JP2010261912A - Immunology-detection method of human influenza virus h3 subtype - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a measuring tool for specifically, speedily, and easily detecting human influenza virus H3 subtypes. <P>SOLUTION: Both of a first antibody which reacts with all human influenza A virus subtypes and a second antibody which reacts with human influenza virus H1 subtypes but does not react with human influenza virus H3 subtypes are used to detect an antigen of which the reatcion with the first antibody is positive and of which the reaction with the second antibody is negative by an immunoassay method in the method for detecting human influenza virus H3 subtypes. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention does not react to human influenza virus H3 subtype, but human influenza virus H3 subtype using an antibody that reacts with human influenza A virus subtypes other than H3 subtype, particularly H1 subtype. Type detection method, in particular, sandwich immunoassay, particularly immunochromatography assay and immunochromatography assay device, useful for rapid and simple diagnosis of infected human influenza virus subtypes Regarding the law.

Influenza virus infects the upper and lower respiratory tracts of humans and causes a headache, low back pain, muscle pain, general malaise, digestive symptoms, etc. along with fever (38-39 ° C.) after a latent period of 1-2 days. 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 using enzyme immunoassay (EIA) or immunochromatography assay as a 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, respectively.
However, current influenza virus antigen tests use antibodies against nucleoproteins common to all type A viruses. Therefore, differential diagnosis from seasonal influenza H1N1 or H3N2 subtype infection is not possible.

In 2009, a large number of human infections with swine-origin influenza virus H1N1 subtype occurred, and a new type of influenza occurred. In April of the same year, WHO declared that the level of influenza pandemic alerts would be raised to Phase 5, as human-to-human transmission continues to be seen.

At present, in the diagnosis of new influenza (H1N1 subtype), conventional influenza antigen rapid diagnostic reagents for those who return to the country from infected countries or who are in contact with infected patients and exhibit influenza-like symptoms such as fever and cough Inspection is carried out by When influenza A infection is confirmed by a rapid test, H1N1 subtype or H3N2 subtype is confirmed by genetic testing (RT-PCR method). If the H1N1 subtype is confirmed, a more detailed examination is performed as suspicion of new influenza. As described above, differential diagnosis between the H1N1 subtype and the H3N2 subtype is currently performed by genetic testing (RT-PCR method), but this is not a simple method because it requires special equipment and technology.

In addition, in the treatment of influenza, early treatment by administration of an anti-influenza drug is performed after diagnosis with an influenza antigen rapid diagnostic reagent. Administration of oseltamivir and zanamivir, which are neuraminidase inhibitors, as anti-influenza drugs.

In recent H1N1 subtypes, a virus resistant to the anti-influenza drug oseltamivir has been frequently reported. On the other hand, no resistant virus has been identified in the H3N2 subtype. Therefore, differential diagnosis of H1N1 subtype and H3N2 subtype infection at the time of initial diagnosis is important in formulating a treatment policy with an anti-influenza drug for a patient, and is important in selecting a drug to be administered.
The nucleoprotein of influenza A virus is thought to be well conserved among the subtypes of its amino acid sequence, and conventionally used mainly for differential diagnosis from influenza B virus (see Patent Document 1). That is, it is considered that antibodies against the nucleoprotein of influenza A virus usually react with all influenza A virus subtypes. Among them, the present applicant has found an antinuclear protein antibody that does not specifically react with an influenza A virus virulence strain, and has already developed an immunological detection method for an influenza A virus virulence strain using the antibody. It has been proposed (see Patent Document 2). However, the existence of antinuclear protein antibodies that do not react with influenza A virus H3 subtype has not yet been reported.

International Publication WO2005 / 007697 Pamphlet International Publication WO2007 / 074811 Pamphlet

An object of the present invention is to provide a detection reagent useful for differential diagnosis of influenza A virus H1N1 and H3N2 subtypes, and a detection method using the same, especially a sandwich immunoassay, particularly an immunochromatographic assay and an immunochromatography. It is to provide a lithographic measurement apparatus.

The present inventors immunize mice with influenza A virus as an immunogen to obtain various antibodies, and react with human influenza virus H1 subtype in the course of testing these antibodies in an immunochromatography measuring device. Succeeded in obtaining a monoclonal antibody that does not react with human influenza virus H3 subtype, and by using the antibody in an immunoassay, particularly a sandwich immunoassay, especially an immunochromatographic assay, human influenza The inventors have found that the virus H3 subtype can be detected, and have completed the present invention.

That is, according to one aspect of the present invention, there is provided a monoclonal antibody that reacts with human influenza virus H1 subtype but does not react with human influenza virus H3 subtype. In particular, the monoclonal antibody reacts with a human influenza virus H1 subtype nucleoprotein but does not react with a human influenza virus H3 subtype nucleoprotein.
Since the majority of influenza A viruses that infect humans are H1 and H3 subtypes, immunoassay using the monoclonal antibodies of the present invention not only identifies H3 subtype infection, The differential diagnosis between the H1 subtype and the H3 subtype can be substantially performed. Here, the influenza A virus H3 subtype is synonymous with the H3N2 subtype since only the H3N2 subtype has been confirmed to be isolated from humans. In addition, the influenza A virus H1 subtype currently infecting humans may be considered substantially synonymous with the H1N1 subtype since the H1N1 subtype is the majority.

Thus, according to another aspect of the present invention, a first antibody that reacts against all human influenza A virus subtypes and a human influenza virus H1 subtype that reacts against human influenza virus H1 subtypes. A human influenza virus comprising a second antibody that does not react with a subtype, and that detects an antigen that is positive with the first antibody and negative with the second antibody by immunoassay Methods for detecting the H3 subtype are provided.

The immunoassay in this detection method is not particularly limited, but a sandwich immunoassay, particularly an ELISA (Enzyme-linked immunosorbent assay) method, an immunochromatography assay, and the like are preferable.
Therefore, according to a preferred embodiment of the detection method of the present invention, the immunoassay method uses the first antibody and a third antibody that reacts with an antigen to which the first antibody reacts. It comprises a sandwich immunoassay and / or a sandwich immunoassay using the second antibody and a fourth antibody that reacts with an antigen to which the second antibody reacts. These sandwich immunoassays are conveniently carried out by immobilizing the first antibody and the second or fourth antibody on a carrier. In this case, when a membrane carrier is used as the carrier, an immunochromatographic measurement method can be carried out.

Thus, according to yet another aspect of the present invention, the first antibody that reacts against all human influenza A virus subtypes and the human influenza virus H1 subtype, A membrane carrier having first and second capture sites formed by previously immobilizing a second antibody that does not react with the H3 subtype in advance is prepared in advance, and the antigen reacts with the first antibody. A first mixed solution of the third antibody that reacts with the test sample, and a second mixed solution of the fourth antibody that reacts with the antigen to which the second antibody reacts and the test sample Are chromatographed on the membrane carrier toward the first and second capture sites, respectively, or the first mixture is chromatographed on the membrane carrier toward both of the capture sites, first The human influenza virus H3 subtype can be detected when the reaction with the first antibody at the capture site is positive and the reaction with the second antibody at the second capture site is negative. An immunochromatographic assay is provided.

According to still another aspect of the present invention, the first antibody that reacts against all human influenza A virus subtypes and the human influenza virus H1 subtype react with human influenza virus H3. A second antibody that does not react to the subtype, a third antibody that reacts to the antigen to which the first antibody reacts, and a fourth antibody that reacts to the antigen to which the second antibody reacts At least an antibody and a membrane carrier, wherein the first and second antibodies are previously fixed at predetermined positions of the membrane carrier to form first and second capture sites, respectively, and the third and fourth antibodies Is labeled with an appropriate labeling substance, and the third and fourth antibodies can be chromatographed on the membrane carrier from positions separated from both of the capture sites toward the first and second capture sites, respectively. Prepared Human influenza virus subtype H3 type detection immunochromatographic assay device is provided comprising.

The membrane carrier may be a single membrane carrier comprising a first capture site and a second capture site spaced apart from each other, or a first antibody on which a first antibody is immobilized. Although it may be composed of one membrane carrier and a second membrane carrier to which the second antibody is immobilized, the latter is preferred because of its excellent reaction specificity. In the latter case, if the third antibody is prepared on the first membrane carrier and the fourth antibody is prepared on the second membrane carrier, the test sample is simply injected into each membrane carrier. It is convenient because it can be measured with. The third and fourth antibodies are conveniently prepared by impregnating the impregnating members disposed on the first and second membrane carriers, respectively.
When the first membrane carrier and the second membrane carrier are provided, both the first membrane carrier and the second membrane carrier have an elongated shape, that is, a band shape, and the impregnating members of both membrane carriers are mutually connected. It can be in the form of a so-called immunotomato method test strip in which both membrane carriers are arranged in parallel with each other with a predetermined interval therebetween. Alternatively, the first membrane carrier and the second membrane carrier both have an elongated shape, that is, a belt-like shape, the impregnated members of both membrane carriers are separated from each other at a predetermined interval, and the two membrane carriers are separated. It can be in the form of a so-called immunotomato test strip or immunotomato test kit which is arranged in the radial direction or in the opposite direction.

All of the methods and apparatuses described above are methods in which the second antibody is immobilized on a carrier. However, according to a preferred embodiment of the present invention, a method in which the second antibody is not immobilized on a carrier can be employed.
That is, according to another aspect of the present invention, the first antibody that reacts against all human influenza A virus subtypes and the human influenza virus H1 subtype, A second antibody that does not react with the mold and a third antibody that reacts with the antigen to which the first antibody reacts, and is formed by fixing the first antibody in place in advance. A membrane carrier having a first capture site is prepared, a first mixed solution containing the second antibody and a test sample, and a second mixed solution containing the third antibody and the test sample. , And chromatographically develop on the membrane carrier toward the first capture site, respectively, and the reaction of the third antibody at the first capture site is positive and the second antibody at the first capture site is positive. The reaction is negative Immunochromatographic assay, characterized in that to be able to detect human influenza virus subtype H3 type is provided with.
In this measurement method, it is sufficient to prepare one type of membrane carrier to which the first antibody is immobilized, but two chromatographic developments of the first mixed solution and the second mixed solution are developed. Development is required, and a total of two membrane carriers are required for each chromatographic development.

According to another aspect of the present invention, the first antibody that reacts against all human influenza A virus subtypes and the human influenza virus H1 subtype react with human influenza virus H3 subtype. At least a second antibody that does not react with the mold, a third antibody that reacts with an antigen with which the first antibody reacts, and a membrane carrier, wherein the first antibody is pre-determined on the membrane carrier Fixed at a position to form a first capture site, the second and third antibodies are labeled with an appropriate labeling substance, and the second and third antibodies are separated from the capture site. There is provided an immunochromatography measuring device for detecting human influenza virus H3 subtype, which is prepared so as to be chromatographed on the membrane carrier toward the first capture site.
In the measurement apparatus, the membrane carrier is preferably composed of at least a first membrane carrier to which the first antibody is immobilized and a second membrane carrier to which the first antibody is immobilized. When the second antibody is prepared on the first membrane carrier and the third antibody is prepared on the second membrane carrier, the measurement can be performed simply by injecting the test sample into both membrane carriers. It is convenient because it can be done. The second and third antibodies are preferably prepared by impregnating impregnation members disposed on the first and second membrane carriers, respectively.

The detection method and measurement method of the present invention include commercially available immunochromatographic test strips or kits for differential measurement of type A and B influenza such as Capilia (registered trademark) FluA + B (manufactured by Towns Co., Ltd.), It can be carried out in combination with a sandwich type immunoassay device using an antibody and a second antibody.
Thus, for such an embodiment, according to another aspect of the invention, the first antibody reacts against all human influenza A virus subtypes and reacts against human influenza virus H1 subtype However, it comprises at least a second antibody that does not react with human influenza virus H3 subtype and a membrane carrier, and the second antibody is previously fixed at a predetermined position of the membrane carrier to form a first capture site. The first antibody is labeled with an appropriate labeling substance, and prepared so that it can be chromatographed on the membrane carrier from a position remote from the first capture site toward the first capture site. An immunochromatography measuring device for detecting human influenza virus H3 subtype is provided.

Also, for similar embodiments, according to another aspect of the invention, a first antibody that reacts against all human influenza A virus subtypes and a reaction against human influenza virus H1 subtype However, it comprises at least a second antibody that does not react with human influenza virus H3 subtype and a membrane carrier, and the first antibody is previously fixed at a predetermined position of the membrane carrier to form a first capture site. The second antibody is labeled with an appropriate labeling substance, and prepared so that it can be chromatographed on the membrane carrier from a position remote from the first capture site toward the first capture site. An immunochromatography measuring device for detecting human influenza virus H3 subtype is provided.

The first and second antibodies used in the present invention may be any antibodies against influenza viruses, but are usually obtained as antibodies against influenza virus nucleoproteins. Each of the first and second antibodies may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity.
As said 1st antibody, the well-known monoclonal antibody with respect to the nucleoprotein of influenza A virus can be normally used.
The second antibody is an antibody that reacts with the human influenza virus H1 subtype but does not react with the human influenza virus H3 subtype. The above-described monoclonal antibody can be used.

In the case of the first antibody, in the case of the first antibody, for example, a DNA fragment corresponding to a portion containing an amino acid residue constituting an epitope in a DNA sequence encoding the amino acid sequence represented by SEQ ID NO: 6 is cloned, The cloned gene can be expressed by genetic engineering in a host such as Escherichia coli, and the expressed protein can be extracted and purified. The purified protein can be used as an antigen to immunize an animal according to a conventional method, and can be obtained from the antiserum. In the case of the second antibody, for example, a DNA fragment corresponding to a portion containing an amino acid residue constituting an epitope different from the influenza virus H3 subtype in the DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 6 is cloned. The cloned gene can be expressed by genetic engineering in a host such as Escherichia coli, and the expressed protein can be extracted and purified. The purified protein can be used as an antigen to immunize the animal according to a conventional method and obtain it from the antiserum. .
In the case of the first and second antibodies, for example, after immunizing an animal such as a mouse using the purified protein obtained as described above as an antigen, spleen cells and myeloma cells of the immunized animal The fused cells obtained by cell fusion are selected with a HAT-containing medium and grown, and the grown strain is selected using the purified protein obtained as described above, for example, by enzyme-labeled immunization. You can get it.

The monoclonal antibody used as the second antibody is a monoclonal antibody that reacts with the human influenza virus H1 subtype but does not react with the human influenza virus H3 subtype. It recognizes epitopes that are conserved in all influenza A viruses, but are mutated and lost in influenza virus H3 subtypes, in particular, epitopes of nucleoproteins of influenza A virus. In the influenza virus H3 subtype, the epitope of the influenza A virus is mutated due to substitution of amino acid sequences or the occurrence of different sugar chain modifications. It is thought that it is not recognized. Such a monoclonal antibody has not been known so far and is novel.

In the present invention, the third antibody used in the sandwich immunoassay method such as an immunochromatographic assay method may be any antibody that reacts with the antigen to which the first antibody reacts, and is the same as the first antibody. It may be an antibody or an antibody other than the first antibody. For example, when the antigen has a plurality of epitopes for the first antibody, the same antibody as the first antibody can be used as the third antibody. Thus, the third antibody may be an antibody that reacts against all human influenza A virus subtypes. The third antibody may be a monoclonal antibody or a polyclonal antibody. From the viewpoint of reaction specificity, the third antibody is preferably a monoclonal antibody, particularly a monoclonal antibody against a nucleoprotein of influenza virus.

In the present invention, the fourth antibody used in the sandwich immunoassay method such as an immunochromatographic assay method may be any antibody that reacts with the antigen to which the second antibody reacts, and is the same as the second antibody. There may be an antibody or an antibody other than the second antibody. For example, when the antigen has a plurality of epitopes for the second antibody, the same antibody as the second antibody can be used as the fourth antibody. Thus, the fourth antibody may be an antibody that reacts against all human influenza A virus subtypes, or reacts against human influenza virus H1 subtype, but against human influenza virus H3 subtype. It may be an antibody that does not react with the antibody. The fourth antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody, particularly a monoclonal antibody against a nucleoprotein of influenza virus, from the viewpoint of reaction specificity. If the fourth antibody is an antibody that reacts against all human influenza A virus subtypes, the fourth antibody may be the same antibody as the third antibody.

In the present specification, “human influenza virus” refers to an influenza virus that has been infected between humans and humans, or an influenza virus that has been isolated from a human after being infected with an influenza virus derived from an animal such as a pig or bird. .

At present, 16 subtypes of H (hemagglutinin) protein and 9 subtypes of N (neuramidase) protein are known as influenza A virus subtypes, and theoretically 16 × 9 = 144 subtypes. Possible types. Among these, influenza A virus subtypes that have been confirmed to be infected between humans and humans and between humans and animals, that is, “human influenza virus subtypes” of type A are H1 subtype, H2 subtype, H3 Subtype, H5 subtype, H7 subtype and H9 subtype. Of these, two types of H1 subtypes and H3 subtypes mostly infect humans. Therefore, in order to substantially achieve the differential diagnosis of H1 and H3 subtypes, as in the present invention, it reacts against human influenza virus H1 subtype but reacts against human influenza virus H3 subtype. Monoclonal antibodies that react against all human influenza A virus subtypes other than the H3 subtype, ie, H1 subtype, H2 subtype, H5 subtype, H7 Monoclonal antibodies that react against all of the subtypes and H9 subtypes are used.

In the present specification, “an antibody that reacts with all human influenza A virus subtypes” refers to H1 subtype, H2 subtype, H3 subtype, H5 subtype, H7 subtype, and H9 subtype. It is an antibody that reacts with all of the above. However, “an antibody that reacts against all human influenza A virus subtypes” may react not only with human influenza virus subtypes but also against other animal type A influenza virus subtypes. It is preferable to react to all representative examples of ˜15 subtypes. As representative examples, A / Puerto Rico / 8/34 (H1N1), A / Hokkaido / 11/02 (H1N1), A / Singapore / 1/57 (H2N2), A / Aichi / 2/68 (H3N2) , A / Hokkaido / 1/03 (H3N2), A / duck / Czech / 56 (H4N6), A / duck / Pennsylvania / 1028/83 (H5N2), A / Hong Kong / 156/97 (H5N1), A / Hong Kong / 483/97 (H5N1), A / turey / Massachusetts / 3740/65 (H6N2), A / seal / Massachusetts / 1/80 (H7N7), A / turey / Ontario / 67 (H8N4), A / turey / Wisconsin / 66 (H9N2), A / chicken / Germany / N / 49 (H10N7), A / duck / England / 56 (H11N6), A / duck / Alberta / 60/76 (H12N5), A / gull / Maryland There are 19 types: / 704/77 (H13N6), A / mallard / Astrakhan / 263/82 (H14N5), A / duck / Australia / 341/83 (H15N8).

The antibody against the nucleoprotein of influenza A virus does not show cross-reactivity with any of the nucleoproteins of influenza B or C virus. Similarly, the first and second antibodies of the present invention No cross-reactivity with any of the nucleoproteins of type B and type C influenza viruses.

In the present specification, “positive reaction with antibody” means that the antigen reacts with the antibody, and “negative reaction with antibody” means that the antigen does not react with the antibody. Means that.
Further, in the present specification, the antibody “does not react with influenza A virus H3 subtype” means that the antibody does not react with influenza A virus H3 subtype at all or does not react even when antigen-antibody reaction occurs. It also includes the case where the antibody does not show an antigen-antibody reaction when it is used, for example, when immobilized on a membrane carrier.

According to the present invention, an immunological measurement is performed using an antibody that reacts with the human influenza virus H1 subtype but does not react with the human influenza virus H3 subtype. Infection can be identified, differential diagnosis of H1N1 and H3N2 subtype infections can be substantially performed, and can also be used for diagnosis of new influenza.

Thus, according to the present invention, an antibody that reacts against all human influenza A virus subtypes is used as the first antibody and reacts against human influenza virus H1 subtype. By using an antibody that does not react with the antibody as the second antibody, it is possible to detect an antigen that is positive with the first antibody and negative with the second antibody by immunoassay. . This detection method can be used to selectively or specifically detect influenza virus H3 subtype, and can be widely applied to the diagnosis of infectious diseases caused by Hong Kong influenza virus (H3 subtype). Can also be used substantially as a means for differentiating between H1N1 subtype and H3N2 subtype.

In addition, according to the immunochromatography measurement method and the immunochromatography measurement apparatus of the present invention, it is possible to detect influenza virus H3 subtype easily and quickly in hospitals and the like without requiring special equipment and skilled techniques, and by using the virus. It becomes possible to diagnose infection.

a is a plan view showing a specific example of the immunochromatography measuring apparatus of the present invention composed of one immunochromatographic test strip, and b is a longitudinal sectional view of the apparatus shown by a. The top view which shows the specific example of the immunochromatography measuring apparatus of this invention comprised from the two immunochromatography test strips. The top view which shows the other specific example of the immunochromatography measuring apparatus of this invention comprised from the two immunochromatography method test strips. a is a plan view showing still another specific example of the immunochromatography measuring apparatus of the present invention composed of two immunochromatographic test strips, and b is a longitudinal sectional view of the apparatus shown by a.

A measuring apparatus that can be used for carrying out the immunochromatographic measuring method of the present invention can be easily carried out according to the configuration of a known immunochromatographic test strip. Specific examples thereof will be described below with reference to the drawings.

(1) Embodiment using a single immunochromatographic test strip As a specific example of the immunochromatographic test strip, there is a test strip 10 shown in FIG. 1, for example. In FIG. 1, numeral 1 is an adhesive sheet, 2 is an impregnation member, 3 is a membrane carrier, 4 is a capture site, 5 is an absorption member, and 6 is a sample addition member.
In the example shown in the figure, the membrane carrier 3 is composed of a strip-shaped nitrocellulose membrane filter having a width of 5 mm and a length of 36 mm, and is attached to the middle of the adhesive sheet 1 having the same width. On the membrane carrier 3, its chromatographic starting point side, that is, the left side in FIG. 1 (hereinafter referred to as “upstream side”. The opposite side, that is, the chromatographic development direction side in FIG. The first antibody is fixed at a position 7.5 mm downstream from the end of the first end, and the first antibody for capturing a complex of the third antibody and a virus antigen such as a nucleoprotein of influenza A virus is captured. A capture site 41a is formed. Further, a second antibody is immobilized at a position 10.5 mm downstream from the upstream end of the membrane carrier 3, and the fourth antibody (in the apparatus of FIG. 1, as described later, is the same as the third antibody. And a second capture site 41b for capturing a complex with a virus antigen such as a nucleoprotein of an influenza A virus other than the H3 subtype. Further, a so-called control line 42 is provided at a position 15.0 mm downstream from the upstream end of the membrane carrier 3. This control line 42 is used to confirm that the reaction has been performed regardless of the presence or absence of the analyte, and usually binds immunologically specifically to the third and / or fourth antibody. It can be formed by immobilizing substances to be analyzed (excluding substances to be analyzed) on the membrane carrier 3. For example, when a mouse antibody is used as the third and / or fourth antibody, an antibody against the mouse antibody can be used.

In the apparatus of FIG. 1, the membrane carrier 3 uses a nitrocellulose membrane filter. However, the membrane carrier 3 is capable of chromatographic development of a substance to be analyzed contained in a test sample and can immobilize an antibody that forms the capture site 4. As long as it is anything, other cellulose membranes, nylon membranes, glass fiber membranes and the like can be used.
In the apparatus of FIG. 1, it is preferable to use a monoclonal antibody that reacts with all human influenza A virus subtype nucleoproteins as the first antibody. As the second antibody, it is preferable to use a monoclonal antibody that reacts with human influenza virus H1 subtype but does not react with human influenza virus H3 subtype.

In the present invention, the third antibody is an antibody that reacts with an antigen to which the first antibody reacts, preferably an antibody that reacts with all human influenza A virus subtype nucleoproteins. In this case, the same antibody as the first antibody can be used. The fourth antibody is an antibody that reacts with an antigen to which the second antibody reacts. For example, a monoclonal antibody that reacts with a nucleoprotein of all human influenza A virus subtypes, and a human influenza virus Monoclonal antibodies that react against H1 subtype nuclear proteins can be used. That is, the fourth antibody may be the same antibody as the first antibody, the same antibody as the second antibody, or the same antibody as the third antibody. May be. In the apparatus of FIG. 1, as the third and fourth antibodies, the same or different antibodies reacting with all human influenza A virus subtypes are used, and in many cases, the third and fourth antibodies are used. As the first antibody, the same monoclonal antibody can be used.

The third and fourth antibodies are mixed with a test sample and a developing solvent in a suitable container separate from the immunochromatographic test strip 10 to obtain a mixed solution, and this mixed solution is used in the illustrated immunochromatographic test strip. The membrane carrier 3 may be chromatographed by injecting it into the sample addition member 6. However, when the mixed solution prepared by mixing the test sample and the developing solvent is injected into the sample addition member 6, the third and fourth antibodies are mixed with the mixed solution and chromatographed on the membrane carrier 3. As described above, it is preferable that the immunochromatographic test strip 10 be integrated with the test strip 10. For this purpose, in the apparatus of FIG. 1, the third and fourth antibodies are impregnated in the impregnation member 2 connected to the upstream end of the membrane carrier 3 in a state of being pre-labeled with an appropriate labeling substance. It constitutes a part of an immunochromatographic test strip 10. In the present specification, the labeled third antibody and / or fourth antibody may be referred to as a labeled antibody.
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 third and fourth antibodies may be any substance that can be used, and examples thereof include a color 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 capture site 4 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 suspension of the labeled antibody into a member such as the glass fiber nonwoven fabric and drying it.

As shown in FIG. 1, the immunochromatographic test strip 10 has a membrane carrier 3 stuck in the middle of the pressure-sensitive adhesive sheet 1, and on the upstream end of the membrane carrier 3 on the downstream side of the impregnating member 2. The ends can be overlapped and connected, and the upstream portion of the impregnating member 2 can be adhered to the pressure-sensitive adhesive sheet 1.
Furthermore, in the apparatus of FIG. 1, while mounting the downstream part of the sample addition member 6 on the upper surface of the impregnation member 2, the upstream part of the sample addition member 6 is stuck to the adhesive sheet 1, In addition, the upstream portion of the absorbing member 5 is placed on the upper surface of the downstream portion of the membrane carrier 3, and the downstream portion of the absorbing member 5 is attached to the adhesive sheet 1 so that the immunochromatographic test strip 10 is attached. It is composed.

As the sample addition member 6, for example, a porous synthetic resin sheet or film such as porous polyethylene and 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 absorbent member 5 may be made 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.

The immunochromatographic test strip 10 of FIG. 1 can be used as it is as a dipstick type measuring device, or alternatively, it can be provided as a dipstick type measuring device lined or sandwiched with a suitable plastic sheet. . Preferably, the immunochromatographic test strip 10 of FIG. 1 is a measuring device housed in a suitable plastic case having a test sample injection part and a judgment part opened above the sample addition member 6 and the capture site 4 respectively. Provided. As described above, in the present invention, the third and fourth antibodies are mixed with a test sample and a developing solvent in an appropriate container separate from the immunochromatographic test strip 10, and then mixed. Since the membrane carrier 3 may be chromatographed by injecting the solution into the sample addition member 6 of the immunochromatographic test strip 10, the third and fourth antibodies can be used when the immunochromatographic test strip 10 is accommodated in the case. May be impregnated in the impregnation member 2 and accommodated in the case, or may be accommodated in an appropriate container separate from the case and accommodated outside the case.

Thus, after a test sample composed of a biological sample of a patient suspected of having influenza A virus infection is mixed with an appropriate developing solvent as necessary to obtain a mixture that can be chromatographed, the mixture is shown in FIG. When injected onto the sample addition member 6 of the immunochromatographic test strip 10 shown, the mixed solution passes through the sample addition member 6 and mixes with the labeled antibody in the impregnation member 2.
At this time, if the influenza A virus H3 subtype is present in the mixed solution, a complex of the virus antigen of the H3 subtype virus and the labeled antibody is formed by the antigen-antibody reaction.
This complex is chromatographed in the membrane carrier 3 to reach the capture site 4, and is captured by an antigen-antibody reaction with the first antibody immobilized on the first capture site 41a, giving a positive result. The second antibody immobilized on the second capture site 41b is not captured and exhibits a negative result. On the other hand, if an influenza A virus other than the H3 subtype is present in the mixed solution, a positive result is shown at both the first capture site 41a and the second capture site 41b. Therefore, by combining the results of the first and second capture sites, it is possible to make a differential determination as to whether the infection is an H3 subtype or an influenza A virus other than the H3 subtype.

In the determination of positive or negative, if a colored labeling substance such as colloidal gold is used as the labeling substance, the first and second capture sites 41a and 41b are colored by the accumulation of the colored labeling substance. It can be judged immediately.
In the apparatus of FIG. 1, since the second capture site 41b is downstream of the first capture site 41a, the detection sensitivity of the second capture site 41b tends to decrease. It is preferable to adjust the sensitivity by a method such as increasing the amount of the antibody immobilized on the first capture site 41a.

The test sample is not particularly limited, and examples thereof include nasal aspirate, nasal swab, throat swab, tracheal swab, saliva, sputum, blood (can be whole blood, serum, or plasma). . The test sample may be diluted with a suitable diluent such as a developing solvent or physiological saline 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.

(2) Embodiment using two immunochromatographic test strips The apparatus of FIG. 2 is an immunochromatographic measuring apparatus comprising two immunochromatographic test strips 10 and 20. Each immunochromatographic test strip 10 and 20 is the same as the configuration of the immunochromatographic test strip 10 of FIG. 1 except for the configuration of the capture site 4 and the labeled antibody. In the immunochromatographic test strip 10 of FIG. 2, the first antibody is immobilized at a position 7.5 mm from the upstream end of the membrane carrier 3, and the first antibody for capturing the complex of the third antibody and the analyte. The point that one capture site 41a is formed is the same as in FIG. 1, but an anti-influenza B virus antibody is immobilized at a position 10.5 mm from the upstream end of the membrane carrier 3 to detect influenza B virus. 1 is different from FIG. 1 in that a capture site 41b is provided. The impregnated member 2 of the immunochromatographic test strip 10 is impregnated with a labeled third antibody and a labeled anti-B influenza virus antibody as a labeled antibody. Here, in many cases, the same antibody as the first antibody can be used as the third antibody. As an anti-type B influenza virus antibody, the antibody currently used for the well-known immunochromatography measuring apparatus can be used.
In the immunochromatographic test strip 20 of FIG. 2, the second antibody is fixed at a position 9.0 mm from the upstream end of the membrane carrier 3, and a second antibody for capturing the complex of the fourth antibody and the analyte. 1 differs from the immunochromatographic test strip 10 of FIG. 1 in that a second capture site 41c is formed. The impregnated member 2 of the immunochromatographic test strip 20 is impregnated with a labeled fourth antibody as a labeled antibody. Here, as the fourth antibody, the same antibody as the second antibody is used, but the same antibody as the first antibody may be used.

Thus, after obtaining a mixed solution containing the test sample in the same manner as described above with reference to FIG. 1, the mixed solution is used for each sample addition member 6 of the immunochromatographic test strips 10 and 20 shown in FIG. When injected above, the mixed solution passes through the sample addition member 6 and is mixed with the labeled antibody in the impregnation member 2.
At this time, if the influenza A virus H3 subtype is present in the mixed solution, a complex of the H3 subtype virus antigen and the third antibody is formed in the immunochromatographic test strip 10 by the antigen-antibody reaction. The This complex is chromatographed in the membrane carrier 3 to reach the capture site 4, and is captured by an antigen-antibody reaction with the first antibody immobilized on the first capture site 41a, giving a positive result. It is not captured by the anti-influenza B virus antibody immobilized on the capture site 41b and gives a negative result. In the immunochromatographic test strip 20, the complex of the H3 subtype virus antigen and the fourth antibody is not formed, and both of them are independently chromatographed in the membrane carrier 3 to reach the second capture site 41c. However, it is not captured by the second antibody immobilized thereon and gives a negative result.

On the other hand, if an influenza A virus other than the H3 subtype is present in the mixed solution, a complex of the virus antigen and the third antibody is formed in the immunochromatographic test strip 10 by the antigen-antibody reaction. The This complex is chromatographed in the membrane carrier 3 to reach the capture site 4, and is captured by an antigen-antibody reaction with the first antibody immobilized on the first capture site 41a, giving a positive result. It is not captured by the anti-influenza B virus antibody immobilized on the capture site 41b and gives a negative result. Also in the immunochromatographic test strip 20, a complex of the virus antigen and the fourth antibody is formed, chromatographed in the membrane carrier 3, reaches the second capture site 41c, and is fixed to the second capture site 41c. It is captured by the antigen-antibody reaction with this antibody and gives a positive result.

If influenza B virus is present in the mixed solution, the immunochromatographic test strip 10 forms a complex of the virus antigen and anti-B influenza virus antibody by the antigen-antibody reaction. This complex is chromatographed in the membrane carrier 3 to reach the capture site 4, and is captured by an antigen-antibody reaction with the anti-influenza B virus antibody immobilized on the capture site 41b, giving a positive result. The first antibody immobilized on one capture site 41a is not captured and exhibits a negative result. In the immunochromatographic test strip 20, a complex of the virus antigen and the fourth antibody is not formed, and both are independently chromatographed in the membrane carrier 3 to reach the second capture site 41c. It is not captured by the second antibody immobilized there and gives a negative result.
Therefore, depending on the results at the first capture site 41a, the second capture site 41c and the capture site 41b, whether the infection is an H3 subtype, an influenza A virus other than the H3 subtype, or an influenza B virus infection Can be identified.

The apparatus of FIG. 2 only needs to include at least two immunochromatographic test strips 10 and 20, even if two strips are accommodated in one case, several strips are accommodated in each case. Further, it may be configured to be taken out and used one by one at the time of use. In the former case, in one case, the impregnated members of both membrane carriers of the immunochromatographic test strips 10 and 20 are separated from each other at a predetermined interval, and the two membrane carriers are arranged in parallel with each other as an integrated product. Also good.
As the immunochromatographic test strip 10 of FIG. 2, a commercially available immunochromatographic test strip or kit for differential measurement of type A and B influenza such as Capilia (registered trademark) FluA + B (manufactured by Towns Co., Ltd.) is used as it is. You can also. Accordingly, only the immunochromatographic test strip 20 of FIG. 2 can be supplied alone so that the detection method or measurement method of the present invention can be carried out in combination with such a commercially available test strip or kit.

The apparatus of FIG. 3 has the same configuration as that of FIG. 2 except that the immunochromatographic test strips 10 and 20 include a single impregnation member 2 and a sample addition member 6 in common. That is, both membrane carriers 3 of the immunochromatographic test strips 10 and 20 are arranged substantially parallel to each other, and a wide single impregnation member 2 (about 12 mm in width) is disposed at the upstream end. It is connected across. The impregnation member 2 is arranged so that the third antibody, the fourth antibody, and the anti-B influenza virus antibody can be chromatographed in the same manner as described above. Preferably, both the third antibody and the fourth antibody are antibodies that react against all human influenza A subtypes, conveniently both are the same as the first antibody. can do.

Thus, after obtaining a mixed solution containing the test sample by the same method as described above with reference to FIG. 2, the sample addition member 6 common to the immunochromatographic test strips 10 and 20 shown in FIG. When injected above, the mixed solution passes through the sample addition member 6 and is mixed with the labeled antibody in the impregnation member 2.
At that time, if influenza A virus H3 subtype is present in the mixed solution, the immunochromatographic test strip 10 gives a positive result at the first capture site 41a as in FIG. Gives a negative result. In the immunochromatographic test strip 20, a complex of the H3 subtype virus antigen and the labeled antibody is formed, but is not captured by the second antibody immobilized on the second capture site 41c and exhibits a negative result. .
If influenza A virus other than H3 subtype is present in the mixed solution, the immunochromatographic test strip 10 gives a positive result at the first capture site 41 as in FIG. 2, but the capture site 41b Shows a negative result. In the immunochromatographic test strip 20, a complex of the viral antigen and the labeled antibody is formed, chromatographed in the membrane carrier 3, reaches the second capture site 41c, and is immobilized on the second antibody. Captured with a positive result.

In addition, if influenza B virus is present in the mixed solution, the immunochromatographic test strip 10 gives a positive result at the capture site 41b, but is negative at the first capture site 41a, as in FIG. Present results. In the immunochromatographic test strip 20, as in FIG. 2, a negative result is exhibited at the second capture site 41c.
Therefore, depending on the results at the first capture site 41a, the second capture site 41c, and the capture site 41b, the infection is an H3 subtype, an influenza A virus other than the H3 subtype, or an influenza B virus infection. Can be identified.
The apparatus of FIG. 3 may be in the form of being housed in a suitable case.

The apparatus of FIG. 4 has the same configuration as that of FIG. 2 except that the immunochromatographic test strips 10 and 20 include a single adhesive tape 1 and a single sample addition member 6. That is, the two membrane carriers 3 of the immunochromatographic test strips 10 and 20 are arranged substantially in a straight line in opposite directions. Specifically, the immunochromatographic test strips 10 and 20 are arranged in a straight line so that the upstream ends of both impregnating members 2 face each other with an appropriate interval, that is, in opposite directions. One sample addition member 6 is arranged so as to be connected across both the impregnation members 2. Alternatively, the membrane supports of the immunochromatographic test strips 10 and 20 may be arranged in a radial direction such that they extend at an angle to each other.

Thus, after obtaining a mixed solution containing the test sample by the same method as described above with reference to FIG. 2, the sample addition member 6 common to the immunochromatographic test strips 10 and 20 shown in FIG. When injected above, the mixed solution passes through the sample addition member 6 and is mixed with the labeled antibody in the impregnating member 2 of both test strips 10 and 20. And, as in FIG. 2, with the results at the first capture site 41a, the second capture site 41c and the capture site 41b, whether the infection is an H3 subtype or an influenza A virus other than the H3 subtype, It can be differentially determined whether it is an influenza B virus infection.

The device of FIG. 4 may be in the form of being housed in a suitable case.
As a modification of the apparatus of FIG. 4, the impregnation member 2 may be common to the test strips 10 and 20 in addition to the adhesive sheet 1 and the sample addition member 6. In this case, the third and fourth antibodies can be constructed according to the embodiment of FIG.
In the apparatus of FIG. 4, since the mixed solution containing the test sample penetrates the test strips 10 and 20 equally, it is necessary to adjust the sensitivity of the first and second capture sites 41a and 41b as in the apparatus of FIG. It is convenient in that there is no.

(3) Embodiment using second antibody as labeled antibody The apparatus of FIG. 2 can be modified to an apparatus for carrying out the embodiment of the present invention using the second antibody as a labeled antibody. 2, the first antibody is fixed instead of the second antibody to the second capture site 41c of the immunochromatographic test strip 20 in the apparatus of FIG. 2, and the impregnation member 2 of the strip 20 is impregnated. It can be constructed by using a second antibody instead of the fourth antibody as a labeled antibody. In such an apparatus, the configuration of the immunochromatographic test strip 10 is exactly the same as that of FIG.
Thus, after obtaining a mixed solution containing the test sample in the same manner as described above with reference to FIG. 2, the mixed solution is added to each of the immunochromatographic test strips 10 and 20 of the modified device of FIG. When injected onto the member 6, the mixed solution passes through the sample addition member 6 and is mixed with the labeled antibody in the impregnating member 2.

At that time, if influenza A virus H3 subtype is present in the mixed solution, the immunochromatographic test strip 10 gives a positive result at the first capture site 41a as in FIG. Gives a negative result. In the immunochromatographic test strip 20, a complex of the H3 subtype virus antigen and the labeled antibody is not formed, and both of them are independently chromatographed in the membrane carrier 3 to reach the second capture site 41c, The H3 subtype virus antigen is captured by the first antibody immobilized at the second capture site 41c, but gives a negative result because the labeled antibody is not bound.
If influenza A virus other than H3 subtype is present in the mixed solution, the immunochromatographic test strip 10 gives a positive result at the first capture site 41 as in FIG. 2, but the capture site 41b Shows a negative result. In the immunochromatographic test strip 20, a complex of the virus antigen and the labeled antibody is formed, chromatographed in the membrane carrier 3 and reaches the capture site 41, and the first antibody is detected at the second capture site 41c. Captured with a positive result.

In addition, if influenza B virus is present in the mixed solution, the immunochromatographic test strip 10 gives a positive result at the capture site 41b as in FIG. 2, but is negative at the first capture site 41a. Present results. In the immunochromatographic test strip 20, a negative result is exhibited at the second capture site 41c.
Therefore, depending on the results at the first capture site 41a, the second capture site 41c, and the capture site 41b, the infection is an H3 subtype, an influenza A virus other than the H3 subtype, or an influenza B virus infection. Can be identified.

The apparatus of FIG. 4 can also be modified to an apparatus for carrying out the embodiment of the present invention using the second antibody as a labeled antibody by making the same changes as described above. That is, in the apparatus of FIG. 4, the labeled antibody that is immobilized on the second capture site 41 c of the immunochromatographic test strip 20 instead of the second antibody and impregnated in the impregnation member 2 of the strip 20. Can be modified by using a second antibody instead of a fourth antibody. The configuration of the immunochromatographic test strip 10 is exactly the same as that shown in FIG.

Thus, after obtaining a mixed solution containing the test sample in the same manner as described above with reference to FIG. 2, the sample addition member common to the immunochromatographic test strips 10 and 20 of FIG. When injected onto the sample 6, the mixed solution passes through the sample addition member 6 and is mixed with the labeled antibody in the impregnated member 2 of both test strips 10 and 20. Then, similar to the modified device of FIG. 2, the results of the first capture site 41a, the second capture site 41c, and the capture site 41b indicate whether the infection is an H3 subtype or an influenza A other than the H3 subtype. It can be discriminated whether it is a virus infection or an influenza B virus infection.

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

Example 1 (Cloning of recombinant nucleoprotein (NP) genes of influenza A and B viruses)
A / Puerto Rico / 8/34 (H1N1), which is influenza virus type A, and B / Lee / 40, which is influenza virus type B, were inoculated into cultured chicken eggs and cultured. Several days later, chorioallantoic fluid was collected to obtain a virus. RNA extraction reagent (TRIzol Reagent, Invitrogen) was added to this virus, and each viral RNA was extracted. Reverse transcription reaction was performed from the extracted RNA to prepare cDNA of NP gene. Uni 12 Primer: 5′-AGCAAAAGCAGG-3 ′ (SEQ ID NO: 1) was used as a primer used for the reverse transcription reaction.

The NP gene was amplified from the obtained A / Puerto Rico / 8/34 and B / Lee / 40 NP gene cDNAs using the polymerase chain reaction (PCR) method. In A / Puerto Rico / 8/34, sense primer: 5′-GGAATTCCATATGGCGTCCCAAGGCACC-3 ′ (SEQ ID NO: 2) and antisense primer: 5′-CCGCTCGAGTTAATTGTCGTACTCCTCTGC-3 ′ (SEQ ID NO: 3) were used as primers. In B / Lee / 40, sense primer: 5′-CGGGATCCATATGTCCAACATGGATATTG-3 ′ (SEQ ID NO: 4) and antisense primer: 5′-CCGCTCGAGTTAATAATCGAGGTCATCATA-3 ′ (SEQ ID NO: 5) were used. The primers used were Nde I and Xho I sites added to the upper primer and lower primer, respectively, for incorporation into the vector pET15b.
After confirming amplification of a part of the obtained PCR product by DNA electrophoresis, each amplified product was cleaved with restriction enzymes NdeI and XbaI and inserted into the vector pET15b cleaved with the same restriction enzymes.

Example 2 (Expression and purification of recombinant nucleoprotein (NP protein))
E. coli BL21 CodonPlus was transformed with the vector pET15b of Example 1 into which the target gene had been inserted, and cultured on ampicillin selective medium to obtain colonies. After overnight culture in L-Broth containing ampicillin (100 μg / ml) and chloramphenicol (25 μg / ml), the culture solution was added to L-broth containing ampicillin (100 μg / ml). After culturing at 37 ° C. for 3 hours, 1 M IPTG was added to a final concentration of 1 mM to induce expression of the recombinant NP protein. Protein expression by IPTG was confirmed by SDS-PAGE.
The cells recovered by centrifugation were suspended in a solubilization buffer. Furthermore, PMSF and lysozyme treatment were performed to completely lyse. Recombinant NP protein was purified from the centrifuged supernatant of the resulting solution through a column using Ni-NTA resin. This purified protein was dialyzed against PBS to obtain the desired recombinant NP protein.

Example 3 (Preparation of monoclonal antibody)
Using the influenza A virus recombinant NP protein (FluA NP) and influenza B virus recombinant NP protein (FluB NP) obtained in Example 2 as antigens, monoclonal antibodies against anti-FluA NP and FluB NP (anti-FluA NP antibody and Anti-FluB NP antibody). Production of the monoclonal antibody was performed according to a conventional method. 100 μg of each recombinant NP protein and 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. .
For cell fusion, Sp2 / 0-Ag14 cells (Shulman et al., 1978), which are mouse myeloma cells, were used. For cell culture, Dulbecco's Modified Eagle Medium (Gibco) is 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 and Sp2 / 0-Ag14 cells of immunized mice and adding polyethylene glycol solution (Sigma) thereto. Cell fusion was cultured in HAT-DMEM (serum-added DMEM containing 0.1 mM Sodium Hypoxanthine, 0.4 μM Aminopeterin and 0.016 mM Tymidine (Gibco)). For screening of anti-FluA NP antibody, inactivated Influenza Virus, Type A ( Antibody production in the culture supernatant was confirmed by enzyme-linked antibody method (ELISA) using a plate on which H1N1) and (H3N2) were immobilized. For screening of anti-FluB NP antibody, an inactivated Influenza B virus-immobilized plate was used, and antibody production in the culture supernatant was confirmed by the same method.

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. Cells with high antibody-producing ability were selected and cloned by ELISA. The cloned cells were inoculated intraperitoneally into mice (BALB / c, Retire, Japan SLC) that had been inoculated with 2,6,10,14-Tetramethylpentadecane (Sigma), and ascites was collected. Furthermore, IgG purification using a protein G adsorbent was performed by a conventional method to obtain a monoclonal antibody. The isotype of the produced monoclonal antibody was identified by ELISA using Mouse Monoclonal Antibody Isotyping Reagents (Sigma).

Finally, 3 clones producing monoclonal antibodies against FluA NP, BLA / 4105, BLA / 3741 and BL / 585, were obtained. Among these, as shown in the Examples below, antibody-producing cells BLA / 585, as a result, react with human influenza virus H1 subtype but do not react with human influenza virus H3 subtype. It was a clone producing (second antibody of the present invention). Two clones of monoclonal antibody-producing cells against FluB NP were obtained. Hereinafter, BLB / 171 and BLB / 3105 were used.

Example 4 (Production of immunochromatography measuring device)
(1) Preparation of anti-FluA NP antibody, anti-FluB NP antibody and anti-FluA NP antibody that does not react with H3N2 subtype Anti-FluA NP antibody-producing cells BLA / 4105 obtained in Example 1 and Each of BLA / 3741 was inoculated into the peritoneal cavity of mice to obtain ascites containing anti-FluA NP antibody. Further, each of the anti-FluB NP antibody-producing cells BLB / 171 and BLB / 3105 was inoculated into the abdominal cavity of the mouse to obtain ascites containing the anti-FluB NP antibody. Furthermore, anti-FluA NP antibody-producing cells BLA / 585 were inoculated into the peritoneal cavity of mice, and ascites containing anti-FluA NP antibody was obtained. Furthermore, IgG purification using a protein G adsorbent was performed by a conventional method to obtain each antibody.

(2) in ultrapure water 99ml boiled by preparative <br/> heating gold colloid solution, a 1% (v / w) aqueous chloroauric acid 1ml was added, further, 1% after the 1 minute (v / w ) After adding 1.5 ml of sodium citrate aqueous solution and heating 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 antibody solution Anti-FluA NP antibody derived from antibody-producing cell BLA / 4105 obtained in (1) above (hereinafter referred to as anti-FluA NP antibody BLA / 4105), antibody-producing cell BLB / 171-derived anti-FluB NP antibody (hereinafter referred to as anti-FluB NP antibody BLB / 171) and antibody-producing cell BLA / 585-derived anti-FluA NP antibody (hereinafter referred to as anti-FluA NP antibody BLA / 585) were respectively used in the following procedures. Gold colloid was labeled.
After mixing 3 μg of the protein equivalent weight of the anti-FluA NP antibody BLA / 4105 and 1 ml of the colloidal gold solution of (2) above, the mixture was allowed to stand at room temperature for 2 minutes to bind all of the antibodies to the surface of the colloidal gold particles. Prepare a colloidal gold-labeled anti-FluA NP antibody BLA / 4105 solution by adding 10% BSA aqueous solution so that the final concentration in the colloidal gold solution is 1% and block all remaining surfaces of the colloidal gold particles with this BSA. did. This solution was centrifuged (5600 × G, 30 minutes) to precipitate the colloidal gold labeled antibody, and the supernatant was removed to obtain a colloidal gold labeled antibody. This gold colloid-labeled antibody is suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 10% saccharose, 1% BSA, 0.5% Triton-X100, and colloidal gold-labeled anti-FluA NP antibody BLA / 4105 solution Was prepared. Further, 1 μg of protein equivalent weight of anti-FluB NP antibody BLB / 171 was bound to the gold colloid particle surface by the above method to prepare a colloidal gold labeled anti-FluB NP antibody BLB / 171 solution. Further, a protein equivalent weight of 1 μg of anti-FluA NP antibody BLA / 585 was bound to the gold colloid particle surface by the above method to prepare a colloidal gold labeled anti-FluA NP antibody BLA / 585 solution.

(4) Production of immunochromatography measurement device The immunochromatography measurement device shown in Fig. 2 was produced by the following procedure.
(4-1) Formation of the first capture site of the membrane carrier An elongated strip-like nitrocellulose membrane having a width of 5 mm and a length of 36 mm was prepared as the membrane carrier 3.
0.5 μl of a solution containing 1.0 mg / ml of anti-FluA NP antibody BLA / 3741 was applied in a line at a position 5.0 mm from the upstream end of the membrane carrier 3 and dried at room temperature. A capture site 41a (first capture site) of a complex of influenza A virus antigen and labeled antibody was formed. In addition, 0.5 μl of a solution containing 1.0 mg / ml of anti-FluB NP antibody BLB / 3105 is applied in a line at a position of 8.0 mm from the upstream end of the membrane carrier 3 and dried at room temperature. Thus, a capture site 41b of a complex of influenza B virus antigen and labeled antibody was formed.

(4-2) The same membrane carrier 3 and the membrane carrier used in the second capturing zone of formation <br/> above membrane carrier (4-1) was another prepared.
0.5 μl of a solution containing 1.0 mg / ml of anti-FluA NP antibody BLA / 3741 was applied in a line at a position 5.0 mm from the upstream end of the membrane carrier 3 and dried at room temperature. A capture site 41c (second capture site) of the complex of influenza virus antigen and labeled antibody was formed.

(4-3) Preparation of colloidal gold labeled antibody impregnated material
A 5 mm × 15 mm strip of glass fiber nonwoven is impregnated with 37.5 μl of a solution obtained by mixing the colloidal gold-labeled anti-FluA NP antibody BLA / 4105 solution and the colloidal gold-labeled anti-FluB NP antibody BLB / 171 solution obtained in (3) above. This was dried at room temperature to obtain a labeled antibody-impregnated member. Further, 37.5 μl of gold colloid-labeled anti-FluA NP antibody BLA / 585 solution was impregnated into another 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric, and this was dried at room temperature to obtain a labeled antibody-impregnated member. The former labeled antibody impregnated member is used as the impregnated member 3 of the test strip 10, the latter labeled antibody impregnated member is used as the impregnated member 3 of the test strip 20, and the membrane carrier obtained in the above (4-1) is used as the test strip 10. The membrane carrier obtained in (4-2) above is used as the membrane carrier 3 of the test strip 20, and a cotton cloth is used as the sample addition member 6 of each test strip as the absorption member 5. Using the filter paper, an immunochromatography measuring apparatus having the same arrangement as that in FIG. 2 was produced.

Reference example (reactivity test with various subtypes of influenza A virus)
Using an immunochromatography measuring apparatus prepared in the same manner as in Example 4 except that the test strip 20 was not provided, reactivity tests against 15 subtypes of influenza A virus were performed. As a test sample, a chorioallantoic fluid obtained by proliferating various subtypes of influenza A virus in growing chicken eggs with a specimen dilution solution was used. Then, 100 μl of the test sample was dropped onto the sample addition member 6 of the test strip obtained as described above with a micropipette and chromatographed, left for 15 minutes at room temperature, and then captured at the first capture site 41a. The captured amount of the complex of the virus antigen and colloidal gold labeled antibody was observed with the naked eye. The amount of capture is red (purple color), ± (weak coloration), + (clear coloration), ++ (clear coloration) Judgment was made by classifying into five levels of ++++ (conspicuous coloring).

As influenza A virus subtypes, A / Puerto Rico / 8/34 (H1N1), A / Hokkaido / 11/02 (H1N1), A / Singapore / 1/57 (H2N2), A / Aichi / 2 / 68 (H3N2), A / Hokkaido / 1/03 (H3N2), A / duck / Czech / 56 (H4N6), A / duck / Pennsylvania / 1028/83 (H5N2), A / Hong Kong / 156/97 (H5N1 ), A / Hong Kong / 483/97 (H5N1), A / turey / Massachusetts / 3740/65 (H6N2), A / seal / Massachusetts / 1/80 (H7N7), A / turey / Ontario / 67 (H8N4) , A / turey / Wisconsin / 66 (H9N2), A / chicken / Germany / N / 49 (H10N7), A / duck / England / 56 (H11N6), A / duck / Alberta / 60/76 (H12N5), A / gull / Maryland / 704/77 (H13N6), A / mallard / Astrakhan / 263/82 (H14N5), and A / duck / Australia / 341/83 (H15N8) were used for the test. The results are shown in Table 1.

As is clear from Table 1, in the first capture site 41a of the test strip 10, remarkable coloring was confirmed in all the virus strains subjected to the test. Therefore, the anti-FluA NP antibody BLA / 3741 and the anti-FluA NP antibody BLA / 4105 used in the test strip 10 are not only reactive against all human influenza A subtypes but also H1-15 subtypes. It was found that all of the representative examples showed reactivity.

Example 5 (Reactivity test with human influenza virus H3N2 subtype)
Influenza virus H1N1 strain (A / New Caledonia / 20/99, New Caledonia strain) and H3N2 strain (separated from clinical samples in 2007 by RT-PCR method using the immunochromatography measuring device prepared in Example 4 A reactivity test against clinical isolates that determined the H3N2 subtype was performed. As a test sample, a culture supernatant obtained by growing each of the above influenza virus strains on MDCK cells and prepared with a sample diluent was used. Then, 100 μl of the test sample is dropped onto the sample addition member 6 of the test strip obtained in Example 4 with a micropipette and chromatographed. After standing at room temperature for 15 minutes, the first capture site 41a and the second capture site 41a The capture amount of the complex of the virus antigen captured at the capture site 41c and the colloidal gold-labeled antibody was observed with the naked eye. The amount of capture is red (purple color), ± (weak coloration), + (clear coloration), ++ (clear coloration) Judgment was made by classifying into five levels of ++++ (conspicuous coloring). The results are shown in Table 2.

From the results shown in Table 2, reactivity to the clinical isolate (H3N2) was not confirmed at the second capture site 41c compared to the New Caledonia strain (H1N1). This shows that the anti-FluA NP antibody BLA / 585 used as the labeled antibody of the test strip 20 does not react with the clinical isolate (H3N2) and does not react with the influenza A virus H3N2 subtype. . Since this anti-FluA NP antibody BLA / 585 is a monoclonal antibody against the nucleoprotein of influenza A virus which is considered to have a highly conserved amino acid sequence among various subtypes, influenza A virus H3N2 It is presumed that it reacts against all human influenza A virus subtypes other than the type, that is, it does not react specifically with the H3N2 subtype. In contrast, at the first capture site 41a, the same reactivity was obtained with the New Caledonia strain (H1N1) and the clinical isolate (H3N2).

Example 6 (Reactivity Test for clinical specimens)
Using the immunochromatography measuring device prepared in Example 4, a reactivity test was performed on a clinical sample (nasal aspirate) collected from an influenza-infected patient. Nasal aspirate was collected from an influenza-infected patient using a suction catheter. The subtypes of 19 collected nasal aspirates were determined by RT-PCR. The RT-PCR method was carried out based on the identification of pathogen detection manual prepared by the National Institute of Infectious Diseases, influenza, and influenza virus by Polymerase Chain Reaction (PCR) method. As a result of RT-PCR, 9 H1N1 subtypes and 10 H3N2 subtypes were identified. A predetermined amount was collected from each nasal aspirate with a cotton swab for sample collection, and extracted with 500 μl of sample dilution solution as a test sample. Then, 100 μl of the test sample is dropped onto the sample addition member 6 of the test strip obtained in Example 4 with a micropipette and chromatographed. After standing at room temperature for 15 minutes, the first capture site 41a and the second capture site 41a The capture amount of the complex of the virus antigen captured at the capture site 41c and the colloidal gold-labeled antibody was observed with the naked eye. The amount of capture is red (purple color), ± (weak coloration), + (clear coloration), ++ (clear coloration) Judgment was made by classifying into five levels of ++++ (conspicuous coloring). The results are shown in Table 3.

As is clear from Table 3, in the first capture region 41a of the test strip 10, coloration was confirmed in all the test samples, and no coloration was observed in all the test samples in the capture region 41b. . On the other hand, in the second capture site 41c of the test strip 20, no color was confirmed in all of the H3N2 subtype nasal aspirate samples subjected to the test, but in all of the H1N1 subtype nasal aspirate samples. Was confirmed. Therefore, the immunochromatography measuring apparatus described in Example 4 shows reactivity to the H1N1 subtype and H3N2 subtype clinical specimens at the first capture site 41a, and to the H3N2 subtype at the second capture site 41c. It was found that there was no reactivity only with respect to it. This is because the anti-FluA NP antibody BLA / 585 used as the labeled antibody of the test strip 20 reacts with the human influenza virus H1 subtype but does not react with the human influenza virus H3 subtype. It is shown.

Therefore, when a sample collected from a patient suspected of having influenza infection is subjected to the immunochromatography measuring apparatus described in Example 4, the coloration at the first capture site 41a and the second capture site 41c is confirmed. Human influenza virus H1N1 infection is suspected. Moreover, although the coloration in the 1st capture | acquisition part 41a is confirmed, when the coloration in the 2nd capture | acquisition part 41c is not confirmed, human influenza virus H3N2 infection is suspected. Therefore, by using this apparatus, it is possible to perform appropriate treatment on the patient according to the type of infecting virus and to work to prevent the spread of infection. In addition, when coloration is recognized in the type B determination part in the capture part 41b, it is determined that influenza virus type B infection is suspected, and when no coloration is recognized in either of the determination parts, influenza infection is negative. is there.

Thus, according to the present invention, the first antibody reacts against all human influenza A virus subtypes and reacts against human influenza virus H1 subtype but reacts against human influenza virus H3 subtype It is possible to detect human influenza virus H3 subtype infection by using a second antibody in combination.

The present invention includes a first antibody that reacts against all human influenza A virus subtypes and a first antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype. A method for detecting human influenza virus H3 subtype comprising detecting two antigens and detecting an antigen positive for reaction with the first antibody and negative for reaction with the second antibody by immunoassay, in particular The present invention provides a sandwich immunoassay method, an immunochromatography measurement method, and a measuring apparatus, and a virus belonging to the human influenza virus H3 subtype can be rapidly detected by a specific and simple method. It is useful for quickly and easily diagnosing this disease.

DESCRIPTION OF SYMBOLS 1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 4 Capture site 5 Absorption member 6 Sample addition member 10 Immunochromatography test strip 20 Immunochromatography test strip

Claims (71)

A monoclonal antibody that reacts against human influenza virus H1 subtype but does not react with human influenza virus H3 subtype. 2. The monoclonal antibody according to claim 1, which reacts with a nucleoprotein of human influenza virus H1 subtype but does not react with a nucleoprotein of human influenza virus H3 subtype. The monoclonal antibody according to claim 2, wherein the human influenza virus H1 subtype is an H1N1 subtype, and the human influenza virus H3 subtype is an H3N2 subtype. The monoclonal antibody according to claim 2, which reacts with all human influenza A virus subtypes other than the H3 subtype. The monoclonal antibody according to claim 4, wherein all human influenza A virus subtypes other than the H3 subtype are H1, H2, H5, H7 and H9 subtypes. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype A method for detecting a human influenza virus H3 subtype comprising detecting an antigen positive for reaction with a first antibody and negative for reaction with a second antibody by immunoassay. The immunoassay is a sandwich immunoassay using the first antibody and a third antibody that reacts with an antigen to which the first antibody reacts, and / or the second antibody The detection method according to claim 6, comprising a sandwich immunoassay method using a fourth antibody that reacts with an antigen to which the second antibody reacts. The detection method according to claim 7, wherein the first antibody and the second or fourth antibody are immobilized on a carrier. The third antibody is an antibody that reacts with all human influenza A virus subtypes, and the fourth antibody reacts with a human influenza virus H1 subtype. The detection method according to claim 8, wherein the antibody does not react with the antibody. 9. The detection method according to claim 8, wherein the third and fourth antibodies are the same or different antibodies that react with all human influenza A virus subtypes. The detection method according to any one of claims 6 to 10, wherein the first, second, third and fourth antibodies are monoclonal antibodies against a nucleoprotein of influenza virus. The detection method according to claim 11, wherein the human influenza virus H1 subtype is an H1N1 subtype, and the human influenza virus H3 subtype is an H3N2 subtype. The detection method according to claim 12, wherein the second antibody is a monoclonal antibody that reacts with all human influenza A virus subtypes other than the H3 subtype. The detection method according to claim 13, wherein all of the human influenza A virus subtypes other than the H3 subtype are H1, H2, H5, H7 and H9 subtypes. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype A membrane carrier having first and second capture sites, each of which is formed in advance by being fixed in place, is prepared, and a third antibody that reacts with an antigen to which the first antibody reacts and a test sample The first mixed solution and the second mixed solution of the fourth antibody that reacts with the antigen to which the second antibody reacts and the test sample are directed to the first and second capture sites, respectively. Chromatographic development on the membrane carrier, or chromatographic development on the membrane carrier with the first mixture directed to both of the capture sites and reaction with the first antibody at the first capture site Is positive And immunochromatographic assay, characterized in that to be able to detect the human influenza virus subtype H3 type with the reaction of the second antibody in the second capturing zone is negative. The third antibody is an antibody that reacts with all human influenza A virus subtypes, and the fourth antibody reacts with a human influenza virus H1 subtype. The measurement method according to claim 15, which is an antibody that does not react with the antibody. 16. The method according to claim 15, wherein the third and fourth antibodies are the same or different antibodies that react with all human influenza A virus subtypes. The method according to any one of claims 15 to 17, wherein the first, second, third and fourth antibodies are monoclonal antibodies against a nucleoprotein of influenza virus. The measurement method according to claim 18, wherein the human influenza virus H1 subtype is an H1N1 subtype, and the human influenza virus H3 subtype is an H3N2 subtype. The method according to claim 19, wherein the second antibody is a monoclonal antibody that reacts with all human influenza A virus subtypes other than the H3 subtype. 21. The detection method according to claim 20, wherein all human influenza A virus subtypes other than the H3 subtype are an H1 subtype, an H2 subtype, an H5 subtype, an H7 subtype, and an H9 subtype. The method according to claim 15, wherein the third and fourth antibodies are labeled with a metal colloid or latex. The measurement method according to claim 22, wherein the membrane carrier is a nitrocellulose membrane. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype; A first antibody that reacts with an antigen to which the first antibody reacts; a fourth antibody that reacts with an antigen with which the second antibody reacts; and a membrane carrier. And the second antibody are respectively fixed in advance on a predetermined position of the membrane carrier to form first and second capture sites, the third and fourth antibodies are labeled with an appropriate labeling substance, and Human influenza virus H3 prepared so that the third and fourth antibodies can be chromatographed on the membrane carrier from positions separated from both of the capture sites toward the first and second capture sites, respectively. Detecting the type for immunochromatographic measuring device. The third antibody is an antibody that reacts with all human influenza A virus subtypes, and the fourth antibody reacts with a human influenza virus H1 subtype. The measurement apparatus according to claim 24, which is an antibody that does not react with the antibody. 25. The measuring apparatus according to claim 24, wherein the third and fourth antibodies are the same or different antibodies that react with all human influenza A virus subtypes. 27. The measuring apparatus according to any one of claims 24 to 26, wherein the first, second, third and fourth antibodies are monoclonal antibodies against influenza virus nucleoprotein. The measurement apparatus according to claim 27, wherein the human influenza virus H1 subtype is an H1N1 subtype, and the human influenza virus H3 subtype is an H3N2 subtype. 29. The measuring apparatus according to claim 28, wherein the second antibody is a monoclonal antibody that reacts with all human influenza A virus subtypes other than the H3 subtype. 30. The measuring apparatus according to claim 29, wherein all human influenza A virus subtypes other than the H3 subtype are an H1 subtype, an H2 subtype, an H5 subtype, an H7 subtype, and an H9 subtype. 25. The measuring apparatus according to claim 24, wherein the membrane carrier comprises a single membrane carrier having a first capture site and a second capture site spaced apart from each other. The measurement apparatus according to claim 24, wherein the membrane carrier includes a first membrane carrier on which a first antibody is immobilized and a second membrane carrier on which a second antibody is immobilized. The measurement apparatus according to claim 32, wherein the third antibody is prepared on the first membrane carrier, and the fourth antibody is prepared on the second membrane carrier. The measurement apparatus according to claim 33, wherein the third and fourth antibodies are prepared by being impregnated in impregnation members disposed on the first and second membrane carriers, respectively. The first membrane carrier and the second membrane carrier both have an elongated shape, and the impregnated members of both membrane carriers are arranged in parallel with each other with a predetermined distance therebetween. The measuring device described in 1. The first membrane carrier and the second membrane carrier each have an elongated shape, and the impregnated members of both membrane carriers are arranged in a radial direction or in opposite directions with a predetermined distance therebetween. Item 35. The measuring device according to Item 34. The third and fourth antibodies are the same or different antibodies that react against all human influenza A virus subtypes and can be chromatographed on the membrane carrier toward the first and second capture sites The measuring device according to claim 32, prepared as described above. 38. The measuring apparatus according to claim 37, wherein the third and fourth antibodies are prepared on a membrane carrier. 39. The measuring apparatus according to claim 38, wherein the third and fourth antibodies are prepared by being impregnated in an impregnating member disposed on a membrane carrier. 40. The measuring apparatus according to claim 39, wherein the third and fourth antibodies are the same antibody, and the impregnating member is connected to both the first membrane carrier and the second membrane carrier. The first membrane carrier and the second membrane carrier both have an elongated shape, and the impregnating members of both membrane carriers are arranged in parallel with each other with a predetermined distance therebetween. The measuring device described in 1. The first membrane carrier and the second membrane carrier each have an elongated shape, and the impregnated members of both membrane carriers are arranged in a radial direction or in opposite directions with a predetermined distance therebetween. Item 40. The measuring device according to Item 39. The measurement apparatus according to claim 24, wherein the third and fourth antibodies are labeled with a metal colloid or latex. 44. The measuring device according to claim 43, wherein the membrane carrier is a nitrocellulose membrane. The measurement apparatus according to claim 24, wherein the membrane carrier is accommodated in a case. The measurement apparatus according to claim 45, wherein the third antibody and / or the fourth antibody is accommodated outside or inside the case. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype; Using a third antibody that reacts with an antigen to which the first antibody reacts, and preparing a membrane carrier having a first capture site formed by preliminarily fixing the first antibody at a predetermined position The first mixed solution containing the second antibody and the test sample, and the second mixed solution containing the third antibody and the test sample are directed to the first capture site, respectively, and the membrane. Human influenza virus having a chromatographic development with a carrier and positive reaction of the third antibody at the first capture site and negative reaction of the second antibody at the first capture site 3 immunochromatographic assay, characterized in that to be able to detect subtypes. 48. The method according to claim 47, wherein the third antibody is an antibody that reacts with all human influenza A virus subtypes. 49. The method according to claim 47 or 48, wherein the first, second and third antibodies are monoclonal antibodies against influenza virus nucleoprotein. The method according to claim 49, wherein the human influenza virus H1 subtype is an H1N1 subtype, and the human influenza virus H3 subtype is an H3N2 subtype. 51. The method according to claim 50, wherein the second antibody is a monoclonal antibody that reacts with all human influenza A virus subtypes other than the H3 subtype. 52. The method according to claim 51, wherein all human influenza A virus subtypes other than the H3 subtype are an H1 subtype, an H2 subtype, an H5 subtype, an H7 subtype, and an H9 subtype. The method according to claim 47, wherein the second and third antibodies are labeled with a metal colloid or latex. The measurement method according to claim 53, wherein the membrane carrier is a nitrocellulose membrane. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype; At least a third antibody that reacts with an antigen to which the first antibody reacts and a membrane carrier, and the first antibody is previously fixed at a predetermined position of the membrane carrier to form a first capture site The second and third antibodies are labeled with an appropriate labeling substance, and the second and third antibodies are respectively separated from the capture site toward the first capture site from the position of the membrane carrier. An immunochromatography measuring device for detecting human influenza virus H3 subtype prepared so that it can be chromatographed on the surface. 56. The measuring apparatus according to claim 55, wherein the third antibody is an antibody that reacts with all human influenza A virus subtypes. 57. The measuring apparatus according to claim 55 or 56, wherein the first, second and third antibodies are monoclonal antibodies against influenza virus nucleoprotein. 58. The measuring apparatus according to claim 57, wherein the human influenza virus H1 subtype is an H1N1 subtype, and the human influenza virus H3 subtype is an H3N2 subtype. 59. The measuring apparatus according to claim 58, wherein the second antibody is a monoclonal antibody that reacts with all human influenza A virus subtypes other than the H3 subtype. 60. The measuring apparatus according to claim 59, wherein all human influenza A virus subtypes other than the H3 subtype are an H1 subtype, an H2 subtype, an H5 subtype, an H7 subtype, and an H9 subtype. 56. The measuring apparatus according to claim 55, wherein the membrane carrier includes a first membrane carrier to which the first antibody is immobilized and a second membrane carrier to which the first antibody is immobilized. The measurement apparatus according to claim 61, wherein the second antibody is prepared on the first membrane carrier, and the third antibody is prepared on the second membrane carrier. 64. The measuring apparatus according to claim 62, wherein the second and third antibodies are prepared by being impregnated in impregnating members disposed on the first and second membrane carriers, respectively. The first membrane carrier and the second membrane carrier both have an elongated shape, and the impregnated members of both membrane carriers are arranged in parallel with each other with a predetermined distance therebetween. The measuring device described in 1. The first membrane carrier and the second membrane carrier each have an elongated shape, and the impregnated members of both membrane carriers are arranged in a radial direction or in opposite directions with a predetermined distance therebetween. Item 64. The measuring device according to Item 63. 56. The measuring device according to claim 55, wherein the second and third antibodies are labeled with a metal colloid or latex. The measurement apparatus according to claim 66, wherein the membrane carrier is a nitrocellulose membrane. 56. The measuring apparatus according to claim 55, wherein the membrane carrier is accommodated in a case. 69. The measuring apparatus according to claim 68, wherein the second antibody and / or the third antibody are accommodated outside or inside the case. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype; At least a membrane carrier, wherein the second antibody is previously fixed at a predetermined position of the membrane carrier to form a first capture site, the first antibody is labeled with an appropriate labeling substance, and the first antibody An immunochromatography measuring apparatus for detecting human influenza virus H3 subtype prepared so as to be chromatographed on the membrane carrier from a position separated from one capture site toward the first capture site. A first antibody that reacts against all human influenza A virus subtypes and a second antibody that reacts against human influenza virus H1 subtype but does not react against human influenza virus H3 subtype; At least a membrane carrier, the first antibody is previously fixed at a predetermined position of the membrane carrier to form a first capture site, the second antibody is labeled with an appropriate labeling substance, and the first antibody An immunochromatography measuring apparatus for detecting human influenza virus H3 subtype prepared so as to be chromatographed on the membrane carrier from a position separated from one capture site toward the first capture site.

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