[go: up one dir, main page]

WO2016080591A1 - Anticorps reconnaissant la nuclécapside du coronavirus du syndrome respiratoire du moyen-orient et son utilisation - Google Patents

Anticorps reconnaissant la nuclécapside du coronavirus du syndrome respiratoire du moyen-orient et son utilisation Download PDF

Info

Publication number
WO2016080591A1
WO2016080591A1 PCT/KR2015/000278 KR2015000278W WO2016080591A1 WO 2016080591 A1 WO2016080591 A1 WO 2016080591A1 KR 2015000278 W KR2015000278 W KR 2015000278W WO 2016080591 A1 WO2016080591 A1 WO 2016080591A1
Authority
WO
WIPO (PCT)
Prior art keywords
mers
cov
antibody
nucleocapsid
antigen
Prior art date
Application number
PCT/KR2015/000278
Other languages
English (en)
Korean (ko)
Inventor
조영식
하건우
정승교
한수호
장성구
김정호
Original Assignee
유한회사 바이오노트
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 유한회사 바이오노트 filed Critical 유한회사 바이오노트
Publication of WO2016080591A1 publication Critical patent/WO2016080591A1/fr

Links

Images

Classifications

    • 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
    • C07K16/1002—Coronaviridae
    • 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

Definitions

  • the present invention is an antibody recognizing a Middle East respiratory syndrome coronavirus (MERS-CoV) nucleocapsid and cells capable of producing the same, a composition, kit for diagnosing a Middle East respiratory syndrome coronavirus comprising the antibody, and the Middle East. It relates to a method for diagnosing a respiratory syndrome coronavirus infection.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • MERS-CoV The Middle East Respiratory Syndrome Corona Virus
  • MERS-CoV has an incubation period of about one week and, like SARS, causes severe respiratory symptoms such as high fever, cough and difficulty breathing. Unlike SARS, however, it is accompanied by acute renal failure. More deadly than the SARS, the findings show that the mortality rate is about six times higher than the SARS. Depending on age, mortality rates are over 50%.
  • the source of infection is not yet known, but camels are pointed out.
  • the virus detected from an infected person coincided with the virus detected from a camel.
  • the practice of eating camel meat or drinking milk is known to be difficult.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • One aspect of the invention provides an antibody that recognizes Middle East respiratory syndrome coronavirus nucleocapsid.
  • Another aspect of the invention provides a hybridoma with accession number KCLRF-BP-00331 or KCLRF-BP-00332.
  • Another aspect of the present invention provides a composition for diagnosing Middle East respiratory syndrome coronavirus.
  • Another aspect of the present invention provides a kit for diagnosing Middle East respiratory syndrome coronavirus.
  • Another aspect of the present invention provides a method for diagnosing a Middle East respiratory syndrome coronavirus infection.
  • One aspect provides antibodies that recognize Middle East respiratory syndrome coronavirus nucleocapsid.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • 'MERS-CoV' Middle East respiratory syndrome coronavirus
  • the MERS-CoV is a species of the genus Betacoronavirus.
  • the MERS-CoV is also known as HCoV-EMC or NcoV.
  • the antibody may specifically bind to nucleocapsid of MERS-CoV.
  • the nucleocapsid of MERS-CoV may be one having an amino acid sequence of SEQ ID NO: 1.
  • Nucleocapsids of MERS-CoV having amino acid sequences of 22-40, 126-146, 164-202, 234-259, 362-388, or combinations thereof in SEQ ID NO: 1 are hydrophilicity Can be.
  • Nucleocapsids of MERS-CoV having amino acid sequences of 22-40, 126-146, 164-202, 234-259, 362-388, or a combination thereof in SEQ ID NO: 1 are respectively SEQ ID NO: 1, Amino acid sequences of 2, 3, 4, and 5.
  • the nucleocapsid of MERS-CoV having the hydrophilicity may comprise an epitope.
  • the inventors investigated the hydrophilicity of the nucleocapsid protein of MERS-CoV, to prepare a polypeptide antigen of the high hydrophilic region of the protein, to prepare an antibody that specifically binds to it. .
  • a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV.
  • a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV is also preferred.
  • a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV, and an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV
  • An antibody is provided that is a combination of monoclonal antibodies that bind specifically.
  • a complete antibody is a structure having two full length light chains and two full length heavy chains, each of which consists of a heavy chain constant region and a light chain constant region divided by a heavy chain and a disulfide bond (SS-bond) antibody.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types, and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma. 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1), and alpha 2 ( ⁇ 2).
  • the constant region of the light chain may have kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • monoclonal antibody means that the antibody molecule is made to consist of a single molecule. Monoclonal antibodies have specificity that responds only to antigens with the same epitope, and also show affinity only for specific epitopes.
  • the monoclonal antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 2 may be one produced in hybridoma cells having accession number KCLRFBP00331.
  • the monoclonal antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 4 may be one produced in hybridoma cells having accession number KCLRFBP00332.
  • the hybridoma cells can be prepared using methods known in the art. Specifically, the hybridoma cells are prepared by immunizing a polypeptide of an immunogen MERS-CoV nucleocapsid to an animal and fusing B cells, which are antibody-producing cells derived from the immunized animal, with myeloma cells. Next, it can be prepared by a method of selecting hybridomas that produce monoclonal antibodies that specifically bind to MERS-CoV nucleocapsid. The immunized animal can use an animal such as a goat, sheep, morph, rat or rabbit as well as the mouse used in the examples.
  • a method for immunizing the immunized animal As a method for immunizing the immunized animal, a method already known in the art may be used. For example, when immunizing mice, 1 to 100 ⁇ g of an immunogen is emulsified with an antigen adjuvant such as physiological saline and / or Freund's adjuvant at one time to subcutaneously subcutaneously in the abdominal cavity of the immunized animal. Or 2-6 inoculations every 2-5 weeks intraperitoneally. After immunizing the immunized animals, spleens or lymph nodes are extracted after 3-5 days of final immunization, and according to cell fusion methods already known in the art, B cells contained in their tissues in the presence of a fusion promoter are myeloma. To fuse with the cell.
  • an antigen adjuvant such as physiological saline and / or Freund's adjuvant
  • the fusion promoter may use a material such as polyethylene glycol (PEG), for example.
  • PEG polyethylene glycol
  • the myeloma cells are described, for example, in P3U1, NS-1, P3x63.
  • Mouse derived cells such as Ag 8.653, Sp2 / 0-Ag14, and rat derived cells such as AG1, AG2 can be used.
  • the cell fusion method known in the art for example, B cells and myeloma cells are mixed at a ratio of 1: 1-10: 1, to which a PEG having a molecular weight of 1,000-6,000 is added at a concentration of 10-80%.
  • the method may be performed by incubating at 30-37 ° C. for 1-10 minutes.
  • hybridomas that produce monoclonal antibodies that specifically bind to the nucleocapsid of MERS-CoV are cultured in a selective medium such as HAT medium, in which only hybridomas can survive, and hybridomas are produced. The antibody activity in the culture supernatant can be measured and selected using methods such as ELISA.
  • hybridomas that produce monoclonal antibodies that specifically bind to nucleocapsids of MERS-CoV may, for example, produce high levels of monoclonal antibodies that specifically bind to nucleocapsids of MERS-CoV. For bridoma, it can be selected by repeating cloning by methods such as limiting dilution. Meanwhile, the monoclonal antibody may be of IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type.
  • Another aspect provides antigen binding fragments according to the monoclonal antibodies described above.
  • antibody is a specific antibody against MERS-CoV, which binds specifically to the nucleocapsid of MERS-CoV, and is a complete antibody, antigen binding fragment of an antibody molecule, synthetic antibody, recombinant antibody, or antibody. It may include a hybrid (antibody hybrid). The description of the complete antibody is as mentioned above.
  • antigen binding fragment refers to a portion of a polypeptide that includes a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin.
  • it can be F (ab ') 2, Fab', Fab, Fv or scFv.
  • Fab of the antigen-binding fragment has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain and a first constant region of the heavy chain (C H1 ).
  • F (ab ') 2 antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds.
  • Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy chain variable regions and light chain variable regions are well known in the art.
  • Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single-chain Fv is generally shared by the variable region of the heavy chain and the short chain variable region through a peptide linker. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv.
  • the antigen-binding fragment can be obtained using proteolytic enzymes (for example, restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') 2 fragment).
  • proteolytic enzymes for example, restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') 2 fragment.
  • the antigen-binding fragment can be produced through genetic recombination technology.
  • Such antibodies include monoclonal antibodies, bispecific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fv (sdFv) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies.
  • hybridoma cells (Accession: KCLRFBP00331) that produce monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV.
  • hybridoma cells (Accession: KCLRFBP00332) that produce monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV.
  • hybridoma cells prepared according to the following examples were deposited on October 14, 2014 to the Korean Collection for Type Cultures, an international depository institution under the Budapest Treaty (Accession No .: KCLRFBP00331 and KCLRFBP00332).
  • the deposited hybridoma cells are stored according to the provisions of the Budapest Treaty for Microbial Deposits and can be distributed to the general public with reference to the accession number.
  • Another aspect provides a composition for diagnosing MERS CoV of an individual comprising said monoclonal antibody or antigen binding fragment and an anti-MERS CoV monoclonal antibody.
  • the composition may be a liquid composition comprising the antibody in a liquid.
  • the liquid may be one capable of dissolving and maintaining the antibody.
  • it may be water or a buffer solution such as PBS.
  • the composition may further include a substance for keeping the antibody stable.
  • Another aspect provides a MERS-CoV diagnostic kit comprising the above-described antibody or antigen-binding fragment thereof.
  • the kit may further comprise a detectable moiety.
  • a detectable moiety can include a moiety in which the presence, relative amount and / or location (eg, location on the array) of the moiety can be determined directly or indirectly.
  • detectable moieties are well known in the art.
  • a detectable moiety can be detected when exposed to certain conditions, such as a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety (eg, a radioactive atom), or an enzyme moiety. It may be selected from the group consisting of.
  • fluorescent moieties may need to be exposed to radiation at certain wavelengths and intensities that cause excitation of the fluorescent moiety, thereby detecting at a particular wavelength that can be detected. It can be made to emit as much fluorescence as possible.
  • the kit includes a sample pad (1) to which a liquid sample including an analyte is applied;
  • a storage pad 2 fluidly connected to the sample pad and movably supported by a conjugate of gold particles, wherein the conjugate of gold particles is a conjugate of a first antibody or fragment thereof and a gold particle Storage pad 2;
  • a chromatographic membrane material (3) in fluid communication with the storage pad and in which the liquid sample is moved by capillary movement, wherein the second antibody or fragment thereof is immobilized downstream of the storage pad.
  • a chromatographic membrane material 3 comprising a detection region;
  • a hygroscopic pad 4 in fluid communication with the chromatography membrane material
  • It may be an analytical device for detecting the Middle East respiratory syndrome coronavirus, including the sample pad, the storage pad, the chromatographic membrane material, and the solid support 5 supporting the hygroscopic pad.
  • the first and second antibodies specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2, for example, in the nucleocapsid of Middle East respiratory syndrome coronavirus (MERS-CoV).
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the first antibody and the second antibody may be different.
  • the first antibody is a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4
  • the second antibody is specifically directed to an epitope represented by the amino acid sequence of SEQ ID NO: 2 It may be a monoclonal antibody that binds.
  • the first antibody is a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2
  • the second antibody is specific to an epitope represented by the amino acid sequence of SEQ ID NO: 4 It may be a monoclonal antibody that binds to.
  • FIG. 1 is a side view showing an example of an analysis device for detecting and detecting the Middle East Respiratory Syndrome Coronavirus according to an embodiment of the present invention.
  • the kit is supported by a chromatography material membrane (3) having a detection region (T) and a control region (C) on which a second antibody is immobilized on a solid support (5), wherein the specimen pad (1)
  • the storage pad 2 and the moisture absorption pad on which the first antibody and the gold conjugate are supported are overlapped and connected.
  • the sample is added to the sample pad 1 in the strip, it moves to the storage pad 2 by capillary action so that the antigen in the sample binds to the conjugate of the gold and the first antibody in the storage pad, Capillary movement is made downstream through the direction of the moisture absorption pad (4).
  • the conjugate and the second antibody specifically bind and develop color, and in the absence of the conjugate, no color development occurs.
  • the conjugate of the first antibody and the gold particles together with the sample passes through the control region (C)
  • the conjugate of the first antibody and the gold particles is specific to the first antibody and the gold particle conjugate in the control region. In combination with the color development, it is possible to check whether the capillary movement is properly performed.
  • the kit may be an immunoassay kit using a porous material as a solid carrier of an immunochemical component such as an antigen or an antibody.
  • Sample pads, storage pads, chromatographic membrane materials and hygroscopic pads used in one embodiment of the invention are materials having a porosity and volume sufficient to contain and contain a liquid sample to be analyzed, for example a microporous membrane. It may be a substance. Examples of such microporous membrane materials include nylon, cellulose materials, polysulfones, polyvinylidene difluorides, polyesters and glass fibers. Preferred examples of the microporous membrane material are nitrocellulose membranes.
  • the analysis device may have the form of a strip.
  • in flow communication with means that a liquid sample is applied to one location (e.g. sample pad) and is available to another location (e.g. storage pad) by capillary movement when the liquid sample is applied.
  • movably supported means that the conjugate of gold particles is immobilized to the storage pad movably with capillary movement of the liquid sample.
  • nondiffusively immobilized means that the second antibody or fragment thereof is immobilized so that it does not diffuse together by capillary movement of the liquid sample in the detection region.
  • the method for non-diffusion immobilizing the antigen and / or antibody can include any method as long as it is capable of non-diffusion immobilizing the chromatography membrane material and the antigen and / or antibody to the detection region of the chromatography membrane material. Examples include, but are not limited to, covalent bonding methods.
  • the detection zone may take any shape, for example rectangular and circular in shape, the area of which is smaller than the area of the chromatography membrane material.
  • Antibodies and gold conjugates used in the kits of one embodiment of the invention can be prepared by methods well known in the art. For example, it can be prepared by making a gold colloidal solution and covalently binding the gold in the solution with the antibody. Antibodies and gold conjugates can be prepared, for example, by the methods disclosed in US Pat. Nos. 5,514,302 and 4,313,734, which are incorporated herein by reference in their entirety.
  • the kit can be used to diagnose MERS-CoV by detecting MERS-CoV according to an immunoassay method.
  • immunoassays can be performed according to various immunoassays or immunostaining protocols developed in the prior art.
  • the immunoassay or immunostaining format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining. And immunoaffinity tablets.
  • the antibody labeled with the radioisotope may be MERS-CoV or a nucleo thereof. It can be used to detect the capsid.
  • certain embodiments of the present invention include the steps of coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; Reacting the cell lysate with a primary antibody against the nucleocapsid of MERS-CoV; Reacting the reaction product of the step of reacting the primary antibody against the nucleocapsid of MERS-CoV and the cell lysate with a secondary antibody to which an enzyme is bound; And measuring the activity of the enzyme.
  • Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, and may be microtiter plates.
  • the enzyme bound to the secondary antibody may include an enzyme catalyzing a color reaction, a fluorescence reaction, a luminescence reaction or an infrared reaction.
  • an enzyme catalyzing a color reaction e.g., alkaline phosphatase, ⁇ -galactosidase, and horse radish fur Oxidase, luciferase and cytochrome P450.
  • alkaline phosphatase When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT) naphthol-AS-B1-phosphate (naphthol-AS-) Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-phosphate) and chromogenic reaction substrates such as enhanced chemifluorescence (ECF) are used and hose radish peroxidase is used.
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • naphthol-AS-B1-phosphate naphthol-AS-
  • Chloronaphthol aminoethylcarbazole
  • aminoethylcarbazole diaminobenzidine
  • one embodiment of the invention comprises coating an anti-MERS-CoV antibody on the surface of a solid substrate as a capturing antibody; Reacting the capture antibody with the sample; The detection result of reacting the capture antibody with the sample is a detection antibody that binds to a label that generates a signal and specifically reacts to MERS-CoV or a nucleocaptide thereof (e.g., one embodiment of the present invention). Antibody or antigen-binding fragment); And measuring the signal generated from the label.
  • the detection antibody carries a label which generates a detectable signal.
  • the label may include chemicals (eg biotin), enzymes (alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125 , P32 and S35), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET).
  • chemicals eg biotin
  • enzymes alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase and cytochrome P450
  • radioactive substances eg C14, I125 , P32 and S35
  • fluorescent materials eg, fluorescein
  • luminescent materials eg, chemiluminescent, and fluorescence resonance energy transfer (FRET).
  • Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of MERS-CoV. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.
  • Another aspect includes contacting a sample with an antibody or antigen-binding fragment thereof as described above to provide information necessary for diagnosing MERS-CoV; And detecting a complex of the antibody or an antigen-binding fragment thereof and MERS-CoV.
  • the method for detecting MERS-CoV may include contacting the sample with the above-described antibody or antigen-binding fragment thereof.
  • the contacting step comprises contacting the sample with a plate coated with the above-described anti-MERS-CoV antibody or antigen-binding fragment thereof (referred to as 'primary antibody or antigen-binding fragment thereof'), followed by anti-MERS-CoV antibody or
  • the antigen binding fragment thereof (referred to as 'secondary antibody or antigen binding fragment thereof') may further comprise the step of contacting the plate.
  • the primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may be the same or different.
  • the primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may be bound to a detectable moiety.
  • the secondary antibody or binding fragment thereof may be one that contains a detectable moiety such as gold.
  • the primary and secondary antibodies or antigen-binding fragments thereof are monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV, and the nucleo of MERS-CoV, respectively. It may be a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the capsid, or vice versa.
  • the sample may be a biological material derived from an individual.
  • the subject may be a vertebrate.
  • the vertebrate may be a mammal.
  • the mammal may be a primate, including a human and a non-human primate, a camel, or a rodent, including a mouse and a rat.
  • the sample may be frozen or left in its natural state.
  • the biological material may be nasal swap, nasal aspirate, nasopharyngeal swab, nasopharyngeal aspirate, blood or blood constituent, body fluid fluid) or a combination thereof.
  • Another aspect includes contacting a sample with an antibody or antigen-binding fragment thereof described above; Detecting a complex of the antibody or antigen-binding fragment thereof with MERS-CoV; And it provides a MERS-CoV diagnostic method comprising the step of detecting whether the sample MERS-CoV infection from the detection result.
  • the MERS-CoV diagnostic method may include determining that an individual from which the sample is derived is infected with MERS-CoV when an antibody or an antigen-binding fragment thereof and a complex of MERS-CoV is detected. In addition, when the complex is not detected, it may include determining that the individual from which the sample is derived is not infected with MERS-CoV.
  • an antibody specifically binding to a nucleocapsid of MERS-CoV and a composition and kit for diagnosing Middle East respiratory syndrome coronavirus comprising the same, and a method for diagnosing Middle East respiratory syndrome coronavirus using the same, Middle East respiratory syndrome Coronavirus infection can be measured efficiently.
  • FIG. 1 is a side view showing an example of an analysis device for detecting and detecting the Middle East Respiratory Syndrome Coronavirus according to an embodiment of the present invention.
  • FIG. 2 is a diagram showing the hydrophilicity of the nucleocapsid protein of MERS-CoV of SEQ ID NO: 1.
  • 3A is a diagram showing the results of SDS-PAGE of polypeptides of MERS-CoV NP P1 to P5.
  • Figure 3b is a view showing the Western blot results of the anti-MERS CoV P1 monoclonal antibody.
  • Figure 3c is a diagram showing the Western blot results of the anti-MERS CoV P3 monoclonal antibody.
  • Figure 4 is a schematic illustration of the operation of the kit used in one embodiment of the present invention.
  • a peptide antigen was synthesized in a hydrophilic site in the nucleocapsid (NP) gene of MERS-CoV and used as an immunogen.
  • the hydrophilicity of the gene encoding the nucleocapsid of MERS-CoV was examined to synthesize peptide antigens for high hydrophilic site portions.
  • 2 is a diagram showing the hydrophilicity of the nucleocapsid protein of MERS-CoV of SEQ ID NO: 1. Parker Hydrophilicity prediction program of Immune epitope database was used.
  • the synthesized peptide antigens are the 22-40 amino acids, 126-146 amino acids, 164-202 amino acids, 234-259 amino acids, and 362-388 amino acids in the nucleocapsid of MERS-CoV of SEQ ID NO: 1, respectively. It is referred to as MERS-CoV NP P1, MERS-CoV NP P2, MERS-CoV NP P3, MERS-CoV NP P4, and MERS-CoV NP P5.
  • immunogens were prepared as follows. Immunogens used in the first immunization were prepared by complete Freud's Adjuvant (Sigma), which was prepared by emulsifying the ratio of antigen to the adjuvant in a 1: 1 ratio, and used for the second to fourth immunizations. The immunogen was prepared by incomplete Freud's Adjuvant (Sigma Co., Ltd.) in a 1: 1 ratio of antigen to the adjuvant.
  • the immunogen prepared by the above method was immunized to BALB / c mice at 6 weeks of age and females as follows. Four intraperitoneal injections at weekly intervals in an amount of 200 ⁇ l / horse and after one week the antigen was diluted three times at a daily interval by diluting the antigen to 20% concentration (v / v) in Phosphate buffered saline (PBS). Intravenously injected 20 ⁇ l / time. After the third immunization, a medium titer test was performed with serum obtained from the tail vein. As a result, mice obtained with sufficient amounts of antibodies were selected to perform a cell fusion process.
  • PBS Phosphate buffered saline
  • spleen cells of the antibody-producing mice were removed and filtered through a 70 ⁇ m pore cell strainer (BD Falcon). The precipitated splenocytes were centrifuged to obtain a precipitate, and 5 ml of red blood cell lysis buffer (Sigma) was mixed and left for 1 minute to remove red blood cells. Spleen cells from which red blood cells were removed were washed three times with DMEM (Dulbecco's Modified Eagle's Medium, Gibco) and subjected to cell counting.
  • DMEM Dulbecco's Modified Eagle's Medium, Gibco
  • the cells to be fused with the splenocytes were SP2 / 0-Ag 14 (ATCC CRL-1581) cell line, a mouse-derived myeloma cell, and the mixing ratio was 10: 1 (splenocytes: SP2 / 0).
  • Mixed cells were washed twice with DMEM and then fused using PEG1500 (Roche).
  • the cell fusion was performed with 1.7 ml of PEG1500 for 1 minute, 1 second for 30 seconds, 1 ml of 1 minute for DMEM, 2 ml of 1 minute for DEME, 6 ml for 30 seconds of DMEM, and finally 30 minutes of DMEM after 30 seconds. 10 ml was dropped for a second.
  • the cell solution after fusion is obtained by centrifugation as a precipitate, and well with DMEM (hereinafter referred to as 'HAT medium') containing 10% of HAT (Gibco), antibiotics (Gibco), and fetal bovine serum (Hyclone). After mixing, the cells were dispensed into 96 well plates at a dose of 200 ⁇ l / well and grown in the incubator for 3 days.
  • DMEM hereinafter referred to as 'HAT medium'
  • HAT HAT
  • antibiotics Gibco
  • Hyclone fetal bovine serum
  • the cell line after the fusion was grown at 37 ° C., 5% CO 2 , and in a cell incubator maintained at humid conditions, and the fused cell line was selected by replacing HAT (hypoxanthine-aminopterin-thymidine medium) for two days at a week.
  • HAT hypoxanthine-aminopterin-thymidine medium
  • the cell lines selected by HAT medium were screened for positive clones by Enzyme-linked immunosorbent assay (ELISA). Specifically, 100 ⁇ l of the fused cell line culture grown in each well of the 96 well plate was placed in a 96 well adsorption plate (Costar) coated with a concentration of 2.5 ug / ml of MERS-CoV nucleocapsid (NP) gene antigen. Dispense at a dose of / well and react for 1 hour at 37 ° C.
  • ELISA Enzyme-linked immunosorbent assay
  • the cells were washed five times, and the anti mouse IgG HRP conjugate and the anti mouse IgM HRP conjugate were dispensed at a dose of 100 ⁇ l / well, and reacted at 37 ° C. for 30 minutes. After the reaction for 30 minutes, it was washed five times, the substrate liquid was dispensed at a dose of 100 ⁇ l / well, and the color reaction was performed for 15 minutes, and the reaction stop solution was added with a dose of 100 ⁇ l / well. After stopping the reaction, the absorbance was measured at 450 nm in the reader to select the positive clones MERS-CoV NP P1 and MERS-CoV NP P3 which showed high values.
  • MERS-CoV NP P1 Selected positive clones were transferred to 24 well tissue culture plates and incubated for 3 days, followed by secondary screening in the same manner as above, that is, by limiting dilution of hybridoma cell lines obtained through repeated selection, the final single positive clone candidate group.
  • MERS-CoV NP P1 Selected positive clones were transferred to 24 well tissue culture plates and incubated for 3 days, followed by secondary screening in the same manner as above, that is, by limiting dilution of hybridoma cell lines obtained through repeated selection, the final single positive clone candidate group.
  • MERS-CoV NP P1 Selected positive clones were transferred to 24 well tissue culture plates and incubated for 3 days, followed by secondary screening in the same manner as above, that is, by limiting dilution of hybridoma cell lines obtained through repeated selection, the final single positive clone candidate group.
  • MERS-CoV NP P1 Selected positive clones were transferred to 24 well tissue culture plates
  • the final selected monoclonal antibody-producing hybridomas were named anti-MERS CoV P1 monoclonal antibodies, and anti-MERS CoV P3 monoclonal antibodies, which were deposited with the Bank of Korea Cell Line and assigned accession numbers.
  • mice The final selected positive clones were transferred to a T75 (SPL) dish and incubated for 3 days, and injected into the abdominal cavity of mice at 1 ⁇ 10 6 cell / ml cell density, and collected one week later.
  • the mouse ascites liquid obtained through the above method was precipitated by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the supernatant was mixed at a 1: 1 ratio with a binding buffer (Binding buffer, Thermo scientific) at room temperature. Reaction was carried out for 1 hour.
  • the suspension was precipitated once again by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the unprecipitated suspension was filtered again using a filter having a 1.2 ⁇ m pore size.
  • Ascites fluid was completely removed from the suspension was placed in a column containing Protein G resin (Pierce) to induce binding between the antibody and Protein G.
  • the plural solution was washed three times with phosphate buffer solution and eluted using an elution buffer (Elution buffer, Thermo scientific).
  • the eluted antibody was again applied to a PD-10 column (GE heathcare) and eluted with phosphate buffer solution for final purification.
  • MERS-CoV NP P1, MERS-CoV NP P2, MERS-CoV NP P3, MERS-CoV NP P4, and MERS-CoV NP P5 conjugated to BSA were each expressed in the nucleocapsid of MERS-CoV of SEQ ID NO: 1.
  • To 40th amino acid 126-146 amino acids, 164-202 amino acids, 234-259 amino acids, and 362-388 amino acids.
  • a 10% polyacrylamide gel was prepared and then 5 MERS-CoV combined with bovine serum albumin (BSA) (EQUITECH-BIO, INC) and BSA prepared above as a control.
  • BSA bovine serum albumin
  • Polypeptides of NP P1 to P5 were each loaded at 1.25 ⁇ g.
  • SDS-PAGE was performed for 1 hour under conditions of 160V and 400 mA, and then, mini-Protean 3 Electrophoresis (BIO-RAD) was used on a nitrocellulose membrane (GE healthcare) under conditions of 160V and 400 mA.
  • the polypeptides were transferred for time.
  • 3A is a diagram showing the results of SDS-PAGE of polypeptides of MERS-CoV NP P1 to P5.
  • lane 1 represents BSA control
  • lane M represents a protein marker
  • lanes 2 to 6 represent polypeptides of MERS-CoV NP P1 to P5, respectively.
  • the SDS-PAGE gel described above was transferred to NC-membrane (200V, 200mA, 1 hour). Placed in 5% skim milk and blocked for 30 minutes at room temperature, then 10 ⁇ g / mL of anti-MERS CoV P1 monoclonal antibody (in 5% Skim milk / TBS) and anti-MERS CoV P3 monoclonal antibody (in 5% Skim milk / TBS) was reacted at room temperature for 1 hour. Thereafter, washing was repeated three times for 5 minutes using a wash solution (1M Tris, 0.0025% Proclin 300, 0.1% Tween 20, and 0.15 mM NaCl) to remove monoclonal antibody that did not bind to the polypeptide on the membrane.
  • a wash solution (1M Tris, 0.0025% Proclin 300, 0.1% Tween 20, and 0.15 mM NaCl
  • FIG. 3B is a diagram showing the Western blot results of the anti-MERS CoV P1 monoclonal antibody.
  • the anti-MERS CoV P1 monoclonal antibody according to one embodiment of the present invention showed color development only in lane 2 and no color development in the remaining lanes 3-6. Therefore, it was confirmed that the anti-MERS CoV P1 monoclonal antibody according to an embodiment of the present invention specifically reacts with a polypeptide including amino acids 22 to 40 of lane 2.
  • the monoclonal antibody that is, anti-MERS CoV P1 monoclonal antibody according to one embodiment of the present invention against MERS-CoV prepared in Example 1 from the above result is the nucleocapsid of MERS-CoV (SEQ ID NO: 1) It was confirmed that the 22-40 amino acid sequence part of (SEQ ID NO: 2) is an epitope.
  • Figure 3c is a diagram showing the Western blot results of the anti-MERS CoV P3 monoclonal antibody.
  • the anti-MERS CoV P3 monoclonal antibody according to one embodiment of the present invention showed distinct color development only in lane 4, and did not show distinct color development in lanes 2, 3, 5, and 6. Therefore, it was confirmed that the anti-MERS CoV P3 monoclonal antibody according to an embodiment of the present invention specifically reacts with a polypeptide including amino acids 164 to 202 of lane 4.
  • the monoclonal antibody according to one embodiment of the present invention the anti-MERS CoV P3 monoclonal antibody according to one embodiment of the present invention against MERS-CoV prepared in Example 1 from the above results It was confirmed that the 164 to 202th amino acid sequence portion (SEQ ID NO: 4) of cleopacapside (SEQ ID NO: 1) was an epitope.
  • an antibody according to one embodiment of the present invention was used to sample 81 camels (Camel) nasal swabs, and the collected body fluid was used as a sample to diagnose MERS-CoV virus.
  • a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV, and an amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV Kits were prepared comprising monoclonal antibodies (called 'P3') that specifically bind to the indicated epitopes.
  • Monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid are those produced in hybridoma cells having accession number KCLRFBP00331.
  • FIG. 4 is a schematic illustration of the operation of the kit used in one embodiment of the present invention.
  • (A), (b) and (c) of FIG. 4 indicate that the analyte 400 is moved by capillary movement in that order, and the analyte 400 operates with a kit according to one embodiment of the present invention. This is a schematic of the principle. In FIG.
  • the second antibody 200 is immobilized in the detection region T of the chromatography membrane material, and the control region C ) Is an antibody 300 specific for the conjugate of the first antibody and the gold particles.
  • a monoclonal antibody of P1 the second antibody 200, is immobilized in the detection region T, and is conjugated with a gold particle and a monoclonal antibody of P3, the first antibody 100.
  • the presence of the MERS-CoV may be determined by detecting the presence of the complex of P1 and P3 in the detection region.
  • a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV was coated on a plate.
  • monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV are conjugated with gold particles.
  • the conjugation with the gold particles was carried out as follows: Gold chloride was dissolved in distilled water at 0.01% and then heated with addition of 0.1% sodium citrate solution. After heating for about 10 minutes, the mixture was cooled and refrigerated to prepare a colloidal gold solution. Thereafter, anti-MERS-CoV P3 monoclonal antibody was reacted with the colloidal gold solution for 10 minutes to the final concentration of 20 ⁇ g / ml to induce conjugation reaction. After stabilizing the gold particles, centrifuge at 10,000 g for 30 minutes to form a precipitate. The precipitate was again dissolved in physiological saline, filtered through a filter of 0.45 ⁇ m, and then absorbed at 540 nm, diluted to a final 20, and conjugated with gold to anti-MERS-CoV P3.
  • a kit comprising a P1 monoclonal antibody immobilized on a plate and a P3 monoclonal antibody conjugated with gold particles was used as a MERS-CoV diagnostic kit.
  • the positive likelihood ratio is 9.14 and the negative likelihood ratio is 0.29.
  • the positive likelihood ratio was calculated by ⁇ (14/19) / (5/62) ⁇ . Since the positive likelihood ratio was 9.14, it was confirmed that the diagnostic kit including the antigen specifically binding to the epitope of the nucleocapsid of MERS-CoV of the present invention has significance as a diagnostic kit.
  • the negative likelihood ratio was calculated by ⁇ (5/19) / (57/62) ⁇ . In addition, the prevalence by MERS-CoV is 23.46%.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des anticorps monoclonaux qui se lient spécifiquement à la nucléocapside du coronavirus du syndrome respiratoire du Moyen-Orient (MERS-CoV), un anticorps qui est une combinaison de ceux-ci, une composition diagnostique du coronavirus du syndrome respiratoire du Moyen-Orient et un kit comprenant l'anticorps, et un procédé de détection du coronavirus du syndrome respiratoire du Moyen-Orient les utilisant.
PCT/KR2015/000278 2014-11-17 2015-01-12 Anticorps reconnaissant la nuclécapside du coronavirus du syndrome respiratoire du moyen-orient et son utilisation WO2016080591A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20140160059 2014-11-17
KR10-2014-0160059 2014-11-17

Publications (1)

Publication Number Publication Date
WO2016080591A1 true WO2016080591A1 (fr) 2016-05-26

Family

ID=55355293

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/KR2015/000278 WO2016080591A1 (fr) 2014-11-17 2015-01-12 Anticorps reconnaissant la nuclécapside du coronavirus du syndrome respiratoire du moyen-orient et son utilisation
PCT/KR2015/012164 WO2016080701A1 (fr) 2014-11-17 2015-11-12 Anticorps reconnaissant la nucléocapside du coronavirus du syndrome respiratoire du moyen-orient et utilisation de celui-ci

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/012164 WO2016080701A1 (fr) 2014-11-17 2015-11-12 Anticorps reconnaissant la nucléocapside du coronavirus du syndrome respiratoire du moyen-orient et utilisation de celui-ci

Country Status (3)

Country Link
KR (1) KR101593641B1 (fr)
SA (1) SA517381545B1 (fr)
WO (2) WO2016080591A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016080701A1 (fr) * 2014-11-17 2016-05-26 주식회사 바이오노트 Anticorps reconnaissant la nucléocapside du coronavirus du syndrome respiratoire du moyen-orient et utilisation de celui-ci
CN111474340A (zh) * 2020-05-18 2020-07-31 博奥赛斯（天津）生物科技有限公司 一种用于新型冠状病毒检测的酶标记的抗原、制备方法及试剂盒与应用
CN111727199A (zh) * 2018-01-31 2020-09-29 赛特瑞恩股份有限公司 针对中东呼吸综合征-冠状病毒具有中和活性的结合分子

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105624335B (zh) * 2016-03-10 2019-08-27 中山大学达安基因股份有限公司 一种基因检测中东呼吸综合征冠状病毒的试剂盒
KR101782862B1 (ko) 2016-04-26 2017-09-29 원광대학교산학협력단 메르스바이러스 진단용 단세포군 항체 및 면역크로마토그래피 진단키트
WO2019066389A1 (fr) * 2017-09-26 2019-04-04 한국생명공학연구원 Protéine de fusion nc comprenant un fragment de domaine n-terminal et un fragment de domaine c-terminal de la protéine de nucléocapside du coronavirus du srmo, et kit de diagnostic d'une infection à coronavirus du srmo utilisant ladite protéine de fusion nc
WO2019151632A1 (fr) 2018-01-31 2019-08-08 (주)셀트리온 Molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient
KR102019008B1 (ko) * 2019-01-31 2019-09-05 대한민국(관리부서 질병관리본부장) 메르스 코로나바이러스 뉴클레오캡시드 융합 단백질을 이용한 메르스 코로나바이러스 검출 방법
KR20200007980A (ko) 2020-01-02 2020-01-22 조한준 중동 호흡기 증후군 및 중증 급성 호흡기 증후군 바이오마커 및 mers 치료제 개발 후보 화합물
KR102351653B1 (ko) 2020-04-29 2022-01-14 한국화학연구원 사스 코로나바이러스 2 스파이크 단백질과 결합하는 수용체와 항체를 포함하는 사스 코로나바이러스 2 진단 키트
DE102020209412B4 (de) 2020-07-02 2024-02-08 Friedrich-Schiller-Universität Jena Mittel und Verfahren zur An- oder Abreicherung und zum Nachweis von Coronaviren
KR102437114B1 (ko) * 2020-10-21 2022-08-26 재단법인 바이오나노헬스가드연구단 스트렙트아비딘 결합 펩타이드가 태그된 중증급성호흡기증후군 코로나바이러스 특이적 항원 및 이의 용도
US20220244257A1 (en) 2021-01-29 2022-08-04 King Abdulaziz University Method for detection of mers-cov with igy antibodies

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070103548A (ko) * 2006-04-19 2007-10-24 대한민국(관리부서 질병관리본부장) 사스 코로나바이러스 뉴클레오캡시드 단백질에 대한 단클론항체 및 이것의 용도
WO2014045254A2 (fr) * 2012-09-23 2014-03-27 Erasmus University Medical Center Rotterdam Lignée c de coronavirus bêta humains et identification de la peptidase dipeptidylique n-terminale en tant que récepteur viral

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014134439A1 (fr) * 2013-03-01 2014-09-04 New York Blood Center, Inc. Composition immunogène pour infection de coronavirus de syndrome respiratoire du moyen orient (mers-cov)
CN103724406A (zh) * 2013-11-20 2014-04-16 中国疾病预防控制中心病毒病预防控制所 一种具有中和活性的MERS-CoV合成肽疫苗及其应用
CN103864924B (zh) * 2014-02-14 2016-09-07 中国科学院微生物研究所 一种中东呼吸综合征冠状病毒抗体及其制备方法
WO2016080591A1 (fr) * 2014-11-17 2016-05-26 유한회사 바이오노트 Anticorps reconnaissant la nuclécapside du coronavirus du syndrome respiratoire du moyen-orient et son utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070103548A (ko) * 2006-04-19 2007-10-24 대한민국(관리부서 질병관리본부장) 사스 코로나바이러스 뉴클레오캡시드 단백질에 대한 단클론항체 및 이것의 용도
WO2014045254A2 (fr) * 2012-09-23 2014-03-27 Erasmus University Medical Center Rotterdam Lignée c de coronavirus bêta humains et identification de la peptidase dipeptidylique n-terminale en tant que récepteur viral

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AGNIHOTHRAM, S. ET AL.: "Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronavuruses.", J. INFECT. DIS., vol. 209, no. 7, 18 November 2013 (2013-11-18) - 18 November 2013 (2013-11-18), pages 995 - 1006, XP055227760, DOI: doi:10.1093/infdis/jit609 *
CORMAN, V. M. ET AL.: "Antibodies against MERS coronavirus in dromedary camels", EMERG. INFECT. DIS ., vol. 20, no. 8, 1992, Kenya, pages 1319 - 1322 *
SHARMIN, R. ET AL.: "A highly conserved WDYPKCDRA epitope in the RNA directed RNA polymerase of human coronaviruses can be used as epitope-based universal vaccine design.", BMC BIOINFORMATICS., vol. 15, 29 May 2014 (2014-05-29), XP021187264, DOI: doi:10.1186/1471-2105-15-161 *
YING, T. ET AL.: "Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies.", J. VIROL., vol. 88, no. 14, pages 7796 - 7805, XP009181617, DOI: doi:10.1128/JVI.00912-14 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016080701A1 (fr) * 2014-11-17 2016-05-26 주식회사 바이오노트 Anticorps reconnaissant la nucléocapside du coronavirus du syndrome respiratoire du moyen-orient et utilisation de celui-ci
CN111727199A (zh) * 2018-01-31 2020-09-29 赛特瑞恩股份有限公司 针对中东呼吸综合征-冠状病毒具有中和活性的结合分子
CN111474340A (zh) * 2020-05-18 2020-07-31 博奥赛斯（天津）生物科技有限公司 一种用于新型冠状病毒检测的酶标记的抗原、制备方法及试剂盒与应用
CN111474340B (zh) * 2020-05-18 2023-05-23 天津博奥赛斯生物科技股份有限公司 一种用于新型冠状病毒检测的酶标记的抗原、制备方法及试剂盒与应用

Also Published As

Publication number Publication date
KR101593641B1 (ko) 2016-02-12
SA517381545B1 (ar) 2023-02-21
WO2016080701A1 (fr) 2016-05-26

Similar Documents

Publication Publication Date Title
WO2016080591A1 (fr) Anticorps reconnaissant la nuclécapside du coronavirus du syndrome respiratoire du moyen-orient et son utilisation
US9823251B2 (en) Anti-Uroplakin II antibodies systems and methods
WO2023036152A1 (fr) Procédé et kit de détection de sars-cov-2
JP7013051B2 (ja) 牛妊娠関連グリコタンパク質１に特異的に結合する抗体及びその用途
US20080254440A1 (en) Anti-Sars Virus Antibody, Hybridoma Producing the Antibody and Immunoassay Reagent Using the Antibody
RU2366663C2 (ru) Устройство для иммунного анализа с использованием антитела к вирусу гриппа типа b
KR101263913B1 (ko) 우결핵 진단 키트 및 이를 이용한 우결핵 진단 방법
US9116152B2 (en) Monoclonal antibodies which specifically recognize human liver-carboxylesterase 1, hybridoma cell lines which produce monoclonal antibodies, and uses thereof
JP7455108B2 (ja) Ｅ型肝炎ウイルスのＯＲＦ２ｉタンパク質に対する特異性を有する抗体及び診断目的のためのその使用
JPH08311100A (ja) ヒト肺腺癌に関するモノクローナル抗体および抗原、及びそれを用いた免疫測定方法
KR20190138442A (ko) 전염성 췌장 괴저 바이러스 검출용 항체 및 그의 용도
CA2281262C (fr) Anticorps monoclonal anti-medullasine humaine, processus de production et dosage immunologique utilisant cet anticorps moinoclonal
EP3306319B1 (fr) Procédé de détection d'objet d'essai, et instrument de dosage immunologique et anticorps monoclonal associés
US20160266124A1 (en) Pre-haptoglobin-2 monoclonal antibodies and uses thereof
CN115850459B (zh) 靶向新型冠状病毒n蛋白的抗体及其应用
KR100493932B1 (ko) 레지스틴에 대한 단클론 항체, 이의 제조 방법, 및 용도
US10316103B1 (en) Systems and methods for anti-Uroplakin III antibodies
CN115894673B (zh) 一种抗新型冠状病毒的抗体及其在检测中的应用
KR20190138443A (ko) 랍도비리대 과 바이러스 검출용 항체 및 그의 용도
KR20200049388A (ko) 페스트균의 f1 캡슐 단백질에 특이적으로 결합하는 단일클론항체, 상기 항체를 생산하는 하이브리도마 세포주 및 이의 용도
JP5448424B2 (ja) ヒトＩｇＧのＦｃを含有するタンパク質の測定試薬
JP2002306164A (ja) エボラウイルスを認識するモノクローナル抗体
CN102597772B (zh) 5.9kDa肽的免疫学测定方法
JP2000125867A (ja) コイのビテロジェニンに対するモノクロ―ナル抗体
JP4851620B2 (ja) Ｎｋ４に対するモノクローナル抗体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15860780

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15860780

Country of ref document: EP

Kind code of ref document: A1