JP4595810B2 - Anti-influenza A virus monoclonal antibody and immunoassay device using the antibody - Google Patents

Description

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

  Influenza has long been a worldwide epidemic, and many people have been killed each time. In 1931, the causative virus was detected and the path to elucidating the cause was opened. Influenza pathogen viruses are classified into three types, A type, B type and C type, depending on the antigenicity of soluble nucleoprotein (NP) inside the virus. Influenza type A is further classified into subtypes based on the antigenicity of two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are present on the surface of the virus.

  Since 1972, vaccines inactivated with formalin after ether treatment have been used to prevent influenza. In recent years, anti-influenza drugs have been found and widely used as therapeutic agents in addition to vaccines used for prevention. These therapeutic agents include amantadine hydrochloride adapted to influenza A virus, zanavir adapted to influenza A virus and B, oseltamivir phosphate, and the like.

  In selecting these therapeutic agents, it is important to detect influenza virus in a specimen and identify whether the infection is caused by influenza virus and the virus type is type A or type B. In addition, influenza A virus is more infectious than influenza virus B and causes serious symptoms. Therefore, identification of infectious virus is more important for early treatment.

  Conventionally, influenza viruses have been detected with anti-influenza virus antibodies. Until now, as an antibody against influenza A virus, in order to identify virus subtypes that are expected to be epidemic and to assist in vaccine production, to specifically recognize virus HA and NA and to identify virus subtypes (See, for example, (Patent Document 1) and (Patent Document 2)). In addition, the amino acid sequence of the nucleoprotein was determined for each subtype of influenza virus A, and reported to National Library of Medicine; Entrez Nucleotides database.

  Furthermore, as an apparatus for detecting influenza virus, for example, a solid phase capable of binding an influenza virus antigen is prepared, and after reacting the influenza virus in a sample, an influenza A virus antibody further bound with a first enzyme In addition, a flow-through type apparatus is known in which an influenza virus type B antibody to which a second enzyme is bound is reacted with the solid phase, a substrate is added to react, and the color on the solid phase is visually observed. (See (Patent Document 3)). Although this apparatus uses an antibody against the nucleoprotein of influenza virus as the antibody in the reagent, the measurement operation is complicated and it is not a simple measurement apparatus. Furthermore, an immunoassay instrument (hereinafter referred to as “immunochromatography instrument”) using a monoclonal antibody against influenza virus nucleoprotein is known (see (Non-patent Document 1)).

  On the other hand, an immunochromatographic instrument using a transmissible band-like matrix has been developed, and by simply spotting a specimen at a predetermined location, an antigen in the specimen can be easily and quickly prepared without requiring a special detection device or skilled measurement technique. Alternatively, antibodies can be measured. The immunochromatography instrument includes an immunochromatography instrument that binds a plurality of syphilis treponema antigens (TP antigens) to a detection zone, for example, and can detect a plurality of anti-TP antibodies in a specimen in separate detection zones depending on the substance to be measured. It has been developed (see (Patent Document 4)). The detection zone of this instrument has a plurality of zones with different TP antigens bound to it, and different anti-TP antibodies are detected separately in a single measurement to identify the time of infection, for selection of therapeutic agents, etc. It's being used.

Japanese Patent Application Laid-Open No. 6-100594 JP-A-7-304799 JP 2001-124775 A JP-A-9-229938 Journal of Infectious Diseases 2001; 75; 792-799

  Conventional anti-influenza A virus monoclonal antibodies, although antibodies against nucleoproteins are used in immunochromatography, etc., have low specificity and reactivity with influenza A viruses and are not satisfactory for highly sensitive immunoassays. . In addition, there is a need for an antibody that reacts with all influenza A viruses including subtypes in which HA and NA of influenza virus are mutated, although it does not react with influenza B virus in order to select an influenza therapeutic agent.

  Furthermore, there is a device that can obtain a measurement result in a short time without distinguishing between influenza A virus and B virus without using special diagnostic equipment or measuring device, and can discriminate between A type and B type in one operation. It was sought after.

  Accordingly, an object of the present invention is to provide an anti-influenza A virus monoclonal antibody with high specificity. Another object of the present invention is to provide an immunoassay instrument that can specifically detect influenza A virus.

  As a result of diligent research, the inventors of the present application have succeeded in producing an anti-influenza A virus monoclonal antibody that specifically reacts with influenza A virus using the influenza A virus nucleoprotein as an antigen. completed. Further, it has been conceived that by using this influenza A virus monoclonal antibody, an immunoassay instrument capable of distinguishing and detecting influenza A virus from influenza B virus can be provided.

That is, the present invention has an antigen-antibody reaction with a nucleoprotein having a molecular weight of 55 to 70 kD of influenza A virus, does not substantially undergo an antigen-antibody reaction with influenza B virus, and has an amino acid sequence 59 of the nucleoprotein of influenza A virus. From H1N1, H2N2, H3N2, H3N8, H4N6, H5N2, H6N2, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8 Provided are anti-influenza A virus monoclonal antibodies or antigen-binding fragments thereof that react with each subtype virus for antigen-antibody reaction. In the present invention, the labeled reagent zone having the movable labeled anti-influenza A virus antibody, the sample spotting zone, the developing solution supply zone, the developing solution absorption zone, and the anti-influenza A virus antibody are immobilized on the matrix. A device having an influenza 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 the monoclonal antibody of the present invention. An immunoassay device is provided.

  The present invention provides an anti-influenza A virus monoclonal antibody that immunoreacts with influenza A virus but does not react with influenza B virus. By 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. The present invention further provides an immunoassay instrument using the anti-influenza A virus monoclonal antibody of the present invention. By using the immunoassay instrument of the present invention, influenza A virus can be detected easily and distinguished from influenza B virus. Therefore, the present invention is expected to greatly contribute to the diagnosis and treatment of influenza.

It is a figure which shows the result when the monoclonal antibody of this invention is made to react about the antigen fractionated by the Western blot method. FIG. 6 is a view showing a restriction enzyme map of an expression plasmid (pW6A) used in Example 4. It is a figure which shows the amino acid of the fragment A1 to A7 which further subdivided the A fragment of influenza H1N1. It is a figure which shows the CBB staining pattern and Western blot which confirm the reactivity with FVA2-11. It is a figure which shows the nucleoprotein amino acid sequence (80-140) of influenza A virus subtype. It is sectional drawing which shows typically an example of the measuring instrument aspect of this invention. It is a top view which shows an example when the measuring instrument of this invention is set to the cassette. It is sectional drawing of AA 'of FIG.

  As described above, the antibody reacts with an influenza A virus nucleoprotein having a molecular weight of 55 to 70 kD. The antigen-antibody reaction with the influenza A virus molecular weight 55-70 kD nucleoprotein was confirmed by Western blotting using influenza A virus as a sample and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). can do. When Western blotting (see Examples below) is performed, the monoclonal antibody of the present invention recognizes a nuclear protein having a molecular weight of 55 to 70 kD.

  As described above, the monoclonal antibody of the present invention does not substantially undergo an antigen-antibody reaction with influenza B virus. Here, “substantially no antigen-antibody reaction” means that even if an antigen-antibody reaction does not occur at a detectable level or an antigen-antibody reaction occurs, the degree of the reaction is less than the antigen-antibody reaction with influenza A virus. Is clearly weak, meaning that no antigen-antibody reaction with influenza A virus reacts to a degree apparent to those skilled in the art. In addition, here, “substantially no antigen-antibody reaction with influenza B virus” means that there is substantially no antigen-antibody reaction with the protein of each component of the virus, including the nucleoprotein of influenza B virus. Meaning. In a preferred form, the monoclonal antibodies of the invention do not substantially react with influenza C virus.

  The monoclonal antibody of the present invention reacts with an epitope within the amino acid region of 130aa from the 59th amino acid residue (hereinafter referred to as “59aa”) of the nucleoprotein of influenza A virus nucleoprotein. The amino acid sequence of the nucleoprotein of influenza A virus is already known, and is described in, for example, GenBank Accession No. ITY238021. The amino acid sequences of 59aa to 140aa of various subtype nucleoproteins of influenza A virus are shown in FIG. Further, in a preferred form, the monoclonal antibody of the present invention reacts with the nucleoprotein of each subtype of influenza A virus, and thus has a common sequence in the amino acid sequence shown in FIG. For example, a region containing 90aa to 95aa, 111aa to 115aa, 120aa to 123aa, or the like, or a region having these common regions and having a common sequence is used as an epitope.

The monoclonal antibody of the present invention undergoes an antigen-antibody reaction with each subtype nucleoprotein of influenza A virus. That is, influenza A virus has hemagglutinin (H) and neuraminidase (N) on the surface of the virus particle, but is classified into various subtypes depending on the difference in the structures of H and N. The monoclonal antibody of the present invention reacts with an antigen-antibody with the following subtype of nucleoprotein. H1N1, H2N2, H3N2, H3N8, H4N6, H5N2, H6N2, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8, H5N1.

  Also, in a preferred form, the monoclonal antibodies of the invention do not substantially antigen-antibody reaction with other infectious pathogens that are at least partially similar in symptoms to influenza. For example, adenovirus (types 1-7), coxsackie virus (types A16, B1-B6), herpes simplex virus type 1, echovirus (type 3, type 4, type 7, type 22, type 30), enterovirus (type 71) ), Mumps virus, poliovirus (type 1 to 3), RS virus (subgroup A, subgroup B), parainfluenza virus (type 1 to 3), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, spirit fungus, epidermis grape Cocci, deformed bacteria, Staphylococcus aureus, corynebacteria, diphtheria, Candida albicans, Streptococcus pyogenes, Streptococcus sp. (Group B, C, G, F), Streptococcus pneumoniae, Haemophilus influenza, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia pneumoniae and substantially no antigen-antibody reaction.

Further, as is well known, antibody fragments having the ability to bind to corresponding antigens such as Fab fragments and F (ab ′) ₂ fragments by degrading antibodies with papain or pepsin (in this specification, “antigen binding”). It is known that an 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, and falls within the scope of the present invention.

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

  The monoclonal antibody is obtained by a hybridoma obtained by immunizing an animal using an antigen containing the nucleoprotein of the influenza A virus as an immunogen and fusing the influenza A virus monoclonal antibody-producing cells with tumor cells. Can be produced.

  The hybridoma can be obtained by the following method. That is, the influenza A virus nucleoprotein of the antigen obtained as described above was divided into several times together with Freund's complete adjuvant, and administered intraperitoneally or intravenously to animals such as mice every 2 to 3 weeks. Immunize by doing. Next, 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).

  The fusion method can be carried out with polyethylene glycol according to the usual method of Kohler and Milstein (Nature, 256, 495, 1975), or with Sendai virus or the like.

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

  In the following examples, a plurality of preferred monoclonal antibodies of the present invention were obtained, and three of them were designated as hybridoma FVA2-3, hybridoma FVA2-6 and hybridoma FVA2-11, respectively. These hybridomas can be found in the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, Tsukuba City, Ibaraki, Japan, 1st, 6th). Hybridoma FVA2-3 has the identification number FVA2-3 and the accession number. As FERM BP-10066 (date of deposit; July 18, 2003, transferred from the domestic deposit FERM P-19437 to the international deposit on July 14, 2004), the hybridoma FVA2-6 has the identification designation FVA2-6, the deposit number FERM BP-10067 (deposit date; July 18, 2003, transferred from the domestic deposit FERM P-19438 to the international deposit on July 14, 2004), and the hybridoma FVA2-11 has the identification indication FVA2-11, Accession number FERM BP-10068 (contract date; July 18, 2003, 2004) It has been an international deposit under the Budapest Treaty, respectively as converted to an international deposit) from domestic deposit FERM P-19439 a month 14 days.

  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 this ascites by administering it to the animal from which the hybridoma is derived.

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

  It was confirmed that the monoclonal antibody produced according to the above method reacted with the nucleoprotein of influenza A virus and reacted with various subtypes of influenza A virus. It reacts with 38 types of influenza A virus stocks (see Table 3 below), such as humans, horses, ducks, turkeys, seals, chickens, seagulls, mallards, etc., which are currently stored and available, and the monoclonal antibodies of the present invention Was detectable. On the other hand, examples of microorganisms that may have cross-reactivity include adenovirus (types 1 to 7), coxsackie virus (types A16 and B1 to B6), herpes simplex virus type 1, and echovirus (types 3, 4, 7, and 22). Type, type 30), enterovirus (type 71), mumps virus, poliovirus (type 1-3), RS virus (subgroup A, subgroup B), parainfluenza virus (type 1-3), E. coli, K. pneumoniae Pseudomonas aeruginosa, Pseudomonas aeruginosa, Staphylococcus epidermidis, deformed bacteria, Staphylococcus aureus, corynebacteria, diphtheria, Candida albicans, Streptococcus pyogenes, Streptococcus sp. (Group B, C, G, F) Reactivity with Streptococcus pneumoniae, Haemophilus influenza, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia pneumoniae, etc. was confirmed, but no reaction was observed. It was.

The monoclonal antibody of the present invention can be used for immunoassay for detection or quantification of influenza A virus. The immunoassay method itself is well known, and any known immunoassay method can be adopted. In other words, there are sandwich methods, competition methods, aggregation methods, Western blot methods, etc. if classified by measurement format, and fluorescent methods, enzyme methods, radiation methods, biotin methods, etc., if classified by the label used. Can also be used. Furthermore, it can be diagnosed by immunohistochemical staining. When a labeled antibody is used for the immunoassay method, the antibody labeling method itself is well known, and any known method can be employed. Further, as is well known, antibody fragments having the ability to bind to corresponding antigens such as Fab fragments and F (ab ′) ₂ fragments by degrading antibodies with papain or pepsin (in this specification, “antigen binding”). The antigen-binding fragment of the antibody of the present invention can be used in the same manner as the antibody of the present invention.

  These immunoassays are well known and need not be described in the present specification, but briefly described, for example, in the sandwich method, the antibody of the present invention or an antigen-binding fragment thereof is used as the first antibody. Immobilized on a solid phase, reacted with a specimen, washed, then reacted with an enzyme of the present invention and a second antibody that reacts with an antigen antibody. After washing, the second antibody bound to the solid phase is measured. The second antibody bound to the solid phase can be measured by labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like. Measure in a plurality of standard samples with known concentrations by the above method, create a calibration curve based on the relationship between the measured labeling amount and the enzyme of the present invention in the standard sample, and obtain the measurement results for the test sample of unknown concentration By applying this calibration curve, the enzyme of the present invention in the test sample can be quantified. The first antibody and the second antibody may be replaced 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 specimen to measure the absorbance. Measure in a plurality of standard samples with known concentrations by the above method, create a calibration curve based on the relationship between the measured labeling amount and the enzyme of the present invention in the standard sample, and obtain the measurement results for the test sample of unknown concentration By applying this calibration curve, the enzyme of the present invention in the test sample can be quantified.

  The present invention also provides an immunoassay instrument that uses the above-described monoclonal antibody of the present invention and can easily detect influenza A virus by distinguishing it from influenza B virus.

  The immunoassay device of the present invention immobilizes a labeled reagent zone having a movable labeled anti-influenza A virus antibody, a specimen spotting zone, a developing solution supply zone, a developing solution absorption zone, and an anti-influenza A virus antibody in a matrix. A device in which an influenza A virus detection zone is provided 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 the monoclonal antibody of the present invention. An immunoassay device that is an antibody. The specimen spotted in the specimen spotting zone undergoes an antigen-antibody reaction with the labeled antibody contained in the labeling reagent zone 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 on the matrix and the labeled antigen-antibody complex react with each other, and the labeled antigen-antibody complex is immobilized on the matrix. Whether or not the labeled antigen-antibody complex is 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 into the developing solution absorption zone. The specimen spotting zone and the labeling reagent zone may be the same (in this case, the specimen is spotted on the labeling reagent zone). In the immunoassay instrument of the present invention, at least one of a labeled antibody or an antibody immobilized on the influenza A virus detection zone is the monoclonal antibody of the present invention. One antibody may be a polyclonal antibody. In addition, since the nucleoprotein of influenza A virus usually has a plurality of molecules associated or attached, both the labeled antibody and the antibody immobilized on the influenza A virus detection zone are the same monoclonal antibody. Can also detect influenza A virus.

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

Matrix The band-shaped matrix in the immunoassay device of the present invention is composed of an absorptive material that can infuse a liquid by capillary action. Examples of the absorptive material include filter papers, membranes, and porous materials manufactured by mixing cellulose or a derivative thereof such as cellulose and nitrocellulose, glass fibers, or the like alone or in combination. Although there is no restriction | limiting in the magnitude | size of this matrix, A strip-shaped thing about 3 mm-10 mm in width and about 30 mm-100 mm in length is easy to handle and preferable. A matrix having a thickness of 100 μm to 1 mm can be used. The matrix is partially or wholly blocked with animal serum such as bovine serum albumin (BSA), casein, sucrose, etc. to prevent adsorption due to non-specific reaction of the protein derived from the sample to the matrix during measurement. Can be used.

Detection Zone In the detection zone, an influenza A virus detection unit in which an anti-influenza A virus antibody is immobilized on the matrix can be provided. The anti-influenza A virus antibody immobilized on the detection part is an anti-influenza A virus monoclonal antibody of the present invention, at least one of them together with a labeled anti-influenza A virus antibody described later. It is preferably a type virus monoclonal antibody. The influenza A virus antibody in the detection unit is on a matrix, and is preferably provided in a line shape in a direction perpendicular to the infusion direction of the liquid for developing the matrix (longitudinal direction of the matrix). The anti-influenza A virus polyclonal antibody can be appropriately selected from commercially available and easily available polyclonal antibodies.

The anti-influenza A virus antibody in this detection zone is the above-described antibody, and a monoclonal antibody can be used alone or in combination. The anti-influenza A virus antibody may be an IgG antibody, an IgM antibody, or Fab, F (ab ′) _2, etc., which are antigen-binding fragments of these antibodies.

  The anti-influenza A virus antibody immobilized on the detection unit may be physically adsorbed directly to the detection zone of the matrix, but can also be provided by immobilization by a chemical bond such as a covalent bond. Alternatively, the anti-influenza A virus antibody may be bound to a water-insoluble carrier and contained in the matrix. Examples of the insoluble carrier include particles obtained by insolubilizing a mixture of gelatin, gum arabic and sodium hexametaphosphate (Japanese Patent Publication No. 63-29223), polystyrene latex particles, and glass fibers. In order to bind the insoluble carrier to the anti-influenza A virus monoclonal antibody, the binding can be performed by the chemical bond or physical adsorption.

  In the detection zone, in addition to an influenza A virus detection unit using an anti-influenza A virus antibody, an influenza B virus detection unit using an anti-influenza B virus antibody can be provided. As long as the influenza B virus detection unit is in the vicinity of the influenza A virus detection unit, it may be upstream or downstream of the influenza A virus detection unit in the infusion direction of the developing solution. The anti-influenza B virus antibody to be immobilized on the influenza B virus detection zone may be a polyclonal antibody or a monoclonal antibody, and is immobilized on the matrix by chemical bonding or physical adsorption in the same manner as the anti-influenza A virus antibody. be able to.

  As described above, the detection zone on the matrix is provided with at least an influenza A virus detection part by anti-influenza A virus antibody, and further provided with an influenza B virus detection part. It is preferable when measuring virus simultaneously. These detection units are downstream of the enzyme labeling reagent zone, the specimen spotting zone, and the developing solution supply zone and upstream of the additive solution absorption zone in the infusion direction of the matrix. The detection unit can be provided with a plurality of lines in proximity to the matrix having a width of about 0.5 mm to 5 mm. In the case of a matrix having a width of about 5 mm, the detection part can be prepared by spotting the antibody and the antigen usually about 0.1 μg to 10 μg, respectively, and drying.

Labeling reagent zone The labeling reagent zone can be provided by spotting a labeled anti-influenza A virus antibody in a zone provided on the matrix so as to be movable. This zone can be provided upstream of the detection zone in the infusion direction of the developing liquid from the developing liquid supply zone. This zone consists of a method of spotting an enzyme labeling reagent on the matrix, a method of laminating a water-absorbing pad containing the enzyme labeling reagent on the matrix, or an enzyme labeling reagent together with the pad on part or all of the matrix part that is in close contact with the pad It is comprised by containing. As the water-absorbing pad, a pad in a specimen spotting zone described later can be used.

  As an antibody of a labeled anti-influenza A virus antibody, at least one is an anti-influenza A virus monoclonal antibody together with the antibody provided in the detection zone, and both antibodies are anti-influenza A virus monoclonal antibodies Is preferred. As the antibody of the labeled anti-influenza A virus antibody, a fragment thereof can be used in the same manner as the antibody in the detection zone.

  The labeled anti-influenza A virus antibody can be produced by binding the antibody and a label. Examples of the label include enzymes, metal colloid particles, colored latex particles, luminescent materials, fluorescent materials, and the like. Examples of the enzyme include various enzymes used in enzyme immunoassay (EIA). Examples of the enzyme include alkaline phosphatase, peroxidase, and β-D-galactosidase. Moreover, as a metal colloid particle, a gold colloid particle, a selenium colloid particle, etc. can be used, for example.

  Moreover, the coupling | bonding method of a label | marker and an anti-influenza A virus antibody can be manufactured using the method of making a well-known covalent bond or a non-covalent bond. Examples of the binding method include the gutalaldehyde method, the periodic acid method, the maleimide method, the pyridyl disulfide method, and a method using various crosslinking agents (for example, “Protein Nucleic Acid Enzyme”, Vol. 31, No. 37-37). 45 (1985)). In the bonding method using a crosslinking agent, examples of the crosslinking agent include N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidohexanoic acid, and N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1. -Carboxylic acid etc. can be used. In the method using covalent bonding, a functional group present in the antibody can be used. For example, a functional group such as a thiol group, amino group, carboxyl group or hydroxyl group is introduced by a conventional method, and then labeled anti-influenza A by the binding method. Type virus antibodies can be produced. Examples of the noncovalent bonding method include a physical adsorption method.

  In addition to detecting influenza A virus as described above, when detecting influenza B virus at the same time, a labeled anti-influenza B virus antibody is added to the labeling reagent zone to produce a device. The labeled anti-influenza A virus antibody and the labeled anti-influenza B virus antibody contained in the labeling reagent zone can be contained in either the matrix or the water-absorbing pad, or both the matrix and the water-absorbing pad can be labeled. Influenza A virus antibodies and labeled anti-influenza B virus antibodies can be included. When performing simultaneous measurement of influenza A virus and influenza B virus, since a large amount of labeling reagent is used, the enzyme labeling reagent can be contained in both the matrix and the pad either alone or in combination. Is advantageous. The amount of the labeled anti-influenza A virus antibody and the amount of the labeled anti-influenza B virus antibody can be appropriately changed depending on the expected amount of the test object, but is usually about 0.01 μg to 5 μg in dry weight. . The labeled anti-influenza A virus antibody and the labeled anti-influenza B virus antibody can be applied together with a reagent stabilizer, a dissolution regulator and the like when contained in the enzyme labeling zone.

Specimen spotting zone The specimen spotting zone can be provided in the matrix at the downstream side in the infusion direction of the development liquid in the development liquid supply zone and without any reagent or the like in the matrix upstream of the detection zone. Further, the sample spotting zone is 1) a predetermined location upstream of the enzyme labeling reagent zone on the downstream side of the developing solution infusion direction of the developing solution zone, 2) a predetermined location upstream of the detection zone on the downstream side of the labeling reagent zone, 3) It can be provided at a predetermined location on the labeling reagent zone. In addition, 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 for efficient analysis. In the device to which this pad is added, a large amount of sample liquid can be spotted, so that a trace component in the sample can be measured with high detection sensitivity. The water-absorbing pad is selected from a labeling reagent and a material that hardly adsorbs influenza virus in a specimen, and is made of, for example, a porous synthetic or natural polymer compound such as polyvinyl alcohol (PVA), non-woven fabric, and cellulose. The material which becomes can be comprised individually or in combination. The size, thickness, density, etc. of the pad are not limited, but it is usually preferable to use a pad having a length and width of about 3 mm to 10 mm and a thickness of about 0.5 mm to 4 mm for efficient measurement. .

Developing Solution Supply Zone The developing solution supply zone is a zone that is provided at one end in the longitudinal direction of the matrix and is supplied with the developing solution. In order to start the measurement, this zone can be immersed in a container containing at least an amount of the developing solution reaching the developing solution absorption zone. Further, for supplying the developing liquid, a liquid tank containing the developing liquid is added to the developing liquid zone, and the measurement can be started by 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. Further, when an enzyme is also used as the label, the substrate can be added to the developing solution together with the substrate reagent zone described later. Examples of the buffer solution containing a buffer include an acetate buffer solution, borate buffer solution, Tris-HCl buffer solution, diethanolamine buffer solution and the like. A developing solution pad can be attached to the developing solution supply zone in order to stably and continuously supply the developing solution to the matrix. As the developing solution pad, for example, filter paper such as cellulose or cellulose derivative can be used.

Developing solution absorption zone The developing solution absorption zone is installed at the other end with respect to the developing solution zone provided at one end of the matrix. This zone is provided in order to absorb the developing solution supplied to the matrix and perform analysis smoothly. The developing solution absorption zone can be secured by forming a long matrix. Further, a water absorbing material can be attached to the matrix to promote the development. As this water-absorbing material, a filter paper, sponge, etc. having high water retention composed of a natural polymer compound, a synthetic polymer compound or the like can be used. The developing fluid absorption zone is a pad-like absorbent material with a volume that absorbs all of the developing fluid, but a miniaturized immunoassay device is manufactured by laminating the absorbent material on or under the matrix. Can do.

Substrate reagent zone Further, when an enzyme is used as the labeling substance 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 in the vicinity of the developing solution supply zone of the matrix. The substrate reagent zone is preferably included in the developing solution pad attached to the developing solution supply zone in order to increase the basic mass and perform high sensitivity measurement.

  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′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) combined with hydrogen peroxide, 3,3 ′, 5,5′-tetra For methylbenzidine (TMB), diaminobenzidine (DAB) alkaline phosphatase: 5-bromo-4-chloro-3-indolyl phosphate (BCIP)
(B) For fluorescent substrate alkaline phosphatase: 4-methylum berylphenyl-phosphate (4MUP) For β-D-galactosidase: 4-methylum berylphenyl-β-D-galactoside (4MUG)
(C) For luminescent substrate alkaline phosphatase: 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD) β- For D-galactosidase: 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -β-D-galactopyranosyl) phenyl-1,2-dioxetane (AMGPD) peroxidase: peroxidation Luminol combined with hydrogen, isoluminol

  When the substrate is provided as a substrate zone, it can usually be formed by dissolving the substrate in an aqueous solution and applying it in a line to a developing solution pad, followed by drying. An agent, a dissolution regulator and the like can also be added. The substrate zone is not particularly limited as long as it is in the developing solution pad attached to the end of the matrix. Although the base mass added to a developing solution and a developing solution pad can be determined by measurement conditions, about 5-500 micrograms can normally be used per instrument.

Arrangement of Zones A preferred example of the immunoassay device of the present invention is schematically shown in FIGS. In FIG. 6, reference numeral 2 is a matrix, 4 is a labeling reagent zone, 8 is a specimen spotting zone, 3 is a developing solution supply zone, 5 is a developing solution absorption zone, 6a is an influenza A virus detection zone, and 7 is a substrate reagent. Zone 9 is the specimen. In this example, the specimen 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).

Method of Using Measuring Instrument Influenza A virus in various specimen samples can be measured with the measuring instrument of the present invention. The measurement is performed by first supplying the specimen to the specimen spotting zone of the measuring instrument of the present invention, and then supplying the developing liquid to the developing liquid pad and developing it in the matrix. Note that a buffer solution containing a surfactant can be used as the sample diluent as the sample diluent. The developing solution moves through the matrix by capillary action, reaches the developing solution absorption zone, and the components in the specimen, enzyme labeling reagent, etc. that are not bound to the detection zone are absorbed, and the development is completed. After a predetermined time (usually 10 to 20 minutes), the detection zone is observed, and the influenza A virus is measured by measuring the label immobilized on the detection part by the influenza A virus in the sample liquid. I can. This detection can be carried out by using a measuring device such as a visual observation or a colorimeter, a fluorometer, a photon counter, a photosensitive film and the like corresponding to the label or the enzyme used. In the measurement, for example, a method of visually measuring the color development in the detection zone is simple. In addition, in this method, a semi-quantitative analysis can be performed by using a color chart corresponding to the concentration of influenza A virus. Further, the coloration in the detection zone can be converted into a numerical value by a colorimeter or the like for quantitative determination.

  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 specimen 9 is spotted on the specimen spotting zone 8 and the developing liquid in the developing liquid tank 11 is supplied to the developing liquid supply zone 3. The developing solution moves in the direction of the horizontal white arrow in the matrix 2 by capillary action. The substrate contained in the substrate reagent zone 7 is dissolved in the developing solution and moves together with the developing solution. On the other hand, the specimen 9 reacts with the labeled antibody in the labeled reagent zone 4 to form a labeled antigen-antibody complex. When the developing solution arrives here, the labeled antigen-antibody complex moves in the matrix 2 while reacting with the substrate in the developing solution, and when reaching the influenza A virus detection zone 6a, it does not move to the influenza A virus detection zone 6a. It reacts with the anti-influenza A virus antibody that has been converted to the influenza A virus detection zone 6a. When influenza A virus is contained in the sample, color develops 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 undergo an antigen-antibody reaction, and the label is not immobilized in the influenza A virus detection zone 6a, so that color development does not occur. Therefore, whether or not influenza A virus is contained in the sample can be determined by whether or not the influenza A virus detection zone 6a is colored. In the developing solution confirmation zone 10, the enzyme that reacts with the reagent in the developing solution to develop color is immobilized, and when the developing solution confirmation zone 10 develops color, it can be seen that the developing solution has reached that point. Alternatively, the antibody against the labeling enzyme is immobilized in the developing solution confirmation zone 10, the labeling reagent that has reached the developing solution confirmation zone 10 is trapped by the antibody, and when the developing solution reaches further, the trapped label and The substrate in the developing solution may react to develop color (in the examples below, the anti-alkaline phosphatase antibody is immobilized). Moreover, if the labeled anti-influenza B virus antibody is included in the labeling reagent zone 4 while the anti-influenza B virus detection zone 6b in which the anti-influenza B virus antibody is immobilized is provided, the influenza B type Virus detection can also be performed at the same time, and it is possible to examine whether the influenza virus is type A or type B by a single measurement operation.

  Further, the matrix can be used by being laminated and fixed on a support member such as plastic, metal or paper. 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 a cover with a case having a hole in each zone portion is configured to constitute an instrument that is easy to handle. be able to. The specimen used in the immunoassay device of the present invention is a specimen considered to contain influenza collected from humans, animals, etc., and various body fluids such as nasal swabs (nasal swabs), nasal aspirates, throat swabs ( And bodily fluid extracts such as (pharyngeal swabs).

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

Example 1 Production of anti-influenza A virus monoclonal antibody An influenza HA vaccine containing an influenza nucleoprotein antigen as an immunogen (Kakekenken: A / New Caledonia / 20/99 (H1N1) (IVR-116) strain, A / Panama / 2007/99 (H3N2) NIB-41) strain). An equal amount of Freund's complete adjuvant (manufactured by Yatron) was added to the immunogen and mixed thoroughly, and then Balb / c mice were immunized 4 times at 2-week intervals. Three days before cell fusion, an immunogen was injected into the abdominal cavity of the mouse, and mouse spleen cells and myeloma cells (P3U1) were fused using PEG. As a culture solution, a HAT selection medium was used, and after about 2 weeks, the culture supernatant was collected and subjected to screening. For primary screening, influenza HA vaccine containing influenza nucleoprotein antigen, influenza A recombinant nucleoprotein antigen (A / New Caledonia / 20/99 origin (r-NP / H1N1), A / Kitakyushu / 159/93 origin Enzyme Linked Immunosorbent Assay (ELISA method) in which (r-NP / H3N2); DDBJ / GeneBank database) was immobilized was used. In other words, influenza HA vaccine was diluted x300 times with 0.1 M carbonate buffer pH 9.6, and A-type recombinant nucleoprotein antigen was diluted to a concentration of 1 μg / ml, and 100 μl was added to each well of a microplate module (manufactured by Nunc). Each was added and incubated at 4 ° C. overnight to immobilize. Next, after washing each well with PBS containing 0.1% Tween 20 (trade name) (PBS-Tween), 300 μl of 1% bovine serum albumin (BSA) diluted with PBS was added and blocked overnight at 4 ° C. went. After removing the blocking solution, 100 μl of the culture supernatant was added and reacted at 37 ° C. for 1 hour. After thoroughly washing with PBS-Tween, 2000 with a 0.05M phosphate buffer pH 7.5 (reaction solution) containing 0.2% BSA, 0.2% emulgen 985, 1% sucrose, 1% KCl. 100 μl of enzyme-labeled anti-mouse Igs antibody (DAKO) diluted 1-fold was added to each well and allowed to react at 37 ° C. for 1 hour. After the reaction, wash well with PBS-Tween, add 100 μl of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to each well and react at room temperature for 30 minutes. 100 μl 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.

  The culture supernatant determined to be positive is subjected to secondary screening by ELISA using influenza B recombinant nucleoprotein (B / Yamanashi / 166/98 (r-NP / B) DDBJ / GeneBank database) as an antigen. Hybridoma 3 strains (hybridoma FVA2-3, hybridoma FVA2-6, and hybridoma FVA2-11) that produce antibodies that react with the influenza A virus nucleoprotein antigen and do not react with the influenza B nucleoprotein antigen were obtained. These hybridomas are deposited at the Patent Microorganism Depositary Center as described above. All subclasses of monoclonal antibodies obtained from the hybridoma 3 strain were IgG1. Table 1 shows the results of the hybridoma reactivity.

Example 2 Reactivity in Western Blotting Method of Monoclonal Antibody Reactivity was confirmed by Western blotting method using a recombinant antigen, influenza HA vaccine (containing nucleoprotein antigen), and virus solution. Each sample was added to 0.01M Tris-HCl (pH 8.0) containing 0.001M EDTA, 1% SDS, 5% 2ME (2-mercaptoethanol), 10% glycerol, 0.005% BPB (Bromophenol Blue). It melt | dissolved and heat-processed to 100 degreeC, Then, it used for electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in accordance with a conventional method using an e-pagel 10% gel concentration (manufactured by Atto Corporation). After electrophoresis, it was transferred to a PVDF membrane (manufactured by Ato) and blocked at 4 ° C. overnight 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 thoroughly washing with PBS-Tween, an enzyme-labeled anti-mouse IgG antibody (manufactured by Cappel) diluted 4000 times with a 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 1 to 15 minutes using an ECL western blotting detection system (manufactured by Amersham Bioscience).

  In the monoclonal antibodies FVA2-3, FVA2-6, and FVA2-11, bands were recognized around 55 to 70 kD for all antigens. The result is shown in FIG.

Example 3 Antigen Measurement by ELISA Using Monoclonal Antibodies Three kinds of purified monoclonal antibodies at a concentration of 2 μg / ml in 0.1 M phosphate buffer pH 7.5, 100 μl in each well of a microplate module (manufactured by Nunc) Each was added and incubated at 4 ° C. overnight to immobilize. Next, after washing each well with PBS containing 0.1% Tween 20 (trade name) (PBS-Tween), 300 μl of 1% bovine serum albumin (BSA) diluted with PBS was added and blocked overnight at 4 ° C. went. 100 μl of influenza A virus solution diluted with the reaction solution was added to each well and reacted at 37 ° C. for 1 hour. After thoroughly washing with PBS-Tween, 100 μl of 3 kinds of alkaline phosphatase-labeled monoclonal antibodies prepared with the reaction solution were added to each well and reacted at 37 ° C. for 1 hour. After the reaction, thoroughly wash with PBS-Tween, add 100 μl of 4-nitrophenyl phosphate (4-NPP) to each well, react for 30 minutes at room temperature, and then add 100 μl of the reaction stop solution to each well. The color development level was measured at 415 nm subwavelength 490 nm. The positive was set to 2.1 times or more of the color development level of the buffer blank, and the strength of reactivity was represented by the final dilution factor that became positive.
In all 9 combinations of the three monoclonal antibodies obtained, the reactivity was 2 to 8 times higher than that of the commercially available anti-influenza A virus monoclonal antibody. The results are shown in Table 2.

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 monoclonal antibody FVA2-11, influenza H1N1 strain consisting of 498 amino acids (New Caledonia / 20/99) By PCR, three fragments of 1 to 159 amino acids (A fragment), 162 to 327 amino acids (B fragment) and 327 to 498 amino acids (C fragment) were amplified. EcoRI and BamHI sites were added to the primer in advance. The amplified fragment was purified by PCR Purification Kit of QIAGEN, inserted into the EcoRI-BamHI site of the expression plasmid pWG6A in which GST was incorporated into the NdeI-EcoRI site of the expression plasmid pW6A shown in FIG. H1N1-A, pWGInf. H1N1-B and pWGInf. H1N1-C was produced. These were used to transform E. coli BL21 (DE3) (obtained from Brookhaven National Laboratory), and ampicillin resistant transformant E. coli BL21 (DE3) pWGInf. H1N1-A, BL21 (DE3) pWGInf. H1N1-B and BL21 (DE3) pWGInf. H1N1-C was obtained.

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 ampicillin. After OD was adjusted to 0.6 to 0.8 at 600 nm in the preliminary culture, 0.4 mM IPTG was added to induce expression, and further cultured for 3 hours. A 1.5 ml amount of the bacterial cell culture solution is centrifuged at 5000 rpm for 2 minutes to collect the bacterial cells, suspended in 100 μl of a buffer (10 mM Tris-HCl, pH 8.0, 0.1 M sodium chloride, 1 mM EDTA), 15 The cells were completely disrupted by ultrasonic disruption for 1 minute. This is used as a cell sample.

  SDS polyacrylamide buffer (0.15 M Tris-HCl, pH 6.8, 6% SDS, 24% glycerol, 6 mM EDTA, 2% 2-mercaptoethanol, 0.03% bromophenol) in 8 μl of bacterial cell sample (Blue) 4 μl was added and stirred well, followed by SDS-polyacrylamide gel electrophoresis. Monoclonal antibody FVA2-11 diluted by 1000 times with a phosphate buffer (10 mM phosphoric acid, pH 7.4, 0.15 M sodium chloride) after blocking by 1% BSA and transferring by Western blotting on a nitrocellulose filter Was reacted. Furthermore, a peroxidase enzyme-labeled anti-mouse immunoglobulin rabbit polyclonal antibody (manufactured by Dako) was reacted, and after washing, 10 ml of a substrate coloring solution (0.01% hydrogen peroxide solution, 0.6 mg / ml 4-chloro-1- Naphthol) was added for color development. As a result, it was found that the monoclonal antibody FVA2-11 reacts only with GST + H1N1-A.

4-3 Preparation of Plasmid Next, fragments A1 (1 to 90 amino acids), A2 (85 to 159 amino acids), A3 (44 to 130 amino acids), A4 (59 to 103 amino acids) obtained by further subdividing the A fragment of influenza H1N1 A5 (44 to 103 amino acids), A6 (59 to 130 amino acids) and A7 (1 to 130 amino acids) (FIG. 3)) were amplified by PCR. The amplified fragment was purified by PCR Purification Kit of QIAGEN, inserted into the EcoRI-BamHI site of the expression plasmid pWG6A, and plasmid pWGInf. H1N1-A1, pWGInf. H1N1-A2, pWGInf. H1N1-A3, pWGInf. H1N1-A4, pWGInf. H1N1-A5, pWGInf. H1N1-A6 was produced. The A7 fragment was inserted into the EcoRI-BamHI site of pW6A shown in FIG. H1N1-A7 was produced. These were used to transform E. coli BL21 (DE3) (obtained from Brookhaven National Laboratory), and ampicillin resistant transformant E. coli BL21 (DE3) pWGInf. H1N1-A1. Bl21 (DE3) pWGInf. H1N1-A2,
BL21 (DE3) pWGInf. H1N1-A3
BL21 (DE3) pWGInf. H1N1-A4, BL21 (DE3) pWGInf. H1N1-A5
BL21 (DE3) pWGInf. H1N1-A6 and BL21 (DE3) pWINF. H1N1-A7 was obtained.

4-4 Expression of Recombinant Proteins (GST + H1N1-A1, GST + H1N1-A2, GST + H1N1-A3, GST + H1N1-A4, GST + H1N1-A5, GST + H1N1-A6 and H1N1-A7) The transformant prepared in 4-3 was treated at 50 μg / The cells were cultured at 37 ° C. in 2 ml of LB medium containing ml of ampicillin. After OD was adjusted to 0.6 to 0.8 at 600 nm in the preliminary culture, 0.4 mM IPTG was added to induce expression, and further cultured for 3 hours. A 1.5 ml amount of the bacterial cell culture solution is centrifuged at 5000 rpm for 2 minutes to collect the bacterial cells, suspended in 100 μl of a buffer (10 mM Tris-HCl, pH 8.0, 0.1 M sodium chloride, 1 mM EDTA), 15 The cells were completely disrupted by ultrasonic disruption for 1 minute. This is used as a cell sample.

  SDS polyacrylamide buffer (0.15 M Tris-HCl, pH 6.8, 6% SDS, 24% glycerol, 6 mM EDTA, 2% 2-mercaptoethanol, 0.03% bromophenol) in 8 μl of bacterial cell sample Add 4 μl of blue, molecular weight marker (117.6, 113.9, 81.2, 60.7, 47.4, 36.1, 25.3, 19.0, 14.7, 6.1 KD)) After stirring, SDS-polyacrylamide gel electrophoresis was performed. Monoclonal antibody FVA2-11 diluted by 1000 times with a phosphate buffer (10 mM phosphoric acid, pH 7.4, 0.15 M sodium chloride) after blocking by 1% BSA and transferring by Western blotting on a nitrocellulose filter Was reacted. Furthermore, a peroxidase enzyme-labeled anti-mouse immunoglobulin rabbit polyclonal antibody (manufactured by Dako) was reacted, and after washing, 10 ml of a substrate coloring solution (0.01% hydrogen peroxide solution, 0.6 mg / ml 4-chloro-1- Naphthol) was added for color development. Each fragment is shown in FIG. The CBB staining pattern after electrophoresis and the results of Western blotting are shown in FIG. From the above results, it can be seen that the recognition site of the anti-influenza A virus monoclonal antibody (FVA2-11) is the amino acid 59-130 region of influenza H1N1. Furthermore, from the influenza A subtype nucleoprotein amino acid sequence shown in FIG. 5, it has a sequence common to the reaction sites of amino acid numbers 59 to 130 and has a hydrophilic structure, for example, 90 to 95, 111 to 115. It is expected that the amino acid sequence such as position No. 120 or 123-123 is an epitope.

Reference Example 1 Preparation 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 ′) ₂ , and then 2ME ( Using 2-mercaptoethanol (manufactured by Nacalai Tesque), F (ab ′) was obtained by cleaving disulfide bonds and exposing free thiol groups. Next, alkaline phosphatase into which maleimide group was introduced and F (ab ′) were coupled, and purified alkaline phosphatase-labeled anti-influenza A virus antibody was obtained by gel filtration.

Reference Example 2 Production of anti-influenza B virus monoclonal antibody)
As an immunogen, an influenza HA vaccine containing an influenza nucleoprotein antigen (B / Yamanashi / 166/98 strain) or an influenza A recombinant nucleoprotein antigen (A / New Caledonia / 20/99 (r-NP / H1N1)), A / Kitakyushu / 159/93 origin (r-NP / H3N2) was used.

  An equal amount of Freund's complete adjuvant (manufactured by Yatron) was added to the immunogen and mixed thoroughly, and then Balb / c mice were immunized 4 times at 2-week intervals. Three days before cell fusion, an immunogen was injected into the abdominal cavity of the mouse, and mouse spleen cells and myeloma cells (P3U1) were fused using PEG. As a culture solution, a HAT selection medium was used, and after about 2 weeks, the culture supernatant was collected and subjected to screening. The primary screening used Enzyme Linked Immunosorbent Assay (ELISA method) in which influenza HA vaccine and influenza B recombinant nucleoprotein (r-NP / B) were immobilized. In other words, influenza HA vaccine was diluted x300 times with 0.1 M carbonate buffer pH 9.6, and recombinant B protein nucleoprotein antigen was diluted to a concentration of 1 μg / ml, and 100 μl was added to each well of a microplate module (manufactured by Nunc). Each was added and incubated at 4 ° C. overnight to immobilize. Next, after washing each well with PBS containing 0.1% Tween 20 (trade name) (PBS-Tween), 300 μl of 1% BSA diluted with PBS was added, followed by blocking at 4 ° C. overnight. After removing the blocking solution, 100 μl of the culture supernatant was added and reacted at 37 ° C. for 1 hour. After thoroughly washing with PBS-Tween, 2000 with a 0.05M phosphate buffer pH 7.5 (reaction solution) containing 0.2% BSA, 0.2% emulgen 985, 1% sucrose, 1% KCl. 100 μl of enzyme-labeled anti-mouse Igs antibody (DAKO) diluted 1-fold was added to each well and allowed to react at 37 ° C. for 1 hour. After the reaction, wash well with PBS-Tween, add 100 μl of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to each well and react at room temperature for 30 minutes. 100 μl 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.

  About the culture supernatant determined to be positive, influenza A recombinant nucleoprotein antigen (A / New Caledonia / 20/99 origin (r-NP / H1N1), A / Kitakyushu / 159/93 origin (r-NP / H3N2) )) As an antigen for secondary screening, and finally hybridoma 3 strains (FrB1-03, FrB1) that produce antibodies that react with influenza B nucleoprotein antigen and do not react with influenza A nucleoprotein antigen -07, FVB2-16). Each of the above hybridomas is based on the Budapest Treaty at the National Institute of Advanced Industrial Science and Technology, based on the Budapest Treaty. Deposited as FERM BP-10069. All subclasses of monoclonal antibodies obtained from the hybridoma 3 strain were IgG1.

(Reactivity in Western blotting of monoclonal antibodies)
Reactivity was confirmed by Western blotting using a recombinant antigen and influenza HA vaccine (containing nucleoprotein antigen). SDS-PAGE followed a conventional method using e-pagel 10% gel concentration (manufactured by Ato). After the electrophoresis, it was transferred to a PVDF membrane (manufactured by Ato) and blocked at 4 ° C. overnight 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 thoroughly washing with PBS-Tween, an enzyme-labeled anti-mouse IgG antibody (manufactured by Cappel) diluted 4000 times with a reaction solution was allowed to stand at room temperature for 45 minutes. After thoroughly washing with PBS-Tween, the signal was detected by exposing to an X-ray film (manufactured by Kodak) for 1 minute using an ECL western blotting detection system (manufactured by Amersham Bioscience).

  In the monoclonal antibodies FrB1-03 and FrB1-07, a strong band was recognized in the vicinity of 60 to 75 kD.

Reference Example 3 Preparation of Alkaline Phosphatase Labeled Anti-Influenza B Virus Antibody By using the anti-influenza B virus monoclonal antibody (FrB1-03) produced in Reference Example 2, the same treatment as in Example Reference Example 1 was repeated, An alkaline phosphatase labeled anti-influenza B virus antibody was obtained.

Example 5 Influenza A virus and influenza B virus simultaneous immunity measuring instrument As shown in FIG. 6, the end of the matrix 2 of a nitrocellulose membrane (made by Millipore) having a width of 5 mm and a length of 50 mm on the developing solution absorption zone 5 side 0.7 μl of an aqueous solution containing the anti-influenza A virus antibody (FVA2-11) produced in Example 2 and the anti-influenza B virus antibody (FrB1-3) produced in Reference Example 2 at positions of 16 mm and 13.5 mm from Were spotted on a nitrocellulose membrane and dried to prepare detection zones 6a and 6b. Furthermore, an anti-alkaline phosphatase antibody (manufactured by Dako) was spotted and dried at a position 11 mm from the end of the matrix 2 on the side of the developing solution absorption zone 5 to prepare a developing solution confirmation unit 10. Next, 5 μl of a mixed aqueous solution of 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 was used as a matrix. Were spotted and dried to create a labeling reagent zone consisting of the enzyme labeling reagent pad 4.

  The developing solution pad 3 is a line having a width of 6.1 mm and 100 μg of 5-bromo-4-chloro-3-indolylphosphoric acid (BCIP) as a substrate on a filter paper (Millipore) having a width of 5 mm and a length of 20 mm. Created by spotting and drying. The matrix 2, the developing solution pad 3, the enzyme labeling reagent pad 4, and the developing solution absorbing pad 5 (width 10 mm, length 20 mm, thickness 1 mm filter paper (Millipore)) are placed in a plastic case having a developing solution tank 11. The influenza A virus and B virus simultaneous immunity measuring instrument 1 shown in FIGS.

Reference Example 4 Influenza A virus and B virus simultaneous immunity measuring instrument (conventional instrument)
Regarding the detection zones 6a and 6b of the influenza A virus and B virus simultaneous immunity measuring instrument 1 produced in Example 3 and alkaline phosphatase-labeled anti-influenza virus antibody, purchased anti-influenza A virus monoclonal antibody and anti-influenza, respectively. An influenza A virus and B virus simultaneous immunity measuring instrument (Espline influenza A & B (manufactured by Fujirebio Inc.); conventional measuring instrument) was prepared using a B-type virus monoclonal antibody.

Example 6 Measurement of influenza A virus and influenza B virus Table 3 shows the sample spotting zone 8 of the influenza A virus and influenza B virus simultaneous measurement instrument 1 manufactured in Example 5 (measurement instrument of the present invention). Subtype samples of the influenza A virus described (using Tris buffer solution (pH 8.0) containing a surfactant as a sample diluent) 30 μl each, and then applying the pushing portion 12 provided on the deformable member It was deformed by applying pressure downward, and the developing solution pad 3 was inserted into the developing solution tank 11 by the protrusion 13 attached to the deformable member, and the developing solution was supplied to the developing solution pad 3 to start the measurement. 15 minutes after the start of the measurement, the development of the developing solution was confirmed by the color development of the target reagent zone 10, and then the color development of the detection zones 6a and 6b was visually measured. The results are shown in Table 3.

  As a control, the virus was similarly detected for 30 μl of the sample shown in Table 3 using the conventional measuring instrument prepared in Reference Example 4. The measurement results are shown in Table 3.

  From the results, it was possible to detect all the subtype specimens of influenza A virus that could not be detected with the conventional measuring instrument with the measuring instrument of the present invention.

Example 7
Using the influenza A virus and influenza B virus simultaneous measurement instrument 1 (measurement instrument of the present invention) produced in Example 5, the same as in Example 6, adenovirus (types 1 to 7), Coxsackie virus (A16, B1-B6 type), herpes simplex virus type 1, echovirus (type 3, type 4, type 7, type 22, type 30), enterovirus (type 71), mumps virus, poliovirus (types 1-3), RS Virus (subgroup A, subgroup B), parainfluenza virus (types 1 to 3), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus, Staphylococcus epidermidis, Deformation, Staphylococcus aureus, Corynebacterium, Diphtheria Albicans, Streptococcus pyogenes, Streptococcus sp. (Group B, C, G, F), cross-reactivity with Streptococcus pneumoniae, Haemophilus influenza, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia pneumoniae was examined. As a result, it did not react with any of these viruses or fungi.

Claims (4)

It reacts with an influenza A virus nucleoprotein having a molecular weight of 55 to 70 kD, does not substantially react with an influenza B virus by an antigen-antibody reaction, and has an amino acid sequence of amino acid sequence 59 to 130 of the influenza A virus nucleoprotein. H1N1, H2N2, H3N2, H3N8, H4N6, H5N2, H6N2, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8 and H5N1 subtypes of H5N1 An anti-influenza A virus monoclonal antibody or antigen-binding fragment thereof that reacts with an antibody.   Labeled reagent zone having a movable labeled anti-influenza A virus antibody, specimen spotting zone, developing solution supply zone, developing solution absorption zone, and influenza A virus detection zone in which anti-influenza A virus antibody is immobilized in a matrix The immunoassay device according to claim 1, 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 a monoclonal antibody. An influenza in which an influenza B virus monoclonal antibody is further immobilized in the vicinity of an influenza A virus detection zone further comprising a labeled anti-influenza B virus antibody in the labeling reagent zone and immobilizing the influenza A virus monoclonal antibody The immunoassay device according to claim 2, further comprising a type B virus detection zone. The immunoassay device according to claim 2 or 3 , wherein the label is an enzyme and contains a substrate for the enzyme in the developing solution and / or in the vicinity of the developing solution supply zone.

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