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WO2003040186A1 - Anticorps monoclonal contre le virus de la maladie de carre et procede de diagnostic de la maladie de carre - Google Patents

Anticorps monoclonal contre le virus de la maladie de carre et procede de diagnostic de la maladie de carre Download PDF

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Publication number
WO2003040186A1
WO2003040186A1 PCT/KR2002/002069 KR0202069W WO03040186A1 WO 2003040186 A1 WO2003040186 A1 WO 2003040186A1 KR 0202069 W KR0202069 W KR 0202069W WO 03040186 A1 WO03040186 A1 WO 03040186A1
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WO
WIPO (PCT)
Prior art keywords
monoclonal antibody
antibody
distemper virus
canine distemper
antigen
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Ceased
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PCT/KR2002/002069
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English (en)
Inventor
Dong-Hyuk Jung
Shien-Young Kang
Kang-Young Kim
In-Joong Yoon
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Definitions

  • the present invention relates to a monoclonal antibody for canine distemper virus and a method of diagnosis of distemper, and more particularly, to a diagnostic kit for quickly detecting canine distemper virus which causes an infection in dogs and frequently leads to decrease of immunity, diarrhea, difficulty in breathing, and fever.
  • BACKGROUND OF THE INVENTION Canine distemper virus frequently causes an infection in dogs, especially pet dogs. Distemper leads to acute or subacute febrile disease and has a high attack rate and mortality (Bush Mitchell, Montali RJ, Brownstein D, James AE, Appel MJG. Vaccine-induced canine distemper in lesser panda. JAVMA 1976; 169: 959-960).
  • Canine distemper virus belongs to the genus Morbillivirus in the family Paramyxovirus (Pringle CR. Paramyxoviridae, Classification and nomenclature of viruses. Arch Virol 1992; 242-246). Canine distemper virus has about 16,000 bp negative single-stranded RNA genomes (Bernard NF, David NK, Peter MH. Fields virology. 3rd ed. Lippincott Raven Publishers, Philadelphia: 1996;1 177-1204). The virus particle has a filament or spherical structure with a diameter of 100 to 700 nm.
  • the virus core contains a nucleocapsid protein, a phosphocapsid protein, a large (L) protein, and a matrix (M) protein, and a fusion (F) protein and an attachment (H) protein are also contained within the envelope (Appel MJCT. Canine distemper virus, In: Virus infections of carnivores. Elsevier Science Publisher,
  • Canine distemper virus is infected through the respiratory organs and is ingested by alveolar macrophages.
  • the macrophages move into the lymph nodes and induce lymph node necrosis, and thereby immunosuppresion, demyelinating encephalitis, and epileptiform convulsions are induced (Comwell HJC, Thomson H, McCandlish IAP, Macartney L and Nash AS: 1988; Encephalitis in dogs associated with a batch of canine distemper (Rockborn) vaccine.
  • Veterinary record 1 12:54-59).
  • dogs After infection, dogs have a low antibody titer against canine distemper virus, but dogs carrying a maternal antibody and vaccinated dogs have a high neutralizing antibody titer (Appel MJG, Gillespie JH. Canine distemper virus. Virol Monogr 1972; 1 1 :1 -94). Therefore, the diagnosis method based on measuring titer of a neutralizing antibody is not suitable for detecting canine distemper virus infection (Shin YS, Mori T, Okita M, Gemma T, Kai C, Mikami T. Detection of canine distemper virus nucleocapsid protein gene in canine peripheral blood mononuclear cells by RT-PCR. J Vet Med Sci 1995; 57:439-445).
  • the object of the present invention is to provide a hybridoma cell line that produces an anti-CDV monoclonal antibody which is capable of specifically binding to an antigen derived from canine distemper virus.
  • Another object of the present invention is to provide an anti-CDV monoclonal antibody.
  • Another object of the present invention is to provide a diagnostic method for canine distemper virus using the monoclonal antibody.
  • Another object of the present invention is to provide a rapid and precise diagnostic kit for canine distemper virus.
  • the present invention provides anti-CDV monoclonal antibody.
  • the present invention also provides a diagnostic kit for canine distemper virus comprising (a) a monoclonal antibody against canine distemper virus, (b) a reactor, and (c) detection means for an antigen- antibody complex.
  • the present invention also provides a diagnostic method comprising mixing a sample solution with a monoclonal antibody against distemper virus and monitoring the formation of antigen-antibody complexes.
  • the inventors isolated a canine distemper virus from feces of animals diagnosed with distemper and produced a monoclonal antibody against the virus.
  • the monoclonal antibody can be produced according to a method as described in Kohler and Milstein's report (Kohler, G. and C. Milstein (1975),
  • the protocol used is as follows: an experimental animal (such as a mouse) is immunized against the antigen of the antibodies to be produced; spleen, lymph node, bone marrow, or tonsil cells of the experimental animal are then fused to cells of a myeloma cell line derived from humans, mice, rats, hamsters, rabbits, or guinea pigs, and the resultant hybridoma fusion cells are plated out on a selective medium; and screening for a specific antibody-producing hybridoma is performed.
  • the screening for a specific antibody-producing hybridoma is undertaken by any suitable technique, for example by confirming antigen- reactivity of a supernatant of a hybridoma culture.
  • a monoclonal antibody can be produced by culturing hybridomas in vitro and isolating the monoclonal antibody from the culture solution or by injecting in vivo. More preferably, the monoclonal antibody is produced by abdominally injecting the hybridoma cells cultured in vitro, collecting the ascites fluids thereof, and purifying the monoclonal antibody according to the general method.
  • the isolation and purification of the monoclonal antibody can be performed thought ion exchange chromatography (DEAE or DE52), and affinity chromatography such as with an anti-immunoglobulin column and a protein A column.
  • the present invention provides a hybridoma cell line capable of expressing and preferably secreting a monoclonal antibody against canine distemper virus.
  • the generation of hybridoma cell lines and the identification of a hybridoma cell line expressing the monoclonal antibody according to the present invention have been described above.
  • the present invention provides a monoclonal antibody which is capable of specifically binding with an antigen derived from canine distemper virus and which is the monoclonal antibody expressed by the hybridoma cell line.
  • the monoclonal antibody of the present invention is highly specific and sensitive.
  • the present invention provides a method for the diagnosis of canine distemper virus infection in animals comprising the step of contacting a test sample of an animals suspected of being infected with a canine distemper virus with a monoclonal antibody, allowing the formation of an antigen-antibody complex, and detecting the presence of the antigen- antibody complex.
  • the test sample is preferably blood, serum, or tissue, and the animals are canines, raccoons, ferrets, and seals.
  • the presence of the antigen-antibody complex is detected by immunoassays which are commonly used in the art for identifying the presence of virus antigens or antibodies in samples taken from humans or animals.
  • the immunoassays include an enzyme-linked immunosorbent assay (EL1SA), an enzyme immunoassay (El A), a florescence immunoassay (FIA), a luminescence immunoassay (LIA), and a radioimmunoassay (RIA).
  • EL1SA enzyme-linked immunosorbent assay
  • El A enzyme immunoassay
  • FIA florescence immunoassay
  • LIA luminescence immunoassay
  • RIA radioimmunoassay
  • the present invention also provides a diagnostic kit comprising a monoclonal antibody and means for detecting whether the antigen is bound to the monoclonal antibody.
  • the diagnostic kit further comprises a reactor, a reaction solution, a buffer, and general tools.
  • the detection means can be selected from the group consisting of an enzyme-linked immunosorbent assay, an enzyme immunoassay, a florescence immunoassay, a luminescence immunoassay, and a radioimmunoassay, but it is not restricted to these.
  • the diagnostic kit can be prepared as an ELISA kit or a strip kit.
  • the ELISA kit comprises a monoclonal antibody which is coated over a surface of a reactor, a buffer, and a means for detecting the presence of an antigen-antibody complex.
  • the detection means comprise a secondary antibody (monoclonal or polyclonal) conjugated with a chromophore or labeled with an isotope, which binds to the canine distemper virus antigens.
  • the chromophore is selected from the group consisting of an enzyme, a florescence compound, and a luminescence compound, and examples of the chromophore are biotin and peroxidase.
  • the substrates for inducing coloring of the chromophore are further contained in the detection means.
  • the strip kit comprises a strip, a monoclonal antibody which is coated at a detection line of the strip, a buffer, and means for detecting the presence of an antigen-antibody complex.
  • the detection means comprise a secondary antibody (monoclonal or polyclonal) conjugated with a chromophore or labeled with an isotope, which binds to the canine distemper virus antigens, or colloidal gold conjugated in a monoclonal antibody.
  • the chromophore is selected from the group consisting of an enzyme, a florescence compound, and a luminescence compound, and examples of the chromophore are biotin and peroxidase.
  • the substrates for inducing coloring of the chromophore are further contained in the detection means.
  • the colloidal gold- labeled monoclonal antibodies were prepared and a control line of a strip was coated with a control antibody (goat anti-mouse IgG), and the detection line was coated with the colloidal gold-labeled monoclonal antibody or attached with a membrane strip including the colloidal gold-labeled monoclonal antibody.
  • a control antibody goat anti-mouse IgG
  • the detection line was coated with the colloidal gold-labeled monoclonal antibody or attached with a membrane strip including the colloidal gold-labeled monoclonal antibody.
  • the diagnostic kit of the present invention can discriminate all positive samples and all negative samples successfully, thereby its specificity and sensitivity values are 100%.
  • EXAMPLE 1 Production of monoclonal antibody against distemper virus 1 . Sample collection
  • Feces were collected from ten dogs suffering from canine distemper at the veterinary hospital of Chung-buk University, and the distemper virus was isolated.
  • the feces sample was diluted in EMEM (Eagles minimum essential medium) by 10% (w/v). The mixture was centrifuged at 5000 rpm for 30 minutes and the supernatant was stored at 20 ° C . 2. Production of monoclonal antibody
  • the collected virus was cultured and purified, and was mixed with incomplete Freunds Adjuvant in ratio of 1 :1 .
  • 0.5 ug of the mixture was injected abdominally to a BALB/C mouse and the mouse was further immunized twice at intervals of 2 weeks in the same manner as above.
  • the spleen of the immunized mouse was aseptically extracted and then transferred to a culture dish.
  • the spleen was caused to pass through a mesh with a spatula.
  • the resultant suspension of spleen cells in the culture dish was collected in a 50 ml centrifugal tube, and Dalbecco's phosphoric acid buffer was added thereto.
  • the resultant mixture was subjected to centrifugation (1000 rpm for 5 minutes) to wash the cells.
  • 1 x 10 6 of the mouse spleen cells were added to about 1 x 10 5 of mouse myeloma SP-2 cells which were prepared beforehand, and then mixed.
  • the medium was removed by centrifuging at 1000 g for 10 minutes and subsequently 2 ml of 50% polyethylene glycol solution were added to the pellet of the mixed spleen and myeloma cells, and the resultant mixture was mixed for 2 minutes.
  • a serum-free medium was added thereto, and a washing operation was carried out once. Further, the same medium was added again, and the resultant mixture was added into respective wells of a 96-well plate for culturing cells so that the number of cells before cell fusion would be 5 to 10 in each well.
  • a HAT medium was added to the wells and the 96-well plate was placed in a 5% CO 2 gas incubator. After the spleen cells and the myeloma cells were fused, the HAT medium was substituted by an HT medium to obtain hybridomas (3C6, 3D10, 6D7, 6E10 and 8B2), and hybridomas generating a desired antibody were selected. The hybridomas were stored in a nitrogen tank.
  • mice The abdominal cavities of BALB/C mice were injected with 1 x 10 6 hybridoma cells cultured in vitro. After about 1 week, the ascites fluids of the mice were collected and then were purified by the general method including affinity chromatography and ion exchange chromatography to obtain the monoclonal antibody.
  • CDV monoclonal antibodies (sample No. 3C6, 3D10, 6D7, 6E10 and 8B2) were obtained.
  • Test Canine parvovirus 780916-LP
  • canine coronavirus CMV
  • A72 cells were infected with canine distemper virus, canine
  • infected cells 24 - 48hrs in CO 2 incubator, infected cells were fixed with 80 % acetone, CDV monoclonal antibodies (MAbs) were added thereto and incubated.
  • MAbs CDV monoclonal antibodies
  • CDV was coated on 96-well plate and each well was added with anti-CDV monoclonal antibodies. After HRP-conjugated goat anti-mouse IgG was added to each well, ABT solution was added thereto and adsorption was measured at 405 nm in spectrophotometer.
  • 1% HAuCI 4 was added to boiling distilled water so that the final concentration of HAuCI 4 was 1.5 v/v% to 3 v/v%. After reaction, 4% HAuCI 4 was slowly added thereto to 0.2 to 0.3 v/v% of the final concentration, and the mixture was cooled at RT. The generated colloidal gold was observed with electron microscopy, and colloidal gold having a diameter of 40 nm was selected.
  • the selected colloidal gold was added to 1 to 10 tg/mL of the
  • distemper virus were prepared, and 100 ⁇ of the samples were
  • the present invention provides a sensitive, rapid, and specific monoclonal antibody against canine distemper virus so that canine distemper virus infection in animals can be easily detected.

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Abstract

Cette invention se rapporte à un anticorps monoclonal contre le virus de la maladie de Carré, à un procédé de diagnostic de la maladie de Carré et à un kit de diagnostic rapide. Cette invention offre un anticorps monoclonal sensible, rapide et spécifique contre le virus de la maladie de Carré, qui permet de détecter aisément toute infection chez des animaux par le virus de la maladie de Carré.
PCT/KR2002/002069 2001-11-07 2002-11-06 Anticorps monoclonal contre le virus de la maladie de carre et procede de diagnostic de la maladie de carre Ceased WO2003040186A1 (fr)

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KR1020010069271A KR20020066171A (ko) 2001-11-07 2001-11-07 개 디스템퍼 바이러스에 대한 모노클로날 항체 및 이를이용한 진단키트
KR2001/69271 2001-11-07

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006109311A3 (fr) * 2005-04-15 2007-05-31 Univ Ramot Biocapteurs electrochimiques bases sur la canalisation d'enzymes
CN102618503A (zh) * 2012-03-26 2012-08-01 江苏省农业科学院 一种分泌高中和活性犬瘟热病毒单克隆抗体的杂交瘤细胞1d7株
CN103992988A (zh) * 2014-04-11 2014-08-20 吉林特研生物技术有限责任公司 一种杂交瘤细胞株及其产生的抗犬瘟热病病毒n蛋白的单克隆抗体
CN111856015A (zh) * 2019-04-24 2020-10-30 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 鸭瘟病毒抗体检测试剂盒及其应用
CN112876561A (zh) * 2021-01-14 2021-06-01 中国农业科学院特产研究所 一种用于检测犬瘟热病毒的抗体对及其应用
CN116462754A (zh) * 2023-06-12 2023-07-21 北京纳百生物科技有限公司 一种识别犬瘟热病毒n蛋白的单克隆抗体、检测试剂及应用
CN119125534A (zh) * 2023-12-20 2024-12-13 中山迈托姆生物技术有限公司 一种犬瘟热疫苗中和抗体检测方法及应用

Families Citing this family (3)

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KR100593286B1 (ko) * 2004-04-14 2006-06-26 (주)에니젠 신속 면역크로마토그라피법에 의한 개 디스템퍼 바이러스 항체 진단키트
KR101595445B1 (ko) * 2013-12-31 2016-02-19 대한민국 수정진동자 바이오센서를 구비하는 개 디스템퍼 바이러스 검출 장치 및 이를 이용한 검출 방법
CN115094043B (zh) * 2022-08-02 2023-08-25 长春西诺生物科技有限公司 一种用于犬冠状病毒与犬细小病毒的杂交瘤细胞株及单克隆抗体和应用

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DATABASE MEDLINE [online] ORVELL C. ET AL.: "Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus", Database accession no. (NLM2579191) *
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006109311A3 (fr) * 2005-04-15 2007-05-31 Univ Ramot Biocapteurs electrochimiques bases sur la canalisation d'enzymes
CN102618503A (zh) * 2012-03-26 2012-08-01 江苏省农业科学院 一种分泌高中和活性犬瘟热病毒单克隆抗体的杂交瘤细胞1d7株
CN103992988A (zh) * 2014-04-11 2014-08-20 吉林特研生物技术有限责任公司 一种杂交瘤细胞株及其产生的抗犬瘟热病病毒n蛋白的单克隆抗体
CN103992988B (zh) * 2014-04-11 2016-08-24 吉林特研生物技术有限责任公司 一种杂交瘤细胞株及其产生的抗犬瘟热病病毒n蛋白的单克隆抗体
CN111856015A (zh) * 2019-04-24 2020-10-30 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 鸭瘟病毒抗体检测试剂盒及其应用
CN111856015B (zh) * 2019-04-24 2023-08-04 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 鸭瘟病毒抗体检测试剂盒及其应用
CN112876561A (zh) * 2021-01-14 2021-06-01 中国农业科学院特产研究所 一种用于检测犬瘟热病毒的抗体对及其应用
CN112876561B (zh) * 2021-01-14 2021-11-16 中国农业科学院特产研究所 一种用于检测犬瘟热病毒的抗体对及其应用
CN116462754A (zh) * 2023-06-12 2023-07-21 北京纳百生物科技有限公司 一种识别犬瘟热病毒n蛋白的单克隆抗体、检测试剂及应用
CN116462754B (zh) * 2023-06-12 2023-09-12 北京纳百生物科技有限公司 一种识别犬瘟热病毒n蛋白的单克隆抗体、检测试剂及应用
CN119125534A (zh) * 2023-12-20 2024-12-13 中山迈托姆生物技术有限公司 一种犬瘟热疫苗中和抗体检测方法及应用

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