JP2006067979A - Immunity detecting method for influenza a type virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and easily detecting a wide range of influenza A type viruses, and to provide an apparatus for the same. <P>SOLUTION: A recombinant NS1 protein and a monoclonal antibody to the same are obtained by cloning a fraction of a gene encoding NS1 protein which is a non structural protein common in various kinds of influenza A type viruses. This invention provides an immunoassay method using an antibody against the NS1 protein of the influenza A type viruses, especially a sandwich type immunoassay method using a first antibody and a second antibody against the NS1 protein, in particular, the invention provides an immunochromatography method and a testing split for immunochromatography. At least one of the first antibody and the second antibody is preferably the monoclonal-antibody. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an immunodetection method for influenza A virus using an antibody against NS1 protein, which is a nonstructural protein of influenza A virus, and in particular, a sandwich immunoassay method, in particular, an immunochromatographic assay method and an immunochromatographic test strip. The present invention relates to a detection method useful for quickly and easily diagnosing a disease caused by influenza A virus. The present invention also relates to a method for immunodetection of antibodies against influenza A virus.

  Influenza viruses infect the upper and lower respiratory tracts of humans, and after a incubation period of 1-2 days, cause fever (38-39 ° C.) and headache, low back pain, muscle pain, general malaise, digestive symptoms, etc. The most common complications of influenza are pneumonia in the elderly and encephalitis / encephalopathy in children. Especially in children, encephalitis / encephalopathy is characterized by high fever, disturbance of consciousness, and convulsions. Rapid diagnosis and treatment is required because the time to onset of symptoms and death is very short.

  Conventionally, it was often diagnosed as an influenza-like disease only with the above clinical symptoms. However, since a kit for rapidly detecting an influenza virus antigen has recently been developed and spread, It has become possible to diagnose early. Examples of such rapid diagnosis kits include those that use enzyme immunoassay (EIA) or immunochromatography assay as the principle, and those that detect only influenza A virus, or those that detect A and B together. And those that detect A type and B type separately.

Anti-influenza virus agents have also been developed for the treatment of influenza virus infections. For example, inhalation zanamivir and oral oseltamivir have been approved as neuraminidase inhibitors effective for both types A and B .
Therefore, appropriate treatment has become possible by combining diagnosis with a rapid diagnosis kit and treatment with an anti-influenza virus agent.

  However, administration of neuraminidase inhibitors should begin within 2 days of the onset of influenza A or B virus cold symptoms. On the other hand, in the conventional rapid diagnosis kit, when nucleoprotein (NP) is used as an antigen to be detected, NP is relatively less susceptible to mutation and is a protein that is widely shared among various viruses of the same type. Although it has excellent specificity for, it has the drawback of not being sufficiently sensitive in the early stages of infection because it is present in the virus particles. Further, when proteins such as hemagglutinin (HA) and neuraminidase (NA) present on the surface of influenza virus are used as antigens to be detected, there is a disadvantage that the specificity is low because these surface proteins vary greatly.

Mitamura, “Fast Diagnosis of Influenza”, Clinical Laboratory, February 2002, Volume 46, No. 2, p. 169-173 Mitamura and two others, “Influenza antigen test using POCT kit”, Test and Technology, May 2002, Volume 30, No. 5, p. 443-448 Minami, “Structure, Function and Expression Control of Influenza Virus NS1 and NS2 Proteins”, Nihon Lin, October 1997, Vol. 55, No. 10, p. 107-111 I. Birch-Machin et al., “Expression of the nonstructural protein NS1 of equine influenza A virus: detection of Anti-NS1 antibody in post infection equine sera”, Journal of Virological Methods, 65 (1997) p.255-263 H. Ozaki et al., “Detection of antibodies to the nonstructural protein (NS1) of influenza A virus allows distinction between vaccinated and infected horses”, Veterinary Microbiology, 82 (2001) p.111-119 K. Nakajima et al., “Evolution of the NS Genes of the Influenza A Viruses. II. Characteristics of the Amino Acid Changes in the NS1 Proteins of the Influenza A Viruses”, VIRUS GENES 4: 1, (1990) p.15-16

Influenza spreads rapidly over a wide area when it is quickly detected and delayed, so it is necessary to detect the infection as soon as possible. In addition, if infection can be detected earlier than before, a therapeutic agent such as an anti-influenza virus agent can be administered early and used more effectively, and early and reliable treatment of influenza virus infection can be expected.
An object of the present invention is to provide a method and a measuring instrument that can detect a wide range of influenza A viruses quickly and easily at an early stage of infection.

  The present inventors have succeeded in cloning a specific fragment of NS1 protein, which is a nonstructural protein of influenza A virus, and expressing it by genetic engineering. It has been found that a wide range of influenza A viruses can be detected at an early stage of infection by using it in an immunoassay, particularly an immunochromatography assay, and the present invention has been completed.

That is, according to one aspect of the present invention, there is provided a method for detecting influenza A virus comprising an immunoassay using an antibody against NS1 protein of influenza A virus.
The immunoassay method in this detection method is not particularly limited, but a sandwich immunoassay method, particularly an ELISA (Enzyme-linked immunosorbent assay) method, an immunochromatographic assay method and the like are preferable.

Therefore, according to another aspect of the present invention, an influenza A virus characterized by comprising a sandwich immunoassay using a first antibody and a second antibody against NS1 protein of influenza A virus. A detection method is provided.
According to a preferred embodiment of the present invention, a membrane carrier having a capture site formed by previously immobilizing a first antibody against the influenza A virus NS1 protein at a predetermined position is prepared, and the first antibody against the NS1 protein is prepared. A mixture of the second antibody and a predetermined amount of the test sample is chromatographed on the membrane carrier toward the capture site, and the influenza A virus-derived NS1 protein and the second sample contained in the test sample An immunochromatographic measurement method is provided, wherein a complex with an antibody is captured at the capture site.
Moreover, according to a preferred embodiment of the present invention, the apparatus comprises at least a first antibody against NS1 protein of influenza A virus, a second antibody, and a membrane carrier, and the first antibody is located at a predetermined position of the membrane carrier. Influenza, which is immobilized in advance to form a capture site, and the second antibody is labeled with an appropriate labeling substance and arranged so as to be chromatographed on the membrane carrier at a position separated from the capture site An immunochromatographic test strip for detecting type A virus is provided.

  The antibody against NS1 protein used in the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity. In the case of a sandwich immunoassay such as an immunochromatography assay, the first antibody and the second antibody used there may be either a polyclonal antibody or a monoclonal antibody. In general, at least one of the antibodies is preferably a monoclonal antibody, and both antibodies are particularly preferably a monoclonal antibody.

A preferred monoclonal antibody in the present invention is a monoclonal antibody against a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and more preferably a monoclonal antibody that reacts with all of influenza A virus subtypes H1 to 15.
An antigenic determinant (epitope) exists in the region consisting of 126 amino acid residues on the C-terminal side of NS1 protein of influenza A / equine 2 / Suffolk / 89, which is an influenza A virus of horse, and encodes this NS1 protein It has been reported in Non-Patent Document 4 that the gene (NS1 gene) has 97% homology with influenza A / equine 2 / Miami / 63, which is an influenza A virus of horses. Although isolation has failed and antisera against NS1 protein have been obtained, nothing is handled about monoclonal antibodies.
The amino acid sequence of SEQ ID NO: 1 in the sequence listing corresponds to the full-length amino acid sequence of the NS1 protein of A / Equine / Miami / 1/63 (H3N8), which is an equine influenza A virus. It is thought to encompass regions that are conserved among a wide range of subspecies. The gene corresponding to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is cloned in the present invention, and the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is expressed and purified from the cloned gene by genetic engineering. It is useful for detecting antibodies against viruses, particularly antibodies against NS1 protein (anti-NS1 antibodies). Various monoclonal antibodies against the peptide consisting of the amino acid sequence of SEQ ID NO: 1 are considered novel.

Therefore, according to the other aspect of this invention, the monoclonal antibody with respect to the peptide which consists of an amino acid sequence of sequence number 1 is provided.
According to still another aspect of the present invention, any one of the following peptides (a) and (b) is provided.
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1,
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and exhibiting the immunogenicity of NS1 protein of influenza A virus.

Furthermore, according to still another aspect of the present invention, a gene encoding any one of the peptides (a) and (b) above and a recombinant vector containing the gene are provided.
Furthermore, according to still another aspect of the present invention, a transformant containing the above recombinant vector, and the immunogenicity of NS1 protein of influenza A virus derived from the recombinant vector expressed by this transformant. A recombinant protein is provided. As described above, this recombinant protein can be used to detect antibodies against influenza A virus, particularly anti-NS1 antibodies.

Therefore, according to the other aspect of this invention, the detection method of the antibody with respect to the influenza A virus which consists of an immunoassay using the said recombinant protein is provided.
According to a preferred embodiment of the present invention, a membrane carrier having a capture site formed by preliminarily immobilizing the above-mentioned recombinant protein in a predetermined position is prepared, and a test sample collected from a living body and an antibody against immunoglobulin in the living body And the mixture of the antibody and the antibody against the NS1 protein of influenza A virus contained in the test sample is captured at the capture site. There is provided an immunochromatographic assay for detecting an antibody against influenza A virus characterized by

  According to another preferred embodiment of the present invention, the apparatus comprises at least the recombinant protein, an antibody against biological immunoglobulin, and a membrane carrier, and the recombinant protein is preliminarily fixed at a predetermined position of the membrane carrier and has a capture site. The anti-influenza type A collected from the living body, wherein the antibody is labeled with an appropriate labeling substance and arranged so as to be chromatographed on the membrane carrier at a position separated from the capture site An immunochromatographic test strip for detecting viral antibodies is provided.

The NS1 protein of the influenza A virus detected in the present invention is accumulated in the nucleus of the cell at the early stage of infection in the influenza A virus-infected cells, and is also accumulated in the cytoplasm after the middle stage of the virus infection. Thus, NS1 protein is found in infected cells, which means that influenza A virus is already expressed in a large amount before it proliferates inside the cell, forms virus particles, and is released outside the cell. is doing. This means that the protein is expressed before the nuclear protein that has been conventionally detected. Therefore, the present invention that uses NS1 protein as a detection target enables determination at an early stage of infection more than conventional influenza antigen detection reagents. Furthermore, since this NS1 protein is a protein common to viruses belonging to influenza A virus, a wide range of influenza A viruses such as human influenza virus, swine influenza virus, avian influenza virus, and equine influenza virus, in particular, A virus It can be used in common for the detection of all subtypes of the above and is widely applicable to the diagnosis of influenza infections in humans and animals.
In addition, the recombinant NS1 protein cloned and expressed in the present invention can be easily prepared and used as an antigen for simple and rapid antibody detection.

In the present invention, the NS1 protein of influenza A virus is synthesized by referring to the information corresponding to the amino acid sequence of SEQ ID NO: 1, for example, the DNA sequence information of SEQ ID NO: 2, and reverse transcription-polymerase The NS1 gene can be obtained from a suitable influenza A virus or an infected cell thereof by a chain reaction (RT-PCR) method, cloned, and expressed by genetic engineering.
Here, it is considered that the influenza A virus that obtains the NS1 gene is common to a wide range of influenza viruses including human influenza, swine influenza, avian influenza, and equine influenza, particularly influenza A viruses. Various viruses belonging to influenza A virus can be used. As such a virus, in addition to various strains stored in the depository, various field strains can be obtained and used. Examples of strains stored in the depository include, for example, A / WS / 33 (H1N1 [tissue culture adapted]) (ATCC number VR-1520), A / Swine / 1976/31 (H1N1) (ATCC number VR-99). ), A / Port Chalmers / 1/73 (ATCC number VR-810), A / NWS / 33 (ATCC number VR-219), A / New Jersey / 8/76 (Hsw N1) (ATCC number VR-897) , A / MaI / 302/54 (H1N1) (ATCC number VR-98), A / Hong Kong / 8/68 (H3N2) (ATCC number VR-544), A2 / Aichi / 2/68 (H3N2) (ATCC In addition to the number VR-547), various strains available from NIBSC (National Institute for Biological Standards and Control) are listed.

In the present invention, each step in the production of an antibody and the detection method and measurement method using the antibody is performed according to a known immunological technique.
In the present invention, the polyclonal antibody is extracted and purified by genetically expressing the NS1 protein extracted or purified from the culture supernatant of influenza A virus-infected cells or the cloned NS1 protein in a host such as Escherichia coli. The obtained NS1 protein is used as an antigen to immunize an animal according to a conventional method, and it can be obtained from the antiserum.

  The monoclonal antibody was obtained, for example, by immunizing an animal such as a mouse using the NS1 protein extracted and purified in the same manner as described above as an antigen, and cell fusion of the spleen cells and myeloma cells of the immunized animal. The fused cells are allowed to grow after selection with HAT-containing medium. Using the NS1 protein obtained as described above for the grown strain, an anti-NS1 protein antibody-producing strain is selected by, for example, enzyme-labeled immunization.

The immunochromatographic measurement method of the present invention for detecting NS1 protein in a test sample can be easily performed according to the configuration of a known immunochromatographic test strip.
In general, such an immunochromatographic test strip is labeled with a first antibody capable of reacting with an antibody at the first antigenic determinant of the antigen and an antibody antigen reactive with the second antigenic determinant of the antigen. At least a second antibody and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody is separated from the capture site. The membrane carrier is arranged and configured to be chromatographed. As described above, each of the first antibody and the second antibody may be a polyclonal antibody or a monoclonal antibody, but at least one of them is preferably a monoclonal antibody. Usually, the first antibody and the second antibody are used in a “hetero” combination, that is, the first antibody and the second antibody, each recognizing each antigenic determinant differing in position and structure on the antigen. These antibodies are used in combination. However, the first antigenic determinant and the second antigenic determinant may be structurally the same as long as the positions on the antigen are different. It may be a “homo” combination of monoclonal antibodies, ie the same monoclonal antibody can be used for both the first antibody and the second antibody.

A specific example of the immunochromatographic test strip is, for example, the test strip shown in FIG. In FIG. 3, numeral 1 is an adhesive sheet, 2 is an impregnating member, 3 is a membrane carrier, 31 is a capture site, 4 is an absorbing member, and 5 is a sample adding member.
In the example shown in the figure, the membrane carrier 3 is made of a strip-like nitrocellulose membrane filter having a width of 5 mm and a length of 36 mm.
The first antibody is fixed to the membrane carrier 3 at a position 7.5 mm from the end on the side of the chromatographic expansion start point, and a specimen capture site 31 is formed.
In the illustrated example, the membrane carrier 3 uses a nitrocellulose membrane filter. However, the membrane carrier 3 should be capable of chromatographic development of the specimen contained in the test sample and immobilize the antibody forming the capture site 31. Any cellulose membrane, nylon membrane, glass fiber membrane, etc. can be used.

The impregnated member 2 is a member impregnated with a second antibody that reacts with the antigen with the second antigen determinant located at a different site from the first antigen determinant to which the first antibody binds. Become. The second antibody is previously labeled with an appropriate labeling substance.
In the illustrated example, a 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric is used as the impregnating member 2, but is not limited thereto, and examples thereof include cellulose cloth (filter paper, nitrocellulose membrane, etc.), polyethylene, Porous plastic cloths such as polypropylene can also be used.

The labeling substance for the second antibody may be any substance as long as it can be used, and examples thereof include a coloring labeling substance, an enzyme labeling substance, and a radiation labeling substance.
Among these, it is preferable to use a color labeling substance from the viewpoint that the color change at the capturing site 31 can be determined quickly and easily by observing with the naked eye.
Examples of the color labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with respective pigments such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloid are particularly preferred.
The impregnated member 2 can be produced by impregnating the labeled second antibody suspension into a member such as the glass fiber non-woven fabric and drying it.

As shown in FIG. 3, the membrane carrier 3 is stuck in the middle of the pressure-sensitive adhesive sheet 1 and the chromatographic development start side of the membrane carrier 3 (that is, the left side of FIG. 3, hereinafter referred to as “upstream side”, The opposite side, that is, the right side of FIG. 3 is hereinafter referred to as the “downstream side”, and the downstream end of the impregnation member 2 is overlapped and connected to the upstream end of the impregnation member 2. Can be attached to the pressure-sensitive adhesive sheet 1 to produce the immunochromatographic test strip of the present invention.
Further, if necessary, the downstream portion of the sample addition member 5 may be placed on the upper surface of the impregnation member 2, and the upstream portion of the sample addition member 5 may be attached to the adhesive sheet 1. Further, the upstream portion of the absorbing member 4 can be placed on the upper surface of the downstream portion of the membrane carrier 3, and the downstream portion of the absorbing member 4 can be attached to the adhesive sheet 1.

As the sample addition member 5, for example, a porous synthetic resin sheet or film such as porous polyethylene or porous polypropylene, and a paper or woven or non-woven fabric made of cellulose such as filter paper and cotton cloth are used. be able to.
The absorbing member 4 may be of any material that can absorb and hold liquid quickly, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., and filter paper is particularly suitable. is there.

  Further, in the case of a commercially available product, the immunochromatographic test strip of FIG. 3 is accommodated in a suitable plastic case in which a test sample injection part and a judgment part are opened above the sample addition member 5 and the capture part 31, respectively. Provided.

Thus, after mixing a test sample made of a biological sample with an appropriate developing solvent as necessary to obtain a mixed solution that can be chromatographed, the mixed solution is used for sample addition of the immunochromatographic test strip shown in FIG. When injected onto the member 5, the mixed solution passes through the sample addition member 5 and is mixed with the labeled second antibody in the impregnating member 2.
At that time, if a sample is present in the mixed solution, a complex of the sample and the second antibody is formed by the antigen-antibody reaction.
This complex is chromatographed in the membrane carrier 3 to reach the capture site 31, where it is captured by an antigen-antibody reaction with the first antibody immobilized thereon.
At this time, if a colored labeling substance such as colloidal gold is used as the labeling substance, the capture site 31 is colored due to the accumulation of the colored labeling substance. Therefore, the sample should be measured qualitatively or quantitatively immediately. Can do.

  Although there is no restriction | limiting in particular as a test sample, A nasal aspirate, a nasal swab, and a throat swab are preferable. The test sample may be diluted with an appropriate diluent such as a developing solvent and injected into the membrane carrier. NS1 protein detected in the present invention is a non-structural protein, hardly exists in virus particles, and is expressed only in cells in which the virus grows.

  In the case of the immunochromatographic assay method of the present invention for detecting anti-influenza A virus antibody, particularly anti-NS1 antibody in a test sample, for example, in FIG. 3 described above, the first antibody immobilized on the capture site 31 Alternatively, NS1 protein can be immobilized on the capture site 31, and instead of the labeled second antibody, an antibody against the biological immunoglobulin from which the test sample was obtained can be labeled and impregnated in the impregnated member. . In this case, a test sample made of serum or the like is mixed with an appropriate developing solvent as needed to obtain a mixed solution that can be chromatographed, and then the mixed solution is used for sample addition of the immunochromatographic test strip shown in FIG. When injected onto the member 5, the mixed solution passes through the sample addition member 5 and is mixed with the labeled antibody in the impregnated member 2. At this time, if an anti-NS1 antibody is present in the mixed solution, the anti-NS1 antibody and the labeled antibody form a complex by an antigen-antibody reaction, and this complex is chromatographed in the membrane carrier 3. It reaches the capture site 31 and is captured by an antigen-antibody reaction with the NS1 protein immobilized thereon. If a colored labeling substance such as colloidal gold is used as the labeling substance, the capture site 31 is colored by the accumulation of the colored labeling substance. Therefore, the presence or absence of the anti-NS1 antibody in the mixed solution can be qualitatively or quantitatively measured by the color density at the capture site 31.

  The antibody against the immunoglobulin used here may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred from the viewpoint of reaction specificity. The monoclonal antibody may be an anti-Igs antibody of an organism from which the test sample is derived. For example, if the organism is a human, a commercially available anti-human Igs antibody such as an anti-human IgG mouse IgG antibody is used. If the horse is a horse, an anti-horse Igs antibody is used. These antibodies can be labeled in the same manner as the second antibody.

The test sample used for detecting the anti-NS1 antibody is not particularly limited, and examples thereof include body fluids collected outside the body such as blood (can be whole blood, serum, or plasma), saliva, urine, and the like. . The test sample may be diluted with an appropriate diluent such as a developing solvent and injected into the membrane carrier.
When whole blood is used as a test sample, and particularly when a colored labeling substance such as gold colloid is used as the labeling substance of the labeled antibody, it is preferable to dispose a blood cell capturing membrane member on the sample addition member. . The blood cell trapping membrane member is preferably laminated between the impregnation member and the sample addition member. This prevents red blood cells from being developed on the membrane carrier, so that it is easy to confirm the accumulation of the colored label at the capture site of the membrane carrier. As the blood cell capturing membrane member, a carboxymethylcellulose membrane is used. Specifically, ion exchange filter paper CM (trade name) sold by Advantech Toyo Co., Ltd. or ion exchange cellulose sold by Whatman Japan Co., Ltd. Paper or the like can be used.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Details of the experimental protocol used in the present invention are as follows.
Fluorescent antibody method The fluorescent antibody method followed the method of Office International Des Epizooties (2000). Lab-Tek II Chamber Slide System (Nunc) with a monolayer formed on the cultured cells was inoculated with virus at moi = 1, cultured at 37 ° C for 48 hours, the culture supernatant was removed, and the infected cells were washed with PBS. Washed twice. The cells were fixed by soaking in 100% cold acetone (Wako Pure Chemical Industries) for 5 to 10 minutes, and then dried. Mouse ascites containing the monoclonal antibody was diluted 2,000 times with an antibody diluent, added as a primary antibody solution, and allowed to react at room temperature for 1 hour. After washing once with PBS, Fluorecein-conjugated Goat IgG Fraction to Mouse IgG (ICN Biomedicals) diluted 1,000-fold with an antibody diluent was added as a secondary antibody solution and reacted at room temperature for 1 hour. After washing twice with PBS, cells were encapsulated with a mounting medium (Glycerol for fluorescence microscopy (Merck) mixed with 0.5 M carbonate bicarbonate buffer (pH 9.5) at a ratio of 3: 1), and then fluorescence microscope (Axiovert 200, ZEISS) and observed with FITC Filter (Chroma).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE followed the method of Laemmli (1970). Sample buffer [0.1M Tris-HCl (pH6.8), 4% SDS (Nacalai Tesque), 10% 2-Mercaptoethanol (Merck), 20% glycerin (Wako Pure Chemicals) and 0.005% bromo-phenol ・Blue (Kishida Kagaku)] was added in an equal amount and boiled for 5 minutes to solubilize. When using virus-infected cells as samples, the solubilized buffer [0.1M Tris-HCl (pH 7.6), 0.1M NaCl, 1% Nonidet P-40 (Nacalai Tesque), 1% Triton X-100 and Complete, Mini, EDTA-free (Roche)] was added, and the same treatment was performed. Solubilized samples were run on 7.5% or 10% polyacrylamide gels. The gel was stained with Coomassie Brilliant Blue R-250 (Nacalai Tesque).

Western blotting Western blotting followed the method of Towbin et al. (1979). First, the protein fractionated by SDS-PAGE was transferred to Immobilon-P Transfer Membrane (Millipore). The membrane was blocked with PBS containing 5% skim milk. Next, mouse ascites containing 6xHis Monoclonal Antibody (Clontech) or monoclonal antibody was diluted 5,000 times with PBS containing 0.5% skim milk and allowed to react with the membrane. After washing, the reaction was similarly performed with Goat Anti-Mouse IgG-HRP Conjugate diluted 5,000 times. After washing, the film was immersed in a luminescent substrate solution (Lumi-Light Western Blotting Substrate, Roche), exposed to a film (Hyperfilm ECL, Amersham) in a dark room, and developed.

Example 1 (Cloning of NS1 gene)
A / Equine / Miami / 1/63 (H3N8), an equine influenza A virus, was inoculated into the amniotic cavity of 11-day-old embryonated chicken eggs and cultured. Several days later, the amniotic fluid was collected to obtain the virus. An RNA extraction reagent (TRIzol Reagent, Invitrogen) was added to the virus to extract viral RNA. The NS1 gene was amplified from the extracted RNA by the reverse transcription-polymerase chain reaction (RT-PCR) method using RNA LA PCR Kit (AMV) Ver.1.1 (Takara Shuzo). NS24- Eco RI (sense): 5′-ACA GAATTC TAATGGATTCCAACAC-3 ′ (SEQ ID NO: 3) and SP6 primer (antisense): 5′-CATACGATTTAGGTGACACTATAG-3 ′ (SEQ ID NO: 4) were used as primers. In the primer base sequence, the underline indicates the restriction enzyme site ( Eco RI; GAATTC).

A part of the obtained PCR product was electrophoresed on a 0.8% agarose gel (Agarose S, Wako Pure Chemical Industries), and after confirming the amplification of NS1 gene, the entire amount was electrophoresed to cut out a gel containing the target band. NS1 gene fragment was purified from this gel using MinElute Gel Extraction Kit (Qiagen) and cloned into a vector using pGEM-T Vector System I (Promega). The plasmid extracted and purified using QIAprep Spin Miniprep Kit (Qiagen) was cleaved with restriction enzyme Eco RI (Toyobo) and then electrophoresed on 0.8% agarose gel to confirm that the target gene was inserted. .

Example 2 (Expression and purification of recombinant NS1 protein)
The plasmid of Example 1 in which the target gene was inserted was purified in large quantities using the QIAGEN Plasmid Midi Kit (Qiagen). This plasmid was cleaved with the restriction enzyme Eco RI, and the target NS1 gene fragment was extracted from the gel and purified using the MinElute Gel Extraction Kit.
Esherichia coli ( E. coli ) expression vector pET-30c (Novagen) was cleaved with the restriction enzyme Eco RI and then dephosphorylated (alkaline phosphatase treatment). The pET-30c vector and the NS1 gene fragment were mixed, and then a ligation reaction was performed using DNA Ligation Kit Ver. 2 (Takara Shuzo). The reaction solution after ligation was introduced into E. coli JM109 strain (Toyobo) and then cultured on LB agar plate medium. A plasmid was extracted from the obtained colony bacterial solution using the QIAprep Spin Miniprep Kit, treated with the restriction enzyme Eco RI, and then a clone into which the target gene had been inserted was selected. A gene map of the prepared expression vector is shown in FIG. The base sequence of the obtained NS1 gene fragment is shown in SEQ ID NO: 2 in the sequence listing.

A recombinant NS1 plasmid (pET-30c / A / Equine / Miami / 1/63 NS1) in which the target gene was cloned was introduced into a recombinant protein expression host E. coli BL21 (DE3) pLysS (Novagen). The introduced bacteria were cultured on LB agar plate medium, and the resulting colonies were cultured on LB liquid medium. After adding 1 mM IPTG (Takara Shuzo) to induce expression of recombinant NS1 protein, E. coli was recovered. The collected bacteria were resuspended in a solubilization buffer [0.5% Triton X-100 (Sigma), 10 mM Imidazole, 20 mM Phosphate and 0.5 M NaCl (pH 7.4) (Amersham)] and solubilized by sonication. Recombinant NS1 protein was purified using HisTrap Kit (Amersham). This purified protein was dialyzed against PBS to obtain the desired recombinant NS1 protein.

In addition, when the solubilized sample was analyzed by SDS-PAGE and Western blotting, a protein of about 30 kDa was predominantly increased (FIG. 2, central lane). Since this protein was not observed before expression induction by IPTG (FIG. 2, rightmost lane), it can be seen that this protein is the desired recombinant NS1 protein.
The amino acid sequence of NS1 protein of A / Equine / Miami / 1/63 is as shown in SEQ ID NO: 1. In this example, the molecular weight of the region expressed in E. coli is expected to be about 26 KDa. Furthermore, since it is expressed as a fusion protein with a His tag sequence of about 4 kDa by using the pET-30c (+) vector, the expected molecular weight of the recombinant NS1 protein is calculated to be about 30 kDa. This is consistent with the molecular weight of the protein detected in the solubilized sample after expression in FIG. When this recombinant NS1 protein was purified and analyzed by SDS-PAGE, it was confirmed as a single band at a position of about 30 kDa (not shown).

Example 3 (Production of monoclonal antibody against NS1 protein)
Using the recombinant NS1 protein obtained in Example 2 as an antigen, a monoclonal antibody against the NS1 protein (anti-NS1 antibody) was produced. Monoclonal antibodies were produced according to a conventional method. 100 μg of recombinant NS1 protein and an equal amount of Adjuvant Complete Freund (Difco) were mixed, and mice (BALB / c, 5 weeks old, Japan SLC) were immunized 3 times, and the spleen cells were used for cell fusion.

  Sp2 / 0-Ag14 cells (Shulman et al., 1978), which are mouse myeloma cells, were used for cell fusion. For cell culture, Dulbecco's Modified Eagle Medium (Gibco) was supplemented with 0.3 mg / ml L-glutamine, 100 units / ml penicillin G potassium, 100 μg / ml streptomycin sulfate, and 40 μg / ml Gentacin (DMEM). A culture solution to which fetal serum (JRH) was added to 10% was used.

  Cell fusion was performed by mixing spleen cells of immunized mice and Sp2 / 0-Ag14 cells and adding polyethylene glycol solution (Sigma) thereto. The fused cells were cultured in HAT-DMEM [serum-added DMEM containing 0.1 mM Sodium Hypoxanthine, 0.4 μM Aminopterin and 0.016 mM Thymidine (Gibco)], and antibody production in the culture supernatant was confirmed by enzyme-linked antibody method (ELISA). . ELISA followed the method of Kida et al. (1982).

  Antibody-positive cells were cultured in HT-DMEM [serum-added DMEM containing 0.1 mM sodium hypoxanthine and 0.016 mM thymidine (Gibco)], and further cultured in serum-added DMEM. Antibody-positive cells were cloned by colonization on soft agar (Kida et al., 1982). The cloned cells were intraperitoneally inoculated into mice (BALB / c, Retire, Japan SLC) inoculated with 2, 6, 10, 14-Tetramethylpentadecane (Sigma), and ascites was collected. This ascites was used as a monoclonal antibody. The isotype of the produced monoclonal antibody was identified by ELISA using Mouse Monoclonal Antibody Isotyping Reagents (Sigma).

The purified recombinant protein is thought to contain a very small amount of E. coli- derived components. Therefore, a protein (ET-30c S) expressed without inserting a gene into the pET-30c vector was purified in the same manner as the recombinant NS1 protein and subjected to ELISA as a control antigen. Thus, antibody-producing cells against S protein or E. coli- derived components were excluded, and only antibody-producing cells against NS1 protein were screened.
Finally, 18 clones producing monoclonal antibody against NS1 protein were obtained. The immunoglobulin isotypes of these monoclonal antibodies were all IgG1.

  The reactivity of the produced 18-clone monoclonal antibody with influenza A virus subtypes H1 to H15 was examined by the fluorescent antibody method. The 18 clones were divided into two groups according to the difference in reactivity (Table 1). Group 1 reacts with all subtypes from H1 to H15 by the fluorescent antibody method, and 5 clones of 67/6, 160/1, 188/5, 204/5 and 440/1 are classified into this. It was. Group 2 was reactive to influenza A virus but could not recognize all subtypes. In addition, all the clones of Group 2 showed no reactivity to the H5 type. Therefore, it was shown that in order to detect a wide range of influenza A viruses, it is preferable to use monoclonal antibodies that react with influenza A virus subtype H5.

In Table 1, Mabs indicates the type of monoclonal antibody clone, Isotype indicates immunoglobulin isotype, Viruses indicates each of subtypes H1 to H15, + indicates a reaction, and-indicates no reaction.
The ELISA titers of the clones 160/1, 188/5 and 440/1 of the obtained Group 1 were as shown in Table 2.

  In Table 2, Mab represents the type of monoclonal antibody clone, Isotype represents the immunoglobulin isotype, and ELISA titer represents the titer of the recombinant NS1 protein obtained in Example 2 by the ELISA method.

Example 4 (Immunochromatography kit using anti-NS1 antibody)
(1) Preparation of anti-NS1 antibody Each of clones 160/1 and 188/5 obtained in Example 3 was further subjected to IgG purification using a protein G adsorbent by a conventional method to obtain anti-NS1 antibody.

(2) Preparation of colloidal gold solution Add 1 ml of 1% (v / w) chloroauric acid aqueous solution to 99 ml of ultrapure water boiled by heating, and 1 minute later 1% (v / w) sodium citrate After 1.5 ml of an aqueous solution was added and heated to boil for 5 minutes, it was allowed to cool to room temperature. Subsequently, 200 mM potassium carbonate aqueous solution was added to this solution to adjust to pH 9.0, and ultrapure water was added thereto to make a total volume of 100 ml to obtain a gold colloid solution.

(3) Preparation of colloidal gold-labeled anti-NS1 antibody solution As anti-NS1 antibody for colloidal gold labeling, clone 160/1 obtained in (1) above was used.
1 μg of protein equivalent weight of anti-NS1 antibody (hereinafter, when the protein equivalent weight of the antibody is indicated, it is simply indicated by weight value of the purified protein by weight analysis) and 1 ml of the colloidal gold solution of (2) above are mixed at room temperature. Let the antibody stand for 2 minutes to bind all of these antibodies to the gold colloid particle surface, and then add 10% bovine serum albumin (hereinafter referred to as “BSA”) aqueous solution so that the final concentration in the gold colloid solution is 1%. And the remaining surface of the colloidal gold particles were blocked with the BSA to prepare a colloidal gold-labeled anti-NS1 antibody (hereinafter referred to as “gold colloid-labeled antibody”) solution. This solution was centrifuged (5600 × G, 30 minutes) to precipitate colloidal gold labeled antibody, and the supernatant was removed to obtain colloidal gold labeled antibody. The colloidal gold labeled antibody was suspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, 0.5% Triton-X100 to obtain a colloidal gold labeled antibody solution.

(4) Preparation of immunochromatographic test strip for NS1 protein measurement The immunochromatographic test strip shown in FIG. 3 was prepared by the following procedure.
(4-1) An elongated strip-shaped nitrocellulose membrane having a capture site width of 5 mm and a length of 36 mm for the complex of NS1 protein and colloidal gold labeled antibody was prepared as a membrane carrier 3 for chromatographic development of a chromatographic medium.
0.5 μl of a solution containing 1.0 mg / ml of anti-NS1 antibody is applied in a line at a position 7.5 mm from the end of the chromatographic development starting point side of the membrane carrier 3 for chromatographic development, and dried at room temperature. Then, it was used as the capture site 31 for the complex of NS1 protein and colloidal gold labeled antibody. As this anti-NS1 antibody, clone 188/5 obtained in (1) above was used.

(4-2) Colloidal gold labeled antibody impregnated member
A 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric was impregnated with 37.5 μl of a colloidal gold labeled antibody solution, which was dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 2.
(4-3) Preparation of immunochromatographic test strip In addition to the membrane carrier 3 for chromatographic development and the labeled antibody impregnated member 2, a cotton cloth and a filter paper as an absorbing member 4 were prepared as the sample adding member 5. Then, using these members, a chromatographic test strip similar to that shown in FIG. 3 was prepared.

(5) The purified recombinant NS1 protein obtained in Test Example 2 was diluted with a sample diluent, adjusted to each concentration, and used as a test sample. Then, 100 μl of the test sample is dropped with a micropipette onto the sample addition member 5 of the test strip obtained in (4) above, chromatographed, left for 15 minutes at room temperature, and then recombined captured at the capture site 31. The captured amount of the complex of NS1 protein and colloidal gold labeled antibody was observed with the naked eye. The captured amount is 4 (colorless), ± (weak coloration), + (clear coloration), and ++ (significant coloration). Judged in stages. The results are shown in Table 3.

  As is apparent from Table 3, it was found that the NS1 protein of influenza A virus can be detected by immunochromatographic assay using two types of anti-NS1 antibodies.

  The present invention provides a sandwich immunoassay method using an antibody against NS1 protein that is commonly present in influenza A viruses, particularly an immunochromatographic assay method and an immunochromatographic test strip. Since a wide range of viruses belonging to influenza A viruses such as avian influenza and equine influenza can be rapidly detected by a simple method, it is useful for quickly and easily diagnosing diseases caused by the virus. In addition, the recombinant NS1 protein of the present invention can be used to detect antibodies against these viruses.

It is the schematic which shows the preparation procedure and gene map of the recombinant NS1 plasmid (pET-30c / A / Equine / Miami / 1/63 NS1) produced in Example 2. 2 is an SDS-PAGE image of the recombinant NS1 protein expressed in Example 2. a is a plan view of an immunochromatographic test strip, and b is a longitudinal sectional view of the immunochromatographic test strip indicated by a.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 31 Capture part 4 Absorption member 5 Sample addition member

Claims (36)

  A method for detecting influenza A virus comprising an immunoassay using an antibody against NS1 protein of influenza A virus.   The detection method according to claim 1, wherein the antibody is a monoclonal antibody.   The detection method according to claim 2, wherein the monoclonal antibody is a monoclonal antibody against a peptide consisting of the amino acid sequence of SEQ ID NO: 1.   The detection method according to claim 3, wherein the monoclonal antibody reacts with all influenza A virus subtypes H1 to H15.   A method for detecting influenza A virus, comprising a sandwich immunoassay using a first antibody and a second antibody against NS1 protein of influenza A virus.   The detection method according to claim 5, wherein at least one of the first antibody and the second antibody is a monoclonal antibody.   The detection method according to claim 6, wherein the monoclonal antibody is a monoclonal antibody against a peptide consisting of the amino acid sequence of SEQ ID NO: 1.   The detection method according to claim 7, wherein the monoclonal antibody reacts with all of influenza A virus subtypes H1 to H15.   The detection method according to any one of claims 5 to 8, wherein either one of the first antibody and the second antibody is immobilized on a carrier.   A membrane carrier having a capture site formed by preliminarily immobilizing a first antibody against the NS1 protein of influenza A virus at a predetermined position, and mixing a second antibody against the NS1 protein with a predetermined amount of a test sample The solution is chromatographed on the membrane carrier toward the capture site, and the complex of NS1 protein derived from influenza A virus and the second antibody contained in the test sample is captured at the capture site. An immunochromatographic measurement method characterized by the above.   The method according to claim 10, wherein at least one of the first antibody and the second antibody is a monoclonal antibody.   The method according to claim 11, wherein the monoclonal antibody is a monoclonal antibody against a peptide consisting of the amino acid sequence of SEQ ID NO: 1.   The method according to claim 12, wherein the monoclonal antibody reacts with all influenza A virus subtypes H1 to H15.   The method according to any one of claims 10 to 13, wherein the second antibody is labeled with a metal colloid or latex.   The measurement method according to claim 10, wherein the membrane carrier is a nitrocellulose membrane.   At least a first antibody against NS1 protein of influenza A virus, a second antibody, and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site; An immunochromatographic test strip for detecting influenza A virus, wherein the second antibody is labeled with an appropriate labeling substance and is arranged so as to be chromatographed on the membrane carrier at a position separated from the capture site.   The immunochromatographic test strip according to claim 16, wherein at least one of the first antibody and the second antibody is a monoclonal antibody.   The immunochromatographic test strip according to claim 17, wherein the monoclonal antibody is a monoclonal antibody against a peptide consisting of the amino acid sequence of SEQ ID NO: 1.   19. The immunochromatographic test strip according to claim 18, wherein the monoclonal antibody reacts with all of influenza A virus subtypes H1 to 15.   The immunochromatographic test strip according to any one of claims 16 to 19, wherein the second antibody is labeled with a metal colloid or latex.   The immunochromatographic test strip according to any one of claims 16 to 20, wherein the membrane carrier is a nitrocellulose membrane.   A monoclonal antibody against the peptide consisting of the amino acid sequence of SEQ ID NO: 1.   The monoclonal antibody according to claim 22, which reacts with all of influenza A virus subtypes H1 to H15. Any one of the following peptides (a) and (b):
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1,
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and exhibiting the immunogenicity of NS1 protein of influenza A virus. A gene encoding any one of the following peptides (a) and (b):
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1,
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and exhibiting the immunogenicity of NS1 protein of influenza A virus.   A recombinant vector containing the gene of claim 25.   A transformant comprising the recombinant vector of claim 26.   A recombinant protein exhibiting the immunogenicity of NS1 protein of influenza A virus derived from the recombinant vector expressed by the transformant of claim 27.   A membrane carrier comprising a capture site formed by preliminarily immobilizing the recombinant protein of claim 28 in a predetermined position is prepared, and a mixed solution of a test sample collected from a living body and an antibody against immunoglobulin in the living body is Influenza characterized in that it is chromatographed on the membrane carrier toward the capture site, and a complex of the antibody and an antibody against NS1 protein of influenza A virus contained in the test sample is captured at the capture site Immunochromatographic assay for detection of antibodies against type A virus.   30. The method according to claim 29, wherein the antibody against immunoglobulin is an antibody against human immunoglobulin.   The measurement method according to claim 29 or 30, wherein the antibody against the immunoglobulin is labeled with a metal colloid or latex.   32. The measuring method according to claim 29, wherein the membrane carrier is a nitrocellulose membrane.   29. The recombinant protein according to claim 28, an antibody against biological immunoglobulin and a membrane carrier at least, wherein the recombinant protein is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the antibody is appropriately labeled An immunochromatographic method for detecting an anti-influenza A virus antibody collected from the living body, which is labeled with a substance and arranged so as to be chromatographed on the membrane carrier at a position separated from the capture site Test strip.   The immunochromatographic test strip according to claim 33, wherein the antibody against immunoglobulin is an antibody against human immunoglobulin.   The immunochromatographic test strip according to claim 33 or 34, wherein the antibody against the immunoglobulin is labeled with a metal colloid or latex. 36. The immunochromatographic test strip according to any one of claims 33 to 35, wherein the membrane carrier is a nitrocellulose membrane.