WO2019156248A1 - チクングニアウイルス検出用免疫クロマト分析装置 - Google Patents

チクングニアウイルス検出用免疫クロマト分析装置 Download PDF

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WO2019156248A1
WO2019156248A1 PCT/JP2019/004755 JP2019004755W WO2019156248A1 WO 2019156248 A1 WO2019156248 A1 WO 2019156248A1 JP 2019004755 W JP2019004755 W JP 2019004755W WO 2019156248 A1 WO2019156248 A1 WO 2019156248A1
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amino acid
acid sequence
seq
following
antibody
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French (fr)
Japanese (ja)
Inventor
達雄 塩田
英美 中山
鈴木　啓太
久彦 岩本
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Tanaka Kikinzoku Kogyo KK
University of Osaka NUC
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Tanaka Kikinzoku Kogyo KK
Osaka University NUC
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Priority to US16/968,467 priority Critical patent/US20200399672A1/en
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/181—Alphaviruses or Group A arboviruses, e.g. sindbis, VEE, EEE, WEE or semliki forest virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates to an immunochromatography analyzer, an immunochromatography analysis kit, and an immunochromatography analysis method for detecting chikungunya virus.
  • Chikungunya virus grows in arthropods such as mosquitoes and ticks, and is transmitted to vertebrates by their blood-sucking activity. Infection with chikungunya virus causes high fever called chikungunya fever and severe joint pain. Sometimes serious symptoms may be caused, and establishment of a rapid diagnostic method for early treatment is desired.
  • Chikungunya virus is classified into the Togaviridae alphavirus genus and is a spherical RNA virus.
  • the genotypes are broadly classified, and it is known that there are three genotypes: ECSA type, WA type, and Asian type.
  • ECSA type the 1/3 region on the 3 ′ end side of genomic RNA encodes five structural proteins of CP, E3, E2, 6K, and E1, and the 2/3 region on the 5 ′ end side is It encodes four nonstructural proteins, nsP1, nsP2, nsP3, and nsP4.
  • Non-Patent Documents 1 and 2 disclose methods for detecting ECSA type and Asian type chikungunya viruses by immunochromatography using a mouse monoclonal antibody that recognizes the E1 protein of chikungunya virus.
  • Non-Patent Document 3 discloses CK47, which is a mouse monoclonal antibody that reacts with the E1 protein of chikungunya virus.
  • Patent Document 1 discloses that an anti-chikungunya virus E2 antibody, which is an antibody against the E2 protein of chikungunya virus, is used to form an immune complex, and the presence or absence of chikungunya virus depends on the presence or absence of the immune complex.
  • a method of detecting is disclosed.
  • Non-Patent Document 2 has good sensitivity in ECSA type chikungunya virus, but has low sensitivity in Asian type chikungunya virus.
  • Non-Patent Document 3 the antibody used in the immunochromatography described in Non-Patent Document 1 has a problem that the activity is significantly lowered due to a point mutation of the viral protein.
  • Patent Document 1 discloses an antibody against E2 protein of chikungunya virus.
  • the antibody has reactivity with various genotypes of chikungunya virus. The genotype reactivity and sensitivity are unknown.
  • the E2 protein of chikungunya virus makes neutralizing antibodies by the host (human).
  • the E2 protein of chikungunya virus is the target of an immunological measurement system, competitive inhibition by the host-derived antibody is inhibited. There was a risk of receiving it.
  • the present invention has been made in view of the above-described technical problems, and an object thereof is to detect promptly and with high sensitivity even between chikungunya viruses of different genotypes. Another object of the present invention is to detect Chikungunya virus in which a part of the structural protein is mutated rapidly and with high sensitivity. Furthermore, it aims at suppressing the cross-reaction with other viruses other than chikungunya virus (for example, Sindbis virus, SINV) and specifically detecting chikungunya virus.
  • viruses other than chikungunya virus for example, Sindbis virus, SINV
  • the present inventors have used the above-mentioned problems by combining a specific antibody against chikungunya virus in the labeling substance holding part and the detection part in the immunochromatography analyzer. It was found that the problem can be solved, and the present invention has been achieved.
  • An immunochromatography analyzer for detecting chikungunya virus comprising a sample addition unit, a labeling substance holding unit, a chromatographic medium unit having a detection unit and an absorption unit,
  • the labeling substance holding part contains the following antibody (A) or an antigen-binding fragment thereof:
  • the detection unit contains the following antibody (B) or antigen-binding fragment thereof, and the following (C) antibody or antigen-binding fragment thereof: Immunochromatographic analyzer.
  • CDRH1 is a polypeptide comprising the following amino acid sequence (H1-A):
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-A):
  • CDRH3 is a polypeptide comprising the following amino acid sequence (H3-A) heavy chain variable region (H1-A) (H1-A1), (H1-A2) or (H1-A3) amino acid sequence (H1 -A1) amino acid sequence of SEQ ID NO: 1 (H1-A2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 1 (H1-A3) one or more amino acid sequences of SEQ ID NO: 1 Amino acid sequence in which several amino acids are deleted, substituted, inserted and / or added (H2-A) The following
  • An immunochromatography analyzer for detecting chikungunya virus comprising a sample addition unit, a labeling substance holding unit, a chromatographic medium unit having a detection unit and an absorption unit,
  • the labeling substance holding part contains the following antibody (A) or an antigen-binding fragment thereof:
  • the detection unit contains the following antibody (B) or antigen-binding fragment thereof, and the following (C) antibody or antigen-binding fragment thereof: Immunochromatographic analyzer.
  • A An antibody against chikungunya virus comprising the following heavy chain variable region (1) and the following light chain variable region (2): (1) a heavy chain comprising a polypeptide comprising the following amino acid sequence (HA) Variable region (HA) amino acid sequence of (HA1), (HA2) or (HA3) below (HA1) amino acid sequence of SEQ ID NO: 13 (HA2) amino acid sequence of SEQ ID NO: 13 Amino acid sequence having 80% identity or more (HA3) amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 13 ( 2) Light chain variable region comprising a polypeptide having the amino acid sequence of (LA) below (LA) Amino acid sequence of (LA), (LA2) or (LA3) below (L- A1) SEQ ID NO: 14 Amino acid sequence (LA2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 14 (LA3) The amino acid sequence of SEQ ID NO: 14 lacks one or several amino acids
  • An immunochromatography analyzer for detecting chikungunya virus comprising a sample addition unit, a labeling substance holding unit, a chromatographic medium unit having a detection unit and an absorption unit,
  • the labeling substance holding part contains the following antibody (A) or an antigen-binding fragment thereof:
  • the detection unit contains the following antibody (B) or antigen-binding fragment thereof, and the following (C) antibody or antigen-binding fragment thereof: Immunochromatographic analyzer.
  • A An antibody against chikungunya virus comprising the heavy chain of (1) below and the light chain of (2) below (1)
  • Heavy chain comprising a polypeptide comprising the amino acid sequence of (HA) below (HA) (HA1 below) ), (HA2) or (HA3) amino acid sequence (HA1) amino acid sequence of SEQ ID NO: 17 (HA2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 17 (HA3)
  • a light chain comprising a polypeptide consisting of the amino acid sequence of (1) the following (LA), wherein one or several amino acids are deleted, substituted, inserted and / or added (LA): (LA1), (LA2) or (LA3) amino acid sequence (LA1) amino acid sequence of SEQ ID NO: 18 (LA2) amino acid sequence of SEQ ID NO: 18 Amino acid sequence having 80% or more identity (LA3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 18
  • the immunochromatographic analyzer according to any one of 1 to 3, for detecting an envelope glycoprotein of chikungunya virus among chikungunya viruses. 5. 5. The immunochromatographic analyzer according to 4, wherein the Chikungunya virus envelope glycoprotein is an E1 protein of Chikungunya virus. 6).
  • the antibody (A) or antigen-binding fragment thereof, the antibody (B) or antigen-binding fragment thereof, and the antibody (C) or antigen-binding fragment thereof are at least ECSA type, Asian type, and WA type. 6.
  • the labeling substance holding part further contains the following antibody (D) or an antigen-binding fragment thereof, 7.
  • the immunochromatographic analyzer according to any one of 1 to 6, wherein the detection unit further contains the following antibody (E) or an antigen-binding fragment thereof.
  • D An antibody against chikungunya virus comprising the following heavy chain variable region (1) and light chain variable region (2): (1) comprising heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3
  • CDRH1 is a polypeptide comprising the following amino acid sequence (H1-D):
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-D):
  • CDRH3 is a polypeptide comprising the amino acid sequence of the following (H3-D) heavy chain variable region (H1-D) amino acid sequence of the following (H1-D1), (H1-D2) or (H1-D3) (H1 -D1) amino acid sequence of SEQ ID NO: 21 (H1
  • the labeling substance holding part further contains the following antibody (D) or an antigen-binding fragment thereof, 7.
  • the labeling substance holding part further contains the following antibody (D) or an antigen-binding fragment thereof, 7.
  • the immunochromatographic analyzer according to any one of 1 to 6, wherein the detection unit further contains the following antibody (E) or an antigen-binding fragment thereof.
  • D An antibody against chikungunya virus comprising the following heavy chain of (1) and light chain of (2) below (1) Heavy chain comprising a polypeptide comprising the amino acid sequence of (HD) below (HD) (HD1 below) ), (HD2) or (HD3) amino acid sequence (HD1) amino acid sequence of SEQ ID NO: 29 (HD2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 29 (HD3) SEQ ID NO: 29
  • a light chain comprising a polypeptide consisting of the amino acid sequence of (1) the following (LA), wherein one or several amino acids are deleted, substituted, inserted and / or added (LA): (LA1), (LA2) or (LA3) amino acid sequence (LA1) amino acid sequence of SEQ ID NO
  • the antibody (D) or antigen-binding fragment thereof, and the antibody (E) or antigen-binding fragment thereof are specific to chikungunya virus having at least one genotype of ECSA type, Asian type, and WA type 10.
  • the immunochromatographic analyzer according to any one of 7 to 9, wherein the immunochromatographic analyzer is bound.
  • the content ratio (mass) of the antibody (A) or antigen-binding fragment thereof to the antibody (D) or antigen-binding fragment thereof in the labeling substance holding part is 1: 2 to 2: 1.
  • the immunochromatographic analyzer according to any one of 1 to 11, wherein the sample added to the sample adding unit is any one of whole blood, serum, plasma, semen, and cerebrospinal fluid of a chikungunya virus infected person. 13.
  • An immunochromatography analysis kit comprising the immunochromatography analyzer according to any one of 1 to 12 above and a sample diluent for diluting and developing a sample. 14 14.
  • a step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to the sample addition unit (2) The chikungunya virus by the antibody or antigen-binding fragment thereof (A) held in the labeling substance holding unit (3) Step of developing the sample and the antibody or antigen-binding fragment thereof (A) in the chromatographic medium part as a mobile phase (4) Chikungunya virus in the developed mobile phase in the detection part The step of detecting with the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof (C)
  • various chikungunya viruses with different genotypes and chikungunya viruses in which a part of the structural protein is mutated can be detected quickly and with high sensitivity.
  • cross-reactivity with other viruses other than chikungunya virus for example, Sindbis virus
  • chikungunya virus can be specifically detected.
  • FIG. 1 is a cross-sectional view for explaining the structure of an immunochromatographic analyzer according to an embodiment of the present invention.
  • FIG. 2 is a graph showing the results of measurement of chikungunya virus (ECSA type, Asian type) using the immunochromatographic analyzers of Examples and Comparative Examples of the present invention.
  • FIG. 3 is a graph showing the results of measurement of chikungunya virus (WA type) using the immunochromatography analyzers of Examples and Comparative Examples of the present invention.
  • FIG. 4 is a graph showing the results of measurement of wild-type (WT) and mutant-type (MT) chikungunya viruses (ECSA type) using the immunochromatography analyzers of Examples and Comparative Examples of the present invention.
  • FIG. 5 is a graph showing the results of measurement of wild-type (WT) and mutant-type (MT) chikungunya viruses (Asian type) using the immunochromatographic analyzers of Examples and Comparative Examples of the present invention.
  • the labeling substance holding unit contains the antibody (A) or antigen-binding fragment thereof
  • the detection unit is the antibody (B) or antigen-binding fragment thereof
  • It contains the antibody (C) or antigen-binding fragment thereof.
  • the immunochromatographic analyzer according to the present invention has the specific combination of antibodies or antigen-binding fragments thereof in the labeling substance holding part and the detection part, so that various chikungunya viruses having different genotypes and part of the structural protein Even if chikungunya virus is mutated, it can be detected quickly and with high sensitivity, and the cross-reactivity with other viruses other than chikungunya virus (for example, Sindbis virus) is low. It can be specifically detected.
  • specimen specimen or simply specimen The origin of the specimen (hereinafter sometimes referred to as specimen specimen or simply specimen) applicable to the immunochromatography analyzer according to the present invention is not particularly limited.
  • examples thereof include humans and non-human animals other than humans.
  • the non-human animal include mammals such as mice, rats, dogs, monkeys, rabbits, sheep, and horses, and arthropods such as Aedes aegypti and Aedes albopictus as vectors.
  • the type of the specimen is not particularly limited, and examples thereof include biological samples such as body fluid, urine, body fluid-derived cells, organs, tissues or cells separated from the living body.
  • the body fluid include body cavity fluid such as blood and synovial fluid, lymph fluid, tissue fluid, and the like, and specific examples include whole blood, serum, plasma, semen, spinal fluid, and the like.
  • the biological sample is preferably whole blood, serum, plasma, semen, or cerebrospinal fluid, and more preferably serum or plasma.
  • the collected sample may be used as it is in the present invention, or may be used after performing other treatment such as dilution with a liquid or the like.
  • the liquid is not particularly limited, and examples thereof include water, physiological saline, buffer solution, medium, and the like.
  • the sample may be acid-treated in advance, for example. Thereby, for example, when the Chikungunya virus and the antibody in the sample form an antigen-antibody complex, the antibody or the like and the Chikungunya virus can be dissociated, so that the Chikungunya virus can be detected with higher sensitivity.
  • the acid used for the acid treatment is not particularly limited, and examples thereof include hydrochloric acid.
  • the labeling substance holding unit contains the antibody (A) or antigen-binding fragment thereof
  • the detection unit is the antibody (B) or antigen-binding fragment thereof
  • It contains the antibody (C) or antigen-binding fragment thereof.
  • the antibody or the like or an antigen-binding fragment thereof (hereinafter also referred to as an antibody or the like) is specific for chikungunya virus having at least one genotype of ESCA type CHIKV, WA type CHIKV, and Asian type CHIKV.
  • the ESCA-type CHIKV, the WA-type CHIKV, and the Asian-type CHIKV can be classified with reference to Reference Document 1 described later, for example.
  • the antibody or the like binds to, for example, an ESCA-type CHIKV envelope glycoprotein (hereinafter also referred to as “Env protein”), a WA-type CHIKV Env protein, and an Asian-type CHIKV Env protein.
  • the Env protein means, for example, a CHIKV envelope protein.
  • the Env protein is also referred to as an E3-E2-6K-E1 protein because it is composed of, for example, a 6K-E1 protein, an E2 protein, and an E3 protein.
  • information registered in an existing database can be referred to.
  • the ESCA-type CHIKV-derived Env protein includes, for example, the following amino acid sequence (SEQ ID NO: 31) from the 268th to the 1247th in the amino acid sequence registered under NCBI accession number BAP74220.1.
  • Examples of the WA type CHIKV-derived Env protein include the following amino acid sequence (SEQ ID NO: 32) from the 268th to the 1247th in the amino acid sequence registered under NCBI accession number AAU43881.1.
  • Asian type CHIKV-derived Env protein includes, for example, the following amino acid sequence from the 268th to the 1247th amino acid sequence (SEQ ID NO: 33) in the amino acid sequence registered under NCBI accession number ADG95938.1.
  • Reference 1 Aekkacha Tuekprakhhon et. al. , “Variation at position 350 in the Chikungunya virus 6K-E1 protein determines the sensitivity of detection in a rapid E1-antigen test”, Scientific. 8, Article Number: 1094, 2018
  • ESCA-type CHIKV-derived Env protein (SEQ ID NO: 31) MCLLANTTFPCSQPPCTPCCYEKEPEETLRMLEDNVMRPGYYQLLQASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDRTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRQGKIHIPFPLANVTCRVPKARNPT TYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHE
  • WA type CHIKV-derived Env protein (SEQ ID NO: 32) LCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVMRPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPT TYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYEL
  • Asian type CHIKV-derived Env protein SEQ ID NO: 33
  • the antibody or the like includes CHIKV, more specifically, in addition to an antibody that binds to a protein consisting of the full-length amino acid sequence of the Env protein, for example, the meaning of an antibody that binds to a peptide fragment of the Env protein.
  • the Env protein includes, for example, an amino acid corresponding to the 826th amino acid in the amino acid sequence of SEQ ID NO: 31 (284th in E1 protein and 350th in 6K-E1 protein) (in SEQ ID NO: 31, the glutamic acid indicated by underlining).
  • E contains a mutated Env protein (E350D) substituted with aspartic acid (D).
  • the Env protein is, for example, an amino acid corresponding to the 826th amino acid in the amino acid sequence of SEQ ID NO: 32 or 33 (284th position of E1 protein, 350th position of 6K-E1 protein) (in SEQ ID NO: 32 or 33,
  • the underlined aspartic acid (D)) contains a mutated Env protein (D350E) substituted with glutamic acid (E).
  • D350E mutated Env protein substituted with glutamic acid
  • the Env protein includes, for example, the meaning of these peptide fragments in addition to the Env protein, the mutant Env protein (E350D), or the mutant Env protein (D350E) consisting of the full-length amino acid sequence.
  • the antibody or the like may be, for example, a so-called “antibody” whose molecular structure is immunoglobulin, or an antigen-binding fragment thereof.
  • the antibody or the like may have the aforementioned heavy chain variable region and light chain variable region.
  • its immunoglobulin class and isotype are not particularly limited.
  • the immunoglobulin class include IgG, IgM, IgA, IgD, IgE and the like.
  • Examples of the IgG include IgG1, IgG2, IgG3, and IgG4.
  • the antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human (eg, fully human) antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody.
  • the “antigen-binding fragment” in the present invention means a part of the antibody, for example, a partial fragment that recognizes (binds) the chikungunya virus.
  • the antigen-binding fragment include Fab, Fab ′, F (ab ′) 2 , variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), bispecific antibody, and polymers thereof. Can be given.
  • the antibody or the like may have, for example, a constant region in addition to the aforementioned heavy chain variable region and light chain variable region, and the constant region is, for example, a human constant region or a mouse constant region.
  • the heavy chain constant region includes, for example, the CH1, CH2, and CH3 regions
  • the light chain constant region includes, for example, the CL region.
  • a heavy chain and a light chain of an antibody molecule each have three complementarity determining regions (CDRs).
  • CDR is also referred to as a hypervariable domain.
  • the CDR is a region having a particularly high primary structure variability even in the variable region of the heavy chain and light chain, and is usually separated into three locations on the primary structure.
  • the three CDRs in the heavy chain are represented as heavy chain CDR1 (CDRH1), heavy chain CDR2 (CDRH2), and heavy chain CDR3 (CDRH3) from the amino terminal side in the heavy chain amino acid sequence.
  • the three CDRs in the chain are represented as light chain CDR1 (CDRL1), light chain CDR2 (CDRL2), and light chain CDR3 (CDRL3) from the amino terminal side in the amino acid sequence of the light chain. These sites are close to each other on the steric structure and determine the specificity for the antigen to bind.
  • the antibody of (A) is an antibody against chikungunya virus, comprising the following heavy chain variable region (1) and light chain variable region (2) below.
  • CDRH heavy chain complementarity determining region
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-A):
  • CDRH3 is a polypeptide comprising the following amino acid sequence (H3-A) heavy chain variable region (H1-A) (H1-A1), (H1-A2) or (H1-A3) amino acid sequence (H1 -A1) amino acid sequence of SEQ ID NO: 1 (H1-A2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 1 (H1-A3) one or more amino acid sequences of SEQ ID NO: 1 Amino acid sequence in which several amino acids are deleted, substituted, inserted and / or added (H2-A) The following amino acid sequence (H2-A1), (H2-A2) or (H2-A3) (H2-A1) The amino acid sequence of
  • identity is, for example, the degree of identity when the sequences to be compared are appropriately aligned, and means the appearance rate (%) of an exact match of amino acids between the sequences.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • the identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • “one or several” related to substitution or the like is, for example, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the amino acid substitution may be, for example, a conservative substitution (hereinafter the same).
  • Said conservative substitution means substitution of one or several amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein.
  • the “substituted amino acid” and the “substituted amino acid” have, for example, similar properties and / or functions.
  • chemical properties such as hydrophobicity and hydrophilicity index (hydropathy), polarity, charge, etc., or physical properties such as secondary structure are similar.
  • Amino acids or amino acid derivatives having similar properties and / or functions are known in the art, for example.
  • nonpolar amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • Polar amino acids neutral amino acids
  • neutral amino acids include glycine, serine, and threonine.
  • positively charged amino acids basic amino
  • negatively charged amino acids include aspartic acid, And glutamic acid.
  • the antibody of (A) is an antibody against chikungunya virus, comprising the following heavy chain variable region (1) and light chain variable region (2) below.
  • Heavy chain variable region comprising a polypeptide having the following amino acid sequence (HA) (HA) Amino acid sequence (H-A1), (H-A2) or (H-A3) -A1) amino acid sequence of SEQ ID NO: 13 (H-A2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 13 (H-A3) in the amino acid sequence of SEQ ID NO: 13, one or A light chain variable region comprising a polypeptide consisting of the amino acid sequence (2) below (LA) in which several amino acids are deleted, substituted, inserted and / or added (LA) below (L- A1), the amino acid sequence of (L-A2) or (L-A3) (L-A1) the amino acid sequence of SEQ ID NO: 14 (L-A2) 80% or more identity to the amino acid sequence of SEQ ID NO: 14
  • LA amino acid sequence
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” relating to substitution and the like is, for example, 1 to 20, 1 to 15, 1 to 10, respectively. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (A) is an antibody against chikungunya virus, comprising a heavy chain of (1) below and a light chain of (2) below.
  • Heavy chain comprising a polypeptide consisting of the following amino acid sequence (HA) (HA) Amino acid sequence of (HA1), (HA2) or (HA3) below (HA1) Amino acid sequence of SEQ ID NO: 17 (HA2)
  • Light chain comprising a polypeptide consisting of the following amino acid sequence (LA) (LA) Amino acid sequence (LA1), (LA2) or (LA3) below (LA1) Amino acid sequence of SEQ ID NO: 18 (LA2) sequence An amino acid sequence having 80% or more identity to the amino acid sequence of No. 18 (LA3) Te, one
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” related to substitution and the like are, for example, 1 to 80, 1 to 60, 1 to 40, and 1 to 30, respectively. 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 One, two, or one.
  • the antibody of (B) is an antibody against chikungunya virus, comprising the heavy chain variable region of (3) below and the light chain variable region having each CDR of (2) above. .
  • CDRH heavy chain complementarity determining region
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-B)
  • CDRH3 is a polypeptide comprising the amino acid sequence of the following (H3-B) heavy chain variable region (H1-B) amino acid sequence of the following (H1-B1), (H1-B2) or (H1-B3)
  • H1 -B1 amino acid sequence of SEQ ID NO: 7 (H1-B2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 7 (H1-B3) one or more amino acid sequences of SEQ ID NO: 7 or Amino acid sequence in which several amino acids are deleted, substituted, inserted and / or added
  • H2-B The following (H2-B1), (H2-B2) or (H2-B3) amino acid sequence (H2-B)
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • “one or several” related to substitution or the like is, for example, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (B) is an antibody against chikungunya virus comprising the following heavy chain variable region (3) and light chain variable region (2).
  • (3) Heavy chain variable region comprising a polypeptide consisting of the following amino acid sequence (HB) (HB) The following (H-B1), (H-B2) or (H-B3) amino acid sequence (H -B1) amino acid sequence of SEQ ID NO: 15 (H-B2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 15 (H-B3) one amino acid sequence in the amino acid sequence of SEQ ID NO: 15 or Amino acid sequences with several amino acids deleted, substituted, inserted and / or added
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” relating to substitution and the like is, for example, 1 to 20, 1 to 15, 1 to 10, respectively. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (B) is an antibody against chikungunya virus, comprising the heavy chain of (3) below and the light chain of (2) above.
  • (3) Heavy chain comprising a polypeptide consisting of the amino acid sequence of (HB) below (HB) Amino acid sequence of (HB1), (HB2) or (HB3) below (HB1) Amino acid sequence of SEQ ID NO: 19 (HB2) SEQ ID NO: Amino acid sequence having 80% or more identity with respect to 19 amino acid sequences (HB3) Amino acid in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 19 Array
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” related to substitution and the like are, for example, 1 to 80, 1 to 60, 1 to 40, and 1 to 30, respectively. 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 One, two, or one.
  • the antibody of (C) is an antibody against chikungunya virus, comprising the heavy chain variable region of (4) below and the light chain variable region having each CDR of (2) above. .
  • CDRH1 is a polypeptide comprising the following amino acid sequence (H1-C):
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-C):
  • CDRH3 is a polypeptide comprising the amino acid sequence of the following (H3-C) heavy chain variable region (H1-C) amino acid sequence of the following (H1-C1), (H1-C2) or (H1-C3)
  • H2-C Amino acid sequence of (H2-C1), (H2-C2) or (H2-C3) below (H2-C1) Amino acid sequence of SEQ ID NO:
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • “one or several” related to substitution or the like is, for example, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (C) is an antibody against chikungunya virus comprising the following heavy chain variable region (4) and light chain variable region (2).
  • Heavy chain variable region comprising a polypeptide consisting of the following amino acid sequence (HC) (HC) Amino acid sequence (H-C1), (H-C2) or (H-C3) -C1) amino acid sequence of SEQ ID NO: 16 (H-C2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 16 (H-C3) one or more in the amino acid sequence of SEQ ID NO: 16 or Amino acid sequences with several amino acids deleted, substituted, inserted and / or added
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” relating to substitution and the like is, for example, 1 to 20, 1 to 15, 1 to 10, respectively. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (C) is an antibody against chikungunya virus, comprising the following heavy chain (4) and light chain (2).
  • Heavy chain comprising a polypeptide consisting of the following amino acid sequence (HC) (HC) The following (HC1), (HC2) or (HC3) amino acid sequence (HC1) SEQ ID NO: 20 amino acid sequence (HC2) SEQ ID NO: Amino acid sequence having 80% or more identity with respect to 20 amino acid sequences (HC3) Amino acid in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 20 Array
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” related to substitution and the like are, for example, 1 to 80, 1 to 60, 1 to 40, and 1 to 30, respectively. 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 One, two, or one.
  • the antibody (A) or antigen-binding fragment thereof, the antibody (B) or antigen-binding fragment thereof, and the antibody (C) or antigen-binding fragment thereof are at least ECSA type, Asian type, and WA type. It specifically binds to chikungunya virus having any one genotype.
  • the antibody of (D) is an antibody against chikungunya virus, comprising the following heavy chain variable region (1) and light chain variable region (2) below.
  • CDRH1 is a polypeptide comprising the following amino acid sequence (H1-D):
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-D):
  • CDRH3 is a polypeptide comprising the amino acid sequence of the following (H3-D) heavy chain variable region (H1-D) amino acid sequence of the following (H1-D1), (H1-D2) or (H1-D3) (H1 -D1) amino acid sequence of SEQ ID NO: 21 (H1-D2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 21 (H1-D3) in the amino acid sequence of SEQ ID NO: 21, or Amino acid sequence in which several amino acids are deleted, substituted, inserted and / or added (H2-D) Amino acid sequence of (H2-D1), (H2-D2) or (H2-D3) below (H2-D)
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • “one or several” related to substitution or the like is, for example, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (D) is an antibody against chikungunya virus, comprising the following heavy chain variable region (1) and light chain variable region (2) below.
  • Heavy chain variable region comprising a polypeptide comprising the amino acid sequence of (HD) below (HD) Amino acid sequence of (HD1), (HD2) or (HD3) below (H -D1) amino acid sequence of SEQ ID NO: 27 (H-D2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 27 (H-D3) one or more in the amino acid sequence of SEQ ID NO: 27
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence of (2) the following (LD) with the amino acid sequence in which several amino acids have been deleted, substituted, inserted and / or added (LD) D1), (L-D2) or (L-D3) amino acid sequence (L-D1) amino acid sequence of SEQ ID NO: 14 (L-D2) 80% or more identity to the amino acid sequence of SEQ ID NO:
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” relating to substitution and the like is, for example, 1 to 20, 1 to 15, 1 to 10, respectively. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (D) is an antibody against chikungunya virus, comprising the following heavy chain of (1) and light chain of (2) below.
  • Heavy chain comprising a polypeptide comprising the following amino acid sequence (HD) (HD) Amino acid sequence (HD1), (HD2) or (HD3) below (HD1) Amino acid sequence of SEQ ID NO: 29 (HD2) SEQ ID NO: Amino acid sequence having 80% or more identity with respect to 29 amino acid sequences (HD3) Amino acid in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 29 Sequence (2) A light chain comprising a polypeptide consisting of the following amino acid sequence (LD) (LD) The following (LD1), (LD2) or (LD3) amino acid sequence (LD1) SEQ ID NO: 18 amino acid sequence (LD2) sequence An amino acid sequence having 80% or more identity to the amino acid sequence of No. 18 (LD3) Te, one or several amino acids are deleted, substitute
  • LD3 amino
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” related to substitution and the like are, for example, 1 to 80, 1 to 60, 1 to 40, and 1 to 30, respectively. 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 One, two, or one.
  • the antibody of (E) is an antibody against chikungunya virus, comprising the heavy chain variable region of (3) below and the light chain variable region having each CDR of (2) above. .
  • CDRH heavy chain complementarity determining region
  • CDRH2 is a polypeptide comprising the following amino acid sequence (H2-E)
  • CDRH3 is a polypeptide comprising the amino acid sequence of the following (H3-E) heavy chain variable region (H1-E) amino acid sequence of the following (H1-E1), (H1-E2) or (H1-E3)
  • H1 -E1 amino acid sequence of SEQ ID NO: 24 (H1-E2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 24 (H1-E3) one or more in the amino acid sequence of SEQ ID NO: 24 or Amino acid sequence in which several amino acids are deleted, substituted, inserted and / or added
  • H2-E Amino acid sequence of (H2-E1), (H2-E2) or (H2-E3) below
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • “one or several” related to substitution or the like is, for example, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (E) is an antibody against chikungunya virus comprising the following heavy chain variable region (3) and light chain variable region (2).
  • Heavy chain variable region comprising a polypeptide comprising the amino acid sequence of (HE) below (HE) Amino acid sequence of (H-E1), (H-E2) or (H-E3) below (H -E1) amino acid sequence of SEQ ID NO: 28 (H-E2) amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 28 (H-E3) one amino acid sequence in the amino acid sequence of SEQ ID NO: 28 or Amino acid sequences with several amino acids deleted, substituted, inserted and / or added
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” relating to substitution and the like is, for example, 1 to 20, 1 to 15, 1 to 10, respectively. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the antibody of (E) is an antibody against chikungunya virus, comprising the heavy chain of (3) below and the light chain of (2) above.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • “one or several” related to substitution and the like are, for example, 1 to 80, 1 to 60, 1 to 40, and 1 to 30, respectively. 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 One, two, or one.
  • the antibody (D) or antigen-binding fragment thereof, and the antibody (E) or antigen-binding fragment thereof are specific for chikungunya virus having at least one genotype of ECSA type, Asian type, and WA type Join.
  • amino acid sequences of SEQ ID NOs: 1 to 30 are, for example, mouse-derived amino acid sequences.
  • the method for producing the antibody or the like is not particularly limited, and can be produced, for example, by genetic engineering based on the amino acid sequence information described above. Specifically, for example, it can be performed as follows. In addition, this invention is not limited to this illustration.
  • a vector containing a nucleic acid sequence encoding the amino acid sequence of each region, heavy chain, and / or light chain in the antibody or the like is introduced into a host to obtain a transformant. Then, the transformant is cultured, and a fraction containing an antibody that binds to the Chikungunya virus, specifically, the Env protein, is collected, and the antibody is isolated or purified from the obtained collected fraction.
  • the vector examples include a vector containing a nucleic acid sequence encoding the heavy chain variable region, a vector containing a nucleic acid sequence encoding the light chain variable region, a vector containing a nucleic acid sequence encoding the heavy chain, and the light chain. And a vector containing a nucleic acid sequence encoding
  • the host is not particularly limited as long as it can introduce the vector and express the nucleic acid sequence in the vector.
  • Examples of the host include mammalian cells such as HEK cells, CHO cells, COS cells, NS0 cells, and SP2 / 0 cells.
  • the method for introducing the vector into the host is not particularly limited, and a known method can be adopted.
  • the culture method of the transformant is not particularly limited, and can be appropriately determined according to the type of the host.
  • the fraction containing the antibody can be collected, for example, by crushing the cultured transformant and collecting it as a liquid fraction.
  • the isolation or purification of the antibody is not particularly limited, and a known method can be adopted.
  • the antibody is, for example, a monoclonal antibody.
  • the monoclonal antibody include monoclonal antibodies obtained by immunization of animals, chimeric antibodies, humanized antibodies, human antibodies (also referred to as fully human antibodies), and the like.
  • the chimeric antibody is an antibody in which a variable region of an antibody derived from an animal other than a human and a constant region of a human antibody are linked.
  • the chimeric antibody can be produced, for example, as follows. First, for a monoclonal antibody derived from a non-human animal, a variable region (V region) gene that binds to Chikungunya virus, specifically, the Env protein, is prepared, and the variable region gene and human antibody constant region ( C region) gene, and this is further linked to an expression vector. Then, the cells transfected with the expression vector are cultured, and the target chimeric antibody secreted into the culture medium is recovered. Thereby, a chimeric antibody can be prepared.
  • V region variable region
  • C region human antibody constant region
  • the animal from which the variable region gene is derived is not particularly limited, and examples thereof include rats and mice.
  • the method for producing the chimeric antibody is not limited thereto, and can be produced by referring to a known method such as the method described in Japanese Patent Publication No. 3-73280.
  • the humanized antibody is an antibody in which only the CDR is derived from a non-human animal and the other region is derived from a human.
  • the humanized antibody can be produced, for example, as follows. First, the CDR gene of a monoclonal antibody derived from a non-human animal is prepared, transplanted to a human antibody gene, for example, a constant region (CDR grafting), and further linked to an expression vector. Then, by culturing the cells transfected with the expression vector, the humanized antibody in which the target CDR is transplanted is secreted into the culture medium. It can be prepared by recovering the secreted humanized antibody.
  • the CDR-derived animal is not particularly limited, and examples thereof include rats and mice.
  • the method for producing a humanized antibody is not limited to this, and for example, a known method such as the method described in Japanese Patent Publication No. 4-506458 and Japanese Patent Application Laid-Open No. Sho 62-296890 can be used. Can be manufactured by reference.
  • the human antibody is a human-derived antibody in all regions.
  • the human antibody can be prepared, for example, by introducing a human antibody gene into a non-human animal.
  • a transgenic animal for human antibody production can be used as the animal into which the human antibody gene is introduced.
  • the kind of the animal is not particularly limited, and examples thereof include mice.
  • the method for producing the human antibody is described in, for example, Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; Japanese National Publication No. 4-504365; Japanese National Publication No. 7-509137; International Publication No. 1994/25585; Nature, Vol. 368, p. 856-859, 1994; and Japanese National Publication No.
  • the human antibody can also be produced, for example, using a phage display method, for example, see Marks, J. et al. D. et al. : J. Mol. Biol. , Vol. 222, p. 581-597, 1991 etc., and can be produced with reference to known methods.
  • the antibody or the like can be prepared, for example, by immunizing an animal with an antigen.
  • the antigen include chikungunya virus, specifically, a protein consisting of the full-length amino acid sequence of Env protein or a peptide fragment thereof.
  • the antigen is preferably immunized multiple times.
  • the antigen to be immunized each time is preferably, for example, Chikungunya virus or Env protein having a different genotype, or a peptide fragment thereof.
  • the peptide fragment may be, for example, a peptide fragment consisting only of an antigenic determinant (epitope) or a peptide fragment containing the antigenic determinant.
  • Monoclonal antibodies obtained by immunization of the animals include, for example, Current Protocols in Molecular Biology (John Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. It can be produced by referring to known methods such as those described in Harlow and David Lane, Cold Spring Harbor Laboratory (1988).
  • an animal is immunized with an antigen, and an antibody-producing cell collected from the immunized animal is fused with a myeloma cell (myeloma cell) lacking autoantibody-producing ability to produce a hybridoma.
  • a myeloma cell myeloma cell
  • antibody-producing cells are screened from the hybridoma, and a single hybridoma clone is prepared by cloning.
  • this hybridoma clone is administered to an animal, and the monoclonal antibody is purified from the obtained abdominal cavity.
  • the hybridoma is cultured, and the monoclonal antibody is purified from the culture solution.
  • the myeloma cell is preferably derived from, for example, mouse, rat, human or the like.
  • the myeloma cell and the antibody-producing cell may be derived from the same or different species, but are preferably the same species.
  • immunochromatography analyzer according to the invention
  • “fixed” means that the antibody or the like is arranged on a carrier such as a membrane so that the antibody or the like does not move
  • “hold” means that the antibody or the like is in the surface of the carrier such as a membrane. It means that it is arranged to be movable.
  • the sample addition unit (1) is a part to which a specimen sample is added in the immunochromatography analyzer.
  • the sample addition section (1) can be formed of a porous sheet that absorbs the specimen sample quickly but has a weak holding power and moves the specimen sample quickly.
  • the porous sheet include cellulose filter paper, glass fiber filter paper, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth.
  • a buffer solution a salt such as sodium caseinate, a nonionic surfactant, a cationic surfactant, an anion Surfactants such as surfactants, polymer compounds such as polyvinyl pyrrolidone (PVP), polyanions, nitrogen atom-containing vinyl-based water-soluble polymers, antibacterial agents, chelating agents, and the like can be contained. These may contain 1 type (s) or 2 or more types.
  • a surfactant having an HLB value of 13 to 17 can be preferably used.
  • Triton X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether, HLB: 13.7)
  • Tween 20 (trade name, polyoxyethylene sorbitan monolaurate, HLB: 16.7)
  • Tween 80 (Pro names, polyoxyethylene sorbitan monooleate, HLB: 15.0), Brij35 (polyoxyethylene glycol dodecyl ether, HLB: 13.7) and the like may be used, or one or more may be added.
  • the content of the surfactant per unit area of the specimen application portion (1) is usually 0.25 ⁇ g / cm 2 ⁇ 8 ⁇ g / cm 2, preferably 0.5 ⁇ g / cm 2 ⁇ 5 ⁇ g / cm 2, More preferably, it is 1 ⁇ g / cm 2 to 4 ⁇ g / cm 2 . By being the said range, promotion of an antigen antibody reaction or a non-specific reaction can be suppressed.
  • examples of the nitrogen atom-containing vinyl water-soluble polymer include polyvinyl pyrrolidone (PVP).
  • PVP polyvinyl pyrrolidone
  • the content of the nitrogen atom-containing vinyl-based water-soluble polymer per unit area in the sample addition part (1) is usually 0.05 ⁇ g / cm 2 to 0.5 ⁇ g / cm 2 , preferably 0.1 ⁇ g / cm 2. a ⁇ 0.5 ⁇ g / cm 2, more preferably 0.2 ⁇ g / cm 2 ⁇ 0.4 ⁇ g / cm 2.
  • the labeling substance holding part (2) contains a labeling substance to be described later, and the labeling substance is held in the labeling substance holding part (2) as a labeled antibody or the like combined with an antibody or the like.
  • the type of antibody or the like that binds to the labeling substance is not limited to one type, but includes at least the antibody (A) or an antigen-binding fragment thereof.
  • the labeled antibody or the like held in the labeling substance holding part is bound to chikungunya virus in the specimen.
  • a glass fiber or cellulose film is usually used for the labeling substance holding part (2).
  • the content of the antibody (A) or antigen-binding fragment thereof in the labeling substance holding part is usually 0.01 ⁇ g to 1 ⁇ g, preferably 0.075 ⁇ g to 0.75 ⁇ g, more preferably 0.1 ⁇ g to 0.5 ⁇ g.
  • the content of the antibody (A) or antigen-binding fragment thereof per unit area of the labeling substance holding part is usually 0.01 ⁇ g / cm 2 to 1.8 ⁇ g / cm 2 , preferably 0.13 ⁇ g / cm 2. a cm 2 ⁇ 1.4 ⁇ g / cm 2, more preferably 0.15 ⁇ g / cm 2 ⁇ 1 ⁇ g / cm 2.
  • the antibody that binds to the labeling substance preferably includes the antibody (D) or an antigen-binding fragment thereof.
  • the content of the antibody (D) or antigen-binding fragment thereof in the labeling substance holding part is usually 0.05 ⁇ g to 1 ⁇ g, preferably 0.075 ⁇ g to 0.75 ⁇ g, more preferably 0 .1 ⁇ g to 0.5 ⁇ g.
  • the content of the antibody (D) or antigen-binding fragment thereof per unit area of the labeling substance holding part is usually 0.01 ⁇ g / cm 2 to 1.8 ⁇ g / cm 2 , preferably 0.13 ⁇ g / cm 2. a cm 2 ⁇ 1.4 ⁇ g / cm 2, more preferably 0.15 ⁇ g / cm 2 ⁇ 1 ⁇ g / cm 2.
  • the content of the antibody (A) or antigen-binding fragment thereof and the antibody (D) or antigen-binding fragment thereof (D) in the labeling substance holding part should be 1: 2 to 2: 1 by mass ratio. Is preferable, and 1: 1 is more preferable.
  • an enzyme or the like is generally used, but it is preferable to use an insoluble carrier as the labeling substance because it is suitable for visually determining the presence of the substance to be detected.
  • an insoluble carrier as a labeling substance, metal particles such as gold, silver or platinum, metal oxide particles such as iron oxide, non-metallic particles such as sulfur and latex particles made of synthetic polymer, or other insoluble carriers are used. be able to.
  • the insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored to facilitate visual determination.
  • the metal particles and the metal oxide particles themselves exhibit a specific natural color corresponding to the particle diameter, and the color can be used as a label.
  • An insoluble carrier as a labeling substance is particularly preferable because gold particles are easy to detect and are difficult to agglomerate and non-specific color development is unlikely to occur.
  • the average particle size of the gold particles is, for example, 10 nm to 250 nm, preferably 35 nm to 120 nm, and more preferably 40 nm to 80 nm.
  • TEM transmission electron microscope
  • JEM-2010 was used to randomly measure the projected area equivalent circle diameter of 100 particles using a photograph taken. It can be calculated from the average value.
  • the content of gold particles in the labeling substance holding section, per unit area of the labeling substance holding portion is usually 0.006 ⁇ g / cm 2 ⁇ 0.42 ⁇ g / cm 2, preferably 0.01 ⁇ g / cm 2 ⁇ 0.3 ⁇ g / Cm 2 , more preferably 0.01 ⁇ g / cm 2 to 0.2 ⁇ g / cm 2 .
  • the labeled particles are developed in a dispersed state, and recognition sites such as antibodies can be increased without increasing the sensitivity.
  • the chromatographic medium part (3) is a developed part of the chromatograph.
  • the chromatographic medium part (3) is an inert film made of a microporous material that exhibits capillary action.
  • the chromatographic medium part (3) includes, for example, a membrane made of nitrocellulose, A membrane made of cellulose acetate can be preferably used. A membrane made of nitrocellulose is particularly preferable. Cellulose membranes, nylon membranes, and porous plastic cloths such as polyethylene and polypropylene can also be used.
  • the membrane made of nitrocellulose only needs to contain nitrocellulose as a main component, and a membrane mainly composed of nitrocellulose such as a pure product or a mixed product of nitrocellulose can be used.
  • the membrane made of nitrocellulose can further contain a substance that promotes capillary action.
  • a substance that lowers the surface tension of the film surface and brings about hydrophilicity is preferable.
  • a substance having an amphipathic action such as saccharides, amino acid derivatives, fatty acid esters, various synthetic surfactants or alcohols, which does not affect the movement of the detected substance and does not affect the coloring of the labeling substance. No material is preferred.
  • Nitrocellulose membrane is porous and exhibits capillary action.
  • the index of the capillary phenomenon can be confirmed by measuring the water absorption rate (water absorption time: capillary flow time).
  • the water absorption rate affects detection sensitivity and inspection time.
  • the form and size of the chromatographic medium part (3) typified by the nitrocellulose membrane and cellulose acetate membrane as described above are not particularly limited, and actual operation points and observation of reaction results are not limited. In this respect, it may be appropriate.
  • the support made of plastic or the like on the back surface of the chromatographic medium part (3).
  • the properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is preferably colorless or white.
  • the chromatographic medium part (3) is blocked by a known method as necessary. Processing can be performed. Generally, proteins such as bovine serum albumin, skim milk, casein, and gelatin are preferably used for the blocking treatment. After such blocking treatment, if necessary, for example, one or a combination of two or more surfactants such as Tween 20, Triton X-100, and SDS may be washed.
  • a surfactant such as Tween 20, Triton X-100, and SDS may be washed.
  • the detection part (4) is formed at an arbitrary position on the chromatographic medium part (3), and is at least the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof. Is included. Immobilization of the antibody to the detection unit (4) can be performed according to a conventional method.
  • chikungunya virus in the specimen that has passed through the chromatographic medium unit as a mobile phase is sandwiched between the antibody immobilized on the detection unit (4), the labeled antibody, and the like. Specific reaction binding.
  • the content of the antibody (B) or antigen-binding fragment thereof contained in the detection part (4) is usually 0.05 ⁇ g to 1 ⁇ g, preferably 0.2 ⁇ g to 0.7 ⁇ g, more preferably 0.2 ⁇ g to 0.6 ⁇ g.
  • the content of the antibody (B) or antigen-binding fragment thereof per unit area of the detection unit (4) is usually 0.05 ⁇ g / cm 2 to 2 ⁇ g / cm 2 , preferably 0.1 ⁇ g / cm 2. a cm 2 ⁇ 1 ⁇ g / cm 2, more preferably 0.2 ⁇ g / cm 2 ⁇ 0.7 ⁇ g / cm 2.
  • the content of the antibody (C) or antigen-binding fragment thereof contained in the detection part (4) is usually 0.1 ⁇ g to 1 ⁇ g, preferably 0.2 ⁇ g to 0.7 ⁇ g, more preferably 0.3 ⁇ g to 0.6 ⁇ g.
  • the content of an antibody or antigen-binding fragment thereof of said (C) is usually 0.15 ⁇ g / cm 2 ⁇ 2 ⁇ g / cm 2, preferably 0.2 [mu] g / a cm 2 ⁇ 1 ⁇ g / cm 2, more preferably 0.2 ⁇ g / cm 2 ⁇ 0.7 ⁇ g / cm 2.
  • the total content of the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof (C) contained in the detection unit (4) is usually 0.2 ⁇ g to 2 ⁇ g, The amount is preferably 0.4 ⁇ g to 1.4 ⁇ g, more preferably 0.6 ⁇ g to 1.2 ⁇ g.
  • the total content of the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof (C) per unit area of the detection unit (4) is usually 0.3 ⁇ g / cm. a 2 ⁇ 4 ⁇ g / cm 2, preferably from 0.4 ⁇ g / cm 2 ⁇ 2 ⁇ g / cm 2, more preferably 0.4 ⁇ g / cm 2 ⁇ 1.4 ⁇ g / cm 2.
  • the content ratio of the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof (C) contained in the detection unit (4) is from 1: 2 to 2: 1 by mass ratio. Is preferable, and 2: 1 is more preferable.
  • the detection unit (4) preferably contains the antibody (E) or antigen-binding fragment thereof.
  • the content of the antibody (E) or antigen-binding fragment thereof in the labeling substance holding part is usually 0.1 ⁇ g to 1 ⁇ g, preferably 0 .2 ⁇ g to 0.7 ⁇ g, more preferably 0.3 ⁇ g to 0.6 ⁇ g.
  • the content of an antibody or antigen-binding fragment thereof of said (E) is usually 0.15 ⁇ g / cm 2 ⁇ 4 ⁇ g / cm 2, preferably 0.2 [mu] g / cm 2 to 3 ⁇ g / cm 2 , more preferably 0.2 ⁇ g / cm 2 to 2.5 ⁇ g / cm 2 .
  • the content of the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof in the labeling substance holding part and the antibody (E) or antigen-binding fragment thereof (E) The ratio is preferably 1: 2 to 2: 1 by weight.
  • the absorption part (5) is installed at the end of the chromatographic medium part (3) in order to absorb a liquid such as a specimen or a developing solution that has passed through the detection part (4).
  • the absorbent portion (5) is, for example, glass fiber, pulp, cellulose fiber, or the like, or a nonwoven fabric containing a hydrophilic agent having a polymer such as an acrylic acid polymer, an ethylene oxide group, or the like. Particularly preferred is glass fiber.
  • the backing sheet (6) is an arbitrary base material. By applying an adhesive on one side or sticking an adhesive tape, one side has adhesiveness, and the sample addition part (1), labeling substance holding part (2), chromatographic medium on the adhesive side Part or all of the part (3), the detection part (4), and the absorption part (5) are provided in close contact with each other.
  • the backing sheet (6) is not particularly limited as long as the backing sheet (6) becomes impermeable and impermeable to the sample solution by the adhesive.
  • the immunochromatography analyzer according to the present invention is usually subjected to a drying treatment before commercialization.
  • the drying temperature is, for example, 20 ° C. to 50 ° C., and the drying time is 0.5 hour to 1 hour.
  • the immunochromatography analysis kit includes the immunochromatography analyzer and a sample diluent for diluting and developing the sample.
  • the specimen diluent can also be used as a developing solution, but usually water is used as a solvent, and a buffer solution, a salt such as sodium caseinate, a nonionic interface, etc.
  • Surfactants such as activators, cationic surfactants, anionic surfactants, polymer compounds such as polyvinylpyrrolidone (PVP), polyanions, nitrogen atom-containing vinyl water-soluble polymers, antibacterial agents, chelating agents Etc. can be contained. These may contain 1 type (s) or 2 or more types.
  • a surfactant having an HLB value of 13 to 17 can be preferably used.
  • Triton X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether, HLB: 13.7)
  • Tween 20 (trade name, polyoxyethylene sorbitan monolaurate, HLB: 16.7)
  • Tween 80 (Pro names, polyoxyethylene sorbitan monooleate, HLB: 15.0), Brij35 (polyoxyethylene glycol dodecyl ether, HLB: 13.7) and the like may be used, or one or more may be added.
  • the content of the nonionic surfactant in the sample diluent is usually 0.5% to 5%, preferably 0.5% to 3%, more preferably 0.5% to 2%. .
  • examples of the nitrogen atom-containing vinyl water-soluble polymer include polyvinyl pyrrolidone (PVP).
  • PVP polyvinyl pyrrolidone
  • the content of the nitrogen atom-containing vinyl water-soluble polymer in the specimen diluent is usually 0.1% to 0.8%, preferably 0.2% to 0.6%, and more preferably 0.8%. 2% to 0.5%.
  • the specimen diluent can be developed by adding a specimen-containing liquid obtained by mixing with a specimen sample in advance to the sample addition section, or after adding the specimen sample to the sample addition section first, It may be added to the sample addition section and developed.
  • the immunochromatographic analysis method includes the following steps (1) to (4), and is a method for detecting chikungunya virus contained in a specimen using the immunochromatographic analysis kit.
  • a step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to the sample addition unit (2) The chikungunya virus by the antibody or antigen-binding fragment thereof (A) held in the labeling substance holding unit (3) Step of developing the sample and the antibody or antigen-binding fragment thereof (A) in the chromatographic medium part as a mobile phase (4) Chikungunya virus in the developed mobile phase in the detection part Steps of detection using the antibody (B) or antigen-binding fragment thereof and the antibody (C) or antigen-binding fragment thereof (C) contained in each step will be described below.
  • Step (1) Step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to the sample addition unit
  • the sample is moved smoothly in the apparatus without reducing the measurement accuracy. It is preferable to adjust or dilute with a sample diluent to a concentration of about a sample-containing solution.
  • the specimen dilution liquid described above can be used.
  • a predetermined amount (usually 0.1 ml to 2 ml) is added onto the sample addition section (1) using the specimen-containing liquid as a sample.
  • movement starts in the sample addition unit (1).
  • those described above can be used.
  • Step (2) The step of recognizing the chikungunya virus by the antibody of (A) or its antigen-binding fragment held in the labeling substance holding part Step (2) was added to the sample addition part in step (1)
  • the sample is moved to the labeling substance holding part (2), and the Chikungunya virus in the specimen is held by the antibody (A) or the antigen-binding fragment thereof bound to the labeling substance held in the labeling substance holding part. It is a process of recognizing.
  • the labeling substance described above can be used.
  • Step (3) is a step (2) in which Chikungunya virus in the sample holds the labeling substance. After being recognized by the antibody (A) or antigen-binding fragment thereof bound to the labeling substance in the part, the specimen and the antibody (A) or antigen-binding fragment thereof (A) are passed through the chromatographic medium part as a mobile phase. It is a process.
  • the Chikungunya virus in the specimen that has passed over the chromatographic medium portion as a mobile phase is immobilized on the detection portion by the antigen-antibody specific binding reaction, or the antigen-binding fragment thereof and Specifically reacting and binding so as to be sandwiched between the antibody or antigen-binding fragment thereof in (C) and the antibody or antigen-binding fragment thereof in (A) to which the labeling substance is bound in the step (2).
  • the detection unit is colored.
  • the labeling reagent dissolved in the sample water does not cause a specific binding reaction even when it passes through the detection section on the chromatographic medium section. do not do.
  • amino acid sequences of the anti-CHIKV antibodies 13H11, 3D11, and 15B2 were determined. These results are shown below. Note that the light chains of 13H11, 3D11, and 15B2 all have the same amino acid sequence.
  • amino acid sequence indicated by underlining corresponds to the amino acid sequences of CDR1, CDR2, and CDR3 from the N-terminus to the C-terminus, respectively.
  • amino acid sequence enclosed in parentheses corresponds to the amino acid sequence of the variable region.
  • 3D11 heavy chain (SEQ ID NO: 19) [QVQLQQSGAELARPGASVKLSCKAS GYTFTSYW MQWVKQRPGQGLEWIGA IYPGDGDTRYT QKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCAR SYDPFDY WGQGTTLTVSS ] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA IERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLD
  • CK- (d) and CK- (e) monoclonal antibodies B7 cells were infected with CHIKV and incubated until a wide range of cytopathic effects were observed. Infected cells were inactivated with phosphate buffered saline (PBS) containing 4% formaldehyde overnight at 4 ° C., harvested, washed 3 times with PBS, and stored at ⁇ 80 ° C. prior to injection. 5 week old female BALB / c mice (National Laboratory Animal Center, Mahidol University, Bangkok, Thailand) vs.
  • PBS phosphate buffered saline
  • inactivated BHI cells in complete Freund's adjuvant (Sigma Aldrich, Saint Louis, MO) 2.5K ⁇ 10 6 infected cells / mouse) were immunized intraperitoneally.
  • booster immunization was performed 3 times every 3 weeks with CHIKV-infected cells without adjuvant.
  • the spleen was removed and spleen cells were obtained and fused with mouse myeloma PAI cells using polyethylene glycol # 1500 (Roche diagnostics, Mannheim, Germany) to prepare hybridomas.
  • Hybridomas were cultured in RPMI 1640 supplemented with 15% FBS, hypoxanthine-aminopterin-thymidine (GIBCO, Grand Island, NY) and 3% BM-Condimed H1 supplement (Roche diagnostics). Antibodies secreted by the hybridomas were screened by an indirect immunofluorescence assay (IFA) using CHIKV infected Vero cells.
  • IFA indirect immunofluorescence assay
  • CK- (d) and CK- (e) strains Two types of hybridomas (CK- (d) and CK- (e) strains) were isolated. Two types of monoclonal antibodies (CK- (d) and CK- (e)) were obtained as anti-CHIKV antibodies produced from these hybridomas.
  • the isotypes of CK- (d) and CK- (e) were IgG1 and IgG2a, respectively.
  • CK- (d) corresponds to the antibody (D)
  • CK- (e) corresponds to the antibody (E).
  • the amino acid sequence indicated by underlining corresponds to the amino acid sequences of CDR1, CDR2, and CDR3 from the N-terminus to the C-terminus, respectively.
  • the amino acid sequence enclosed in parentheses corresponds to the amino acid sequence of the variable region.
  • CK- (d) heavy chain [ESGGGLVKLGGSLKLSCAAS GFTFSTYY MSWVRQTPEKRLELVAA INSNGGST YYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALYYC ARHELVGGWFVY WGQGTLVTVSA ] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQK
  • CK- (e) heavy chain (SEQ ID NO: 30) [QVQLQQSGAELVRPGTSVKMSCKAA GYTFTNYW IGWIKQRPGHGLEWIGD VYPGGGST YYNEKFKAKATLTADTSSSTAYMQLSRLTSEDSAIYYC SRVTSTTGWYFDV WGAGTTVTVSS ] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNY
  • An immunochromatographic analyzer including a sample diluent, a sample addition unit (1), a labeling substance holding unit (2), a chromatographic medium (3) having a detection unit (4), and an absorption unit (5)
  • An immunochromatographic analysis kit consisting of
  • sample Addition Part A non-woven fabric made of glass fiber (Millipore: 300 mm ⁇ 30 mm) was used as the sample addition part.
  • the labeling substance solution was prepared by the above procedure. After adding 300 ⁇ L of the prepared labeling substance solution 300 ⁇ L of 10% by mass trehalose aqueous solution and 1.8 mL of distilled water to a 16 mm ⁇ 300 mm glass fiber pad (Millipore) uniformly, It dried with the vacuum dryer and produced the labeling substance holding
  • chromatographic medium part and detection part A sheet made of nitrocellulose (trade name: HF120, 300 mm x 25 mm, manufactured by Millipore) was used as a membrane. Next, a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol was used to adjust the concentration of the 3D11 monoclonal antibody to 0.3 mg / ml and the 15B2 monoclonal antibody to 0.6 mg / ml.
  • 150 ⁇ L of the antibody solution was applied to the detection part on the dried membrane in a line shape with an amount of 1 ⁇ L / mm (25 ⁇ L per sheet) using an immunochromatographic dispenser “XYZ3050” (manufactured by BIODOT) with a width of 1 mm.
  • XYZ3050 manufactured by BIODOT
  • a goat-derived antiserum having a wide affinity for the gold nanoparticle labeling substance and phosphate buffer solution (pH 7.4) downstream of the detection unit in order to confirm the presence / absence and development speed of the gold nanoparticle labeling reagent was applied to the control site (control line). Then, it was dried at 50 ° C. for 30 minutes and dried overnight at room temperature to prepare a chromatographic medium part and a detection part.
  • the content of 3D11 monoclonal antibody in the detection part was 0.14 ⁇ g (0.25 ⁇ g / cm 2 ), and the content of 15B2 monoclonal antibody was 0.14 ⁇ g (0.25
  • the substrate made by Kuramoto Sangyo Co., Ltd.
  • the substrate consisting of a backing sheet absorbs the sample addition part, the labeling substance holding part, the chromatographic medium part having the detection part, the developed sample and the labeling substance.
  • Glass fiber non-woven fabrics were bonded together as an absorbing part for the purpose. And it cut
  • the length of the labeling substance holding part in the sample developing direction was 16 mm.
  • specimen diluent 50 mM HEPES buffer (pH 7.5) containing 1% by mass of a nonionic surfactant (1: 1 mixture of NP-40 manufactured by Nacalai Tesque and Nonidet MN-811 manufactured by NOF Corporation) was prepared as a specimen diluent for subjecting the specimen to dilution treatment.
  • a nonionic surfactant 1: 1 mixture of NP-40 manufactured by Nacalai Tesque and Nonidet MN-811 manufactured by NOF Corporation
  • Example 2 ⁇ Example 2>
  • the CK- (d) monoclonal antibody was used in addition to the 13H11 monoclonal antibody in the labeling substance holding part
  • the CK- (e) monoclonal antibody was used in addition to the 3D11 monoclonal antibody and 15B2 monoclonal antibody in the detection part.
  • the immunochromatographic analysis kit of Example 2 was produced in the same manner as in Example 1 except that the content of each antibody in the labeling substance holding part and the detection part was as described below.
  • the content of the 13H11 monoclonal antibody in the labeling substance holding part is 0.05 ⁇ g (0.09 ⁇ g / cm 2 ), and the content of the CK- (d) monoclonal antibody is 0.05 ⁇ g (0.09 ⁇ g / cm 2 ).
  • the content of 3D11 monoclonal antibody in the part is 0.06 ⁇ g (0.69 ⁇ g / cm 2 ), the content of 15B2 monoclonal antibody is 0.1 ⁇ g (1.1 ⁇ g / cm 2 ), and the content of CK- (e) monoclonal antibody was 0.2 ⁇ g (2.29 ⁇ g / cm 2 ).
  • Example 3 the content of the 13H11 monoclonal antibody and the CK- (d) monoclonal antibody in the labeling substance holding part was 0.03 ⁇ g (0.05 ⁇ g / cm 2 ) so that the mass ratio was 1: 2. ), And the immunochromatographic analysis kit of Example 3 was prepared in the same manner as in Example 2 except that the CK- (d) monoclonal antibody was changed to 0.06 ⁇ g (0.1 ⁇ g / cm 2 ).
  • Example 1 In Example 1, only the CK- (d) monoclonal antibody was used instead of the 13H11 monoclonal antibody, and only the CK- (e) monoclonal antibody was used instead of the 3D11 monoclonal antibody and the 15B2 monoclonal antibody.
  • the content of the CK- (d) monoclonal antibody was 0.1 ⁇ g (0.18 ⁇ g / cm 2 ), and the content of the CK- (e) monoclonal antibody in the detection part was 0.29 ⁇ g (3.3 ⁇ g / cm 2 ). Except for this, an immunochromatographic analysis kit of Comparative Example 1 was prepared in the same manner as Example 1.
  • Example 1 the immunochromatography analysis kits of Examples 1 to 3 and Comparative Example 1 were examined to determine whether there was a difference in reactivity between chikungunya viruses with different genotypes. Specifically, using the immunochromatography analysis kits of Examples 1 to 3 and Comparative Example 1 prepared above, measurement was performed using the following samples. ECSA type (autologous culture), Asian type (autologous culture) and WA type (autologous culture) were used as the chikungunya virus of the specimen. For the WA type (autologous culture), a pseudotype virus having an envelope derived from chikungunya virus was prepared and used for the HIV lentivector.
  • ECSA type autologous culture
  • Asian type autologous culture
  • WA type autologous culture
  • each sample virus For ECSA type and Asian type, prepare each sample virus by diluting each virus to 1 ⁇ 10 4 pfu / mL, 1 ⁇ 10 5 pfu / mL, or 1 ⁇ 10 6 pfu / mL. This was used as a specimen sample. As a negative control sample, a sample diluent containing no virus was used. In addition, for the WA type (autologous culture), each virus is diluted with a sample diluent so as to be 6 ng / mL, 30 ng / mL, and 150 ng / mL, and a sample-containing solution is prepared. did. As a negative control sample, a sample diluent containing no virus was used.
  • Test Example 2 In this test, in the immunochromatography analysis kits of Examples 1 to 3 and Comparative Example 1, differences in reactivity depending on the presence or absence of mutation of chikungunya virus were examined. Specifically, using the immunochromatography analysis kits of Examples 1 to 3 and Comparative Example 1 prepared above, measurement was performed using the following samples. As specimen Chikungunya virus, ECSA wild type (WT) and mutant type (MT), Asian wild type (WT) and mutant type (MT) of autologous culture were used, and each virus was 6 ng / mL, 30 ng / mL. , 150 ng / mL was diluted with the sample diluent to prepare a sample-containing solution, which was used as the sample.
  • ECSA wild type (WT) and mutant type (MT) Asian wild type (WT) and mutant type (MT) of autologous culture were used, and each virus was 6 ng / mL, 30 ng / mL.
  • 150 ng / mL was
  • the CSA mutant is obtained by substituting the 350th glutamic acid in the amino acid sequence of the ESA protein of ECSA wild type with aspartic acid, and the Asian mutant is the 350th aspartic acid in the amino acid sequence of the Asian wild type E1 protein. Is substituted with glutamic acid.
  • Example 3 In this test, the cross-reactivity with viruses other than chikungunya virus was examined in the immunochromatography analysis kits of Examples 1 to 3 and Comparative Example 1. Specifically, Sindbis virus (SINV, self-culture) belonging to the same Togaviridae alphavirus genus as Chikungunya virus was used as a sample using the immunochromatography analysis kits of Examples 1 to 3 and Comparative Example 1 prepared above. The virus is diluted with a sample diluent to 1 ⁇ 10 5 pfu / mL, 1 ⁇ 10 6 pfu / mL, or 1 ⁇ 10 7 pfu / mL to prepare a sample-containing solution, which is used as a sample sample. It was.
  • Sindbis virus SINV, self-culture
  • the virus is diluted with a sample diluent to 1 ⁇ 10 5 pfu / mL, 1 ⁇ 10 6 pfu / mL, or 1 ⁇ 10 7

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US11560421B2 (en) 2018-02-09 2023-01-24 Osaka University Broad-spectrum monoclonal antibodies against chikungunya virus E1 structural protein
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