JP6824026B2 - Influenza virus H5 subtype immunoassay - Google Patents

Description

The present invention is an immunodetection method for influenza virus H5 subtype using an antibody against hemagglutinin (HA) protein, which is a molecule on the envelope surface of influenza virus H5 subtype, specifically, a sandwich immunoassay method, particularly immunochromatography. A measurement method and an immunochromatography test strip, which relates to a detection method useful for quickly and easily diagnosing an influenza virus H5 subtype infection such as a highly pathogenic avian influenza virus.

Highly pathogenic avian influenza is an infectious disease caused by influenza virus that is highly lethal to chickens, etc., and is also called poultry plague. In Japan, it is designated as a legally infectious disease under the Domestic Animal Infectious Diseases Control Law, and is designated worldwide. It is listed as a List A disease by the World Organization for Animal Health (OIE).

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 virulent, but in Japan, if livestock are infected with these subtypes regardless of whether they are highly virulent or weakly virulent, they are all slaughtered. ..

Highly pathogenic avian influenza caused by the H5N1 subtype spread in Hong Kong in 1997, and further spread in several Asian countries from 2003 to 2004, with human infections and deaths reported. In 2016, a large-scale epidemic was confirmed in wild birds and poultry due to the H5N6 subtype in South Korea, and infection in wild birds and poultry was also confirmed in Japan.
Therefore, rapid detection of highly pathogenic avian influenza infections is an important issue in preventing the spread of large-scale influenza infections in birds, and is also an important issue in the rapid detection of new influenza infections in humans. Is.

Currently, highly pathogenic avian influenza diagnosis is a method of collecting tracheal swabs or cloaca swabs (cloaca swabs) from diseased birds suspected of being infected with the virus, inoculating them into developing chicken eggs, culturing them, and then isolating the virus. Is done by. However, this method is disadvantageous in that it takes several days to obtain the result and the result cannot be obtained quickly. In addition, by using genetic testing (PCR method, LAMP method), the time required to obtain results can be significantly shortened, but since such genetic testing requires special equipment and technology, testing at poultry farms, etc. Cannot be done. Furthermore, a simple rapid test (immunochromatography) using an antibody against the nucleoprotein of influenza virus is also used, and although influenza virus infection can be confirmed, it is not possible to identify the subtype of the infected virus. It is not possible to diagnose highly pathogenic avian influenza.

In addition, there is a report on a simple detection kit for influenza virus H5 subtype, but hemagglutinin of influenza virus is always exposed to neutralizing antibody, always causes minor mutations, and the antibody used may not react. It has been suggested. Under such circumstances, an antibody capable of reliably detecting a mutated virus and a simple detection kit using the antibody have been required.

Japanese Unexamined Patent Publication No. 2008-196967

Yoshimi Tsuda et al., Development of rapid diagnostic kit for H5 influenza virus infection, Japanese Society of Virology Academic Program / Abstracts, November 1, 2006, Vol. 54, p. 199

Therefore, the present invention has produced an antibody that recognizes an epitope that has not been previously reported for the hemagglutinin (HA) protein of influenza virus H5 subtype, and an immunoassay method for influenza virus H5 subtype using the antibody, particularly, By providing sandwich immunoassays, in particular immunochromatography and immunochromatography test strips, it is intended to enable reliable, rapid and convenient definitive diagnosis of influenza virus H5 subtype. ..

In order to detect the influenza virus H5N6 subtype, which is currently spreading in Asian countries, with high sensitivity, the present inventors immunize mice with the H5 subtype as an immunogen and the hemagglutinin (HA) protein of the virus. We succeeded in obtaining an antibody against a new epitope, and obtained an antibody that recognizes an epitope different from the previously reported antibody. In addition, by using the antibody in immunoassays, especially sandwich immunoassays, especially immunochromatography, it enables highly sensitive and highly specific detection compared to conventional H5 subtype detection methods, and influenza virus. We have found that the H5 subtype can be detected specifically, and have completed the present invention.

That is, according to one aspect of the present invention, it comprises an immunoassay method using an antibody against the hemagglutinin protein of influenza virus H5 subtype, and the antibody comprises the 172nd amino acid of the hemagglutinin protein amino acid sequence shown in SEQ ID NO: 1. A method for detecting an influenza virus H5 subtype, which comprises an antibody against an epitope containing the present, is provided.
The immunoassay method in this detection method is not particularly limited, but a sandwich-type immunoassay method, particularly an ELISA (Enzyme-linked immunosorbent assay) method, an immunochromatography measurement method, or the like is preferable.

Therefore, according to another aspect of the present invention, it comprises a sandwich immunoassay using a first antibody and a second antibody against the hemagglutinin protein of influenza virus H5 subtype, wherein the first antibody and the first antibody. A method for detecting an influenza virus H5 subtype is provided, wherein at least one of the two antibodies contains an antibody against an epitope containing the 172nd amino acid in the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1.

Further, according to a preferred embodiment of the present invention, a membrane carrier having a capture site formed by previously immobilizing a first antibody against the hemagglutinin protein of influenza virus H5 subtype at a predetermined position is prepared, and influenza virus H5 subtype is prepared. A mixed solution of a second antibody against the hemagglutinin protein of the type and a predetermined amount of the test sample was chromatographed on the membrane carrier toward the capture site, and the influenza virus H5 subtype contained in the test sample and the test sample were described. An immunochromatographic measurement method in which a complex containing a second antibody is captured at the capture site, wherein at least one of the first antibody and the second antibody is hemagglutinin shown in SEQ ID NO: 1. An immunochromatographic measurement method for influenza virus H5 subtype is provided, which comprises an antibody against an epitope containing the 172nd amino acid in the amino acid sequence of the protein.

Further, according to a preferred embodiment of the present invention, at least a first antibody against the hemagglutinin protein of influenza virus H5 subtype, a second antibody and a membrane carrier are provided, and the first antibody is a predetermined position of the membrane carrier. The second antibody is labeled with a suitable labeling substance and is arranged so that it can be chromatographed on the membrane carrier at a position separated from the capture site. In an immunochromatography test strip, at least one of the first antibody and the second antibody contains an antibody against an epitope containing the 172nd amino acid of the hemagglutinin protein amino acid sequence shown in SEQ ID NO: 1. An immunochromatographic test strip for detecting influenza virus H5 subtypes is provided.

The antibody against the hemagglutinin protein of the influenza virus H5 subtype essentially used in the present invention is an antibody against the epitope containing the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1. According to the method for describing the hemagglutinin amino acid sequence of influenza virus (Wilson et al.'S method), the recognition site of this antibody is the 160th of the H5 subtype hemagglutinin molecule when compared based on the hemagglutinin molecule amino acid sequence of influenza virus H3 subtype. Located in. Unless otherwise specified in the present specification, the amino acid position number compared with the hemagglutinin amino acid sequence of SEQ ID NO: 1 is used. The antibody may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of reaction specificity, it is preferable to use a monoclonal antibody. In the case of sandwich immunoassays such as immunochromatography, the first and second antibodies used therein may be polyclonal or monoclonal, respectively, but of reaction specificity. From the viewpoint, it is generally preferable that at least one antibody is a monoclonal antibody, and it is particularly preferable that both antibodies are monoclonal antibodies. In addition, the antibody may be a mixture with an antibody that recognizes other epitopes of the hemagglutinin protein shown in SEQ ID NO: 1.

The antibody used in the present invention is an antibody against the hemagglutinin protein of influenza virus H5 subtype, and therefore reacts specifically with influenza virus H5 subtype and does not react with influenza viruses other than influenza virus H5 subtype. is there. The entire amino acid sequence of the hemagglutinin protein of the H5N6 subtype represented by the influenza virus H5N6 subtype (A / duck / Vietnam / HU1-115 / 2014 (H5N6)) is known as shown in SEQ ID NO: 1. The epitope containing the 172nd amino acid in the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1 is an epitope present in the HA1 region of the hemagglutinin protein. These epitopes are considered to be composed of several amino acid residues before and after the 172nd amino acid residue of the above amino acid sequence, and are usually centered on each amino acid residue at the 172nd position. It is thought to be composed of 2 to 10 amino acid residues. Of these, the preferred antibody used in the present invention comprises an antibody that recognizes an epitope containing the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1, particularly a monoclonal antibody. The epitope containing the 172nd amino acid is considered to be an epitope conserved in the H5 subtype of influenza virus, and is suitable for detecting a wide range of H5 subtypes of influenza virus without mutation of the hemagglutinin molecule of the virus.

Thus, according to another aspect of the invention, there is provided a monoclonal antibody that recognizes an epitope containing amino acid 172 of the amino acid sequence of the hemagglutinin protein set forth in SEQ ID NO: 1.

Thus, according to yet another aspect of the invention, there is provided a monoclonal antibody that recognizes an epitope comprising the 172nd amino acid of the amino acid sequence of the hemagglutinin protein set forth in SEQ ID NO: 1 and consisting of the amino acid sequence set forth in SEQ ID NO: 2. To. Since the monoclonal antibody reacts not only with the recently isolated H5N6 subtype but also with a wide range of influenza virus H5 subtypes including a virulent strain of the conventional H5N1 subtype, the influenza virus H5 subtype is widely detected as described above. It is suitable for detecting highly virulent strains.

The monoclonal antibody of the present invention does not react with influenza viruses other than influenza virus H5 subtype, and therefore is an antibody that specifically reacts with the hemagglutinin protein of influenza virus H5 subtype.

According to the present invention, in the detection method by immunoassay, an antibody against a specific epitope present in the HA1 region of the hemagglutinin protein of influenza virus H5 subtype is used. Therefore, hemagglutinin of influenza virus H5 subtype of the antibody is used. The influenza virus H5 subtype can be selectively detected by utilizing the specific reactivity to the protein, and can be widely applied to the diagnosis of infections caused by the influenza virus H5 subtype in birds, humans and the like.

In addition, according to the immunochromatographic measurement method and the immunochromatographic test strip of the present invention, influenza virus H5 subtype can be detected easily and quickly at poultry farming sites, medical sites, etc. without the need for special equipment and skilled techniques. And it becomes possible to diagnose the infection by the virus.

a is a plan view of the immunochromatographic test strip, and b is a vertical sectional view of the immunochromatographic test strip shown by a.

In the present invention, each step in the production of an antibody and the detection and measurement methods using the antibody is carried out in accordance with a known immunological method per se.
In the present invention, for example, the polyclonal antibody clones a DNA fragment corresponding to a portion of the DNA sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 containing the 172nd amino acid residue of the amino acid sequence. The cloned gene can be genetically engineered in a host such as Escherichia coli to extract and purify the expressed protein, and the purified protein can be used as an antigen to immunize an animal according to a conventional method and obtain it from its antisera.

In the present invention, for example, a monoclonal antibody is obtained by immunizing an animal such as a mouse with the purified protein obtained in the same manner as described above as an antigen, and then fusing the spleen cells and myeloma cells of the immunized animal. The resulting fusion cells are selected in a HAT-containing medium and then proliferated, and the proliferated strain is obtained by using the purified protein obtained as described above and selecting, for example, by an enzyme-labeled immunological method or the like. be able to.

Alternatively, the monoclonal antibody is obtained, for example, by immunizing an animal such as a mouse with the H5 subtype influenza virus itself as an antigen, and then fusing the spleen cells and myeloma cells of the immunized animal. The fused cells were selected in a HAT-containing medium and then proliferated, and from the proliferated strains, strains that react with H5 subtype influenza virus but do not react with all influenza viruses other than H5 subtype are selected. By doing so, you can get it. Furthermore, among H5 subtype influenza viruses, strains with higher sensitivity and specificity can be selected by selecting virus strains belonging to specific subtypes such as H5N6 subtype, H5N8 subtype, and H5N1 subtype. Can be sorted.

The immunochromatographic measurement method of the present invention for detecting influenza virus H5 subtype in a test sample can be easily carried out according to the structure of a known immunochromatographic test strip.
In general, such immunochromatographic test strips are labeled with a first antibody capable of antibody-antigen reaction with the first antigenic determinant of the antigen and an antibody-antigen-reactable and labeled with the second antigenic determinant of the antigen. It comprises at least a second antibody and a membrane carrier, the first antibody is preliminarily immobilized at a predetermined position on the membrane carrier to form a capture site, and the second antibody is at a position separated from the capture site. It is configured so that it can be chromatographed on the membrane carrier. As described above, the first antibody and the second antibody may be polyclonal antibodies or monoclonal antibodies, respectively, but it is preferable that at least one of them is a monoclonal antibody. Usually, the first and second antibodies are used in a "hetero" combination, i.e., the first and second antibodies that recognize each antigenic determinant, which differs in position and structure on the antigen, respectively. 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, and a plurality of antigens are expressed in the detection target. If multiple are known to be present, the first and second antibodies may be monoclonal antibodies of a "homo" combination, i.e. identical to both the first and second antibodies. Monoclonal antibodies can be used.

Specific examples of the immunochromatography test strip include the test strip shown in FIG. In FIG. 1, the number 1 is an adhesive sheet, 2 is an impregnating member, 3 is a membrane carrier, 31 is a trapping site, 4 is an absorbing member, and 5 is a sample addition member.
In the illustrated example, the membrane carrier 3 is made of an elongated strip of nitrocellulose membrane filter 5 mm wide and 36 mm long.
The first antibody is immobilized on the membrane carrier 3 at a position 7.5 mm from the end on the chromatographic expansion start point side, and a sample capture site 31 is formed.
In the illustrated example, the membrane carrier 3 uses a membrane filter made of nitrocellulose, but the sample contained in the test sample can be expanded by capillarity and the antibody forming the capture site 31 can be fixed. Anything may be used as long as it is used, and other cellulose membranes, nylon membranes, glass fiber membranes and the like can be used.

The impregnating member 2 is generally impregnated with a second antibody that reacts with the antigen by a second antigenic determinant located at a site different from the first antigenic determinant to which the first antibody binds. It consists of a member that has been squeezed. The first antibody and the second antibody may be the same antibody. The second antibody is pre-labeled with a suitable labeling substance.
In the illustrated example, a 5 mm × 15 mm strip-shaped glass fiber non-woven fabric is used as the impregnating member 2, but the present invention is not limited to this, and for example, cellulose cloth (filter paper, nitrocellulose film, etc.), polyethylene, etc. Porous plastic cloth such as polypropylene can also be used.

The labeling substance of the second antibody may be any substance as long as it can be used, and examples thereof include a coloring labeling substance, an enzyme labeling substance, and a radiation labeling substance.
Of these, it is preferable to use a color-labeling substance because it can be quickly and easily determined by visually observing the color change at the capture site 31. It is also possible to use a so-called immunochromatographic reader to quantitatively measure the color change of the capture site 31, but it is also possible to use a color labeling substance to quantitatively measure the color change. preferable.
Examples of the color-developing labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with each pigment such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloids are particularly preferable.
The impregnated member 2 can be produced by impregnating a member such as the glass fiber non-woven fabric with a suspension of the labeled second antibody and drying the impregnated member 2.

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

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

Further, in the case of a commercially available product, the immunochromatographic test strip of FIG. 1 is housed in a suitable plastic case in which a test sample injection part and a judgment part are opened above the sample addition member 5 and the capture part 31, respectively. Provided.

Thus, a test sample consisting of a biological sample or the like is mixed with an appropriate developing solvent as needed to obtain a chromatographically developable mixed solution, and then the mixed solution is used for sample addition of the immunochromatography test strip shown in FIG. When injected onto the member 5, the mixture passes through the sample addition member 5 and mixes with the labeled second antibody in the impregnated member 2.
At that time, if the 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 chromatographically expanded in the membrane carrier 3 to reach the capture site 31, and is captured by an antigen-antibody reaction with the first antibody immobilized therein.
At this time, if a color labeling substance such as gold colloid is used as the labeling substance, the capture site 31 develops color due to the accumulation of the color labeling substance. Therefore, immediately measure the sample qualitatively or quantitatively. Can be done.

The test sample is not particularly limited, but includes, for example, cloaca swab, tracheal swab, feces, nasal aspirate, nasal swab and pharyngeal swab, blood (whole blood, serum, plasma), saliva, urine, etc. Organ emulsion 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, and particularly when a coloring labeling substance such as gold colloid is used as a labeling substance for a labeled antibody, it is preferable to dispose a blood cell trapping membrane member on the sample addition member. .. The blood cell trapping membrane member is preferably laminated between the impregnating member and the sample addition member. This prevents red blood cells from deploying on the membrane carrier, facilitating confirmation of the accumulation of color markers at the capture site of the membrane carrier. As the blood cell capture membrane member, a material capable of capturing blood cells such as a carboxymethyl cellulose membrane can be used.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to this example.

Example 1 (Creation of anti-influenza virus H5 subtype monoclonal antibody)
Influenza virus H5 subtype A / duck / Pennsylvania / 10128/84 (H5N2) strains were inoculated into the amnion cavity of 11-day-old hatched chicken eggs and cultured. A few days later, 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.
That is, 100 μg of viral antigen and an equal amount of Adjuvant Complete Freund (Difco) were mixed, and mice (BALB / c, 5 weeks old, Japan SLC) were immunized three times, and the spleen cells were used for cell fusion. For cell fusion, mouse myeloma cells Sp2 / 0-Ag14 cells (Shulman et al., 1978) were used.

The obtained fusion cells were selected in a HAT-containing medium and then proliferated, and two clones of monoclonal antibody-producing cells reacting with the above strain of influenza virus H5 subtype were finally obtained from the proliferating fusion cells. The antibodies produced from the clones obtained below are referred to as HTA001 and HTA002.

Example 2 (Preparation of immunochromatography kit using anti-influenza virus H5 subtype antibody)
(1) Preparation of anti-influenza virus H5 subtype antibody (anti-H5 antibody) Each of the two clones obtained in Example 1 was inoculated into the abdominal cavity of a mouse to obtain ascites containing the anti-H5 antibody. Furthermore, IgG purification using a protein G adsorbent was carried out by a conventional method to obtain an anti-H5 antibody.

(2) Preparation of platinum-gold colloidal particle solution All the glassware used was washed with aqua regia. 390 ml of ultra-pure water is placed in a flask and boiled, and 30 ml of a gold chloride aqueous solution (1 g of gold per liter of the aqueous solution, manufactured by Katayama Chemical Industry Co., Ltd.) is added to the boiling water, and then a 1 wt% sodium citrate aqueous solution is added. 60 ml was added, and after 6 minutes and 45 seconds, 30 ml of a platinum chloride aqueous solution (1 g as platinum per 1 liter of the aqueous solution, manufactured by Wako Pure Chemical Industries, Ltd.) was added. Five minutes after the addition of the platinum chloride aqueous solution, 60 ml of a 1 wt% sodium citrate aqueous solution was added and heated under reflux conditions for 4 hours to obtain a platinum-gold colloidal particle solution.

(3) Preparation of platinum-gold colloid-labeled anti-H5 antibody solution The anti-H5 antibodies of HTA001 and HTA002 derived from the two clones obtained in (1) above were respectively platinum-gold colloid-labeled according to the following procedure.
The protein equivalent weight of the anti-H5 antibody is 1 μg (hereinafter, when the protein equivalent weight of the antibody is shown, it is simply indicated by the weight value by weight analysis of the purified protein) and 1 ml of the platinum-gold colloidal solution of (2) above are mixed. After allowing this antibody to bind to the surface of platinum-gold colloidal particles by allowing it to stand at room temperature for 2 minutes, 10% bovine serum albumin (hereinafter referred to as "BSA") so that the final concentration in the platinum-colloidal gold solution is 1% An aqueous solution is added, and the entire remaining surface of the platinum-gold colloidal particles is blocked with this BSA to prepare a platinum-gold colloidal-labeled anti-H5 antibody (hereinafter referred to as "platinum-gold colloidal-labeled antibody") solution. Prepared. This solution was centrifuged (5600 × G, 30 minutes) to precipitate a platinum-gold colloid-labeled antibody, and the supernatant was removed to obtain a platinum-gold colloid-labeled antibody. This platinum-gold colloid-labeled antibody was suspended in 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 10% saccharose, 1% BSA, and 0.5% Triton-X100 to obtain a platinum-gold colloid-labeled antibody solution.

(4) Preparation of Immunochromatography Test Strip for Anti-Influenza Virus H5 Subtype Measurement The immunochromatography test strip shown in Fig. 1 was prepared by the following procedure.
(4-1) Capture site of complex of anti-H5 antibody and platinum-gold colloid-labeled antibody An elongated strip-shaped nitrocellulose membrane with a width of 5 mm and a length of 36 mm was prepared as a membrane carrier 3 for chromatographic development of a chromatographic medium.
0.5 μl of a solution containing 1.0 mg / ml of anti-H5 antibody was applied in a line at a position 7.5 mm from the end on the chromatographic development start point side of the chromatographic development membrane carrier 3, and this was dried at room temperature. The capture site 31 of the complex of influenza virus H5 subtype and platinum-gold colloid-labeled antibody was used. HTA001 and HTA002 monoclonal antibodies were used as anti-H5 antibodies.

(4-2) Platinum-gold colloidal labeled antibody impregnated member
A 5 mm × 15 mm strip-shaped glass fiber non-woven fabric was impregnated with 37.5 μl of a platinum-gold colloid-labeled antibody solution, and this was freeze-dried to obtain a platinum-gold colloid-labeled antibody-impregnated member 2. As the platinum-gold colloid-labeled antibody, a platinum-gold colloid-labeled antibody composed of HTA001 and HTA002 monoclonal antibodies was used.
(4-3) Preparation of Immunochromatography Test Strip In addition to the above-mentioned membrane carrier 3 for chromatography development and the above-mentioned labeled antibody impregnated member 2, a cotton cloth was prepared as a sample addition member 5, and a filter paper was prepared as an absorption member 4. Then, using these members, a chromatographic test strip similar to that shown in FIG. 1 was prepared.

(5) Test Two types of immunochromatography test strips using HTA001 or HTA002 monoclonal anti-H5 antibody were prepared as platinum-gold colloid-labeled antibody and capture site-forming antibody (membrane-immobilized antibody), respectively.
Influenza virus H5 subtype attenuated strain A / duck / Pennsylvania / 10128/1984 (H5N2) strain and virulent strain A / black swan / Akita / 1/12016 (H5N6) were used for evaluation. Each virus solution was serially diluted with a sample diluent to prepare a test sample. Then, 100 μl of the test sample is dropped onto the sample addition member 5 of the test strip obtained in (4) above with a micropipette, chromatographically developed, left at room temperature for 15 minutes, and then the coloration intensity of the capture site 31 is immunochromatographically measured. The color intensity was measured by measuring with a reader (manufactured by Otsuka Electronics Co., Ltd.). Tables 1 and 2 show the reactivity results with the A / duck / Pennsylvania / 10128/1984 (H5N2) strain, and Tables 3 and 4 show the reactivity results with A / black swan / Akita / 1/22016 (H5N6). It is shown in each.

Reactivity test with A / duck / Pennsylvania / 10128/1984 (H5N2)

Reactivity test with A / duck / Pennsylvania / 10128/1984 (H5N2)

As is clear from Tables 1 and 2, it was found that the H5 detection kit consisting of HTA001 and HTA002 anti-H5 antibodies can detect the influenza virus H5 subtype A / duck / Pennsylvania / 1984 (H5N2) attenuated strain. In addition, an immunochromatography kit (manufactured by Towns Co., Ltd., hereinafter referred to as NP detection kit) using an antibody against the nucleoprotein of influenza virus used as a control was also able to detect the virus strain with high sensitivity.

Reactivity test with A / black swan / Akita / 1/2016 (H5N6)

Reactivity test with A / black swan / Akita / 1/2016 (H5N6)

On the other hand, as is clear from Tables 3 and 4, when HTA001 was used as an anti-H5 antibody, it could be detected in the same manner as the influenza virus H5 subtype attenuated strain, whereas when HTA002 was used, it could be detected. A / black swan / Akita / 1/12016 (H5N6) virulent strain could not be detected. The NP detection kit used as a control showed the same reactivity as the influenza virus H5 subtype attenuated strain.

These results suggest that the HTA001 anti-H5 antibody is an antibody that recognizes a conserved region in the hemagglutinin molecule of influenza virus H5 subtype, while the HTA002 anti-H5 antibody is an attenuated strain of influenza virus H5 subtype. Although it is possible to recognize the region present in the hemagglutinin molecule of influenza virus, it was suggested that the antibody may not be able to recognize the same region of the hemagglutinin molecule of the H5 virulent strain isolated in recent years.

Example 3 (Epitope analysis of anti-H5 antibody)
The HTA001 and HTA002 anti-H5 antibodies prepared in Example 1 and the A / duck / Pennsylvania / 10128/84 (H5N2) strain were mixed and inoculated into the amniotic fluid cavity of an 11-day-old hatched chicken egg, cultured, and numbered. A day later, amniotic fluid was collected to obtain the virus. The hemagglutinin gene of the virus obtained from each sheep water was sequence-analyzed, the amino acid sequence was compared with the sequence of SEQ ID NO: 1, and the amino acid mutation in the hemagglutinin molecule of influenza virus resulting from selective pressure due to the neutralizing activity of the monoclonal antibody. Was analyzed to determine the epitope that is the binding site for the anti-H5 antibody. In addition to the above A / duck / Pennsylvania / 10128/84 (H5N2), A / duck / Vietnam / HU1-1151 / 2014 (H5N6), A / black swan / Akita / 1/22016 (H5N6), A / whooper swan / Similar analysis using influenza virus H5 subtypes of Hokkaido / 4/2011 (H5N1), A / chicken / Kumamoto9 / 1-7 / 2014 (H5N8) and A / duck / Vietnam / HU3-16 / 2015 (H5N1) Was carried out. Table 5 shows the results of reactivity with each virus strain. Table 6 shows the results of epitope analysis of each anti-H5 antibody in the hemagglutinin molecule of influenza virus H5 subtype.

Reactivity test with each influenza virus H5 subtype strain. In Table 5, high reactivity is indicated by “◯”, moderate to slight reactivity is indicated by “Δ”, and no reactivity is indicated by “x”.

As is clear from the results in Table 5 above, the HTA001 anti-H5 antibody showed good reactivity with all strains of all influenza virus H5 subtypes tested. On the other hand, the HTA002 anti-H5 antibody showed good reactivity only to A / duck / Pennsylvania / 10218/1984 (H5N2), but A / chicken / Kumamoto 9 / 1-7 / 2014 (H5N8) and A / Shows slight reactivity to duck / Vietnam / HU3-16 / 2015 (H5N1), other A / duck / Vietnam / HU1-1151 / 2014 (H5N6), A / black swan / Akita / 1 / The results showed no reactivity with 2016 (H5N6) and A / whooper swan / Hokkaido / 4/2011 (H5N1). Furthermore, in H5 subtypes other than A / duck / Pennsylvania / 10218/1984 (H5N2), it is strongly suggested that the epitope recognized by the HTA002 anti-H5 antibody was mutated and the amino acid sequence was changed, so that the antibody could not be recognized. Was done.

The results of the epitope analysis in Example 3 showed that the HTA001 anti-H5 antibody recognizes the epitope containing the 172nd amino acid residue of SEQ ID NO: 1, and the HTA002 anti-H5 antibody is the 158th of SEQ ID NO: 1. It has been shown to recognize epitopes containing amino acid residues.
In addition, the epitope containing the 172nd amino acid residue of SEQ ID NO: 1 recognized by the HTA001 anti-H5 antibody was widely conserved in the H5 subtype of influenza virus tested, and the results support the reactivity with each strain. .. On the other hand, the HTA002 anti-H5 antibody had an epitope containing the 158th amino acid residue of SEQ ID NO: 1 in A / duck / Pennsylvania / 10218/1984 (H5N2), but it was a slight reaction. In / Kumamoto9 / 1-7 / 2014 (H5N8) and A / duck / Vietnam / HU3-16 / 2015 (H5N1), the reactivity decreased due to some amino acid mutations, and no reactivity was shown. In / duck / Vietnam / HU1-1151 / 2014 (H5N6), A / black swan / Akita / 1/22016 (H5N6) and A / whooper swan / Hokkaido / 4/2011 (H5N1), in front of the epitope center It was strongly suggested that the antibody could not be recognized because the amino acid was mutated to proline and the structure around the epitope recognized by the antibody changed significantly, and this result was a kit using the HTA002 anti-H5 antibody. This proved to be the result of the reactivity of.
From the above analysis results, the antibody that recognizes the epitope containing the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1 recognized by the HTA001 anti-H5 antibody is suitable for detecting a wide range of influenza virus H5 subtypes. Was shown to be.

The present invention provides a sandwich-type immunoassay using an antibody against the hemagglutinin protein of influenza virus H5 subtype, particularly immunochromatography assay and immunochromatography test strip, for viruses belonging to influenza virus H5 subtype. Since it can be detected rapidly by a specific and simple method, it is useful for quickly and easily diagnosing diseases such as birds and humans caused by the virus.

1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 31 Capture site 4 Absorption member 5 Sample addition member

Claims (19)

It consists of an immunoassay using an antibody against the hemagglutinin protein of influenza virus H5 subtype, and the antibody is an antibody against the epitope shown in SEQ ID NO: 2 containing the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1. A method for detecting influenza virus H5 subtype, which is contained. The detection method according to claim 1 , wherein the antibody is a monoclonal antibody. The detection method according to claim 2 , wherein the monoclonal antibody does not react with influenza viruses other than influenza virus H5 subtype. It consists of a sandwich immunoassay using a first antibody and a second antibody against the hemagglutinin protein of influenza virus H5 subtype, and at least one of the first antibody and the second antibody has a SEQ ID NO: A method for detecting an influenza virus H5 subtype, which comprises an antibody against the epitope shown in SEQ ID NO: 2, which contains the 172nd amino acid of the hemagglutinin protein amino acid sequence shown in 1. The detection method according to claim 4 , wherein at least one of the first antibody and the second antibody is a monoclonal antibody. The detection method according to claim 5 , wherein the monoclonal antibody does not react with influenza viruses other than influenza virus H5 subtype. The detection method according to claim 4 , wherein either one of the first antibody and the second antibody is immobilized on a carrier. Prepare a membrane carrier having a capture site formed by previously fixing the first antibody against influenza virus H5 subtype hemagglutinin protein in a predetermined position, and prepare a second antibody against influenza virus H5 subtype hemagglutinin protein and a predetermined amount. The mixed solution with the test sample was chromatographed on the membrane carrier toward the capture site, and a complex containing the influenza virus H5 subtype contained in the test sample and the second antibody was obtained. An immunochromatographic measurement method in which a capture site is captured, wherein at least one of the first antibody and the second antibody contains the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1. An immunochromatographic measurement method for influenza virus H5 subtype, which comprises an antibody against the epitope shown in No. 2. The immunochromatographic measurement method according to claim 8 , wherein at least one of the first antibody and the second antibody is a monoclonal antibody. The immunochromatographic measurement method according to claim 9 , wherein the monoclonal antibody does not react with influenza viruses other than influenza virus H5 subtype. The immunochromatographic measurement method according to claim 8 , wherein the second antibody is labeled with a metal colloid or latex. The immunochromatographic measurement method according to claim 11 , wherein the membrane carrier is a nitrocellulose membrane. It comprises at least a first antibody, a second antibody and a membrane carrier against the hemagglutinin protein of influenza virus H5 subtype, and the first antibody is preliminarily fixed at a predetermined position on the membrane carrier to form a capture site, and the said The second antibody is an immunochromatographic test strip that 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 . Influenza virus H5, wherein at least one of the antibody and the second antibody contains an antibody against the epitope shown in SEQ ID NO: 2, which contains the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1. Immunochromatography test strip for subtype detection . The immunochromatographic test strip according to claim 13 , wherein at least one of the first antibody and the second antibody is a monoclonal antibody. The immunochromatographic test strip according to claim 14 , wherein the monoclonal antibody does not react with influenza viruses other than influenza virus H5 subtype. The immunochromatographic test strip according to claim 13 , wherein the second antibody is labeled with a metal colloid or latex. The immunochromatographic test strip according to claim 16 , wherein the membrane carrier is a nitrocellulose membrane. A monoclonal antibody that recognizes the epitope shown in SEQ ID NO: 2, which contains the 172nd amino acid of the amino acid sequence of the hemagglutinin protein shown in SEQ ID NO: 1. The monoclonal antibody according to claim 18 , which does not react with influenza viruses other than influenza virus H5 subtype.

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