WO2007021002A1 - Immunodetection method for influenza virus h5 subtype - Google Patents

Abstract

It is intended to provide a method capable of detecting an influenza virus H5 subtype specifically, rapidly and simply and an assaying device. An antibody, particularly a monoclonal antibody, specifically reacting with a hemagglutinin protein of the influenza virus H5 subtype was obtained. Accordingly, an immunoassay method using the antibody, particularly a sandwich immunoassay method using a primary antibody and a secondary antibody against the hemagglutinin protein, particularly an immunochromatography assay method and a test strip for the immunochromatography method are provided. At least either of the primary antibody and the secondary antibody is preferably the monoclonal antibody.

Description

 Specification

 Immunodetection of influenza virus H5 subtype

 Technical field

 [0001] The present invention relates to an influenza virus H5 subtype immune detection method using an antibody against hemadalchun (HA) protein, which is a molecule on the envelope surface of influenza virus H5 subtype, and more specifically, a sandwich immunoassay method. In particular, the present invention relates to an immunochromatographic measurement method and an immunochromatographic test strip, and relates to a detection method useful for quickly and easily diagnosing infection of influenza virus H5 subtype such as highly pathogenic avian influenza virus.

 Background art

 [0002] Highly pathogenic avian influenza is an infectious disease caused by influenza virus that is highly lethal to chickens, and is also called poultry plague. In Japan, it has been designated as a legal infectious disease for the prevention of livestock infectious diseases. It is listed as a List A disease by the World Veterinary Secretariat (OIE).

 [0003] To date, the only influenza viruses that have caused highly pathogenic avian influenza are the H5 and H7 subtypes of influenza A virus. Not all of these subtypes are highly toxic, but in Japan, if livestock is infected with these subtypes, regardless of whether they are toxic or weakly toxic, Have been!

 [0004] Highly pathogenic avian influenza caused by H5N1 subtype was epidemic in Hong Kong in 1997, and was also pandemic in several Asian powers from 2003 to 2004, and reported cases of human infection and death. 7 mouths.

 Therefore, the rapid detection of highly pathogenic avian influenza infection is an important issue in preventing the spread of large-scale influenza infection.

[0005] At present, highly pathogenic avian influenza diagnosis is performed by collecting tracheal or cloacas (total excretory cavity) from diseased birds with suspected viral infection, inoculating them into growing chicken eggs, Is done by the method of separating. However, this method is disadvantageous in that it takes several days for results to be obtained and results cannot be obtained quickly. Also, By using genetic tests (PCR method and LAMP method) developed in recent years, the time required to obtain results is greatly shortened, but such genetic testing requires special equipment and technology, so chicken farming sites, etc. It is not possible to inspect with

[0006] Patent Literature 1: Japanese Translation of Special Publication 2004-509648

 Patent Document 2: Japanese Patent Laid-Open No. 11-108932

 Patent Document 3: Japanese Patent Laid-Open No. 2001-215228

 Non-Special Reference 1: Hinshaw VS et al., Specinc antibody responses and generation of antigen ic variants in chickens immunized against a virulent avian influenza virus, Avian Di s. 1990, 34 (1): 80-6

 Patent Document 2: Kaverin NV et al., “Structure of antigenic sites on the haemagglutinin mol ecule of H5 avian influenza virus and phenotypic variation escape mutantsj, J. Gen. Virol. 2002, 83 (ptl0): 2497-505

 Disclosure of the invention

 Problems to be solved by the invention

[0007] Therefore, the present invention relates to an immunodetection method for influenza virus H5 subtype, particularly a sandwich immunoassay method using an antibody against hemadalchun (HA) protein which is a molecule on the envelope surface of influenza virus H5 subtype. In particular, it is an object to provide a rapid and simple diagnosis of avian influenza by providing an immunochromatographic measurement method and an immunochromatographic test strip. Means for solving the problem

[0008] The present inventors succeeded in obtaining an antibody against hemagglutinin (HA) protein of the virus by immunizing a mouse using the influenza virus H5 subtype as an immunogen, and immunoassay the antibody. In particular, the present inventors have found that influenza virus H5 subtype can be specifically detected by using sandwich immunoassay, particularly immunochromatography, and have completed the present invention.

[0009] That is, according to one aspect of the present invention, there is provided an influenza virus H5 subtype detection method capable of immunoassay using an antibody against hemaggluten protein of influenza virus H5 subtype. The immunoassay for this detection method is not particularly limited, but a sandwich immunoassay, particularly an ELISA (enzyme-linked immunosorbent assay) method, an immunochromatography assay, etc. are preferred.

 [0010] Therefore, according to another aspect of the present invention, there is provided a sandwich immunoassay using a first antibody and a second antibody against influenza virus H5 subtype hemagtinin protein. Methods for detecting influenza virus H5 subtype are provided. Further, according to a preferred embodiment of the present invention, there is provided a membrane carrier comprising a capture site formed by preliminarily fixing a first antibody against influenza virus H5 subtype hemadalchun protein in a predetermined position. A mixture of a second antibody against the virus H5 subtype hemadalchun protein and a predetermined amount of the test sample is chromatographed on the membrane carrier toward the capture site, and influenza virus contained in the test sample is obtained. There is provided an immunochromatographic assay characterized in that a complex comprising an H5 subtype and the second antibody is captured at the capture site.

 [0011] Further, according to a preferred embodiment of the present invention, the apparatus comprises at least a first antibody, a second antibody, and a membrane carrier against influenza virus H5 subtype of hematodalchus protein, wherein the first antibody is the above-mentioned Preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody is labeled with an appropriate labeling substance and can be chromatographed on the membrane carrier at a position separated from the capture site. An immunochromatographic test strip for detecting influenza virus H5 subtype is provided.

 [0012] The antibody against the influenza virus H5 subtype hemagglutinin protein used in the present invention may be a polyclonal antibody or a monoclonal antibody. From the viewpoint of reaction specificity, the antibody may be a monoclonal antibody. preferable. In the case of a sandwich immunoassay such as an immunochromatography method, the first antibody and the second antibody used there may be either a polyclonal antibody or a monoclonal antibody. From the viewpoint of reaction specificity, in general, at least one of the antibodies is preferably a monoclonal antibody, and both antibodies are particularly preferably monoclonal antibodies.

[0013] The antibody used in the present invention is a hemagglutinin protein of influenza virus H5 subtype Therefore, it reacts specifically with the influenza virus H5 subtype and does not react with influenza viruses other than the influenza virus H5 subtype !. The entire amino acid sequence of the hemagglutin protein of influenza virus H5N1 (A / Hong Kong / 156/97 (H5Nl)) is known as shown in SEQ ID NO: 1 (Science 279 (5349), 393-396 (1998). A preferred antibody for use in the present invention is an antibody that recognizes an epitope comprising the amino acid at position 168 of the amino acid sequence of the hemadalchun protein shown in SEQ ID NO: 1, particularly a monoclonal antibody. It is considered to be composed of several amino acid residues before and after the 168th amino acid residue of the sequence, and usually 6 to: centering on the 168th amino acid residue: L0 amino acid residues It is thought that it consists of

 [0014] Forcibly, according to another aspect of the present invention, a monoclonal antibody that recognizes an epitope containing the 168th amino acid of the amino acid sequence of the hemagglutinin protein represented by SEQ ID NO: 1 (hereinafter referred to as "first Also referred to as “monoclonal antibodies”). However, according to the fluorescent antibody method, the first monoclonal antibody is capable of reacting generally against influenza virus H5 attenuated strains A / tn / S. Africa / 61, A / swan / Shima / 449/83 (24a5b), A / HongKong / 156/97, A / HongKong / 483/97, A / duck / Yokohama / aq-10 / 2003 and A / chicken / Yamaguchi / 7/04 Does not respond to at least one of the highly virulent strains or is less reactive than the attenuated strain.

[0015] Forcibly, according to still another aspect of the present invention, influenza virus H5 subtypes A / tn / S. Africa / 61, A / swan / Shima / 449/83 (24a5b), A / Monoclonal antibodies that react with all of HongKong / 156/97, A / Hong Kong / 483/97, A / duck / Yokohama / aq-10 / 2003 and A / chicken / Yamaguchi / 7/04 A second monoclonal antibody). According to the fluorescent antibody method, the second monoclonal antibody is clearly distinguished from the first monoclonal antibody in that it reacts strongly with all of the above virulent strains. It is thought that it recognizes different epitopes. The second monoclonal antibody reacts against a wide range of virulent and attenuated strains and is therefore suitable for the wide detection of influenza virus H5 subtypes, especially for the detection of virulent strains. . [0016] It should be noted that neither the first monoclonal antibody nor the second monoclonal antibody reacts with influenza viruses other than influenza virus H5 subtype, and thus against hemagglutinin protein of influenza virus H5 subtype. Antibody that reacts specifically.

 The invention's effect

 [0017] According to the present invention, since the antibody against influenza virus H5 subtype of hemagglutin protein is used in the detection method by immunoassay, the antibody is specific to hemagglutinin protein of influenza virus H5 subtype. Using the reactivity, the influenza virus H5 subtype can be selectively detected, and can be widely applied to the diagnosis of infections caused by influenza virus H5 subtypes such as birds and humans.

 [0018] Further, according to the immunochromatography measurement method and the immunochromatography test strip of the present invention, it is possible to easily and quickly detect avian influenza virus at a poultry farming site or the like that requires special equipment and skilled techniques. It becomes possible to diagnose infection by the virus.

 BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, each step in the production of an antibody and the detection method and measurement method using the antibody is performed according to a known immunological technique.

 In the present invention, for example, a polyclonal antibody clones a DNA fragment corresponding to a portion containing the 168th amino acid residue of a DNA sequence encoding the amino acid sequence described in SEQ ID NO: 1, The cloned 匕 gene can be genetically expressed 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 can be obtained from the antiserum. In addition, a DNA sequence encoding an amino acid sequence corresponding to the epitope of madaltinin protein is obtained in an influenza virus virulent strain recognized by the second monoclonal antibody, and may be used as an immunizing antigen in the same manner as described above. .

[0020] In the present invention, the monoclonal antibody is used, for example, after immunizing an animal such as a mouse using the purified protein obtained in the same manner as described above as an antigen, and then spleen cells and mice of the immunized animal. For example, the fused cells obtained by cell fusion with the erythroid cells are selected with a HAT-containing medium, and then proliferated, and the proliferated strain is purified using the purified protein obtained as described above. It can be obtained by sorting by a labeled immunization method or the like.

 Alternatively, the monoclonal antibody can be obtained, for example, by immunizing an animal such as a mouse using the H5 subtype influenza virus itself as an antigen, and then cell-splitting the spleen cells and myeloma cells of the immunized animal. The resulting fused cells were grown after selection in a HAT-containing medium and reacted with the H5 subtype influenza virus from the proliferated strain, but did not react with all of the influenza viruses other than the H5 subtype. It can be acquired by selecting stocks.

[0021] The immunochromatographic assay method of the present invention for detecting influenza virus H5 subtype in a test sample can be easily performed according to the configuration of a known immunochromatographic test strip.

 In general, a powerful immunochromatographic test strip comprises a first antibody capable of reacting with an antigen at the first antigenic determinant of the antigen, an antibody antigen-reactive with the second antigenic determinant of the antigen, and A labeled second antibody and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody has the capture site force It is arranged so that it can be chromatographed on the membrane carrier at a separated position. As described above, each of the first antibody and the second antibody may be a polyclonal antibody or a monoclonal antibody, but at least one of them is preferably a monoclonal antibody. Usually, the first antibody and the second antibody are used in a “hetero” combination, that is, the first antibody and the second antibody, each recognizing each antigenic determinant that differs in position and structure on the antigen. These antibodies are used in combination. However, the first antigenic determinant and the second antigenic determinant may be structurally the same as long as their positions on the antigen are different. Homo ”combination monoclonal antibodies, which means that the same monoclonal antibody can be used for both the first antibody and the second antibody.

[0022] As a specific example of an immunochromatographic test strip, for example, a test strip shown in FIG. Lips are listed. In FIG. 1, numeral 1 is an adhesive sheet, 2 is an impregnated member, 3 is a membrane carrier, 31 is a capture site, 4 is an absorbing member, and 5 is a sample adding member.

 In the example shown in the figure, the membrane carrier 3 is made of an elongated strip-trocellulose membrane filter having a width of 5 mm and a length of 36 mm.

 The first antibody is fixed to the membrane carrier 3 at a position 7.5 mm from the end on the chromatographic start point side, and a sample capture site 31 is formed.

 In the example shown in the figure, the membrane carrier 3 is a force using a membrane filter made of nitrocellulose as long as it can develop a sample contained in the test sample and can immobilize the antibody forming the capture site 31. Any other cellulose film, nylon film, glass fiber film, etc. can be used.

[0023] The impregnating member 2 is impregnated with a second antibody that reacts with the antigen with the second antigenic determinant located at a site different from the first antigenic determinant to which the first antibody binds. It also becomes a member. The second antibody is previously labeled with an appropriate labeling substance.

 In the example shown in the figure, a force using a glass fiber nonwoven fabric of 5 mm X 15 mm as the impregnating member 2 is not limited to this. For example, cellulose cloth (filter paper, nitrocellulose membrane, etc.), polyethylene, polypropylene, etc. Other porous plastic cloths can also be used.

 [0024] Examples of the labeling substance for the second antibody include any colorable labeling substance, enzyme labeling substance, and radiation labeling substance, as long as they are usable.

 Of these, it is preferable to use a colored labeling substance because it can be quickly and easily determined by observing the color change at the capturing site 31 with the naked eye.

 Examples of the coloring labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with pigments such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloid are particularly preferred.

 The impregnated member 2 can be produced by impregnating the labeled second antibody suspension into a member such as the glass fiber nonwoven fabric and drying it.

[0025] As shown in FIG. 1, the membrane carrier 3 is stuck in the middle of the pressure-sensitive adhesive sheet 1, and the chroma of the membrane carrier 3 is Impregnation on the end of the starting point of the unfolding (that is, the left side of FIG. 1, hereinafter referred to as “upstream side”, and the opposite side, that is, the right side of FIG. The downstream end of the member 2 is overlapped and connected, and the upstream portion of the impregnated member 2 is adhered to the adhesive sheet 1 to produce the immunochromatographic test strip of the present invention.

 Furthermore, if necessary, the downstream portion of the sample addition member 5 may be placed on the upper surface of the impregnation member 2, and the upstream portion of the sample addition member 5 may be adhered to the adhesive sheet 1. Further, the upstream portion of the absorbing member 4 can be placed on the upper surface of the downstream portion of the membrane carrier 3 and the downstream portion of the absorbing member 4 can be attached to the adhesive sheet 1.

[0026] The sample addition member 5 includes, for example, porous synthetic resin sheets or films such as porous polyethylene and porous polypropylene, and cellulose paper or woven cloth such as filter paper and cotton cloth. Or a nonwoven fabric can be used. Examples of the absorbent member 4 include cotton cloth, filter paper, and porous plastic non-woven fabric that has strength such as polyethylene and polypropylene, as long as the material can absorb and hold liquid quickly. Filter paper is particularly suitable. It is.

[0027] In the case of Sarako, a commercially available product, the immunochromatographic test strip shown in FIG. 1 is made of a suitable plastic having a test sample injection part and a judgment part opened above the sample addition member 5 and the capture part 31, respectively. Provided housed in a case.

[0028] After a powerful test sample such as a biological sample is mixed with an appropriate developing solvent as necessary to obtain a liquid mixture that can be chromatographed, the mixed liquid is subjected to the immunochromatographic test shown in FIG. When injected onto the sample addition member 5 of the strip, the mixed solution passes through the sample addition member 5 and mixes with the labeled second antibody in the impregnation member 2.

 At that time, if a sample is present in the mixed solution, a complex of the sample and the second antibody is formed by the antigen-antibody reaction.

 This complex is chromatographed in the membrane carrier 3 to reach the capture site 31, and is captured by an antigen-antibody reaction with the first antibody immobilized thereon.

At this time, if a colored labeling substance such as colloidal gold is used as the labeling substance, the capture site 31 develops color due to the accumulation of the colored labeling substance, so immediately measure the sample qualitatively or quantitatively. be able to. [0029] The test sample is not particularly limited, but, for example, cloacas tube, tracheal tube, feces, nasal aspirate, nasal wipe and throat swab, blood (whether whole blood, serum or plasma), Saliva, urine, organ emulsions and the like can be mentioned. The test sample may be diluted with an appropriate diluent such as a developing solvent and injected into the membrane carrier.

 When whole blood is used as a test sample, particularly when a colored labeling substance such as gold colloid is used as the labeling substance of the labeled antibody, a blood cell capturing membrane member may be disposed on the sample addition member. preferable. The blood cell trapping membrane member is preferably laminated between the impregnation member and the sample-added calorie member. This prevents the red blood cells from being spread on the membrane carrier, thereby facilitating confirmation of the accumulation of the colored label at the capture site of the membrane carrier. A carboxymethyl cellulose membrane is used as the blood cell capturing membrane member. Specifically, the ion exchange filter paper CM (trade name) sold by Advantech Toyo Co., Ltd. or the Whatman Japan Co., Ltd.! An exchange cellulose paper etc. can be used.

 Example

 [0030] The ability to explain the present invention more specifically by the following examples The present invention is not limited to these examples.

[0031] (Production of anti-influenza virus H5 subtype monoclonal antibody)

 Influenza virus H5 subtype A / duck / Pennsylvania / 10128/84 (H5N2) strain 1

Inoculated into the amniotic cavity of 1-day-old embryonated chicken eggs and cultured. Several days later, the amniotic fluid was collected to obtain the virus.

 Using the obtained virus as an antigen, a monoclonal antibody against the virus was produced. Monoclonal antibodies were produced according to a conventional method.

 In other words, 100 g of virus antigen 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. It was. Sp2 / 0-Agl4 cells (Shulman et al., 1978), mouse myeloma cells, were used for cell fusion.

[0032] The obtained fused cells were grown after being selected in a HAT-containing medium, and then the monoclonal cells that reacted with the above strain of influenza virus H5 subtype from the grown fused cells. Finally, 15 antibody-producing cells were obtained.

 The reactivity of the 15 monoclonal antibodies produced and various influenza virus H5 subtypes was confirmed by the fluorescent antibody method, and the results are shown in Table 1. The fluorescent antibody method followed the following procedure.

[0033] Fluorescent antibody method

 A fluorescent antibody method was performed using a cell line derived from a kidney kidney (Madin-Darby canine kidney cell: MDCK cell). For culture of MDCK cells, 10% Bovine calf serum (Roche), 0.3 mg / ml L-glutamine, 100 units / mL penicillin G, heat-immobilized at 56 ° C for 30 minutes in Eagle's minimum essential medium (Nissui Pharmaceutical) After preparing potassium and 100 g / ml streptomycin sulfate, a medium whose pH was adjusted with sodium bicarbonate was used.

The fluorescent antibody method followed the method of Office International Des Epizooties (2000). 10 ⁴ TCID / ml in MDCK cells with monolayers formed on Lab-Tek II Chamber Slide System (Nunc)

 After inoculating 50 viruses and culturing at 35 ° C for 8 hours, the culture supernatant was removed, and the infected cells were washed once with PBS. The cells were fixed by soaking in 100% cold acetone (Wako Pure Chemical Industries) for 20 minutes and then dried. Mouse ascites containing the monoclonal antibody or culture supernatant was diluted 800 times with antibody diluent (PBS containing 1% BSA and 0.05% Tween20) and added as the primary antibody solution, and then at room temperature for 1 hour. Reacted. After washing once with PBS, Fluerescen-conjugated Goat IgG Fraction to Mouse IgG (ICN Biomedicals) diluted 1000-fold with an antibody diluent was added as a secondary rod and allowed to react at room temperature for 1 hour. After washing once with PBS, the cells were sealed with a mounting medium (Glycerol for fluerescence microscopy (Merck) mixed with 0.5 M carbonate bicarbonate notifier (pH 9.5) at a ratio of 3: 1), followed by fluorescence microscopy. (Axiovert 200, ZEISS) was used and observed with FITC Filter (Chroma). The determination was positive (+) by visually confirming the specific intracellular fluorescence of green in the nucleus and cytoplasm.

[0034] [Table 1] (X // (X //////// / okkaido67okkaido5196H5N3 AswanHHokkaido496H5N3 AswanH

 yggX (/ (/////// 2 AswanduckPennslvania1012884H5NuckHonKon69879H5N3 A d

 gg (/, (//// ¾ //// ssυH5N2 AH5N3 AduckHonKon34278 AduckMi5476ow π pi Monoclonal antibody and influenza virus H5 subtype reactivity

Monon antagonist

 Virus 25 40 48 64 A25 Β16β D31 145 Β220 3 Α27 Β9 Α310 Β29 Β59

A duck Mi / 54/76 (H5N3) + + + + + + + + + + + + + + +!

A duck / HongKong / 342 78 (H5N2) + + + + Ten + + + + + + + + + +

A / ducl ^ HongKong / 698/79 (H5N3) + + + + + + + + + + + + + + +

A duck Pennsylvania / 10128/84 (H5N2) + + + + + + + + + + + + + + +

A swan Hokkaido / 4/96 (H5N3) + + + + + + + + + + + + + +

A swan Hokkaido / 51/96 (H5N3) + + + + + + + + + + + + +

 +

 A swan / Hok / kaido / 67/96 (H5N3) + + + + + + + + + + + + + + +

A / duck Hokkaido / 69/00 (H5N3) + + + + + + + + + + + + + + +

A duck / Hokkaido / 447/00 (H5N3) + + + + + + + + + + + + + + +

A duck / ongolia / 54/01 (H5N2) + + + + + + + + + + + + + + +

A / duck / Mongolia / 500/01 (H5N3) + + + + + + + + + + + + + + +

A / duck MongoliQ / 596/01 (H5N3) + + + + + + + + + + + + + +:

A / duck / Hokkaido / 84/02 (H5N3) + + + + + + + + + + + + + + +:

A / tn / 5.Africa 61 (H5N3) + + + + + + +--+ Uniform + +

A swan / Shima / 449/83 (24a5b) (H5N3) + + + + + + + + + + + + + + +

A / HonqKong / 156/97 (H5N1) + + + + + + + + + Ten + + One +

A Hon Konq / 483/97 (H5N1) + + + + + + + + +-+ + +

A / duck / Yokohama aq-10 / 2003 (H5N1) + + + + + + + + + + + + One +:

A chicken Yamaguchi / 7/04 (H5N1) + + + + + + + One + + + + + + I —:

/ 96 (H5N3), A / duck / Hokkaido / 69/00 (H5N3), A / duck / Hokkaido / 447/00 (H5N3), A / duck / Mongolia / 54/01 (H5N2), A / duck / Mongolia 13 species of / 500/01 (H5N3), A / duck / Mon golia / 596/01 (H5N3) and A / duck / Hokkaido / 84/02 (H5N3) are influenza virus H5 attenuated strains, A / tn / S.Africa/61 (H5N3), A / swan / Shima / 449/83 (24a5b) (H5N3), A / HongKong / 156/97 (H5N1), A / HongKong / 483/97 (H5N1), A / duck Six species of / Yo kohama / aq-10 / 2003 (H5N1) and A / chicken / Yamaguchi / 7/04 (H5N1) are influenza virus H5 virulent strains.

 In Table 1, A / duck / Mongolia / 500/01 (H5N3) and A / duck / Mongolia / 596/01 (H5N3) are A / duck / Mongolia / 3/01 (H5N3), respectively. And is known to be equivalent to A / duck / Mongolia / 10/01 (H5N3)!

 [0036] From Table 1, clones Nos. 25, 40, 48, 64, A25, B168, and D31 are the 13 attenuated strains and 6 strong strains shown in Table 1 according to the fluorescent antibody method. It turns out that it reacts to all of the toxic strains. In contrast, clones Nos. 145, B220, 3, A27, B9, A310 B29 and B59 respond to all 13 attenuated strains shown in Table 1 in the fluorescent antibody method. It can be seen that it does not react to at least one of the highly virulent strains of the species or is less reactive than the attenuated strain.

 [0037] Twelve clones No.3, A27, B29, 25, 40, 48, A25, 64, D3 1, B9, A310 and B168 were selected from the 15 clones shown in Table 1. Clone Nos. 3, A27 and B29 are referred to as clones 3/3, A27 / 1 and B29 / 1, respectively), these 12 clone isotypes and A / duck / Pennsylvania / 10128/84. The titer in the ELISA method using the (H5N2) strain as an immobilized antigen was examined. The results are shown in Table 2. In Table 2, Mab indicates the type of clone, Isotype indicates the immunoglobulin isotype of the monoclonal antibody, and ELISA titer indicates the strength of the A / duck / Pennsylvania / 10128/84 (H5N2) strain by the ELISA method. Indicates the value. The monoclonal antibodies produced have 7 isotypes for Mouse Monoclonal Antibody Isotyping Reagents (bigma).

[0038] [Table 2] Influenza "T virus H5 subtype reactive monoclonal antibody

Isotype

 A duck / pennsylvania 1012B / 84

 3/3 102,400

 A27 / 1 102,400

G

[0039] Furthermore, the reactivity of the 12 selected clones with influenza viruses other than influenza virus H5 subtype was confirmed by ELISA, and the results are shown in Table 3. The results in Table 3 are common to 12 clones.

[0040] [Table 3]

Reactivity with various subtype viruses

表 3から明らかなように、上記 12種のクローンはインフルエンザウイルス H5亜型以 外のインフルエンザウイルスとは反応せず、したがって、インフルエンザウイルス H5 亜型と特異的に反応するものであることがわ力つた。

As is clear from Table 3, the above 12 clones do not react with influenza viruses other than the influenza virus H5 subtype, and thus are specifically reactive with the influenza virus H5 subtype. I got it.

 In addition, among the above clones, the monoclonal antibodies obtained from clones 3/3, A27 / 1 and B29 / 1 were mixed with A / duck / Pennsylvania / 10128/84 (H5N2) strain, and 11 days old were mixed. Virus was obtained by inoculating the amniotic cavity of a hatched chicken egg, culturing, and collecting amniotic fluid several days later. The resulting virus is highly likely to be mutated at the site where the monoclonal antibody binds because the selective pressure of the monoclonal antibody is strong.

 Therefore, when the hemagglutinin gene of the obtained virus was sequenced and its amino acid sequence was compared with the sequence of SEQ ID NO: 1, the former amino acid sequence was the 168th amino acid residue of the amino acid sequence of SEQ ID NO: 1. I found it mutated.

Therefore, the above three clones 3/3, A27 / 1 and B29 / 1 specifically bind to the hemadalchun protein of influenza virus H5 subtype, and further, the 168th amino acid of SEQ ID NO: 1. It was shown to recognize epitopes containing residues. [0042] (Preparation of immunochromato kit using anti-influenza virus H5 subtype antibody)

(1) Preparation of anti-influenza virus H5 subtype antibody (anti-H5 antibody)

 Each of the 12 clones obtained in Example 1 was inoculated into the abdominal cavity of mice to obtain ascites containing anti-H5 antibody. Furthermore, IgG was purified using a protein G adsorbent by a conventional method to obtain an anti-H5 antibody.

 [0043] (2) Preparation of colloidal gold solution

 Add 99 ml of l% (v / w) chloroauric acid aqueous solution to 99 ml of ultrapure water boiled by heating, and then add 1.5 ml of l% (v / w) sodium quenate aqueous solution after 1 minute. After boiling for 5 minutes, it was allowed to cool to room temperature. Next, a 200 mM potassium carbonate aqueous solution was added to this solution to prepare PH 9.0, and ultrapure water was added thereto to make up a total volume of 100 ml to obtain a colloidal gold solution.

 [0044] (3) Preparation of colloidal gold labeled anti-H5 antibody solution

 The anti-H5 antibodies derived from the 12 clones obtained in (1) above were labeled with colloidal gold according to the following procedure.

 1 g of the protein equivalent weight of the anti-H5 antibody (hereinafter, the protein equivalent weight of the antibody is simply indicated by the weight value of the purified protein by gravimetric analysis) and lml of the colloidal gold solution of (2) above are mixed. After allowing the antibody to bind to the surface of the gold colloid particles by allowing to stand at room temperature for 2 minutes, 10% ushi serum albumin (hereinafter referred to as “BSA”) is prepared so that the final concentration in the gold colloid solution is 1%. An aqueous solution was prepared, and all the remaining surfaces of the colloidal gold particles were blocked with the BSA to prepare a colloidal gold-labeled anti-H5 antibody (hereinafter referred to as “gold colloid-labeled antibody”) solution. This solution was centrifuged (5600 × G, 30 minutes) to precipitate colloidal gold labeled antibody, and the supernatant was removed to obtain colloidal gold labeled antibody. This gold colloid-labeled antibody was suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 10% saccharose · 1% BSA ′ 0.5% Triton-X100 to obtain a colloidal gold-labeled antibody solution.

(4) Preparation of immunochromatographic test strip for anti-influenza virus H5 subtype measurement The immunochromatographic test strip shown in FIG. 1 was prepared by the following procedure. (4 1) Capture site of complex of anti-H5 antibody and colloidal gold labeled antibody

5mm wide and 36mm long! /, Strip-trocellulose membrane is chromatographic chroma Prepared as a membrane carrier 3 for development.

 A solution 0.51 containing 1.0 mg / ml of anti-H5 antibody was applied in a line at a position 7.5 mm from the end of the chromatographic development start side of this membrane carrier 3 for chromatographic development, and this was applied at room temperature. It was dried and used as the capture site 31 of the complex of influenza virus H5 subtype and colloidal gold labeled antibody. As the anti-H5 antibody, monoclonal antibodies derived from the clones shown in Table 4-1 and Table 4-2 were used.

 [0046] (4 2) Colloidal gold labeled antibody impregnated member

 A 5 mm × 15 mm belt-shaped glass fiber nonwoven fabric was impregnated with 37.5 μ1 of a colloidal gold labeled antibody solution, and dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 2. As colloidal gold labeled antibodies, the colloidal gold colloid labeled antibodies listed in Tables 41 and 42 were used.

 (4-3) Preparation of immunochromatographic test strip

 In addition to the chromatographic development membrane carrier 3 and the labeled antibody impregnated member 2, a cotton cloth was prepared as the sample addition member 5, and a filter paper was prepared as the absorption member 4. Using these members, a chromatographic test strip similar to that shown in Fig. 1 was prepared.

[0047] (5) Test 1 (Attenuated strain detection)

 A total of 5 types of immunochromatographic test strips using the gold colloid-labeled antibodies and the capture site-forming antibodies (anti-antibody 5 antibodies) listed in Table 41 were prepared.

 The A / duck / Pennsylvania / 10128/84 (H5N2) strain was diluted with a specimen diluent to adjust to 2.5 μg / mL, and used as a test sample. The test sample 1001 is dropped with a micropipette onto the sample addition member 5 of the test strip obtained in (4) above, chromatographed, allowed to stand at room temperature for 15 minutes, and then captured at the capture site 31. The captured amount of the complex of the A / duck / Pennsylvania / 10128/84 (H5N2) strain and the colloidal gold-labeled antibody was observed with the naked eye. The amount of reddish purple that increases or decreases in proportion to the amount captured is visually (4) (no coloring), 2 (weak coloring), + (clear coloring), + + (significant coloring). Judgment was made by dividing into stages (where w is weak). As a control, the specimen dilution solution 100 ^ 1 was chromatographed in the same manner, and the degree of coloration was observed. The results are shown in Table 41.

[0048] [Table 4-1] Examination of antibody combinations

[0049] As is clear from Table 41, it was found that the influenza virus H5 subtype can be detected in any combination of Table 41.

 [0050] (6) Test 2 (Strongly virulent strain detection)

 A total of 144 types of immunochromatographic test strips using the colloidal gold-labeled antibodies and the capture site-forming antibodies (anti-H5 antibodies) listed in Table 4-2 were prepared. In Table 4-2, the capture site-forming antibody is expressed as a membrane-bound antibody.

 A / Chicken / Yamaguchi / 7/04 (H5N1) strain (HA value: 1024HA), an influenza virus H5 virulent strain, was diluted 625 times with a sample diluent to prepare a test sample. Then, 100 μl of the test sample is dropped with a micropipette onto the sample addition member 5 of the test strip obtained in (4) above, chromatographed, left at room temperature for 15 minutes, and then captured at the capture site 31. The amount of captured complex of the A / Chicken / Yamaguchi / 7/04 (H5Nl) strain and colloidal gold labeled antibody was observed with the naked eye. The amount of capture was determined by classifying the degree of red-purple coloration that increases or decreases in proportion to the amount into three levels: one (no coloring), one (weak coloring), and + (clear coloring). As a control, the specimen dilution solution 1001 was chromatographed in the same manner to observe the degree of coloration. The results are shown in Table 42.

[0051] [Table 4-2]

Anti-phased solid

Gold colloid-labeled antibody

[0052] As is clear from Table 42, it was found that four types of clones 64, A25, 25, and 48 were suitable for detection of influenza virus H5 virulent strains. In addition, clones A27 and A310 did not react with at least one influenza virus H5 virulence strain based on the results shown in Table 1, and thus were judged unsuitable for detection of influenza virus H5 virulence strains. .

 [0053] Nao ^ ffi (specificity test using similar pathogens in chickens)

 Using the immunochromatography kit prepared in Example 2 (using B29 / 1 as the anti-H5 antibody immobilized on the capture site and A27 / 1 as the anti-H5 antibody of the colloidal gold-labeled antibody), Specificity testing for similar disease pathogens was performed. The test was carried out in the same manner as in Example 2, except that each pathogen virus was diluted with a sample diluent and adjusted to 2.5 gZmL to obtain a test sample.

 [0054] Pathogen Newcastle disease virus (strain name NDV / Mongoria / 705/02 (APMV- 1)), Avian p aramyxovirus (serotype 2) (strain name Chicken / California / Yucaipa / 56 (APMV-2)), Avian p aramyxovirus (serotype 3) (strain name Turkey / Wisconsin / 68 (APMV- 3)), Avian paramyxovi rus (serotype 4) (strain name Duck / Mississippi / 320/75 (APMV— 4)), Avian paramyxovirus ( Serotype 5) (strain name Budgerigar / Kunitachi / 74 (APMV—5)), Avian paramyxovirus (serotype 6) (strain name Duck / HongKong / 199/77 (APMV-6)), Avian paramyxovirus (serotype 7) (Strain name Dove / Tennessee / 4/75 (APMV-7)), Coronavirus (strain name B-42), Herpesvirus (strain name NS1751), Poxvirus (salted water), Pasteurella multocida (serotype 5A, 8A, 9A), Haemo philus Paragallinarum (strain name HK-1), Mycoplasma gallisepticum, M. synoviae (strain name 5PT), Aspergillus lumigates, A. flavus (strain name KI-102162) were used for the test. As shown, the coloration at the capture site 31 is not observed for any pathogen. Nakatsu was. Therefore, it was confirmed that in all the pathogens listed in Table 5, the capture site of the influenza virus の 5 subtype detection immunochromatography kit of the present invention was not colored and did not react. The disease names, pathogen names, and strain names used are also shown in Table 5. In Table 5, one indicates that there is no coloration of the capture site.

[0055] [Table 5] Similar disease pathogens and reactivity results in chickens

[0056] Emperor M (Reactivity experiment with influenza viruses H5N1 and H9N2 for infection experiments) Imunochromatography kit prepared in Example 2 (B29 / 1 was used as an anti-H5 antibody immobilized on the capture site, and colloidal gold labeling) Using A27 / 1 as an antibody anti-H5 antibody, reactivity experiments of influenza viruses H5N1 and H9N2 for infection experiments were performed. Strain name A / Ck / Yamaguchi / 7/ 04 as Infuruen The virus H5N1 (H5N1), using a HA titer 1024 as a stock solution, prepared in the stock solution and dilutions (10 10 ^2, 10 ^3, 10 ^"4) The diluted solution was used as a test sample for the reaction, and the stock name A / Ck / Y-55 / 01 (H9N 2) and HA value 4096 were used as the stock solution for influenza virus H9N2, and the stock solution and dilution (10 — The diluted solution prepared in 10 " ² ) was used for the reaction as a test sample. The results are shown in Table 6. In Table 6, one indicates that the capture site is not colored, and + indicates that the capture site is colored.

As Table 6 clearly, from a stock solution in influenza virus H5N1 until dilution ^10-3 is reactivity was confirmed, mosquitoes such be confirmed reactive in dilution ^10-4 isゝivy. Influenza virus H9N2 showed no reactivity in the stock solution and all dilutions.

 [0057] [Table 6] Reactivity with infection viruses H5N1 and H9N2 H 驗

 Strain name HA value Dilution degree Reactivity

A / Ck / Yamaguchi / 7/04 (H5N1) 1024 Stock solution +

10 in ¹ +

 10 +

10 one ³ +

10 " ⁴ ―

A / Ck / Y-55 / 01 (H9N2) 4096 Stock solution

 Ten-' -

I t) - ² -

^ S (Influenza virus H5N1 inoculation-detection of virus antigens from chickens such as Chloascus tube)

An influenza virus H5N1 was inoculated into a bird, and the virus was detected by collecting the black locust of the bird that was infected with the virus. A white leghorn species was used as the target bird. The strain A / chicken / Yamag uchi / 7/04 (H5Nl) was used as the test virus, and the virus solution was inoculated nasally into the pupa, and crocus koji was collected every day until death. This bird died 3 days after infection, and immediately after that tracheal tube and various organs were collected.

 The tracheal tube and cloaca tube were suspended in the specimen diluent and used as test samples. Each organ was sampled aseptically, and 9% PBS was added to the weight of each organ collected and ground to make a 10% organ emulsion. The emulsion was then centrifuged, and the centrifuged supernatant was used as a test sample. These test samples were prepared using the immunochromatography kit prepared in Example 2 (B29 / 1 was used as the anti-H5 antibody immobilized on the capture site and A27 / 1 was used as the anti-H5 antibody of the colloidal gold-labeled antibody). It used for the test. In addition, virus isolation was performed on various organs. The results are shown in Table 7 and Table 8. In Tables 7 and 8, one indicates that the capture site is not colored, and + indicates that the capture site is colored.

 [0059] From Table 7, antigen detection was observed on days 1 and 2 after virus inoculation. Therefore, according to the present invention, it is possible to detect virus antigens from cloacacus ribs in the case of infected birds several days after infection. It was shown that

 [0060] [Table 7]

Chicken black fever inoculated with H5N1 virus

 Mul of Pulse from the sub

 Number of days after virus inoculation

 0 曰 1 曰 2 曰 3 曰

+ + One death

[0061] From Table 8, according to the present invention, virus antigens such as tracheal tubes and all organ emulsions were obtained. It was shown that the detection results were consistent with the virus separation results for organ emulsions. From the above results, according to the present invention, it was confirmed that viral antigens can be detected with high sensitivity using specimens collected from various site forces of infected birds.

The values shown in Table 8 for the virus isolation results are the virus infectivity values (10 ^x EID / m

 50

1 or g), and a 10-fold dilution series of the above 10% organ emulsion is prepared. After inoculating each diluted solution into a 9-11 day old chicken egg and culturing for 2 days, urine fluid is collected from the grown chicken egg. It was used for the HA test and calculated from the results.

[0062] [Table 8]

Chicken subsidized with H5N1 virus,

 Virus out of ^

Spa Breast

 After inoculation

 Until death

 Days of trachea trachea lung liver fiber n fiber Km

3 NT / + 8.50 / + 8.50 / + 7.60 / + 7.00 / + 7.25 / + β.50 / + Virus isolation menochromato kit

[0063] Example 6 (Reactivity test with influenza virus H5N1)

Influenza virus H5N1 using the immunochromatography kit prepared in Example 2 (No. 64 was used as the anti-antibody 5 antibody immobilized on the capture site and No. 64 was used as the anti-H5 antibody of the colloidal gold-labeled antibody). And the reactivity test was conducted. As influenza virus H5N1 stock solution, A / Chicken / Yamaguchi / 7/04 (HA value: 1024HA), A / Hongkong / 483/97 (HA value: 512HA), A / Chicken / Suphanburi / 1/04 (HA value: 512HA) ), A / Vietnam / 1194/04 (HA value: 512HA), A / Whooperswan / Mongolia / 3/05 (HA value: 16HA). A / Swan / Hokkaido / 51/96 (H5N3) (HA value: 256HA), A / Chicken / Ibaraki / 1/05 (H5N2) (HA value: 512HA) and A / Chicken as control influenza virus stock solutions / Italy / 99 (H7N1) ( HA value: 512HA), A / Chicken / Netherlands / 03 (Η7Ν7) (HA value: 512HA) was used. A test sample was prepared by diluting these virus stock solutions with a specimen diluent and subjected to the reaction. For A / Chicken / Yamaguchi / 7/04, the virus stock solution was diluted 625 times to obtain a test sample, and other strains were diluted 125 times to obtain test samples.

 The results are shown in FIG. As is apparent from FIG. 2, this immunochromatography kit showed reactivity to all H5 subtypes and no reactivity to the control H7 subtype.

 [0064] Further, the above immunochromatography kit (No. 64 was used as an anti-H5 antibody immobilized on a capture site V, No. 64 was used as an anti-H5 antibody of a colloidal gold labeled antibody) Prepare a chromato kit (No. B29 / 1 was used as an anti-H5 antibody to be immobilized at the capture site and No. A27 / 1 was used as an anti-H5 antibody for colloidal gold-labeled antibody). A / Chicken / Yamaguchi / 7 / 04, A / Hong kong / 483/97, A / Chicken / Suphanburi / 1/04, A / Vietnam / 1194/04, A / Whooperswan / Mongolia / 3/05, A / Swan / Hokkaido / 51/96 ( Test samples were prepared by diluting the H5N3) and A / Chicken / ¾araki / l / 05 (H5N2) virus stock solutions 25, 125, and 625 times with the sample diluent, and subjected to the reaction.

 The results are shown in Table 9. As is clear from Table 9, the immunochromatography kit using No. 64 as an anti-H5 antibody reacted well with influenza virus H5 and attenuated strains, especially for detection of influenza virus H5 strains. Is preferred.

[0065] [Table 9]

Reactivity against influenza virus H5 subtype

 [0066] Using the above immunochromatography kit (No. 64 was used as the anti-H5 antibody immobilized on the capture site and No. 64 was used as the anti-H5 antibody of the gold colloid-labeled antibody), the animal-derived influenza was used. Reactivity tests against each virus HI to H15 subtype were performed. Each virus strain was prepared by diluting with a sample diluent and used as a test sample. Then, the test sample 1001 was dropped and chromatographed, and left to stand at room temperature for 15 minutes for visual judgment. The results are shown in Table 10.

[0067] [Table 10]

/ qlvz¾zv 卜 c5Je>

 / ll9 / 0 / pl / v

 9i 9 inch l

[0068] As is apparent from Table 10, this immunochromatography kit did not react with influenza viruses other than influenza virus H5 subtype, but was shown to react specifically with H5 subtype.

 [0069] Example 7 (Inoculation with influenza virus H5N1-detection of virus antigens from cloacacus in birds, etc.)

Influenza virus H5N1 (A / Chicken / Yamaguchi / 7/04, abbreviated as "Yamaguchi strain") ) Was inoculated into chickens, and tracheal and clocus tubes from chickens infected with the virus were collected, and virus antigens were detected with an immunochromatography kit. In addition, trachea, kidney, and colon were collected from dead birds, organ emulsions were prepared, and viral antigens were detected using an immunochromatography kit.

 A police brown species was used as a bird to be infected. Viral solution was inoculated intranasally in chickens, and cloacas and tracheal tubes were inoculated daily until death.

 The tracheal tube and cloaca tube were suspended in the specimen diluent and used as test samples. Various organs were collected aseptically, and 9% PBS was added to each organ weight collected and ground to prepare a 10% organ emulsion. The emulsion was centrifuged and the centrifuged supernatant was used as a test sample. Test of these test samples using the immunochromatography kit prepared in Example 2 (No. 64 was used as the anti-H5 antibody immobilized on the capture site and N 0.64 was used as the anti-H5 antibody of the gold colloid-labeled antibody). It was used for. Then, the degree of coloration of the trapped part can be visually checked: — (no coloration), Shi (weak coloration), + (clear coloration), + + (significant coloration), +++ (more noticeable coloration) (Coloring) was evaluated in five stages. The results are shown in Tables 11 and 12. In Tables 11 and 12, d indicates the number of days, and (D) means that the person died on that day.

In addition, the virus infectivity titer (10 ^× EID / ml or g) was calculated in the same manner as in Example 5 using the tracheal tube, the crocus tube and the 10% organ emulsion of various organs,

 50

The results are shown in Tables 11 and 12.

[Table 11]

[0071] [Table 12]

[0072] From Table 11, antigen detection was confirmed in the tracheal tube and clocus tube 2 days after the virus inoculation. Therefore, according to the present invention, it is possible to detect a viral antigen from the trachea and clonal power in the case of an infected bird several days after the infection. In addition, it can be seen from Table 11 that the detection result by the immunochromatography method of the present invention matches the virus infectivity value by virus separation.

 [0073] From Table 12, it can be seen that according to the present invention, virus antigens can be detected from all organ emulsions. From the above results, it was confirmed that according to the present invention, viruses can be detected with high sensitivity using samples collected from various parts of infected birds. Further, from Table 12, it is clear that the detection result by the immunochromatography method of the present invention matches the virus infection value by virus separation.

 Industrial applicability

[0074] The present invention provides a sandwich immunoassay method using an antibody against hemagglutin protein of influenza virus H5 subtype, in particular, an immunochromatographic assay method and an immunochromatographic test strip, and provides an influenza virus Since a virus belonging to the H5 subtype can be detected quickly and specifically by a simple method, it is useful for quickly and easily diagnosing diseases such as birds and humans caused by the virus.

 Brief Description of Drawings

 [0075] [Fig. L] a is a plan view of an immunochromatographic test strip, and b is a longitudinal sectional view of the immunochromatographic test strip indicated by a.

 FIG. 2 is a graph showing the results of Example 6.

 Explanation of symbols

[0076] 1 Adhesive sheet

 2 Impregnated parts

 3 Membrane carrier

 31 Capture site

 4 Absorbing material

 5 Sample addition material

Claims

 The scope of the claims  [I] A method for detecting influenza virus H5 subtype comprising an immunoassay using an antibody against hemadalchun protein of influenza virus H5 subtype.  2. The detection method according to claim 1, wherein the antibody is a monoclonal antibody.  [3] The detection method according to claim 2, wherein the monoclonal antibody is a monoclonal antibody that recognizes an epitope containing the 168th amino acid of the amino acid sequence of the hemadalchun protein represented by SEQ ID NO: 1. [4] Monoclonal antibody strength A / tn / S.Africa / 6 which is influenza virus H5 subtype 1, A / swan / Shima / 449/83 (24a5b), A / HongKong / 156/97, A / HongKong / 483/97, The detection method according to claim 2, which is a monoclonal antibody that reacts with all of A / duck / Yokohama / aq-10 / 2003 and A / chicken / Yamaguchi / 7/04. 5. The detection method according to any one of claims 1 to 4, wherein the antibody does not react with an influenza virus other than influenza virus H5 subtype. [6] A method for detecting influenza virus H5 subtype, characterized by having a sandwich immunoassay capability using a first antibody and a second antibody against hemadalchun protein of influenza virus H5 subtype. 7. The detection method according to claim 6, wherein at least one of the first antibody and the second antibody is a monoclonal antibody. [8] The detection method according to claim 7, wherein the monoclonal antibody is a monoclonal antibody that recognizes an epitope comprising the 168th amino acid of the amino acid sequence of the hemadalchun protein represented by SEQ ID NO: 1. [9] A / tn / S. Africa / 6, which is the monoclonal antibody strength influenza virus H5 subtype 1, A / swan / Shima / 449/83 (24a5b), A / HongKong / 156/97, A / HongKong / 483/97, The detection method according to claim 7, which is a monoclonal antibody that reacts with all of A / duck / Yokohama / aq-10 / 2003 and A / chicken / Yamaguchi / 7/04. 10. The detection method according to any one of claims 6 to 9, wherein the antibody does not react with influenza viruses other than influenza virus H5 subtype. [II] Any one of the first antibody and the second antibody is fixed to a carrier. The detection method according to any one of 1 to 10. [12] A membrane carrier having a capture site formed by preliminarily immobilizing a first antibody against influenza virus H5 subtype hemagglutinin protein in place, and preparing a second carrier against influenza virus H5 subtype hemagglutin protein The mixture of the antibody and a predetermined amount of the test sample is chromatographed on the membrane carrier toward the capture site, and the influenza virus H5 subtype contained in the test sample and the second antibody are mixed together. An immunochromatographic assay method comprising capturing the complex at the capture site [13] The method according to claim 12, wherein at least one of the first antibody and the second antibody is a monoclonal antibody. [14] The method according to claim 13, wherein the monoclonal antibody is a monoclonal antibody that recognizes an epitope containing the 168th amino acid of the amino acid sequence of the hemadalchun protein represented by SEQ ID NO: 1. [15] A / tn / S. Africa / 6, which is the monoclonal antibody strength influenza virus H5 subtype 1, A / swan / Shima / 449/83 (24a5b), A / HongKong / 156/97, A / HongKong / 483/97, The method according to claim 13, which is a monoclonal antibody that reacts with all of A / duck / Yokohama / aq-10 / 2003 and A / chicken / Yamaguchi / 7/04. 16. The method according to any one of claims 12 to 15, wherein the antibody does not react with influenza viruses other than influenza virus H5 subtype. [17] The method according to any one of claims 12 to 16, wherein the second antibody is labeled with a metal colloid or latex. 18. The measurement method according to any one of claims 12 to 17, wherein the membrane carrier is a trocellulose membrane. [19] At least a first antibody against the influenza virus H5 subtype hemadalchun protein, a second antibody, and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier and is captured. The second antibody is labeled with an appropriate labeling substance, and is arranged so that it can be chromatographed on the membrane carrier at a position separated from the capture site force. For detection of influenza virus H5 subtype Immunochromatographic test strip 20. The immunochromatographic test strip according to claim 19, wherein at least one of the first antibody and the second antibody is a monoclonal antibody.  [21] The immunochromatographic test strip according to claim 20, which is a monoclonal antibody that recognizes an epitope containing the 168th amino acid of the amino acid sequence of the hemagglutinin protein represented by SEQ ID NO: 1.  [22] A / tn / S.Africa / 6 1, A / swan / Shima / 449/83 (24a5b), A / HongKong / 156/97, A / HongKong / The immunochromatographic test strip according to claim 20, which is a monoclonal antibody that reacts with all of 483/97, A / duck / Yokohama / aq-10 / 2003 and A / chicken / Yamaguchi / 7/04.  23. The immunochromatographic test strip according to any one of claims 19 to 22, wherein the antibody does not react with influenza viruses other than influenza virus H5 subtype.  24. The immunochromatographic test strip according to any one of claims 19 to 23, wherein the second antibody is labeled with a metal colloid or latex. 25. The immunochromatographic test strip according to any one of claims 19 to 24, wherein the membrane carrier is a -trocellulose membrane.  [26] A monoclonal antibody that recognizes an epitope containing the 168th amino acid of the amino acid sequence of the hemagglutinin protein represented by SEQ ID NO: 1.  27. The monoclonal antibody according to claim 26, which does not react with influenza virus other than influenza virus H5 subtype! [28] Influenza virus H5 subtype A / tn / S. Africa / 61, A / swan / Shima / 449/83 (2 4a5b), A / HongKong / 156/97, A / HongKong / 483/97, A / duck / Yokohama / aq- 10/20 Monoclonal antibody that reacts with all of 03 and A / chicken / Yamaguchi / 7/04. [29] The monoclonal antibody according to claim 28, which does not react with influenza viruses other than influenza virus H5 subtype!