WO2005007697A1

WO2005007697A1 - Anti-influenza type a virus monoclonal antibody and immunoassay instrument using the antibody

Info

Publication number: WO2005007697A1
Application number: PCT/JP2004/010475
Authority: WO
Inventors: Jun-Ichi Azumi; Takashi Yamada; Tomoe Honda; Nobuyuki Fujii
Original assignee: Fujirebio Inc.
Priority date: 2003-07-23
Filing date: 2004-07-23
Publication date: 2005-01-27
Also published as: KR101133910B1; HK1105005A1; RU2366662C2; RU2006103301A; CN1934133A; JP4595810B2; CN1934133B; JPWO2005007697A1; KR20060054332A

Abstract

An anti-influenza type A virus monoclonal antibody of high specificity; and an immunoassay instrument comprising the same that is capable of specifically detecting influenza type A viruses. This anti-influenza type A virus monoclonal antibody performs an antigen antibody reaction with the nucleoprotein of 55 to 70 kD molecular weight of influenza type A virus but substantially does not perform any antigen antibody reaction with influenza type B viruses.

Description

Specification

Anti-influenza A virus monoclonal antibody and immunoassay instrument using the antibody

Technical field

The present invention relates to an anti-influenza A virus monoclonal antibody and an immunoassay device using the antibody.

Background art

[0002] Influenza has repeatedly repeated a global epidemic since ancient times, causing many deaths each time. However, in 1931, the causative virus was detected, paving the way for elucidation of the cause. Influenza pathogen viruses are classified into three types, A, B or C, depending on the antigenicity of soluble nucleoprotein (NP) in the virus. Influenza A is further classified into subtypes according to the antigenicity of hemagglutinin (HA), neuraminidase (NA) and two envelope fe proteins present on the virus surface.

[0003] Since 1972, for influenza prevention, actin treated with ether and inactivated with formalin has been used. In recent years, in addition to vaccines used for prevention, anti-influenza drugs have been found and widely used as therapeutic drugs. These treatments include amantadine hydrochloride for influenza A virus, xanavir and oseltamivir phosphate for influenza A and B viruses.

[0004] In selecting these therapeutic agents, it is important to detect the influenza virus in the sample and identify whether the infection is due to the influenza virus and the virus type is A or B. It is. In addition, influenza A virus causes severe symptoms that are more infectious than influenza virus B, and identification of the infectious virus is even more important for early treatment.

[0005] Conventionally, influenza viruses have been detected with anti-influenza virus antibodies. Up to now, antibodies against influenza A virus have been used to determine the subtype of the virus that is expected to spread and use it for vaccine production. Antibodies that specifically recognize DNA and NA and identify virus subtypes have been known (for example, see (Patent Document 1) and (Patent Document 2)). The amino acid sequence of the nucleoprotein for each influenza virus type A subtype has been determined and reported to the National Library of Medicine; Entrez Nucleotides database.

[0006] Further, as a device for detecting influenza virus, for example, a solid phase capable of binding to an influenza virus antigen is prepared, and after reacting the influenza virus in a sample, the influenza virus further bound to a first enzyme is used. A flow-through type device is used in which an influenza virus B antibody to which an A virus antibody and a second enzyme are bound is reacted with the solid phase, a substrate is added and the reaction is performed, and the color development on the solid phase is visually observed. Known (see (Patent Document 3)). Although this device uses an antibody against the nuclear protein of influenza virus as the antibody in the reagent, the measurement operation was complicated and was not a simple measurement device. Furthermore, an immunoassay device using a monoclonal antibody against influenza virus nucleoprotein (hereinafter referred to as "immunochromatography device") is known (see (Non-Patent Document 1)).

[0007] On the other hand, an immunochromatographic instrument using a strip-shaped matrix capable of infusion has been developed, and only a sample is spotted on a predetermined location, and a special detecting device and a skilled measuring technique are not required, so that it can be performed in a short time. It has become possible to easily measure an antigen or an antibody in a sample. Depending on the substance to be measured, for example, the immunochromatography device can bind multiple treponema pallidum antigens (TP antigens) to the detection zone and detect multiple anti-TP antibodies in the sample in separate detection zones. Instruments have been developed (see (Patent Document 4)). The detection zone of this device has multiple zones to which different TP antigens are bound.In one measurement, different anti-TP antibodies are separately detected to identify the time of infection and to select therapeutic agents. It is used for such purposes.

[0008] Patent Document 1: JP-A-6-100594

Patent Document 2: JP-A-7-304799

Patent Document 3: JP-A-2001-124775

Patent Document 4: JP-A-9-229938

Non-Patent Document 1: Journal of Infectious Diseases 2001; 75; 792-799 Disclosure of the invention

Problems to be solved by the invention

[0009] Conventional anti-influenza A virus monoclonal antibodies are satisfactory for high-sensitivity immunoassay with low specificity and reactivity against influenza A virus, although antibodies against nucleoprotein are used in immunochromatography and the like. I couldn't do it. In addition, antibodies that respond to all influenza A viruses including influenza virus HA and NA mutated subtypes are required, although they do not react with influenza B virus in order to select a therapeutic agent for influenza. Was.

[0010] Furthermore, the determination of influenza A virus or B virus can be performed in a short time without using any special diagnostic equipment or measuring equipment, and it is possible to determine A or B in one operation. There was a need for a device that could do it.

Therefore, an object of the present invention is to provide an anti-influenza A virus monoclonal antibody having high specificity. Another object of the present invention is to provide an immunoassay device capable of specifically detecting influenza A virus.

Means for solving the problem

[0012] As a result of intensive studies, the present inventors have succeeded in producing an anti-influenza A virus-less monoclonal antibody that specifically reacts with influenza A virus using the nucleoprotein of influenza A virus as an antigen. Thus, the present invention has been completed. Also, the present inventors have conceived that it is possible to provide an immunoassay device capable of distinguishing and detecting influenza A virus from influenza B virus by using the influenza A virus monoclonal antibody.

[0013] That is, the present invention relates to an anti-influenza A virus, which reacts with an influenza A virus nuclear protein having a molecular weight of 5570 kD as an antigen-antibody and does not substantially react with an influenza B virus as an antigen-antibody. Provide a monoclonal antibody or an antigen-binding fragment thereof. Further, the present invention immobilizes a labeled reagent zone having a movable labeled anti-influenza A virus antibody, a sample spotting zone, a developing solution supply zone, a developing solution absorption zone, and an anti-influenza A virus antibody to a matrix. An influenza A virus-free detection zone provided in a matrix, wherein the labeled anti-influenza A virus is provided. The present invention provides an immunoassay device in which at least one of the virus antibody and the anti-influenza A virus antibody immobilized in the detection zone is the monoclonal antibody of the present invention.

According to the present invention, an anti-influenza A virus monoclonal antibody that does not react with influenza B virus is provided. By performing an immunoassay using the anti-influenza A virus monoclonal antibody of the present invention, influenza A virus can be distinguished from influenza B virus and detected or quantified. According to the present invention, there is further provided an immunoassay device using the anti-influenza A virus monoclonal antibody of the present invention. By using the immunoassay device of the present invention, influenza A virus can be easily and distinctively detected from influenza B virus. Therefore, the present invention is expected to greatly contribute to the diagnosis and treatment of influenza.

Brief Description of Drawings

FIG. 1 is a view showing the results obtained when an antigen fractionated by Western blotting was reacted with the monoclonal antibody of the present invention.

FIG. 2 is a view showing a restriction map of an expression plasmid (pW6A) used in Example 4.

FIG. 3 is a view showing the amino acids A1 to A7 obtained by further subdividing the influenza H1N1 A fragment.

FIG. 4 is a view showing a CBB staining pattern and a Western blot for confirming the reactivity with FVA2_11.

FIG. 5 is a view showing a nucleoprotein amino acid sequence (80-140) of influenza A virus subtype.

FIG. 6 is a cross-sectional view schematically showing one example of a measuring instrument embodiment of the present invention.

FIG. 7 is a plan view showing an example when the measuring instrument of the present invention is set in a cassette.

FIG. 8 is a sectional view taken along line AA ′ of FIG. 7.

BEST MODE FOR CARRYING OUT THE INVENTION

[00161] As described above, influenza A virus has a molecular weight of 55 70 kD It reacts with the original antibody. The antigen-antibody reaction with the influenza A virus nuclear protein with a molecular weight of 55-70 kD can be achieved by using influenza A virus as a sample and incorporating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into the sample. It can be confirmed by the plotting method. When a Western plot (see Examples below) is performed, the monoclonal antibody of the present invention recognizes a nucleoprotein having a molecular weight of 5570 kD.

[0017] As described above, the monoclonal antibody of the present invention does not substantially react with an influenza B virus. Here, "substantially no antigen-antibody reaction" means that there is no antigen-antibody reaction at a detectable level, or even if an antigen-antibody reaction occurs, the degree of the antigen-antibody reaction with influenza A virus The fact that the antigen-antibody reaction with the influenza A virus, which is clearly weaker than that of the influenza A virus, means that it reacts only to a degree apparent to those skilled in the art. In addition, here, "there is substantially no antigen-antibody reaction with influenza B virus" means "nucleoprotein of influenza B virus, and proteins of each component of the virus and substantially antigen-antibody. Means not responding

. In a preferred form, the monoclonal antibodies of the present invention also do not substantially react with influenza C virus.

[0018] In a preferred embodiment, the monoclonal antibody of the present invention has an amino acid sequence within the amino acid region from the 59th amino acid residue (hereinafter, referred to as "59aa") to 130aa in the amino acid sequence of the nucleoprotein of influenza A virus. Reacts with epitope. The amino acid sequence of the nuclear protein of influenza A virus is already known, for example, GenBank Accession No.

It is described in ITY238021 and the like. The amino acid sequences of 59aa 140aa of the nuclear proteins of various subtypes of influenza A virus are shown in FIG. Further, in a preferred embodiment, the monoclonal antibody of the present invention has a common sequence in the amino acid sequences shown in FIG. A region having a structure, for example, 90aa 95aa, 11laa-115aa, or 120aa-123aa, or a region containing each of these regions and having a common sequence can be used as an epitope.

[0019] In a preferred form, the monoclonal antibody of the present invention comprises an influenza A virus An antigen-antibody reaction with each subtype of nuclear protein. That is, influenza A virus has hemagnoretinin (H) and neuraminidase (N) on the surface of the virus particle, and is classified into various subtypes depending on the difference in the structure of H and N. The monoclonal antibody of the present invention preferably has at least an antigen-antibody reaction with a nucleoprotein of the following subtype, and more preferably has an antigen-antibody reaction with all known influenza A virus subtype nucleoproteins. . H1N1, H2N2, H3N2, H3N8, H4N6, H5N2, H6N2, H7N7, H band, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8, H5N1.

[0020] In a preferred embodiment, the monoclonal antibody of the present invention does not substantially react with an infectious agent that is at least partially similar to influenza in antigen-antibody reactions. For example, adenovirus (type 17), coxsacki virus (type A16, B1-B6), simple virus 1 and echovirus (type 3, 4, 7, 22, 22 and 30) , Enterovirus (type 71), mumps virus, poliovirus (type 13), RS virus (subgroup 8, subgroup B), nora influenza virus (type 13), Escherichia coli, Klebsiella pneumoniae, green Pseudomonas aeruginosa, S. aureus, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus aureus, Corynebacterium, Diphtheria, Candida albicans, Streptococcus pyogenes, Streptococcus sp. (Groups B, C, G, F), Streptococcus pneumoniae, It does not substantially react with antigenic antibodies with Mofilus influenza, Listeria monocytogenes, Mycoplasma pneumonia, Chlamydia trachomatis, and Chlamydia pneumoniae.

[0021] As is well known, an antibody fragment having a binding property to a corresponding antigen, such as a Fab fragment ゃ F (ab ') fragment, is obtained by degrading an antibody with papain or pepsin (the present invention).

2

The antigen binding fragment of the monoclonal antibody of the present invention can be used in the same manner as the monoclonal antibody of the present invention. Get in.

[0022] The monoclonal antibody of the present invention can be prepared by using the nucleoprotein of influenza A virus as an immunogen and the conventional hybridoma method. As the influenza A nucleoprotein used as an immunogen, any of the viral virus solution, influenza HA vaccine, HA antigen for HI, and recombinant antigen can be used if the nucleoprotein is present in a large amount and can exert its immunogenic effect. It is possible. Immunogenicity contained in antigen In order to suppress the action of high HA and NA, nuclear protein purification by ultracentrifugation (for example, see J. Biochem .; 102: 1241-1249, 1987) and protease treatment (for example, J. Immunol. Methods; 180: 107-116) , 1995) can also be used.

[0023] The monoclonal antibody is used to immunize an animal using an antigen containing the nucleoprotein of the influenza A virus as an immunogen, and to perform cell fusion between the influenza A virus monoclonal antibody-producing cells and tumor cells. Can be produced by the hybridoma obtained by the above method.

[0024] The above-mentioned hybridoma can be obtained by the following method. That is, the influenza A virus nucleoprotein of the antigen obtained as described above, together with Freund's complete adjuvant, is divided into several times and injected into animals such as mice intraperitoneally or intravenously every 2-3 weeks. Immunize by giving. Next, the antibody-producing cells derived from the spleen and the like are fused with tumor cells that can grow in a test tube such as cells from the myeloma line (myeloma cells).

[0025] The fusion method described above can be carried out with polyethylene glycol according to Koehler and Milstein's ordinary method (Nature, 256, 495, 1975), or by Sendai virus or the like. Can be.

[0026] A method for selecting a hybridoma that produces an antibody that recognizes the influenza A virus nucleoprotein from the fused cells can be performed, for example, as follows. That is, surviving cells can be selected as hybridomas in the HAT medium and / or HT medium from the fused cells by the limiting dilution method. Next, the above hybridoma culture medium is reacted on an Atsushi plate on which highly purified influenza A virus nucleoprotein is immobilized, and further reacted with anti-mouse immunoglobulin (Ig) or the like. Hybridomas that produce monoclonal antibodies that specifically recognize influenza A virus nuclear protein can be selected by the EIA method or the like.

In the following examples, a plurality of preferred monoclonal antibodies of the present invention were obtained, and three of them were named as hybridoma FVA2_3, hybridoma FVA2-6, and hybridoma FVA2-11, respectively. These hybridomas are available from Industrial Technology At the Research Institute for Patent Organism Depositary (Tsukuba-Higashi, Ibaraki Pref., Japan 1-1, 1-1, Chuo No. 6), Hypridoma FVA2-3 is identified by identification number FVA2_3, Accession No. FERM BP-1 0066 (Accepted date: 2003) (Transferred from FERM P—19437 to domestic deposit on July 18, 2004, July 14, 2004), Hypridoma FVA2-6 was identified by FVA2_6, accession number FERM BP-10067 (date of deposit; As of July 18, 2003, July 14, 2004, the domestic deposit was transferred from FERM P-19438 to the international deposit), and the hybridoma FVA2-11 was identified by the identification number FVA2_11, the deposit number FERM BP_10068 (the date of deposit; 2003). July 18, 2004, and July 14, 2004, transferred to domestic deposit FERM P—19439, and transferred to international deposit) according to the Budapest Treaty.

[0028] Each of the above hybridomas is usually cultured in a medium used for cell culture, and the monoclonal antibody can be recovered from the culture supernatant. In addition, ascites can be stored and recovered from ascites by administering it to the animal from which the hybridoma originated.

As the method for recovering the monoclonal antibody, a commonly used purification method can be used, and examples include gel filtration chromatography, ion exchange chromatography, affinity chromatography using protein A, and the like. .

[0030] It was confirmed that the monoclonal antibody produced according to the above method reacts with the nucleoprotein of influenza A virus and reacts with various subtypes of influenza A virus. Reacts with 38 conserved strains of influenza A virus (see Table 3 below), such as humans, pests, ducks, turkeys, seals, chickens, gulls, mallards, etc., that are currently stored and available. It was detectable with a monoclonal antibody. On the other hand, microorganisms that may be cross-reactive include, for example, adenovirus (type 17), Koksatsu virus (A16, B1-B6), simple herpes virus 1 and echovirus (types 3 and 4). Type 7, type 22, type 22, type 30), enterovirus (type 71), mumps virus, poliovirus (type 13), RS virus (subgroup A, subgroup B), parainfluenza virus (1 Type 3), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, Staphylococcus aureus, Corynebacterium, Diphtheria, Candida albicans, Streptococcus pyogenes, Streptococcus sp. , G, F), Streptococcus pneumoniae, Hemophilus influenza, Listeria · No force reaction was observed that confirmed its reactivity with Monocytogenes, Mycoplasma pneumoniae, Chlamydia 'Tracomatis, Chlamydia' pneumoniae, etc.

[0031] The monoclonal antibody of the present invention can be used for immunoassay for detecting or quantifying influenza A virus. The immunoassay method itself is well-known, and any of the well-known immunoassay methods can be employed. That is, if classified according to the measurement format, there are the sandwich method, the competitive method, the agglutination method, the Western plot method, and the like, and if the label used is classified, there are the fluorescence method, the enzyme method, the radiation method, the biotin method, etc. Any of these can be used. Furthermore, the diagnosis can be made by immunohistological staining. When a labeled antibody is used for the immunoassay, the method of labeling the antibody itself is well known, and any of the well-known methods can be adopted. In addition, as is well known, antibodies are decomposed with papain or pepsin to bind to the corresponding antigen, such as Fab fragment or F (ab ') fragment.

2

It is known that an antibody-binding fragment (hereinafter referred to as “antigen-binding fragment”) can be obtained, but an antigen-binding fragment of the antibody of the present invention can be used in the same manner as the antibody of the present invention.

[0032] Note that these immunoassays are well known and need not be described in the present specification. However, in brief, for example, in the sandwich method, the antibody of the present invention or an antigen-binding fragment thereof (1) Immobilized as an antibody on a solid phase, reacted with a sample, washed, reacted with a second antibody that reacts with the enzyme of the present invention as an antigen-antibody, and after washing, the second antibody bound to the solid phase is measured. By labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like, the second antibody bound to the solid phase can be measured. In a plurality of standard samples with known concentrations, measurement was performed by the above method, and a calibration curve was created based on the relationship between the measured amount of label and the enzyme of the present invention in the standard sample. By applying the measurement results to the calibration curve, the enzyme of the present invention in the test sample can be quantified. Note that the first antibody and the second antibody may be interchanged with the above description. In the agglutination method, the antibody of the present invention or an antigen-binding fragment thereof is immobilized on particles such as latex, and reacted with a sample to measure the absorbance. A plurality of standard samples with known concentrations were measured by the above method, and a calibration curve was created based on the relationship between the measured label amount and the enzyme of the present invention in the standard sample. By fitting to this calibration curve, The enzyme of the present invention in the test sample can be quantified.

[0033] The present invention also provides an immunoassay device that can easily detect influenza A virus and distinguish it from influenza B virus using the monoclonal antibody of the present invention. I do.

[0034] The immunoassay device of the present invention comprises a labeled reagent zone having a movable labeled anti-influenza A virus antibody, a sample spotting zone, a developing solution supply zone, a developing solution absorption zone, and an anti-influenza A virus antibody. An instrument comprising a matrix provided with an influenza A virus detection zone immobilized in a matrix, wherein at least one of the labeled anti-influenza A virus antibody and the anti-influenza A virus antibody immobilized in the detection zone is as described above. An immunoassay device that is the monoclonal antibody of the present invention. The sample spotted in the sample spotting zone reacts with the labeled antibody contained in the labeling reagent zone by an antigen-antibody reaction to form a labeled antigen-antibody complex. The antigen-antibody complex is flowed by the developing solution supplied from the developing solution supply zone, and reaches the influenza A virus detection zone. Here, the anti-influenza A virus antibody immobilized in the matrix reacts with the labeled antigen-antibody complex to cause an antigen-antibody reaction, and the labeled antigen-antibody complex is immobilized in Matritas. Whether or not the labeled antigen-antibody complex has been immobilized in the influenza A virus detection zone is determined by the presence or absence of the label. The developing solution that has passed through the influenza A virus detection zone is absorbed by the developing solution absorption zone. The sample spotting zone and the labeling reagent zone may be the same (in this case, the sample is spotted on the labeling reagent zone). In the immunoassay device of the present invention, at least one of a labeled antibody and an antibody immobilized in the influenza A virus detection zone is a monoclonal antibody of the present invention. Also, one of the antibodies may be a polyclonal antibody. Since multiple molecules of the influenza A virus nucleoprotein are usually associated or attached, both the labeled antibody and the antibody immobilized in the influenza A virus detection zone are the same monoclonal antibody. Can detect influenza A virus.

Hereinafter, each component of the immunoassay device of the present invention will be described separately.

[0036] Matrix The band-shaped matrix in the immunoassay device of the present invention is made of an absorbent material capable of injecting a liquid by capillary action. Examples of the absorbent material include a filter paper, a membrane, and a porous material manufactured by using cellulose or derivatives thereof such as nitrocellulose, nitrocellulose and the like, and glass fiber alone or in combination. Although the size of the matrix is not limited, a strip having a width of about 3 mm and 10 mm and a length of about 30 mm and 100 mm is preferred because it is easy to handle. A matrix having a thickness of 100 zm lmm can be used. In addition, the matrix is blocked with animal serum such as bovine serum albumin (BSA), casein, sucrose, etc., in order to prevent non-specific reaction of protein from the sample to the matrix during measurement, in part or in whole. Can be used.

[0037] Detection zone

The detection zone may be provided with an influenza A virus detection unit in which an anti-influenza A virus antibody is immobilized on the matrix. The anti-influenza A virus antibody to be immobilized in the detection section is, together with a labeled anti-influenza A virus antibody described later, at least one of them is an anti-influenza A virus monoclonal antibody of the present invention, and both antibodies are anti-influenza A virus antibodies. It is preferably a type virus monoclonal antibody. The influenza A virus antibody in the detection section is on a matrix, and is preferably provided in a line in a direction perpendicular to the infusion direction of the liquid for developing the matrix (the longitudinal direction of the matrix) in order to measure with high sensitivity. . As the anti-influenza A type vinores polyclonal antibody, a commercially available and readily available polyclonal antibody can be appropriately selected and used.

[0038] The anti-influenza A virus antibody in this detection zone is the aforementioned antibody, and a monoclonal antibody may be used alone or in combination. The anti-influenza A virus antibody may be an IgG antibody, an IgM antibody, or an antigen-binding fragment of these antibodies, such as Fab, F (ab ').

[0039] The anti-influenza A virus antibody immobilized in the detection unit may be physically adsorbed directly to the detection zone of the matrix, or may be provided by immobilizing it with a chemical bond such as a covalent bond. Further, an anti-influenza A virus antibody may be bound to a water-insoluble carrier, and this may be contained in the matrix. As this insoluble carrier, Particles obtained by insolubilizing a mixture of gelatin, gum arabic and sodium hexametaphosphate (JP-B-63-29223), polystyrene latex particles, glass fibers and the like can be mentioned. In order to bind the insoluble carrier to the anti-influenza A virus monoclonal antibody, the carrier can be bound by the chemical bond or physical adsorption.

[0040] In the detection zone, an influenza A virus detection unit using an anti-influenza A virus antibody and an influenza B virus detection unit using an anti-influenza B virus antibody can be provided. This influenza B virus detection section may be located upstream or downstream of the influenza A virus detection section in the infusion direction of the developing solution as long as it is near the influenza A virus detection section. ,. The anti-influenza B virus antibody to immobilize the influenza B virus detection zone is applied to the matrix by chemical bonding or physical adsorption similarly to the anti-influenza A virus, which may be a polyclonal antibody or a monoclonal antibody. Can be immobilized.

As described above, the detection zone on the matrix is provided with at least an influenza A virus detection unit using an anti-influenza A virus antibody, and further provided with an influenza B virus detection unit. And influenza B virus at the same time. These detectors are located downstream of the enzyme labeling reagent zone, sample spotting zone, and developing solution supply zone and upstream of the additive solution absorption zone in the matrix infusion direction. The detection unit can provide a plurality of lines close to a matrix having a width of about 0.5 mm to 5 mm in the matrix. If Ma Toritasu about width 5 mm, the antibody and antigen usually wear 10 mu g about point from each 0. 1 _beta g, it is possible to create a detector by drying.

[0042] Labeling reagent zone

The labeled reagent zone can be provided by movably spotting a labeled anti-influenza A virus antibody on a zone provided on the matrix. This zone can be provided on the upstream side of the detection zone in the direction of infusion of the developing solution from the developing solution supply zone. This zone can be formed by spotting the enzyme labeling reagent on the matrix, by laminating a water-absorbent pad containing the enzyme labeling reagent on the matrix, or by forming a part of the matrix in close contact with the pad. It is constituted by including an enzyme labeling reagent together with a pad in part or all of the pad. As the water-absorbing pad, a pad in a sample spotting zone described later can be used.

[0043] As the antibody of the labeled anti-influenza A virus antibody, together with the antibody provided in the detection zone, at least one is an anti-influenza A virus monoclonal antibody, and both antibodies are anti-influenza A virus antibodies. Preferably, it is a monoclonal antibody. As the antibody of the labeled anti-influenza A virus antibody, a fragment thereof can be used similarly to the antibody of the detection zone.

[0044] The labeled anti-influenza A virus antibody can be produced by binding the antibody to a label. Labels include enzymes, metal colloid particles, colored latex particles, luminescent substances, fluorescent substances and the like. Examples of the enzyme include various enzymes used in an enzyme immunoassay (EI A). Examples of the enzyme include alkaline phosphatase, peroxidase, and j3_D_galactosidase. Further, as the metal colloid particles, for example, gold colloid particles, selenium colloid particles, and the like can be used.

[0045] In addition, a method for binding a labeled substance to an anti-influenza A virus antibody can be produced by using a known method for forming a covalent bond or a non-covalent bond. Examples of the binding method include a phthalaldehyde method, a periodic acid method, a maleimide method, a pyridyl 'disulfide method, and a method using various cross-linking agents (for example, “Protein Nucleic Acid Enzyme”, Supplement No. 31, 37, 37). — See page 45 (1985)). In the bonding method using a crosslinking agent, examples of the crosslinking agent include N-succinimidyl-14-maleimidobutyric acid (GMBS), N-succinimidyl-16-maleimidohexanoic acid, and N-succinimidyl-14- (N-maleimidomethyl) cyclo. Xan-1-carbonic acid or the like can be used. In the covalent bond method, a functional group present in the antibody can be used.In addition, after a functional group such as a thiol group, an amino group, a carboxyl group, or a hydroxyl group is introduced by a conventional method, the labeled anti-influenza antibody is labeled by the above-described bonding method. An A-type viral antibody can be produced. In addition, as a method using a non-covalent bond, a physical adsorption method or the like can be used.

[0046] As described above, in addition to detecting influenza A virus and detecting influenza B virus at the same time, labeled anti-influenza B virus is added to the labeling reagent zone. The body is added to make the device. Labeled anti-influenza A virus antibody and labeled anti-influenza B virus antibody contained in the labeling reagent zone can be contained in either the matrix or the water-absorbent pad, or labeled on both the matrix and the water-absorbent pad An anti-influenza A virus antibody and a labeled anti-influenza B virus antibody can be included. When performing simultaneous measurement of influenza A virus and influenza B virus, since a large amount of labeling reagent is used, it is necessary to include an enzyme labeling reagent alone or in a mixture with both the matrix and the pad for high-sensitivity measurement. It is advantageous. The amount of labeled anti-influenza A virus antibody and the amount of labeled anti-influenza B virus antibody can be changed as appropriate according to the expected amount of the test sample. It is about μg. When the labeled anti-influenza A virus antibody and the labeled anti-influenza B virus antibody are contained in the enzyme labeling zone, they can be applied together with a reagent stabilizing agent, a dissolution regulator and the like.

[0047] Sample spotting zone

The sample spotting zone can be provided in the matrix on the downstream side of the developing solution supply zone in the direction of infusion of the developing solution and on the upstream side of the detection zone without particularly including a reagent or the like. Further, the sample spotting zone is: 1) a predetermined location on the downstream side of the developing solution zone in the developing solution infusion direction and upstream of the enzyme-labeled reagent zone; 2) a predetermined location on the downstream side of the labeling reagent zone and upstream of the detection zone. 3) It can be provided at a predetermined location on the labeling reagent zone. In an apparatus in which a sample spotting zone is provided in the enzyme labeling reagent zone, it is preferable to attach a water-absorbing pad containing an enzyme label as described above, in order to perform analysis efficiently. In the device to which the pad is added, a large amount of the sample liquid can be spotted, so that a trace component in the sample can be measured with high detection sensitivity. This water-absorbing pad is selected from materials and materials that are less likely to adsorb the labeling reagent and influenza virus in the sample.For example, a porous synthetic or natural material such as polybutyl alcohol (PVA), nonwoven fabric, or cellulose is used. The above-mentioned polymer compounds can be used alone or in combination. The size, thickness, density, and the like of the pad are not limited, but it is preferable to use a pad having a length and width of about 3 mm and 10 mm and a thickness of about 0.5 mm to 4 mm for efficient measurement.

[0048] Developing liquid supply zone The developing liquid supply zone is a zone provided at one end in the longitudinal direction of the matrix and supplied with the developing liquid. To start the measurement, this zone can be immersed in a container containing at least an amount of the developing solution that reaches the developing solution absorption zone. Further, for the supply of the developing liquid, the measurement can be started by adding a liquid tank containing the developing liquid to the developing liquid zone, breaking the cover of the liquid tank, and bringing the developing liquid into contact with the matrix. The developing solution can appropriately contain a surfactant, a buffer, a stabilizer, an antibacterial agent, and the like. When an enzyme is also used as a label, a substrate can be added to the developing solution together with a substrate reagent zone described later. Examples of the buffer containing a buffer include an acetate buffer, a borate buffer, a Tris-HCl buffer, a diethanolamine buffer and the like. Further, a developing liquid pad can be provided in the developing liquid supply zone in order to stably and continuously supply the developing liquid to the matrix. As the developing liquid pad, for example, filter paper such as cellulose or a cellulose derivative can be used.

[0049] Developing liquid absorption zone

The developing liquid absorption zone is provided at the other end with respect to the developing liquid zone provided at one end of the matrix. This zone is provided to absorb the developing solution supplied to the matrix and to perform the analysis smoothly. The developing liquid absorption zone may be formed by forming a long matrix to secure this zone. Further, the matrix can be provided with a water-absorbing material to promote the development. As the water-absorbing material, a highly water-retentive filter paper, sponge, or the like made of a natural polymer compound, a synthetic polymer compound, or the like can be used. The developing solution absorption zone is made of a pad-shaped absorbent material with a volume that absorbs all the developing solution, but by laminating the absorbing material on or under the matrix, a shaped immunoassay device is manufactured. can do.

[0050] Substrate reagent zone

Further, when an enzyme is used as a label in the labeling reagent zone, the substrate can be contained in the developing solution as described above, or the substrate reagent zone can be provided near the developing solution supply zone of the matrix. The substrate reagent zone is preferably contained in the developing solution pad provided in the developing solution supply zone in order to increase the base mass and perform high-sensitivity measurement.

[0051] As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates and the like shown below corresponding to the enzyme of the labeling reagent can be used. (A) For chromogenic substrate peroxidase: 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in combination with hydrogen peroxide, 3, 3 ′, 5, 5 '—Tetramethylbenzidine (TMB), diaminobenzidine (DAB) For alkaline phosphatase: 5-bromo-4-chloro-3_indolyl phosphate (BCIP)

(b) Fluorescent substrate for alkaline phosphatase: 4-methyl-mbellifene perfluorophosphate (4 MUP) For β_D_galactosidase: 4-methyl-mbellifenyl-β_D_galactoside (4 MUG)

(c) Luminescent substrate For alkaline phosphatase: 3- (2-spiroadamantane) -1-methoxy-4- (3 "-phosphoryloxy) phenyl _1,2-dioxetane disodium salt (AMPPD) β _D_galactosidase : 3_ (2 spiro adamantane) _4-methoxy _4_ (3 "_ j3-D-galactopyranosinole) feninole 1,2-dioxetane (AMGPD) for peroxidase: Luminol, isoluminol combined with hydrogen peroxide

When the substrate is provided as a substrate zone, the substrate can be usually formed by dissolving the substrate in an aqueous solution, applying the solution in a line to a developing solution pad, and then drying the solution. An enhancer, a stabilizer, a dissolution regulator and the like can be added. The substrate zone is not particularly limited as long as it is within the developing solution pad attached to the end of the matrix. The base weight to be added to the developing solution and the developing solution pad can be determined according to the measurement conditions, but usually about 5 to 500 per instrument can be used.

[0054] Arrangement of each zone

One preferred example of the immunoassay device of the present invention is schematically shown in FIGS. In Fig. 6, reference numbers 2 are the matrix, 4 is the labeling reagent zone, 8 is the sample spotting zone, 3 is the developing solution supply zone, 5 is the developing solution absorption zone, 6a is the influenza A virus detection zone, and 7 is the substrate. Reagent zone, 9 is the specimen. In this example, the sample spotting zone 8 and the labeling reagent zone 4 are the same. Reference numeral 6b is an influenza B virus detection zone, 10 is a developing solution confirmation zone, and 11 is a developing solution tank (see Example 6 below).

[0055] How to use the measuring instrument

With the measuring instrument of the present invention, influenza A virus in various sample samples can be measured. First, the sample is supplied to the sample spotting zone of the measuring instrument of the present invention. After that, the developing solution is supplied to the developing solution pad, and the developing solution is developed on a matrix. As a sample diluent, a buffer containing a surfactant can be used as a sample diluent. The developing solution moves through the matrix by capillary action and reaches the developing solution absorption zone, where components in the sample, enzyme-labeled reagents, and the like that are not bound to the detection zone are absorbed, and the developing is completed. After the lapse of a predetermined time (usually 10 to 20 minutes), the detection zone is observed, and the labeling substance immobilized on the detection site by the influenza A virus in the sample solution is measured, thereby detecting the influenza A virus. Measurements can be made. This detection can be carried out visually or by using a measuring device such as a colorimeter, a fluorometer, a photon counter, a photosensitive film or the like corresponding to the label or the label and the enzyme to be used. In the measurement, for example, a method of visually measuring the color development of the detection zone is simple. This method also enables semi-quantitative analysis by using a color chart (color chart) corresponding to the concentration of influenza A virus. Further, it is possible to quantify the coloration of the detection zone by using a colorimeter or the like to quantify the color.

The measurement principle will be further described by taking a preferred example of the immunoassay device of the present invention shown in FIGS. 6 to 8 as an example. The sample 9 is spotted on the sample spotting zone 8, and the developing solution in the developing solution tank 11 is supplied to the developing solution supply zone 3. The developing solution moves in the matrix 2 in the direction of the white arrow by the capillary action. The substrate contained in the substrate reagent zone 7 is dissolved in the developing solution and moves with the developing solution. On the other hand, the sample 9 reacts with the labeled antibody in the labeling reagent zone 4 to form a labeled antigen-antibody complex. When the developing solution arrives here, the labeled antigen-antibody complex moves through the matrix 2 while reacting with the substrate in the developing solution, and when it reaches the influenza A virus detection zone 6a, the influenza A virus detection zone It reacts with the anti-influenza A virus antibody immobilized in 6a and is immobilized in the influenza A virus detection zone 6a. When the influenza A virus is contained in the sample, color development occurs due to the reaction between the labeling enzyme and the substrate. On the other hand, when the influenza A virus is not contained in the sample, the labeled antibody cannot react with the antigen-antibody, and the label is not immobilized in the influenza A virus detection zone 6a, so that no coloring occurs. . Therefore, whether or not the sample contains influenza A virus depends on whether or not the influenza A virus detection zone 6a develops color. You can know. In the developing solution confirmation zone 10, an enzyme that reacts with a reagent in the developing solution and develops a color is immobilized. When the developing solution confirmation zone 10 develops color, it is understood that the developing solution has reached that point. Alternatively, an antibody against the labeling enzyme was immobilized in the developing solution confirmation zone 10, and the labeling reagent that reached the developing solution confirmation zone 10 was trapped by the antibody, and was trapped when the developing solution further reached. The label may react with the substrate in the developing solution to form a color (in the following example, the anti-alkaline phosphatase antibody is immobilized).

). In addition, if the labeled anti-influenza B virus antibody is included in the labeled reagent zone 4, the anti-influenza B virus detection zone 6b in which the anti-influenza B virus antibody is immobilized is provided, Detection of type B virus can be performed at the same time, and it is possible to check whether the influenza virus is type A or type B by a single measurement operation.

Further, the matrix can be laminated and fixed on a support member such as plastic, metal, paper or the like and used. The matrix is fixed to a case made of plastic or the like, a liquid tank containing a developing solution is attached to the developing solution supply zone, and the zone is covered with a case having a hole in each zone, thereby forming an instrument which is easy to handle. be able to. The specimens used in the immunoassay device of the present invention are specimens which are considered to contain influenza collected from humans, animals, etc., and include various body fluids such as nasal swabs (nasal swabs), nasal aspirates, pharyngeal swabs And body fluid extracts such as liquids (pharyngeal swabs).

Hereinafter, the present invention will be described in more detail by reference examples and examples, but the present invention is not limited to these examples.

Example

Example 1 Preparation of Anti-Influenza A Virus Monoclonal Antibody

Influenza HA vaccine containing influenza nucleoprotein antigens (manufactured by I-Dark Blood: A / New Caledonia / 20/99 (HINl) (IVR-116) (IVR-116), A / Panama / 2007/99 (H3N2) NIB -41) strain) was used. After adding an equal volume of Freund's complete adjuvant (manufactured by Jatron) to the immunogen and mixing thoroughly, BallVc mice were immunized four times at 2-week intervals. Three days before cell fusion, an immunogen was injected intraperitoneally into mice, and mouse splenocytes were fused with myeloma cells (P3U1) using PEG. Use a HAT selection medium for the culture, and after about 2 weeks The culture supernatant was collected and provided for screening. Primary screening includes influenza HA vaccine containing influenza nucleoprotein antigen, influenza A recombinant nucleoprotein antigen (from A / New Caledonia / 20/99 (r-NP / HlNl), A / Kitakyushu / 159/93 An Enzyme Linked Immunosobent Assay (ELISA method) on which the source (r-NP / H3N2); DDBJ / GeneBank database) was immobilized was used. That is, the influenza HA-pactin was diluted x300-fold in 0.1 M carbonate buffer pH 9.6, and the A-type recombinant nucleoprotein antigen was also diluted to a concentration of 1 μg / ml. ΙΟΟμΙ was added to each well, and the mixture was incubated at 4 ° C and immobilized. Next, each well was washed with PBS (PBS-Tween) containing 0.1% Tween 20 (trade name) and diluted with PBS1. / ₀ © shea serum Anorebumin the (BSA) 300 μ ΐ was 4 ° C over晚blocking added. After removing the blocking solution, the culture supernatant was reacted at 37 ° C for 1 hour. After sufficient washing with PBS-Tween, enzyme labeling diluted 2000-fold with 0.05M phosphate buffer PH7.5 (reaction solution) containing 0.2% BSA, 0.2% Emulgen 985, 1% sucrose, and 1% KC1 100 μl of an anti-mouse Igs antibody (manufactured by DAKO) was added to each well, and the mixture was reacted at 37 ° C. for 1 hour. After the reaction, wash well with PBS-Tween, add 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to each well at 100 / l, and react at room temperature for 30 minutes. A stop solution was added to each well in an amount of 100 μl, and the color development level was measured at a main wavelength of 415 nm and a sub wavelength of 490 nm.

[0060] The culture supernatant that was determined to be positive was subjected to a secondary ELISA using an influenza B recombinant nuclear protein (B / Yamanashi / 166/98 (r-NP / B) DDBJ / GeneBank database) as an antigen. After screening, the three hybridoma strains that produce antibodies (reactively react with the influenza A virus nucleoprotein antigen and not with the influenza B nucleoprotein antigen) (anti- hybridoma FVA2_3, hybridoma FVA2 6 and hybridoma FVA2-11) were obtained. These hybridomas have been deposited at the Patented Microorganisms Depositary Center as described above. The subclasses of the monoclonal antibodies obtained from the three hybridoma strains were all IgGl. Table 1 shows the results of the reactivity of the hybridoma.

[Table 1] Table 1 Reactivity of monoclonal antibodies

丄

mm

Monoclonal antibody Γ-ΝΡ / Η Ν1 r-NP / H3N2 Vaccine r-NP / B

FVA2-3 +++ 2.206 0.018

FVA2-6 +++ 2.183 0.017

FVA2-1 1 +++ 2.223 0.018

+++, range over

Example 2 Reactivity of Monoclonal Antibody in Western Blotting Method

Reactivity was confirmed by the Western blotting method using a recombinant antigen, an influenza HA vaccine (containing a nucleoprotein antigen), and a virus solution. Dissolve each sample in 0.01M Tris_HCl (pH 8.0) containing 0.001M EDTA, 1% SDS, 5% 2ME (2-mercaptoethanol), 10% glycerol, 0.005% ΒΡΒ (Bromophenol Blue), 100 After heat treatment at ° C, it was subjected to electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), e-Bagel10. /. Using a gel concentration (manufactured by Atoka Co., Ltd.), a conventional method was used. After swimming, it was transferred to a PVDF membrane (manufactured by Atoka), and subjected to 4 ° C-blocking with Block Ace (manufactured by Dainippon Pharmaceutical). After removing the blocking solution and washing with PBS_Tween, a monoclonal antibody adjusted to a concentration of 10 x g / ml was added and reacted at room temperature for 45 minutes. After washing thoroughly with PBS-Tween, an enzyme-labeled anti-mouse IgG antibody (Cappel) diluted 4000 times with the reaction solution was allowed to stand at room temperature for 45 minutes. After sufficiently washing with PBS_Tween, the signal was detected by exposing to an X-ray film (manufactured by Kodak) for 115 minutes using an ECL ゥヱ stamp lotting detection system (manufactured by Amersham Bioscience).

[0063] With the monoclonal antibodies FVA2-3, FVA2_6, and FVA2-11, bands were observed around 55-7 OkD for all antigens. The result is shown in FIG.

Example 3 Measurement of Antigen by ELISA Using Monoclonal Antibody

Three purified monoclonal antibodies were added to each well of a microplate module (Nunc) at a concentration of 2 μg / ml at a concentration of 2 μg / ml in 0.1 M phosphate buffer, pH 7.5, and added to each well at 4 ° C. It was coated and immobilized. Next, after washing each well with PBS (PBS-Tween) containing 0.1% Tween 20 (trade name), add 300% / l of 1% serum albumin (BSA) diluted with PBS and add 4 ° C 晚 Blocking was performed. The influenza A virus solution diluted with the reaction solution was allowed to react with each of the tubes for 1 hour at 37 ° C at 37 ° C. After thorough washing with PBS-Tween, 100 µl of each of the three alkaline phosphatase-labeled monoclonal antibodies prepared in the reaction solution was added to each well and reacted at 37 ° C for 1 hour. After the reaction, wash well with PBS-Tween, add 100 μl of 4-nitrophenyl phosphate (4-NPP) to each well, incubate at room temperature for 30 minutes, and add 100 μl of stop solution to each well. The coloring level was measured at a main wavelength of 415 nm and a sub wavelength of 490 nm. Positive values were 2.1 times or more higher than the color development level of the buffer blank, and the reactivity was expressed by the final dilution ratio that became positive.

In all nine combinations of the three types of monoclonal antibodies obtained, the reactivity was 28-fold higher than that of the commercial anti-influenza A virus monoclonal antibody. Table 2 shows the results.

[0065] [Table 2] Table 2 Antigen measurement EUSA monoclonal-null antibody

A Hand 21 A / Beijing A / Ni--Caledonia A / Panama Enzyme-labeled anti-solid phase antibody Z5 / 97/352/89/20/99/2007/99

FVA2-3 FVA2 3 x640 x80 x320 x640

FVA2-6 x640 xl 60 x320 x640

FVA2-1 1 x640 x160 x320 x640

FVA2-6 FVA2-3 x640 x80 x320 x640

FVA2-6 x640 x160 x320 x640

FVA2-1 1 x640 x80 x320 x640

FVA2-1 1 FVA2-3 x640 x80 x320 x640

FVA2-6 x640 x160 x320 x640

FVA2-1 1 x640 x160 x320 x640 Commercial antibody * Commercial antibody * x320 x40 x80 x80

* Commercially available monoclonal antibody

Example 4 Measurement of Reaction Site of Anti-Influenza A Virus Monoclonal Antibody 4-1 Preparation of Plasmid

In order to determine the recognition site of the monoclonal antibody FVA2-11, the influenza H1N1 strain (New Caledonia / 20/99) consisting of 498 amino acids was subjected to the PCR method using 11159 amino acids (A fragment) and 162 327 amino acids. (B fragment) and three fragments of 327-498 amino acids (C fragment) were amplified. EcoRI and BamHI sites were previously added to the primers. The amplified fragment was purified using QIAGEN's PCR Purification Kit, and the expression plasmid pW6A shown in Figure 2 was used.

Plasmids pWGInf.HlNl-A, pWGInf.HlNl-B and pWGInf.HlNl-C were inserted into the expression plasmid pWG6A having the GST incorporated into the Nde-EcoRI site at the EcoR-BamHI site. Using these, E. coli BL21 (DE3) (obtained from Brookhaven National Laboratory) was transformed, and transformed into an ampicillin-resistant transformant E. coli.

BL21 (DE3) pWGInf.HlNl-A, BL21 (DE3) pWGInf.HlNl_B and

BL21 (DE3) pWGInf.HINl-C was obtained.

[0067] 4-2 Expression of recombinant proteins (GST + H1N1-A, GST + H1N1-B and GST + H1N1-C)

The transformant prepared in 4_1 was cultured at 37 ° C. in 2 ml of LB medium containing 50 μg / ml of ampicillin. After the OD was adjusted to 0.6 0.8 at 600 nm in the preliminary culture, the expression was induced by adding 0.4 mM IPTG, and the cells were further cultured for 3 hours. A 1.5 ml cell culture was centrifuged at 5000 rpm for 2 minutes to collect the cells, and the cells were suspended in ΙΟΟμΙ of buffer (10 mM Tris-HCl, pH 8.0, 0.1 M sodium chloride, ImMEDTA). The cells were completely disrupted by sonication for 15 minutes. This is used as the bacterial sample.

[0068] Three-fold concentration of SDS polyacrylamide buffer (0.15M Tris-HCl, ρΗ6.8,

6% SDS, 24% glycerol, 6 mM EDTA, 2% 2_mercaptoethanol, 0.03% bromophenol blue) 41 were added, and the mixture was sufficiently stirred, followed by SDS-polyacrylamide gel electrophoresis. Transfer by western blotting on a nitrocellulose filter, block with 1% BSA, and dilute 1000-fold with phosphate buffer (10 mM phosphate, ρΗ7.4, 0.15 M sodium chloride) 1000-fold. FVA2-11 Was reacted. Furthermore, a peroxidase enzyme-labeled anti-mouse immunoglobulin / sagi polyclonal antibody (manufactured by Dako) was reacted, washed, and then washed with 10 ml of a substrate coloring solution (0.01% hydrogen peroxide solution, 0.6 mg / ml 4 Naphthol) was added to develop the color. As a result, it was found that the monoclonal antibody FVA2-11 reacted only with GST + H1N1-A.

[0069] 4-3 Preparation of Plasmid

Next, the A fragment of influenza H1N1 was further subdivided into fragments Al (l 90 amino acids), A2 (85 159 amino acids), A3 (44-130 amino acids), A4 (59 103 amino acids), and A5 (44-103 amino acids). Acid), A6 (59-130 amino acids) and A7 (1-130 amino acids) (FIG. 3)) were amplified by PCR. The amplified fragment was purified using QIAGEN's PCR Purification Kit, inserted into the EcoR to BamHI site of the expression plasmid pWG6A, and inserted into plasmids pWGInf.HlNl-Al, pWGInf.HlNl-A2, pWGInf.HlNl-A3, pWGInf.HlNl. -A4, pWGInf.HlNl_A6, and pWGInf.HlNl_A6 were prepared. The A7 fragment was inserted into the EcoR to BamHI site of pW6A shown in Fig.

pWInf.HINl-A7 was prepared. Using these, Escherichia coli BL21 (DE3) (obtained from Brookhaven National Laboratory) was transformed and transformed into an ampicillin-resistant transformant Escherichia coli.

BL21 (DE3) pWGInf.HlNl-Al.B121 (DE3) pWGInf.HlNl-A2,

BL21 (DE3) pWGInf.HlNl-A3,

BL21 (DE3) pWGInf.HlNl-A4, BL21 (DE3) pWGInf.HlNl-A5,

BL21 (DE3) pWGInf.HlNl-A6 and BL21 (DE3) pWInf.HlNl-A7 were obtained.

[0070] 4—4 recombined proteins (GST + H1N1-Al, GST + H1N1-A2, GST + H1N1-A3,

Expression of GST + H1N1-A4, GST + H1N1-A5, GST + H1N1-A6 and H1N1-A7)

4. The transformant prepared in 1-3 was cultured at 37 ° C. in 2 ml of LB medium containing 50 μg / ml of ampicillin. After the OD was adjusted to 0.6-0.8 at 600 nm in the preliminary culture, the expression was induced by adding 0.4 mM IPTG, and the cells were further cultured for 3 hours. A 1.5 ml cell culture was centrifuged at 5000 rpm for 2 minutes to collect the cells, and the cells were suspended in ΙΟΟμΙ of buffer (10 mM Tris-HCl, pH 8.0, 0.1 M sodium chloride, ImMEDTA). The cells were completely disrupted by sonication for 15 minutes. This is used as the cell sample.

[0071] Eight microliters of the bacterial cell sample were added to a 3x concentration of SDS polyacrylamide buffer (0.15 M Tris-HCl, ρΗ6.8, 6% SDS, 24% glycerol, 6 mM EDTA, 2% 2_mercaptoethanol, 0.03% Bromophenol blue, molecular weight markers (117.6, 113.9, 81.2, 60.7, 47.4, 36.1, 25.3, 19.0, 14.7, 6.1KD)) . Perform the transfer by Western blotting on a nitrocellulose filter, block with 1% BSA, and dilute 1000 times with monoclonal antibody FVA2 in phosphate buffer (10 mM phosphoric acid, pH 7.4, 0.15 M sodium chloride). —11 was reacted. Furthermore, a peroxidase enzyme-labeled anti-mouse immunoglobulin / sagi polyclonal antibody (manufactured by Dako) is reacted, washed, and then washed with 10 ml of a substrate color developing solution (0.01% hydrogen peroxide solution, 0.6 mg / ml). Naft ) And colored. Each fragment is shown in FIG. Figure 4 shows the CBB staining pattern after electrophoresis and the results of the stamp lot. These results indicate that the recognition site of the anti-influenza A virus monoclonal antibody (FVA2-11) is in the amino acid 59-130 region of influenza H1N1. Furthermore, based on the influenza A subtype nuclear protein amino acid sequence shown in FIG. 5, the amino acid sequence has a common sequence at the reactive site at amino acids 59 and 130, and has a hydrophilic structure, for example, at positions 90 and 111-111. It is expected that the amino acid sequence at the 120th or the 123rd position is an epitope.

Reference Example 1 Production of Alkaline Phosphatase Labeled Anti-Influenza A Virus Monoclonal Antibody

Anti-influenza A virus monoclonal antibody (FVA2-11) is treated with pepsin to cleave the Fc region to obtain F (ab '), and then 2ME (2_mercaptoethanol, Nacalai Tester)

2

Was used to cleave the disulfide bond to obtain F (ab ') exposing a free thiol group. Next, alkaline phosphatase, into which a maleimide group was introduced, was coupled with F (ab '), and purified by gel filtration to obtain a purified alkaline phosphatase-labeled anti-influenza A virus antibody.

Reference Example 2 Preparation of Anti-Influenza B Virus Monoclonal Antibody)

The immunogen may be influenza HA lactoprotein containing influenza nucleoprotein antigen (B / Yamanashi / 166/98 strain) or influenza A recombinant nucleoprotein antigen (A / New Caledonia / 20/99 derived (r-NP / HlNl) A / Kitakyushu / 159/93 derived (Γ-ΝΡ / Η3Ν2) was used.

[0074] After an equal amount of Freund's complete adjuvant (manufactured by Jatron) was added to the immunogen and thoroughly mixed, Balb mice were immunized four times at 2-week intervals. Three days before cell fusion, an immunogen was injected intraperitoneally into mice, and mouse splenocytes were fused with myeloma cells (P3U1) using PEG. A HAT selection medium was used as a culture solution, and a culture supernatant was collected about 2 weeks later and subjected to screening. For the primary screening, an Enzyme Linked Immunosobent Assay (ELISA) in which influenza HA vaccine and influenza B recombinant nucleoprotein (r-NP / B) were immobilized was used. That is, the influenza HA vaccine was x300-fold with 0.1M carbonate buffer PH9.6, and the B-type recombinant nucleoprotein antigen was also 1 μg / ml. And added to each well of a microplate module (manufactured by Nunc) in an amount of 100 μl, and incubated at 4 ° C. to solidify. Next, each well was washed with PBS (PBS-Tween) containing 0.1% Tween 20 (trade name), and 300 μl of 1% BSA diluted with PBS was added to perform 4 ° C. blocking. After removing the blocking solution, the culture supernatant was reacted at 100 ° C for 1 hour at 37 ° C. After thorough washing with PBS_Tween, 0.2. /. BSA, 0.2% Emalgen 985, 1 Q / o sucrose, l. Enzyme-labeled anti-mouse Igs antibody (manufactured by DAKO) diluted 2000-fold with 0.05M phosphate buffer pH7.5 (reaction solution) containing / oKCl was added to each well in a volume of ΙΟΟμΙΟΟ, and allowed to react at 37 ° C for 1 hour. Was. After the reaction, wash thoroughly with PBS_Tween, add 2,2 azinobis-3_ethylbenzothiazoline_6-sulfonic acid (ABTS) to each well in a volume of ΙΟΟμΙ, stop the reaction at room temperature for 30 minutes, and stop the reaction. 100 μl of the solution was added to each well, and the color development level was measured at a main wavelength of 415 nm and a sub wavelength of 490 nm.

[0075] With respect to the culture supernatant determined to be positive, influenza A recombinant nuclear protein antigen (from A / New Caledonia / 20/99 (Γ-ΝΡ / Η1Ν1), A / Kitakyushu / 159/93 (スクリーニング -ΝΡ / Η3Ν2)) Secondary screening by ELISA using antigen as antigen, and finally produce antibodies that react with influenza type I nucleoprotein antigen and do not react with influenza type II nucleoprotein antigen Three hybridoma strains (FrBl_03, FrBl_07, FVB2-16) were obtained. Each of the above hybridomas is registered with the National Institute of Advanced Industrial Science and Technology (AIST) under the Budapest Treaty based on the Hybridoma FrBl-03, the deposit number FERM BP-10070, the hybridoma FrBl-07, the deposit number FERM BP_10071, the hybridoma FVB2_16, Deposit number FERM BP-10069 has been deposited. The subclasses of the monoclonal antibodies from which the three hybridoma strains were also obtained were all IgGl.

(Reactivity of Monoclonal Antibody in Western Blotting Method)

The reactivity was confirmed by a stamp lotting method using a recombinant antigen and an influenza HA vaccine (containing a nucleoprotein antigen). SDS-PAGE was carried out according to a conventional method using e-Padier 10% gel concentration (manufactured by Atoka). After the electrophoresis, the DNA was transferred to a PVDF membrane (manufactured by Atoka) and blocked at 4 ° C- 晚 with Block Ace (manufactured by Dainippon Pharmaceutical). After removing the blocking solution and washing with PBS_Tween, a monoclonal antibody adjusted to a concentration of 10 μg / ml was added and reacted at room temperature for 45 minutes. After washing thoroughly with PBS-Tween, dilute 4000 times with the reaction solution The enzyme-labeled anti-mouse IgG antibody (Cappel) was allowed to stand at room temperature for 45 minutes. After thorough washing with PBS_Tween, X-ray film (Kodak) was exposed for 1 minute using an ECL western blotting detection system (Amersham Bioscience) to detect signals.

[0077] With the monoclonal antibodies FrBl_03 and FrBl-07, a strong band was observed at around 6075 kD.

Reference Example 3 Preparation of Alkaline Phosphatase-Labeled Anti-Influenza B Virus Antibody Influenza B virus monoclonal antibody (FrBl-

03), the same treatment as in Example Reference Example 1 was repeated to obtain an alkaline phosphatase-labeled anti-influenza B virus antibody.

Example 5 Simultaneous immunoassay device for influenza A virus and influenza B virus

As shown in Figure 6, the nitrocellulose membrane (Millipore) with a width of 5 mm and a length of 50 mm was manufactured in Example 2 at positions 16 mm and 13.5 mm from the end of the developing solution absorption zone 5 side of Matritas 2. 0.7 μ1 of an aqueous solution containing the anti-influenza A virus antibody (FVA2-11) and the anti-influenza B virus antibody (FrBl_3) produced in Reference Example 2 was spotted on a nitrocellulose membrane, dried, and detected. Zones 6a and 6b were created. Further, an anti-alkaline phosphatase antibody (manufactured by Dako) was spotted at a position of 1 mm from the end of the developing solution absorption zone 5 side of the matrix 2 and dried to prepare a developing solution confirmation section 10. Next, the matrix was coated with the alkaline phosphatase-labeled anti-influenza A virus antibody (5 μg / ml) produced in Reference Example 1 and the alkaline phosphatase-labeled anti-influenza B virus antibody (5 μg / ml) produced in Reference Example 3. 5 μl of a mixed aqueous solution of the above was spotted and dried to form a labeling reagent zone comprising enzyme labeling reagent pad 4.

[0080] The developing solution pad 3 is a filter paper (manufactured by Millipore) having a width of 5 mm and a length of 20 mm, and 100 µg of 5_ bromo-4-black mouth-3-indolyl phosphoric acid (BCIP) as a substrate is applied to a width of 6 mm. It was prepared by spotting on a lmm line and drying. The matrix 2, the developing solution pad 3, the enzyme labeling reagent pad 4 and the developing solution absorbing pad 5 (filter paper (manufactured by Millipore) having a width of 10 mm, a length of 20 mm and a thickness of lmm) are placed in a plastic case having a developing solution tank 11. Immobilized, the influenza A virus and B virus simultaneous immunoassay device 1 shown in FIGS. 7 and 8 was produced. Reference Example 4 Influenza A virus and B virus simultaneous immunoassay device (conventional method device)

Regarding the detection zones 6a and 6b and the alkaline phosphatase-labeled anti-influenza virus antibody of the influenza A virus and B virus simultaneous immunoassay instrument 1 prepared in Example 3, the purchased anti-influenza A virus monoclonal antibody, respectively. And an anti-influenza B virus monoclonal antibody to prepare an influenza A and B virus simultaneous immunoassay instrument (Espline Influenza A & B (Fujirebio); conventional instrument).

Example 6 Measurement of Influenza A Virus and Influenza B Virus Sample spotting zone 8 of the influenza A virus and influenza B virus simultaneous measurement device 1 (the measurement device of the present invention) produced in Example 5 above After injecting 30 _{β1 of} each of the influenza A virus subtypes listed in Table 3 (using Tris buffer (PH8.0) containing a surfactant as a sample diluent), the deformed member The pressing portion 12 provided at the bottom is deformed by pressing downward, and the developing solution pad 3 is inserted into the developing solution tank 11 by the protrusion 13 attached to the deformable member, and the developing solution is supplied to the developing solution pad 3. Measurement was started. Fifteen minutes after the start of the measurement, after the development of the developing solution was confirmed by the color development of the target reagent zone 10, the color development of the detection zones 6a and 6b was visually measured. The results are shown in Table 3.

[0083] As a control, virus was detected in the same manner for the sample 30 / il described in Table 3 using the conventional measuring instrument prepared in Reference Example 4. Table 3 shows the measurement results.

[Table 3]

Table 3 Results of measurement of influenza A virus subtype strains

[0085] From the results, all of the influenza A virus subtype samples that could not be detected by the conventional measuring instrument were detectable by the measuring instrument of the present invention.

[0086] Example 7 Using the influenza A virus and influenza B virus simultaneous measuring device 1 (the measuring device of the present invention) produced in Example 5 above, adenovirus (type 17), Koxatsuki virus, in the same manner as in Example 6 (Types A16, B1—B6), simple herpesvirus 1, ecovirus (types 3, 4, 7, 22, and 30), enterovirus (type 71), mumps virus, polio virus (1 one type 3), RS virus (subgroup A, subgroup B), parametric influenza virus (1 one type _3), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus epidermidis, Myxomycetes, Staphylococcus aureus, Corynebacterium, Diphtheria, Candida 'albicans, Streptococcus pyogenes, Streptococcus sp. (Groups B, C, G, F), Streptococcus pneumoniae, Hemophilus influenza, Listeria' mono rhinoceros Genesu, Mycoplasma pneumoniae, lightheadedness di § trachomatis was investigated cross-reactivity with Kuramijia 'pneumoniae. As a result, it did not react with any of these viruses or bacteria.

Claims

The scope of the claims

[1] Influenza A virus reacts with nucleoprotein with molecular weight of 55-70 kD by antigen-antibody.

An anti-influenza A virus monoclonal antibody or an antigen-binding fragment thereof which does not substantially react with an influenza B virus;

[2] The monoclonal antibody or the antigen-binding fragment thereof according to claim 1, which reacts with an epitope in the amino acid sequence of amino acids 59 to 130 of the nucleoprotein of influenza A virus.

[3] The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 or 2, which reacts with each subtype of influenza A virus by an antigen-antibody reaction.

[4] Based on the Budapest Treaty, hybridoma FVA2-3 deposited internationally under accession number FERM BP-10066, hybridoma FVA2-6 deposited internationally under accession number FERM BP-10067, or internationally deposited under accession number FERM BP-10068 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is produced by the hybridoma FVA2-11.

[5] Influenza in which a labeled reagent zone having a movable labeled anti-influenza A virus antibody, a sample spotting zone, a developing solution supply zone, a developing solution absorption zone, and an anti-influenza A virus antibody immobilized in a matrix A device provided with a type A virus detection zone in a matrix, wherein at least one of the labeled anti-influenza A virus antibody and the anti-influenza A virus antibody immobilized in the detection zone is an antibody. An immunoassay device which is the monoclonal antibody according to item 4.

[6] The labeling reagent zone further contains a labeled anti-influenza B virus antibody, and an influenza B virus monoclonal antibody is further placed near the influenza A virus detection zone in which the influenza A virus monoclonal antibody has been immobilized. 6. The immunoassay device according to claim 5, which has an immobilized influenza B virus detection zone.

[7] The immunoassay device according to claim 5, wherein the labeled substance is an enzyme, and the substrate for the enzyme is contained in the developing solution and / or in the vicinity of the developing solution supply zone.