Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5302—Apparatus specially adapted for immunological test procedures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

• the present invention relates to an anti-influenza B virus monoclonal antibody and an immunoassay device using the antibody.
• 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.
• HA hemagglutinin
• NA neuraminidase
• influenza virus 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.
• influenza viruses have been detected with anti-influenza virus antibodies.
• 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 Documents 1 and 2).
• 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. It has been 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 is low and the measurement operation is complicated, and it 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).
• immunoassay devices (hereinafter referred to as "immunochromatography devices") using a strip-shaped matrix capable of infusion have been developed, and a special detection device, It has become possible to easily and simply measure antigens or antibodies in a sample in a short time without the need for sophisticated measurement techniques.
• the immunochromatography instrument 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 instrument has multiple zones to which different TP antigens are bound, and different anti-TP antibodies are detected separately to identify the time of infection and to be used for selection of therapeutic agents, etc. I have.
• 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
• an antibody that does not react with influenza A virus to select a therapeutic agent, but reacts with influenza B virus including a mutant strain of influenza virus is used. Therefore, there is no need for special diagnostic equipment or measuring equipment for influenza A virus or B virus discrimination, a measurement result can be obtained in a short time, and a device that can discriminate A or B in one operation. Was sought.
• an object of the present invention is to provide an anti-influenza B virus monoclonal antibody having high specificity.
• Another object of the present invention is to provide an immunoassay device capable of specifically detecting influenza B virus.
• the present inventors have succeeded in producing an anti-influenza B virus-less monoclonal antibody that specifically reacts with influenza B virus using the nucleoprotein of influenza B virus as an antigen.
• the present invention has been completed.
• an immunoassay device capable of distinguishing and detecting influenza B virus from influenza A virus by using the influenza B virus monoclonal antibody can be provided.
• the present invention provides an antigen-antibody reaction with a nuclear protein having a molecular weight of 60 to 75 kD of influenza B virus, which does not substantially react with an influenza A virus.
• a virus monoclonal antibody or an antigen-binding fragment thereof is provided.
• the present invention immobilizes a labeled reagent zone having a movable labeled anti-influenza B virus antibody, a sample spotting zone, a developing solution supply zone, a developing solution absorption zone, and an anti-influenza B virus antibody to a matrix.
• Influenza B wi A device provided with a Nores detection zone in a matrix, wherein at least one of the labeled anti-influenza B virus antibody and the anti-influenza B virus antibody immobilized in the detection zone is the monoclonal antibody of the present invention.
• the present invention provides an immunoassay instrument.
• an anti-influenza B virus monoclonal antibody which does not react with the ability to immunoreact with influenza B virus S and the influenza A virus.
• the immunoassay using the anti-influenza B virus monoclonal antibody of the present invention can discriminate and detect or quantify influenza B virus from influenza A virus.
• an immunoassay device using the anti-influenza B virus-free monoclonal antibody of the present invention By using the immunoassay device of the present invention, influenza B virus can be detected easily and indistinguishably from influenza A virus. Therefore, the present invention is expected to greatly contribute to diagnosis and treatment of influenza.
• 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 cross-sectional view schematically showing one example of a measuring instrument embodiment of the present invention.
• FIG. 3 is a plan view showing an example when the measuring instrument of the present invention is set in a cassette.
• FIG. 4 is a sectional view taken along line AA ′ of FIG. 3.
• the antibody reacts with a nucleoprotein of influenza B virus having a molecular weight of 60 to 75 kD by an antigen antibody.
• the antigen-antibody reaction with the influenza B virus nuclear protein with a molecular weight of 60-75 kD can be achieved by using influenza B virus as a sample and incorporating dodecyl sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into a Western blot. It can be confirmed by the plotting method.
• SDS-PAGE dodecyl sodium sulfate-polyacrylamide gel electrophoresis
• the monoclonal antibody of the present invention recognizes a nuclear protein having a molecular weight of 6075 kD.
• the monoclonal antibody of the present invention does not substantially react with an influenza A virus in an antigen-antibody reaction.
• 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 B virus.
• the antigen-antibody reaction with influenza B virus which is clearly weaker than that of the influenza B virus, means that it reacts only to a degree apparent to those skilled in the art.
• “there is substantially no antigen-antibody reaction with influenza A virus” means "influenza A virus nucleoprotein and each component protein of the virus and antigen antigen antibody substantially. It means not reacting.
• the monoclonal antibodies of the present invention also do not substantially react with influenza C virus.
• the monoclonal antibody of the present invention performs an antigen-antibody reaction with a nucleoprotein of each subtype of influenza B virus.
• influenza B virus has hemagnoretinin (H) and neuraminidase (N) on the surface of the virus particle, but 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 nuclear protein of a subtype (see Table 3) described in the following Examples, and more preferably a subtype of all known influenza B viruses. Reacts with the type of nuclear protein.
• 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 reaction.
• an infectious agent that is at least partially similar to influenza in antigen-antibody reaction.
• 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
• RS virus subgroup 8, subgroup B
• parainfluenza virus type 13
• Escherichia coli Klebsiella pneumoniae, Pseudomonas Fungi, germs, S. epidermidis, Staphylococcus epidermidis, S.
• an antibody fragment having a binding property to a corresponding antigen such as a Fab fragment (F (ab ') fragment, is obtained by decomposing the antibody with papain or pepsin (the present invention).
• 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.
• the monoclonal antibody of the present invention can be prepared by using the nucleoprotein of influenza B virus as an immunogen and the conventional hybridoma method.
• influenza B nucleoprotein used as an immunogen any of the 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 effect. It is possible.
• nucleoprotein purification by ultracentrifugation for example, see J. Biochem .; 102: 1241-1249, 1987
• protease treatment for example, J. Immunol. Methods; 180: 107-116, 1995
• the monoclonal antibody is obtained by immunizing an animal using an antigen containing the nucleoprotein of the influenza B virus as an immunogen, and fusing the influenza B virus monoclonal antibody-producing cells with tumor cells. Can be produced by the hybridoma obtained by the above method.
• the above-mentioned hybridoma can be obtained by the following method. That is, the influenza B virus nucleoprotein of the antigen obtained as described above and the complete adjuvant of Freund are 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).
• 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.
• a method for selecting a hybridoma that produces an antibody that recognizes the above-mentioned fused cell force influenza B virus nucleoprotein for example, the following method is used. It can. 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 hybridoma culture medium described above was reacted on an atsay plate on which highly purified influenza B-type virulent nucleoprotein was immobilized, followed by further reaction with anti-mouse immunoglobulin (Ig) and the like. Hybridomas that produce monoclonal antibodies that specifically recognize the influenza B virus nuclear protein can be selected by the EIA method or the like.
• hybridoma FrBl_03 hybridoma FrBl_07
• hybridoma FVB2-16 hybridoma FVB2-16
• FERM BP-10071 (Deposit date; transferred from FERM P-19442 on July 18, 2003 and July 14, 2004 to the International Deposit), and the hybridoma FVB2_16 has the identification number FVB2_16, Deposit number FERM BP-10069 (Deposit date: July 18, 2003, transferred to domestic deposit FERM P-19440 power on July 14, 2004 according to the Budapest Treaty) .
• 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.
• ascites can be stored and recovered from ascites by administering it to the animal from which the hybridoma originated.
• a commonly used purification method can be used, and examples thereof include gel filtration chromatography, ion exchange chromatography, affinity chromatography using protein A, and the like. .
• the monoclonal antibody produced according to the above method reacts with the nucleoprotein of influenza B virus and reacts with various virus strains of influenza B virus. confirmed. It reacted with the currently preserved and available influenza B virus stock (see Table 3 below) and was detectable with the monoclonal antibodies of the present invention.
• 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).
• enterovirus type 71
• mumps virus type 13
• respiratory syncytial virus subgroup A, subgroup B
• parainfluenza virus type 11
• Escherichia coli Klebsiella pneumoniae, Pseudomonas aeruginosa
• S. aureus Staphylococcus epidermidis
• S. aureus Staphylococcus aureus
• Corynebacterium Diphtheria, Candida albicans
• S. pyogenes Streptococcus sp .
• the monoclonal antibody of the present invention can be used for immunoassay for detecting or quantifying influenza B 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 a sandwich method, a competitive method, an agglutination method, a Western plot method, etc., and if classified according to the label used, there are a fluorescence method, an enzyme method, a radiation method, a biotin method, etc. Any of these can be used. Furthermore, the diagnosis can be made by immunohistological staining.
• a labeled antibody 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.
• an antibody-binding fragment (hereinafter referred to as “antigen-binding fragment”) can be obtained, but 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. it can.
• the antibody of the present invention or an antigen-binding fragment thereof is used. Is immobilized on a solid phase as a first antibody, reacted with a sample, washed, and reacted with a second antibody that reacts with the enzyme of the present invention as an antigen-antibody.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, A second antibody bound to the phase can be measured.
• the enzyme of the present invention in the test sample can be quantified.
• the first antibody and the second antibody may be interchanged with the above description.
• 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 applying the calibration curve, the enzyme of the present invention in the test sample can be quantified.
• the present invention also provides an immunoassay device capable of easily detecting influenza B virus and discriminating it from influenza A virus using the monoclonal antibody of the present invention. I do.
• the immunoassay device of the present invention comprises a labeled reagent zone having a movable labeled anti-influenza B virus antibody, a sample spotting zone, a developing solution supply zone, a developing solution absorption zone, and an anti-influenza B virus antibody.
• 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 caused to flow by the developing solution supplied from the developing solution supply zone, and reaches the influenza B virus detection zone.
• the anti-influenza B virus antibody immobilized on the matrix reacts with the labeled antigen-antibody complex to cause an antigen-antibody reaction, and the labeled antigen-antibody complex is immobilized on matrix. Whether or not the labeled antigen-antibody complex has been immobilized in the influenza B virus detection zone is determined by the presence or absence of the label.
• the developing solution that has passed through the influenza B 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 in the drug zone).
• the immunoassay device of the present invention at least one of the labeled antibody and the antibody immobilized in the influenza B virus detection zone is the monoclonal antibody of the present invention.
• One of the antibodies may be a polyclonal antibody. Since the influenza B virus nucleoprotein usually has multiple molecules associated with or attached to it, both the labeled antibody and the antibody immobilized in the influenza B virus detection zone are the same monoclonal antibody. Influenza B virus can be detected.
• 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.
• the absorbent material include a filter paper, a membrane, and a porous material produced by using cellulose or a derivative thereof such as cellulose or nitrocellulose, glass fiber, or the like, alone or in combination.
• the size of the matrix is not limited, a strip having a width of about 3 mm to 10 mm and a length of about 30 mm to 100 mm is preferable because it is easy to handle.
• a matrix having a thickness of 100 / im-1 mm can be used.
• 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.
• the detection zone can be provided with an influenza B virus detection unit in which an anti-influenza B virus antibody is immobilized on the matrix.
• At least one of the anti-influenza B virus antibodies immobilized in the detection portion is an anti-influenza B virus monoclonal antibody of the present invention together with a labeled anti-influenza B virus antibody described below, and both antibodies are anti-influenza B viruses.
• It is preferably a type B virus monoclonal antibody.
• the influenza B virus antibody in the detection section is provided on a matrix and provided in a line perpendicular to the infusion direction of the liquid for developing the matrix (the longitudinal direction of the matrix).
• Anti-influenza B virus-free polyclonal antibodies include commercially available and readily available polyclonal antibodies. Can be appropriately selected from these.
• the anti-influenza B virus antibody in this detection zone is the aforementioned antibody, and a monoclonal antibody may be used alone or in combination.
• the anti-influenza B virus antibody may be an IgG antibody, an IgM antibody, or an antigen-binding fragment of these antibodies, such as Fab, F (ab ').
• the anti-influenza B 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.
• an anti-influenza B virus antibody may be bound to a water-insoluble carrier, and this may be contained in the matrix.
• the insoluble carrier include 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.
• the binding can be carried out by the chemical bonding or physical adsorption.
• an influenza A virus detection unit using an anti-influenza A virus antibody in addition to an influenza B virus detection unit using an anti-influenza B virus antibody, an influenza A virus detection unit using an anti-influenza A virus antibody can be provided. If the influenza A virus detection unit is located near the influenza B virus detection unit, the influenza A virus detection unit may be located upstream or downstream in the infusion direction of the developing solution.
• the influenza A virus detection zone immobilized anti-influenza A virus antibody is immobilized on the matrix by chemical bonding or physical adsorption similarly to the anti-influenza B virus antibody, which may be a polyclonal antibody or a monoclonal antibody. Can be immobilized.
• the detection zone on the matrix is provided with at least an influenza B virus detection unit using an anti-influenza B virus antibody, and further provided with an influenza A virus detection unit. And influenza A 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 detector is 5 to 5 mm wide from the matrix.
• a plurality of lines can be provided close to a line of about mm. 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.
• the labeled reagent zone can be provided by movably spotting a labeled anti-influenza B 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 prepared 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 enzymatic labeling together with the pad on part or all of the matrix portion that adheres to the pad It is constituted by containing a reagent.
• a pad in a sample spotting zone described later can be used as a water-absorbing node.
• the antibody of the labeled anti-influenza B virus antibody together with the antibody provided in the detection zone, at least one is an anti-influenza B virus monoclonal antibody, and both antibodies are anti-influenza B virus monoclonal antibodies.
• it is an antibody.
• the antibody of the labeled anti-influenza B virus antibody a fragment thereof can be used similarly to the antibody in the detection zone.
• the labeled anti-influenza B 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.
• the enzyme include various enzymes used in an enzyme immunoassay (EIA), and examples of the enzyme include alkaline phosphatase, peroxidase, 1-D-galactosidase and the like.
• EIA enzyme immunoassay
• the metal colloid particles for example, gold colloid particles, selenium colloid particles, and the like can be used.
• a method for binding a labeled substance to an anti-influenza B virus antibody can be produced by using a known method for forming a covalent bond or a non-covalent bond.
• the binding method include a phthalaldehyde method, a periodic acid method, a maleimide method, a pyridyl disulfide method, and a method using various crosslinking agents (for example, “Protein Nucleic Acid Enzyme”, Supplement No. 31, 37 — See page 45 (1985)).
• a cross-linking agent for example, N-succinimidyl-14-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidohexanoic acid, N-succinimidyl-14- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, etc. can be used. .
• GMBS N-succinimidyl-14-maleimidobutyric acid
• N-succinimidyl-6-maleimidohexanoic acid N-succinimidyl-14- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, etc.
• a functional group present in the antibody can be used.
• a functional group such as a thiol group, an amino group, a carboxyl group, or a hydroxyl group
• the labeled anti-influenza antibody is labeled by the above-described bonding method.
• Type B virus antibodies can be produced.
• a physical adsorption method or the like can be used as a method using a non-covalent bond.
• a labeled anti-influenza B virus antibody is added to the labeling reagent zone to prepare a device.
• Labeled anti-influenza B 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 B virus antibody and a labeled anti-influenza A virus antibody can be included.
• the amount of the labeled anti-influenza B virus antibody and the amount of the labeled anti-influenza A virus antibody can usually be appropriately changed according to the expected amount of the test object, but is usually 0.01 ⁇ g in dry weight. It is about 5 ⁇ g.
• the labeled anti-influenza B virus antibody and the labeled anti-influenza A virus antibody are contained in the enzyme-labeled zone, they can be applied together with a reagent stabilizer, a dissolution regulator and the like.
• 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. Further, in the apparatus in which the sample spotting zone is provided in the enzyme labeling reagent zone, the water absorption containing the enzyme label as described above is used.
• a conductive pad in order to perform analysis efficiently.
• 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.
• a porous synthetic or natural material such as polybutyl alcohol (PVA), nonwoven fabric, or cellulose is used.
• PVA polybutyl alcohol
• 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.
• 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.
• a substrate can be added to the developing solution together with a substrate reagent zone described later.
• the buffer containing a buffer examples include an acetate buffer, a borate buffer, a Tris-HCl buffer, a diethanolamine buffer and the like.
• 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.
• filter paper such as cellulose or a cellulose derivative can be used.
• 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.
• the matrix can be provided with a water-absorbing material to promote the development.
• 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 liquid absorption zone contains all the developing liquid. There is a pad-shaped absorbent material with a capacity to absorb, but by stacking the absorbent material on or under the matrix, a shaped immunoassay device can be manufactured.
• the substrate 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.
• 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-16-sulfonic acid) (ABTS) in combination with hydrogen peroxide, 3,3,5,5 For tetramethylbenzidine (TMB), diaminobenzidine (DAB) alkaline phosphatase: 5-bromo-4-chloro-3-indolyl phosphate (BCIP)
• the substrate 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 5500 x g per instrument can be used.
• 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
• 7 is the substrate.
• 9 is the specimen.
• 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
• 11 is a developing solution tank (see Example 4 below).
• influenza B virus in various sample samples can be measured.
• the measurement is performed by first supplying the sample to the sample spotting zone of the measuring instrument of the present invention, then supplying the developing solution to the developing solution pad, and developing the matrix on the matrix.
• 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.
• the detection zone After a lapse of a predetermined time (usually 10 to 20 minutes), the detection zone is observed, and the labeling substance immobilized on the detection section by the influenza B virus in the sample solution is measured, whereby the influenza virus B virus is detected. 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.
• 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 B 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. 2 to 4 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.
• the sample 9 reacts with the labeled antibody in the labeling reagent zone 4 to form a labeled antigen-antibody complex.
• the labeled antigen-antibody complex moves through matrix 2 while reacting with the substrate in the developing solution, and when it reaches influenza B virus detection zone 6b, it is immobilized in influenza B virus detection zone 6b. Reacts with anti-influenza B virus antibody and becomes immobilized in influenza B virus detection zone 6b.
• influenza B virus detection zone 6b Reacts with anti-influenza B virus antibody and becomes immobilized in influenza B virus detection zone 6b.
• the sample contains influenza B virus
• the color develops due to the reaction between the labeling enzyme and the substrate.
• the labeled antibody cannot react with the antigen, and the labeling power is not immobilized in the influenza B virus detection zone 6b, so that the color is developed. Wake up.
• the developing solution confirmation zone 10 an enzyme that reacts with a reagent in the developing solution and develops a color is immobilized.
• an 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.
• the substrate in the developing solution may react to develop color (in the following examples, the anti-alkaline phosphatase antibody is immobilized).
• influenza A The detection of influenza virus can be performed at the same time, and whether the influenza virus is type B or A can be determined in a single measurement operation.
• 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).
• the immunogen may be an influenza HA peptide containing influenza nucleoprotein antigen (B / Yamanashi / 166/98 strain) or an influenza B recombinant nucleoprotein (from B / Yamanashi / 166/98 (r-NP / B); DDBJ / GeneBank database).
• influenza HA vaccine was diluted x300-fold with 0.1 M carbonate buffer PH9.6, and the B-type recombinant nucleoprotein antigen was also diluted to a concentration of 1 ⁇ g / ml, and added to each well of a microplate module (Nunc). 100 ⁇ l each was added, and the mixture was incubated at 4 ° C- ⁇ to immobilize it. Next, each well was washed with PBS containing 0.1% Tween 20 (trade name) (PBS-Tween), and diluted with PBS. / 0 ⁇ shea serum albumin (BSA) 300 ⁇ ⁇ added was 4 ° C over ⁇ blocking.
• PBS-Tween 0.1% Tween 20 (trade name)
• the culture supernatant was allowed to react for 1 hour at 37 ° C. After thorough washing with PBS_Tween, enzyme-labeled anti-mouse 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 Add 100 ⁇ l of Igs antibody (manufactured by DAKO) to each well 37. The reaction was performed for C1 hour.
• the culture supernatant determined to be positive was derived from influenza A recombinant nuclear protein antigen (from A / New Caledonia / 20/99 ( ⁇ - ⁇ / ⁇ 1 ⁇ 1), from A / Kitakyushu / 159/93 ( ⁇ - ⁇ / ⁇ 3 ⁇ 2); Secondary screening by ELISA using DDBJ / GeneBank database) as antigen And hybridomas that eventually react with influenza B nucleoprotein antigen and produce antibodies that do not react with influenza A nucleoprotein antigen (Hybridoma FrBl_03, hybridoma FrBl_07, hybridoma FVB2-16) Got.
• the subclasses of the monoclonal antibodies obtained from the three hybridoma strains were all IgGl. Table 1 shows the results.
• the DNA was transferred to a PVDF membrane (manufactured by Atto), 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 / ig / ml was added and reacted at room temperature for 45 minutes. After sufficient washing with PBS-Tween, an enzyme-labeled anti-mouse IgG antibody (manufactured by Cappel) diluted 4000 times with the reaction solution was allowed to stand at room temperature for 45 minutes.
• Table 2 shows the results of a 14-fold higher reactivity with the Lenza B virus monoclonal antibody.
• the anti-influenza B virus monoclonal antibody (FrBl_03) is treated with pepsin to cleave the Fc region to obtain F (ab '), and then using 2MEA (2-mercaptoethylamine hydrochloride, manufactured by Nacalai Testa).
• F (ab ') in which the free thiol group was exposed was obtained by cleaving the disulfide bond.
• alkaline phosphatase having a maleimide group introduced therein was coupled with F (ab '), and purified by gel filtration to obtain a purified alkaline phosphatase-labeled anti-influenza B type virus antibody.
• Influenza HA vaccine containing influenza nucleoprotein antigen as immunogen (manufactured by Kasei Ken: A / New Caledonia / 20/99 (H1N1) (IVR-116) strain, A / Panama / 2007/99 (H3N2)
• influenza HA vaccine included influenza A recombinant nucleoprotein antigen (A / New Caledonia / 20/99 derived (r-NP / HINl), A / Kitakyushu / 159/93 derived ( ⁇ Enzyme Linked Immunosobent Assay (ELISA method) on which - ⁇ / ⁇ 3 ⁇ 2)) was immobilized was used. That is, the influenza HA vaccine was diluted x300-fold with 0.1 M carbonate buffer PH9.6, and the type A recombinant nucleoprotein antigen was also diluted to a concentration of 1 ⁇ g / ml. 100 were added to each well, and the mixture was incubated at 4 ° C for 1 hour to solidify.
• influenza A recombinant nucleoprotein antigen A / New Caledonia / 20/99 derived (r-NP / HINl)
• a / Kitakyushu / 159/93 derived ⁇ Enzyme Linked Immunoso
• Enzyme-labeled anti-mouse Igs antibody (manufactured by DAKO), diluted 2000-fold with 0.05M phosphate buffer pH7.5 (reaction solution) containing / oKCl, is added to each well in a volume of ⁇ ⁇ ⁇ , and incubated at 37 ° C for 1 hour I let it. After the reaction, wash well with PBS-Tween, add 2,2'-azinobis-3_ethylbenzothiazoline-6-sulfonic acid (ABTS) to each well 100 ⁇ l each, and react at room temperature for 30 minutes. Then, 100 ⁇ l of a reaction stop 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.
• reaction solution 2,2'-azinobis-3_ethylbenzothiazoline-6-sulfonic acid
• the culture supernatant that was determined to be positive was subjected to secondary screening by ELISA using influenza B recombinant nuclear protein (B / Yamanashi / 166/98 (r-NP / B)) as an antigen.
• influenza B recombinant nuclear protein B / Yamanashi / 166/98 (r-NP / B)
• r-NP / B influenza B recombinant nuclear protein
• Reference Example 3 Preparation of Alkaline Phosphatase-Labeled Anti-Influenza A Virus Antibody Influenza A virus monoclonal antibody (FVA2 _11) and the same treatment as in Reference Example 1 was repeated to obtain an alkaline phosphatase-labeled anti-influenza A virus antibody.
• a nitrocellulose membrane manufactured by Millipore
• Reference Example 2 a nitrocellulose membrane (manufactured by Millipore) with a width of 5 mm and a length of 50 mm was manufactured in Reference Example 2 at positions 16 mm and 13.5 mm from the end of the developing solution absorption zone 5 side of Matrix 2.
• 0.7 ⁇ l of an aqueous solution containing the anti-influenza A virus antibody (FVA2-11) and the anti-influenza B virus antibody (FrBl_03) produced in Example 2 was spotted on a nitrocellulose membrane, dried, and detected. Zones 6a and 6b were created.
• 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.
• the matrix was treated with the alkaline phosphatase-labeled anti-influenza A virus antibody produced in Reference Example 3 (5 ag / ml) and the alkaline phosphatase-labeled anti-influenza B virus antibody produced in Reference Example 1 (7.5 ⁇ g / ml). 5 ⁇ l of an aqueous solution mixed with the above was spotted and dried to create a labeling reagent zone having four enzyme labeling reagent pads.
• 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 ⁇ g of 5_ bromo-4-black mouth-3 -indolinolenic acid (BCIP) as a substrate having 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 Whatman) 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. 3 and 4 was produced.
• the purchased anti-influenza A virus monoclonal antibody was respectively obtained.
• anti-influenza B virus monoclonal antibody, and an influenza A and B virus simultaneous immunoassay B (manufactured by Fujirebio); a conventional measuring instrument was prepared.
• Influenza A virus and influenza B virus simultaneous measurement device 1 (the measurement device of the present invention) produced in Example 3 above, the sample spotting zone 8 of the influenza type B virus subtype sample described in Table 3 ( As a sample diluent, a Tris buffer solution (PH8.0) containing a surfactant was used.) After 30 ⁇ l of each sample was spotted, the push-in section 12 provided on the deformable member was pressed down to deform and deformed. The developing solution pad 3 was introduced into the developing solution tank 11 by the projection 13 attached to the member, and the developing solution was supplied to the developing solution pad 3 to start the measurement. After 15 minutes from 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.
• influenza A virus and influenza B virus simultaneous measuring device 1 (the measuring device of the present invention) produced in the above-mentioned Example 3, the adenovirus (type 17) and the Koksatsu virus were used in the same manner as in Example 4.

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