WO2005007698A1

WO2005007698A1 - Monoclonal antibody against influenza b virus and immunoassay instrument using the antibody

Info

Publication number: WO2005007698A1
Application number: PCT/JP2004/010476
Authority: WO
Inventors: Jun-Ichi Azumi; Takashi Yamada; Tomoe Honda; Nobuyuki Fujii
Original assignee: Fujirebio Inc.
Priority date: 2003-07-23
Filing date: 2004-07-23
Publication date: 2005-01-27
Also published as: CN100441597C; CN1826355A; JPWO2005007698A1; HK1096975A1; RU2006103302A; RU2366663C2; KR20060054333A

Abstract

A highly specific monoclonal antibody against influenza B virus and an immunoassay instrument using the same whereby influenza B virus can be specifically detected. This monoclonal antibody against influenza B virus undergoes an antigen-antibody reaction with a nucleoprotein of influenza virus B having a molecular weight of from 60 to 75 kD but substantially shows no antigen-antibody reaction with influenza A virus.

Description

Specification

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

Technical field

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

Background art

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

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

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

[0005] Conventionally, influenza viruses have been detected with anti-influenza virus antibodies. Up to now, antibodies against influenza A virus have been used to determine the subtype of the virus that is expected to spread and use it for vaccine production. Antibodies that specifically recognize DNA and NA and identify virus subtypes have been known (for example, see Patent Documents 1 and 2).

[0006] Further, as a device for detecting influenza virus, for example, a solid phase capable of binding to an influenza virus antigen is prepared, and after reacting the influenza virus in a sample, the influenza virus further bound to a first enzyme is used. A flow-through type device is used in which an influenza virus B antibody to which an A virus antibody and a second enzyme are bound is reacted with the solid phase, a substrate is added and the reaction is performed, and the color development on the solid phase is visually observed. 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).

[0007] On the other hand, 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. Depending on the substance to be measured, the immunochromatography instrument, for example, 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.

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

Patent Document 2: JP-A-7-304799

Patent Document 3: JP-A-2001-124775

Patent Document 4: JP-A-9-229938

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

Disclosure of the invention

Problems the invention is trying to solve [0009] Many of the conventional anti-influenza B virus monoclonal antibodies recognize the surface antigens HA and NA of the virus, and are satisfactory for high-sensitivity immunoassay with low specificity and reactivity. Was not. In addition, in the flow-through type apparatus, although the antibody in the reagent uses an antibody against the nucleoprotein of influenza virus, the measurement operation is complicated, and false positives are not detected in the detection of influenza B virus, which is not possible with a simple measurement apparatus. Was high (see J. Clin. Microbiol; 40: 1675-1680, 2002)

[0010] On the other hand, 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.

Therefore, 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.

Means for solving the problem

[0012] As a result of intensive studies, 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. Thus, the present invention has been completed. In addition, they have conceived that 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.

[0013] That is, 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. In addition, 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.

According to the present invention, there is provided 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. According to the present invention, there is further provided 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.

Brief Description of Drawings

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

FIG. 2 is a 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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0016] As described above, 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. When a Western plot (see Examples below) is performed, the monoclonal antibody of the present invention recognizes a nuclear protein having a molecular weight of 6075 kD. [0017] As described above, the monoclonal antibody of the present invention does not substantially react with an influenza A virus in an antigen-antibody reaction. Here, “substantially no antigen-antibody reaction” means that there is no antigen-antibody reaction at a detectable level, or even if an antigen-antibody reaction occurs, the degree of the antigen-antibody reaction with influenza B virus The fact that 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. In addition, here, "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. In a preferred form, the monoclonal antibodies of the present invention also do not substantially react with influenza C virus.

[0018] In a preferred embodiment, the monoclonal antibody of the present invention performs an antigen-antibody reaction with a nucleoprotein of each subtype of influenza B virus. In other words, 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.

[0019] In a preferred embodiment, the monoclonal antibody of the present invention does not substantially react with an infectious agent that is at least partially similar to influenza in antigen-antibody reaction. For example, adenovirus (type 17), coxsacki virus (type A16, B1-B6), simple virus 1 and echovirus (type 3, 4, 7, 22, 22 and 30) , Enterovirus (type 71), mumps virus, poliovirus (type 13), RS virus (subgroup 8, subgroup B), parainfluenza virus (type 13), Escherichia coli, Klebsiella pneumoniae, Pseudomonas Fungi, germs, S. epidermidis, Staphylococcus epidermidis, S. aureus, Staphylococcus aureus, Corynebacterium, Diphtheria, Candida 'albicans, Streptococcus pyogenes, Streptococcus sp. (Groups B, C, G, F), Streptococcus pneumoniae, Hemophilus Genoinfluenza, Listeria monocytogenes, Mycoplasma pneumonia, Chlamydia trachomatis, Chlamydia pneumoniae do not substantially react with antigenic antibodies. [0020] Also, as is well known, 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).

2

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

[0021] 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. As the 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. In order to suppress the effects of highly immunogenic HA and NA contained in the antigen, nucleoprotein purification by ultracentrifugation (for example, see J. Biochem .; 102: 1241-1249, 1987) and protease treatment (for example, J. Immunol. Methods; 180: 107-116, 1995) can also be used.

[0022] 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).

[0024] 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.

[0025] As 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.

In the following examples, a plurality of preferred monoclonal antibodies of the present invention were obtained, and three of them were named hybridoma FrBl_03, hybridoma FrBl_07, and hybridoma FVB2-16, respectively. These hybridomas are registered with the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (1-1, Tsukuba East, Ibaraki, Japan, No. 6, Central No. 6). As BP-10070 (acceptance date; July 18, 2003, transferred from FERM P-19441 to the International Depositary on July 14, 2004), the hybridoma FrBl-07 was identified by the identification number FrBl-07, and was accepted as a deposit. No. 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) .

[0027] Each of the above hybridomas is usually cultured in a medium used for cell culture, and the monoclonal antibody can be recovered from the culture supernatant. In addition, ascites can be stored and recovered from ascites by administering it to the animal from which the hybridoma originated.

[0028] As a method for recovering the above monoclonal antibody, 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. On the other hand, microorganisms that may be cross-reactive include, for example, adenovirus (type 17), Koksatsu virus (A16, B1-B6), simple herpes virus 1 and echovirus (types 3 and 4). , Type 7, 22 and 30), enterovirus (type 71), mumps virus, poliovirus (type 13), respiratory syncytial virus (subgroup A, subgroup B), parainfluenza virus (type 11) Type 3), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, S. aureus, Staphylococcus epidermidis, S. aureus, Staphylococcus aureus, Corynebacterium, Diphtheria, Candida albicans, S. pyogenes, Streptococcus _sp . (Group B, C, G, F), S. pneumoniae, Hemophilus influenza, Listeria; monocytogenes, Mycoplasma pneumonia, Chlamydia trachomatis, Chlamydia pneumoniae, etc. The force of the reaction was confirmed with S, the reaction is observed, such a force, ivy.

[0030] 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. When a labeled antibody is used for the immunoassay, the method of labeling the antibody itself is well known, and any of the well-known methods can be adopted. In addition, as is well known, antibodies are decomposed with papain or pepsin to bind to the corresponding antigen, such as Fab fragment or F (ab ') fragment.

2

It is known that an antibody-binding fragment (hereinafter referred to as “antigen-binding fragment”) can be obtained, but 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.

[0031] These immunoassays are well known and need not be explained in the present specification. However, in brief, for example, in the sandwich method, the antibody of the present invention or an antigen-binding fragment thereof 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. In a plurality of standard samples with known concentrations, measurement was performed by the above method, and a calibration curve was created based on the relationship between the measured amount of label and the enzyme of the present invention in the standard sample. By applying the measurement results to the calibration curve, the enzyme of the present invention in the test sample can be quantified. Note that the first antibody and the second antibody may be interchanged with the above description. In the agglutination method, the antibody of the present invention or an antigen-binding fragment thereof is immobilized on particles such as latex, and reacted with a sample to measure the absorbance. A plurality of standard samples with known concentrations were measured by the above method, and a calibration curve was created based on the relationship between the measured label amount and the enzyme of the present invention in the standard sample. By applying the calibration curve, the enzyme of the present invention in the test sample can be quantified.

[0032] 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. A device provided in a matrix with an influenza B virus detection zone immobilized 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 as described above. An immunoassay device that is the monoclonal antibody of the present invention. The sample spotted in the sample spotting zone reacts with the labeled antibody contained in the labeling reagent zone by an antigen-antibody reaction to form a labeled antigen-antibody complex. The antigen-antibody complex is caused to flow by the developing solution supplied from the developing solution supply zone, and reaches the influenza B virus detection zone. Here, 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. Note that the sample spotting zone and the labeling reagent zone may be the same (in this case, the sample is Spotted in the drug zone). In 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.

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

[0035] Matrix

The band-shaped matrix in the immunoassay device of the present invention is made of an absorbent material capable of injecting a liquid by capillary action. Examples of the absorbent material include a filter paper, a membrane, and a porous material produced by using cellulose or a derivative thereof such as cellulose or nitrocellulose, glass fiber, or the like, alone or in combination. Although 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. In addition, the matrix is blocked with animal serum such as bovine serum albumin (BSA), casein, sucrose, etc., in order to prevent non-specific reaction of protein from the sample to the matrix during measurement, in part or in whole. Can be used.

[0036] Detection zone

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. In order to measure with high sensitivity, it is preferable that 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). preferable. Anti-influenza B virus-free polyclonal antibodies include commercially available and readily available polyclonal antibodies. Can be appropriately selected from these.

[0037] 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 ').

[0038] 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. Further, an anti-influenza B virus antibody may be bound to a water-insoluble carrier, and this may be contained in the matrix. Examples of 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. In order to bind the insoluble carrier to the anti-influenza B virus monoclonal antibody, the binding can be carried out by the chemical bonding or physical adsorption.

[0039] In the detection zone, 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. Yeah. 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.

[0040] As described above, 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.

[0041] Labeling reagent zone

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. As a water-absorbing node, a pad in a sample spotting zone described later can be used.

[0042] As 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. Preferably, it is an antibody. As the antibody of the labeled anti-influenza B virus antibody, a fragment thereof can be used similarly to the antibody in the detection zone.

[0043] 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. Examples of 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. Further, as the metal colloid particles, for example, gold colloid particles, selenium colloid particles, and the like can be used.

[0044] In addition, 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. Examples of the binding method include a phthalaldehyde method, a periodic acid method, a maleimide method, a pyridyl disulfide method, and a method using various crosslinking agents (for example, “Protein Nucleic Acid Enzyme”, Supplement No. 31, 37 — See page 45 (1985)). In the bonding method using a cross-linking agent, as the 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. . In the covalent bond method, a functional group present in the antibody can be used.In addition, after a functional group such as a thiol group, an amino group, a carboxyl group, or a hydroxyl group is introduced by a conventional method, the labeled anti-influenza antibody is labeled by the above-described bonding method. Type B virus antibodies can be produced. In addition, as a method using a non-covalent bond, a physical adsorption method or the like can be used.

As described above, in addition to detecting influenza B virus, when detecting influenza B virus simultaneously, 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. When performing simultaneous measurement of influenza B virus and influenza A virus, since a large amount of labeling reagent is used, it is necessary to include an enzyme labeling reagent alone or in a mixture with both the matrix and the pad for high sensitivity measurement. It is advantageous. The amount of 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 μg in dry weight. It is about 5 μg. When 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.

[0046] Sample spotting zone

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

[0047] Developing liquid supply zone

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

[0048] Developing liquid absorption zone

The developing liquid absorption zone is provided at the other end with respect to the developing liquid zone provided at one end of the matrix. This zone is provided to absorb the developing solution supplied to the matrix and to perform the analysis smoothly. The developing liquid absorption zone may be formed by forming a long matrix to secure this zone. Further, the matrix can be provided with a water-absorbing material to promote the development. As the water-absorbing material, a highly water-retentive filter paper, sponge or the like made of a natural polymer compound, a synthetic polymer compound or the like can be used. The developing 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.

[0049] Substrate reagent zone

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

[0050] As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates and the like shown below corresponding to the enzyme of the labeling reagent can be used.

(A) For chromogenic substrate peroxidase: 2,2′-azinobis (3-ethylbenzothiazoline-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)

(b) Fluorescent substrate for alkaline phosphatase: 4-Methyl-mbellifueru-ru-phosphate (4 MUP) For β _D-galactosidase: 4-Methyl-mumbellifeel-β_D_galactoside (4 MUG)

(c) Luminescent substrate For alkaline phosphatase: 3_ (2 spiroadamantane) 4-methoxy 4- (3 "-phosphoryloxy) phenyl 1,2, -dioxetane disodium salt (AMPPD) For β-D_galactosidase : 3— (2-spiroadamantane) —4-methoxy_4— (3, shi—D—galactopyranosinole) phenyl 1,1,2-dioxetane (AMGPD) Peroxidase: Luminol combined with hydrogen peroxide , Isoluminol

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.

[0053] Arrangement of each zone One preferred example of the immunoassay device of the present invention is schematically shown in FIGS. In Fig. 6, reference numbers 2 are the matrix, 4 is the labeling reagent zone, 8 is the sample spotting zone, 3 is the developing solution supply zone, 5 is the developing solution absorption zone, 6a is the influenza A virus detection zone, and 7 is the substrate. Reagent zone, 9 is the specimen. In this example, the sample spotting zone 8 and the labeling reagent zone 4 are the same. Reference numeral 6b is an influenza B virus detection zone, 10 is a developing solution confirmation zone, and 11 is a developing solution tank (see Example 4 below).

[0054] How to use the measuring instrument

With the measuring instrument of the present invention, 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. As a sample diluent, a buffer containing a surfactant can be used as a sample diluent. The developing solution moves through the matrix by capillary action and reaches the developing solution absorption zone, where components in the sample, enzyme-labeled reagents, and the like that are not bound to the detection zone are absorbed, and the developing is completed. After 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. In the measurement, for example, a method of visually measuring the color development of the detection zone is simple. This method also enables semi-quantitative analysis by using a color chart (color chart) corresponding to the concentration of influenza 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.

[0055] 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. On the other hand, the sample 9 reacts with the labeled antibody in the labeling reagent zone 4 to form a labeled antigen-antibody complex. The developing liquid arrives here Then, 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. When the sample contains influenza B virus, the color develops due to the reaction between the labeling enzyme and the substrate. On the other hand, when the sample does not contain influenza B virus, 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. Therefore, it is possible to know whether or not the sample contains influenza B virus based on whether or not the influenza B virus detection zone 6b develops color. In the developing solution confirmation zone 10, an enzyme that reacts with a reagent in the developing solution and develops a color is immobilized. When the developing solution confirmation zone 10 develops color, it can be seen that the developing solution has reached there. Alternatively, an antibody against the labeling enzyme is immobilized in the developing solution confirmation zone 10, and the labeling reagent that has reached the developing solution confirmation zone 10 is trapped by the antibody. And the substrate in the developing solution may react to develop color (in the following examples, the anti-alkaline phosphatase antibody is immobilized). In addition, if labeled anti-influenza A virus antibody is included in labeled reagent zone 4 and anti-influenza A virus detection zone 6a in which anti-influenza A virus antibody is immobilized is provided, 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.

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

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples. There is no limitation.

Example

Example 1 Preparation of Anti-Influenza B Virus Monoclonal Antibody

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).

[0059] An equal volume of Freund's complete adjuvant (manufactured by Jatron) was added to the immunogen and mixed thoroughly, and then BallVc mice were immunized four times at 2-week intervals. Three days before cell fusion, an immunogen was injected intraperitoneally into mice, and mouse splenocytes were fused with myeloma cells (P3U1) using PEG. A HAT selection medium was used as a culture solution, and a culture supernatant was collected about 2 weeks later and subjected to screening. For the primary screening, an Enzyme Linked Immunosobent Assay (ELISA) in which influenza HA vaccine and influenza B recombinant nucleoprotein (r-NP / B) were immobilized was used. That is, the influenza HA vaccine was 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 μg / ml, and added to each well of a microplate module (Nunc). 100 µl each was added, and the mixture was incubated at 4 ° C- 晚 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 μ ΐ added was 4 ° C over晚blocking. After removing the blocking solution, 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 μl of Igs antibody (manufactured by DAKO) to each well 37. The reaction was performed for C1 hour. After the reaction, wash thoroughly with PBS-Tween, add 100 μl of 2.2'-azinobis-3_ethylbenzothiazoline-6-sulfonic acid (ABTS) to each well, react at room temperature for 30 minutes, and add the reaction stop solution. 100 μl was added to each well, and the color development level was measured at a primary wavelength of 415 nm and a secondary wavelength of 490 nm.

[0060] The culture supernatant determined to be positive was derived from influenza A recombinant nuclear protein antigen (from A / New Caledonia / 20/99 (Γ-ΝΡ / Η1Ν1), from A / Kitakyushu / 159/93 ( Γ-ΝΡ / Η3Ν2); 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.

[0061] [Table 1] Table 1 Monoclonal Antibody Reactivity

Antibody r-NP / B Vaccine Γ-ΝΡ / Η1Ν1 Γ-ΝΡ / Η3Ν2

ο

FrB1-03 1.682 0.57 CO8 0.020 0.022

FrB1 -07 1.721 0.598 0.024 0-023

FVB2-16 1.404 0.016

ο

Example 2 Reactivity of Monoclonal Antibody in ゥヱ Stamp Lotting Method

Reactivity was confirmed by a stamp lotting method using a recombinant antigen and an influenza vaccine (containing a nucleoprotein antigen). Each sample was dissolved in 0.01 M Tris-HCl (pH 8.0) containing 0.001 M EDTA, 1% SDS, 5% 2ME (2-mercaptoethanol), 10% glycerol, 0.005% BPB (Bromophenol Blue), and 100 After heat treatment at ° C, it was subjected to electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to a conventional method using e-Padier 10% gel concentration (manufactured by Ato Ichisha). After the electrophoresis, 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.

After washing thoroughly with PBS-Tween, the signal was detected by exposing to X-ray film (Kodak) for 1 minute using ECL Western Blotting Detection System (Amersham Bioscience).

[0063] For the monoclonal antibodies FrBl_03 and FrBl-07, a band was observed around 60 75 kD. . The result is shown in FIG.

Example 3 Measurement of Antigen by ELISA Using Monoclonal Antibody

100 μl of each of the three purified monoclonal antibodies was added to each well of a microplate plate module (manufactured by Nunc) at a concentration of 2 μg / ml in 0.1 M phosphate buffer ρΗ7.5, and incubated at 4 ° C. The solid phase was solidified. Next, wash each well with PBS (PBS-Tween) containing 0.1% Tween 20 (trade name), add 300 μl of 1% BSA diluted with PBS, and perform 4 ° C blocking. Was. 100 μl of the influenza B virus solution diluted with the reaction solution was added to each well and reacted at 37 ° C. for 1 hour. After thorough washing with PBS-Tween, 100 μl of each of the three kinds of alkaline phosphatase-labeled monoclonal antibodies diluted and adjusted with the reaction solution was added to each well and reacted at 37 ° C. for 1 hour. After the reaction, wash well with PBS-Tween, add 100 μl of 4-nitrophenylphosphate (4-NPP) to each well, incubate for 30 minutes at room temperature, and add 100 μl of the reaction stop solution to each well. The coloring level was measured at a primary wavelength of 415 nm and a secondary wavelength of 490 nm by adding μ1 each. The positivity was 2.1 times or more higher than the color development level of the buffer blank.

[0065] In all nine combinations of the three types of monoclonal antibodies obtained,

Table 2 shows the results of a 14-fold higher reactivity with the Lenza B virus monoclonal antibody.

[0066] [Table 2]

Table 2 Antigen measurement ELISA

Z '>> One-null antibody

Enzyme Marking Bong Jin B / Yamanashi / 166/98 8 / Ξ Ξ 重 / ΐ / 93

FrB1-03 FrBl-03 x40 x80

FrB1-07 x20 x80

FVB2-16 x40 x80

FrB1-07 FrB1 ~ 03 x40 x80

FrB1-07 x40 x80

FVB2-16 x40 x160

FVB2-16 * FiB1 ~ 03 x80 x320

FrB1-07 x80 x320

FVB2-16 x80 x160

Commercial antibody ** Commercial antibody ** x20 x80

*: Fab 'sign

Commercial monoclonal antibody

Reference Example 1 Preparation of Alkaline Phosphatase Labeled Anti-Influenza 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). As a result, F (ab ') in which the free thiol group was exposed was obtained by cleaving the disulfide bond. Next, 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.

Reference Example 2 Preparation of anti-influenza A virus monoclonal 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)

(NIB-41 strain, B / Yamanashi / 166/98 strain).

[0069] An equal amount of Freund's complete adjuvant (manufactured by Jatron) was added to the immunogen and mixed thoroughly, and then BallVc mice were immunized four times at 2-week intervals. Three days before cell fusion, an immunogen was injected intraperitoneally into mice, and mouse splenocytes were fused with myeloma cells (P3U1) using PEG. . A HAT selection medium was used as a culture solution, and a culture supernatant was collected about 2 weeks later and subjected to screening. The primary screening included influenza HA vaccine, influenza A recombinant nucleoprotein antigen (A / New Caledonia / 20/99 derived (r-NP / HINl), A / Kitakyushu / 159/93 derived (由来Enzyme Linked Immunosobent Assay (ELISA method) on which -ΝΡ / Η3Ν2)) 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 μg / ml. 100 were added to each well, and the mixture was incubated at 4 ° C for 1 hour to solidify. Next, wash each well with PBS (PBS-Tween) containing 0.1% Tween 20 (trade name), add 300 μl of 1% BSA diluted with PBS, and perform 4 ° C blocking. Was. After removing the blocking solution, the culture supernatant was reacted at 100 ° C for 1 hour at 37 ° C. After thorough washing with PBS_Tween, 0.2. /. BSA, 0.2% Emalgen 985, 1 Q / o sucrose, l. Enzyme-labeled anti-mouse Igs antibody (manufactured by DAKO), diluted 2000-fold with 0.05M phosphate buffer pH7.5 (reaction solution) containing / oKCl, is added to each well in a volume of Ιμ 反応, 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 μl each, and react at room temperature for 30 minutes. Then, 100 µl 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.

[0070] 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. , And three hybridomas that eventually produce antibodies that react with the influenza A virus nucleoprotein antigen and do not react with the influenza B nucleoprotein antigen (hybridoma FVA2_3, hybridoma FVA2-6, and hybridoma FVA2— 1 1) was obtained. Based on the Budapest Treaty, each of the above hybridomas was registered with the National Institute of Advanced Industrial Science and Technology under the Patent Organism Depositary, under the Budapest Treaty. Deposited as BP-10068.

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.

Example 3 Simultaneous immunoassay device for influenza A virus and influenza B virus

As shown in Fig. 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 μl 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. Further, an anti-alkaline phosphatase antibody (manufactured by Dako) was spotted at a position of 1 mm from the end of the developing solution absorption zone 5 side of the matrix 2 and dried to prepare a developing solution confirmation section 10. Next, the matrix was 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 μg / ml). 5 μl of an aqueous solution mixed with the above was spotted and dried to create a labeling reagent zone having four enzyme labeling reagent pads.

[0073] The developing solution pad 3 is a filter paper (manufactured by Millipore) having a width of 5 mm and a length of 20 mm, and 100 µg of 5_ bromo-4-black mouth-3 -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.

Reference Example 4 Influenza A and B virus simultaneous immunoassay devices (conventional measurement devices)

Regarding the detection zones 6a and 6b of the influenza A virus and B virus simultaneous immunoassay measurement device 1 produced in Example 3 and the alkaline phosphatase-labeled anti-influenza virus antibody, the purchased anti-influenza A virus monoclonal antibody was respectively obtained. And 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.

Example 4 Measurement of Influenza A Virus and Influenza B Virus

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 μl 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.

[Table 3] Table 3 Measurement results of influenza B virus strain

[0077] As a control, the test described in Table 3 was performed using the conventional measuring instrument prepared in Reference Example 4. Virus detection was performed in the same manner for 30 / il. Table 3 shows the measurement results.

[0078] From the results, all the influenza B virus samples that could not be detected by the conventional measuring instrument were all detectable by the measuring instrument of the present invention.

Example 5

Using the 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. (Types A16, B1—B6), simple herpesvirus 1, ecovirus (types 3, 4, 7, 22, and 30), enterovirus (type 71), mumps virus, polio virus (1 one type 3), RS virus (subgroup A, subgroup B), parametric influenza virus (1 one type _3), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus epidermidis, Myxomycetes, Staphylococcus aureus, Corynebacterium, Diphtheria, Candida 'albicans, Streptococcus pyogenes, Streptococcus sp. (Groups B, C, G, F), Streptococcus pneumoniae, Hemophilus influenza, Listeria' mono rhinoceros Genesu were examined Mycoplasma pneumoniae, lightheadedness di §. Trachomatis, cross-reactivity with Kuramijia 'pneumoniae. As a result, it did not react with any of these viruses or bacteria.

Claims

The scope of the claims

[1] An antigen-antibody reaction with a nucleoprotein having a molecular weight of 60 75 kD of influenza B virus and no substantial antigen-antibody reaction with influenza A virus, an anti-influenza B virus monoclonal antibody or an antigen-binding fragment thereof .

[2] Based on the Budapest Treaty, hybridoma FrBl_03 deposited internationally under accession number FERM BP-10070, hybridoma FrBl_07 deposited internationally under accession number FERM BP-10071, or hybridoma FVB2 deposited internationally under accession number FERM BP-10069— 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is produced by (16).

[3] Labeled reagent zone with movable labeled anti-influenza B virus antibody, specimen spotting zone, developing solution supply zone, developing solution absorption zone, and influenza B immobilized with anti-influenza B virus antibody in matrix A device provided with a matrix-type virus 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 an antibody according to claim 1 or 2. An immunoassay device which is the monoclonal antibody according to the above.

[4] The labeled reagent zone further contains a labeled anti-influenza A virus antibody, and the influenza A virus monoclonal antibody is further immobilized near the influenza B virus detection zone where the influenza B virus monoclonal antibody has been immobilized. 4. The immunoassay device according to claim 3, which has an influenza A virus detection zone.

5. The immunoassay device according to claim 3, wherein the labeled substance is an enzyme, and the substrate for the enzyme is contained in the developing solution and / or in the vicinity of the developing solution supply zone.