification method such as PCR by amplifying a gene specific for the H1N1 subtype (2009). On the other hand, an immunological test can be easily performed by using the antibody of the present invention. In addition, by including antibodies specific for other types, such as seasonal influenza A virus H1N1 subtype and influenza A virus H3N2 subtype, in the test system, It can be distinguished and detected.

For example, it is possible to easily distinguish existing seasonal influenza from so-called new influenza that was prevalent in 2009 and easily detect it.

Claims

Anti-influenza A virus H1N1 subtype (2009) -specific monoclonal antibody using influenza A virus H1N1 subtype (2009) as an antigen.
The monoclonal antibody according to claim 1, which is produced from a hybridoma identified by international deposit receipt number FERM ABP-11329.
The monoclonal antibody according to claim 1, which is produced from a hybridoma specified by international deposit receipt number FERM ABP-11330.
The anti-influenza A virus H1N1 subtype according to any one of claims 1 to 3, which is capable of antigen-antibody reaction with a hemagglutinin region or a nucleoprotein of influenza A virus H1N1 subtype (2009). 2009) Monoclonal antibody.
A hybridoma for producing a monoclonal antibody identified by international deposit receipt number FERM ABP-11329.
A hybridoma for producing a monoclonal antibody identified by international deposit receipt number FERM ABP-11330.
A device for detecting influenza A virus H1N1 subtype (2009), comprising at least one monoclonal antibody according to any one of claims 1 to 4.
The device for detecting influenza A virus H1N1 subtype (2009) according to claim 7, wherein the device according to claim 7 is a carrier for immunochromatography.
An influenza A virus H1N1 subtype (2009) detection kit comprising the influenza A virus H1N1 subtype (2009) detection device according to claim 7 or 8.
Method for detecting influenza A virus H1N1 subtype (2009) comprising the following steps:
1) contacting a specimen collected from a subject with at least one monoclonal antibody according to any one of claims 1 to 4;
2) a step of reacting an antigen in the sample with an anti-influenza A virus H1N1 subtype (2009) monoclonal antibody by an antigen-antibody reaction;
3) A step of detecting an antigen-antibody reaction product.