WO2007021002A1 - Méthode d’immunodétection pour le sous-type h5 du virus de la grippe - Google Patents

Méthode d’immunodétection pour le sous-type h5 du virus de la grippe Download PDF

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Publication number
WO2007021002A1
WO2007021002A1 PCT/JP2006/316243 JP2006316243W WO2007021002A1 WO 2007021002 A1 WO2007021002 A1 WO 2007021002A1 JP 2006316243 W JP2006316243 W JP 2006316243W WO 2007021002 A1 WO2007021002 A1 WO 2007021002A1
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antibody
influenza virus
subtype
monoclonal antibody
amino acid
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PCT/JP2006/316243
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English (en)
Japanese (ja)
Inventor
Hiroshi Kida
Yoshihiro Sakoda
Yasuharu Namba
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Bl Co., Ltd.
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Priority to JP2007531039A priority Critical patent/JP5525688B2/ja
Publication of WO2007021002A1 publication Critical patent/WO2007021002A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • 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.
  • 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.
  • 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).
  • OIE World Veterinary Secretariat
  • 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!
  • 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
  • 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.
  • HA hemadalchun
  • influenza virus H5 subtype can be specifically detected by using sandwich immunoassay, particularly immunochromatography, and have completed the present invention.
  • 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.
  • 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.
  • 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.
  • an immunochromatographic assay characterized in that a complex comprising an H5 subtype and the second antibody is captured at the capture site.
  • 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.
  • the antibody against the influenza virus H5 subtype hemagglutinin protein used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may be a monoclonal antibody. preferable.
  • the first antibody and the second antibody used there may be either a polyclonal antibody or a monoclonal antibody.
  • at least one of the antibodies is preferably a monoclonal antibody, and both antibodies are particularly preferably monoclonal antibodies.
  • 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
  • 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”).
  • first monoclonal antibody is capable of reacting generally against influenza virus H5 attenuated strains A / tn / S.
  • 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).
  • the second monoclonal antibody is clearly distinguished from the first monoclonal antibody in that it reacts strongly with all of the above virulent strains.
  • 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. .
  • 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.
  • influenza virus H5 subtype of hemagglutin protein 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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
  • 5 is a sample adding member.
  • 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.
  • 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.
  • 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.
  • a force using a glass fiber nonwoven fabric of 5 mm X 15 mm as the impregnating member 2 is not limited to this.
  • cellulose cloth filter paper, nitrocellulose membrane, etc.
  • polyethylene polypropylene, etc.
  • Other porous plastic cloths can also be used.
  • 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.
  • 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.
  • coloring labeling substance examples 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.
  • 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.
  • upstream side the left side of FIG. 1, hereinafter referred to as “upstream side”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mixed liquid is subjected to the immunochromatographic test shown in FIG.
  • the mixed solution passes through the sample addition member 5 and mixes with the labeled second antibody in the impregnation member 2.
  • 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.
  • 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.
  • a blood cell capturing membrane member 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.
  • 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.
  • Monoclonal antibodies were produced according to a conventional method.
  • mice 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.
  • 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.
  • a fluorescent antibody method was performed using a cell line derived from a kidney kidney (Madin-Darby canine kidney cell: MDCK cell).
  • MDCK cell a cell line derived from a kidney kidney (Madin-Darby canine kidney cell: MDCK cell).
  • 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.
  • 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 /
  • 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)!
  • 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.
  • 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.
  • Mab indicates the type of clone
  • Isotype indicates the immunoglobulin isotype of the monoclonal antibody
  • 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).
  • 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.
  • 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.
  • Example 2 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.
  • anti-H5 antibodies derived from the 12 clones obtained in (1) above were labeled with colloidal gold according to the following procedure.
  • the protein equivalent weight of the anti-H5 antibody 1 g
  • the protein equivalent weight of the antibody is simply indicated by the weight value of the purified protein by gravimetric analysis
  • lml of the colloidal gold solution of (2) above 1 g
  • 10% ushi serum albumin 10 ushi serum albumin
  • 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.
  • 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.
  • a 5 mm ⁇ 15 mm belt-shaped glass fiber nonwoven fabric was impregnated with 37.5 ⁇ 1 of a colloidal gold labeled antibody solution, and dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 2.
  • colloidal gold labeled antibodies the colloidal gold colloid labeled antibodies listed in Tables 41 and 42 were used.
  • 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.
  • the A / duck / Pennsylvania / 10128/84 (H5N2) strain was diluted with a specimen diluent to adjust to 2.5 ⁇ g / 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.
  • influenza virus H5 subtype can be detected in any combination of Table 41.
  • 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 ⁇ l 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).
  • the specimen dilution solution 1001 was chromatographed in the same manner to observe the degree of coloration. The results are shown in Table 42.
  • Example 2 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.
  • 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 NDV
  • 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.
  • 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.
  • 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
  • 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.
  • 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 ( ⁇ 7 ⁇ 7) (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.
  • this immunochromatography kit showed reactivity to all H5 subtypes and no reactivity to the control H7 subtype.
  • 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)
  • 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).
  • Test samples were prepared by diluting the H5N3) and A / Chicken / 3 ⁇ 4araki / l / 05 (H5N2) virus stock solutions 25, 125, and 625 times with the sample diluent, and subjected to the reaction.
  • Example 7 Immunofection 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.
  • 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,
  • 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.
  • 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.
  • 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.

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Abstract

L’invention concerne une méthode permettant de détecter spécifiquement, rapidement et simplement un sous-type H5 du virus de la grippe, et un appareil de dosage. Un anticorps, en particulier un anticorps monoclonal, réagissant spécifiquement avec une protéine hémagglutinine du sous-type H5 du virus de la grippe a été obtenu. En conséquence, on propose une méthode d'immunodosage qui utilise l'anticorps, en particulier une méthode d'immunodosage sandwich qui utilise un anticorps primaire et un anticorps secondaire contre la protéine hémagglutinine, en particulier une méthode de dosage par immunochromatographie et une bandelette de test pour la méthode par immunochromatographie. Au moins l'un des anticorps primaire et secondaire est de préférence l'anticorps monoclonal.
PCT/JP2006/316243 2005-08-19 2006-08-18 Méthode d’immunodétection pour le sous-type h5 du virus de la grippe WO2007021002A1 (fr)

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WO2009099394A1 (fr) * 2008-02-05 2009-08-13 Temasek Life Sciences Laboratory Limited Protéine de liaison et elisa de blocage d'épitope pour la détection universelle de virus de la grippe de sous-type h5
WO2009119722A1 (fr) * 2008-03-28 2009-10-01 国立大学法人北海道大学 Anticorps monoclonal anti-(hémagglutinine du sous-type h5 du virus de la grippe de type a)
JP2010261912A (ja) * 2009-05-11 2010-11-18 Bl:Kk ヒトインフルエンザウイルスｈ３亜型の免疫学的検出法
US8124092B2 (en) 2007-03-13 2012-02-28 Institute For Research In Biomedicine Antibodies against H5N1 strains of influenza A virus
US8383121B2 (en) 2007-09-13 2013-02-26 Temasek Life Sciences Laboratory Limited Monoclonal antibodies specific to hemagglutinin and neuraminidase from influenza virus H5-subtype or N1-subtype and uses thereof
JP2013087069A (ja) * 2011-10-17 2013-05-13 Toyobo Co Ltd Ｈ５亜型インフルエンザウイルスを特異的に認識するモノクローナル抗体
WO2015186721A1 (fr) * 2014-06-03 2015-12-10 国立研究開発法人農業・食品産業技術総合研究機構 Anticorps ou fragment d'anticorps comprenant une région variable de celui-ci, polypeptide antigénique et utilisations de ceux-ci
JP2018105662A (ja) * 2016-12-24 2018-07-05 株式会社タウンズ インフルエンザウイルスｈ５亜型の免疫検出法

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8124092B2 (en) 2007-03-13 2012-02-28 Institute For Research In Biomedicine Antibodies against H5N1 strains of influenza A virus
US8540996B2 (en) 2007-09-13 2013-09-24 Temasek Life Sciences Laboratory Limited Monoclonal antibodies specific to hemagglutinin and neuraminidase from influenza virus H5-subtype or N1-subtype and uses thereof
US8574581B2 (en) 2007-09-13 2013-11-05 Temasek Life Sciences Laboratory Limited Monoclonal antibodies specific to hemagglutinin and neuraminidase from influenza virus H5-subtype or N1-subtype and uses thereof
US8383121B2 (en) 2007-09-13 2013-02-26 Temasek Life Sciences Laboratory Limited Monoclonal antibodies specific to hemagglutinin and neuraminidase from influenza virus H5-subtype or N1-subtype and uses thereof
AU2008349862B2 (en) * 2008-02-05 2014-02-06 Temasek Life Sciences Laboratory Limited Binding protein and epitope-blocking ELISA for the universal detection of H5-subtype influenza viruses
JP2011514321A (ja) * 2008-02-05 2011-05-06 テマセック・ライフ・サイエンシズ・ラボラトリー・リミテッド Ｈ５亜型インフルエンザウイルスの普遍的な検出のための結合タンパク質およびエピトープブロッキングｅｌｉｓａ
WO2009099394A1 (fr) * 2008-02-05 2009-08-13 Temasek Life Sciences Laboratory Limited Protéine de liaison et elisa de blocage d'épitope pour la détection universelle de virus de la grippe de sous-type h5
US8574830B2 (en) 2008-02-05 2013-11-05 Temasek Life Sciences Laboratory Limited Binding protein and epitope-blocking ELISA for the universal detection of H5-subtype influenza viruses
JPWO2009119722A1 (ja) * 2008-03-28 2011-07-28 国立大学法人北海道大学 抗ｈ５亜型ａ型インフルエンザウイルスヘマグルチニンモノクローナル抗体
WO2009119722A1 (fr) * 2008-03-28 2009-10-01 国立大学法人北海道大学 Anticorps monoclonal anti-(hémagglutinine du sous-type h5 du virus de la grippe de type a)
US8658354B2 (en) 2008-03-28 2014-02-25 National University Corporation Hokkaido University Anti-(influenza a virus subtype H5 hemagglutinin) monoclonal antibody
JP5586064B2 (ja) * 2008-03-28 2014-09-10 国立大学法人北海道大学 抗ｈ５亜型ａ型インフルエンザウイルスヘマグルチニンモノクローナル抗体
JP2010261912A (ja) * 2009-05-11 2010-11-18 Bl:Kk ヒトインフルエンザウイルスｈ３亜型の免疫学的検出法
JP2013087069A (ja) * 2011-10-17 2013-05-13 Toyobo Co Ltd Ｈ５亜型インフルエンザウイルスを特異的に認識するモノクローナル抗体
WO2015186721A1 (fr) * 2014-06-03 2015-12-10 国立研究開発法人農業・食品産業技術総合研究機構 Anticorps ou fragment d'anticorps comprenant une région variable de celui-ci, polypeptide antigénique et utilisations de ceux-ci
JPWO2015186721A1 (ja) * 2014-06-03 2017-04-20 国立研究開発法人農業・食品産業技術総合研究機構 抗体またはその可変領域を含む抗体断片、抗原ポリペプチド、およびその利用
JP2018105662A (ja) * 2016-12-24 2018-07-05 株式会社タウンズ インフルエンザウイルスｈ５亜型の免疫検出法

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