KR20020066171A - Monoclonal antibody for canine distemper virus and test kit for canine distemper virus using the same - Google Patents
Monoclonal antibody for canine distemper virus and test kit for canine distemper virus using the same Download PDFInfo
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Abstract
Description
[발명이 속하는 기술분야][TECHNICAL FIELD OF THE INVENTION]
본 발명은 개 디스템퍼 바이러스에 대한 모노클로날 항체 및 이를 이용한 개 디스템퍼 바이러스 진단키트에 관한 것으로, 보다 상세하게는 개의 면역저하를 유발하여 2차 감염에 의한 설사, 호흡장애, 발열 등의 다양한 임상증상을 유발하는 개 디스템퍼 바이러스를 신속하게 진단하는 진단키트에 관한 것이다.The present invention relates to a monoclonal antibody against the dog distemper virus and a dog distemper virus diagnostic kit using the same. More specifically, the present invention relates to a dog distemper virus diagnosis kit. The present invention relates to a diagnostic kit for quickly diagnosing a dog distemper virus.
[종래기술][Private Technology]
애완견에서 빈발하는 바이러스 감염성 질병의 하나인 개 디스템퍼 바이러스(Canine distemper virus)는 전염성이 매우 높은 급성 또는 아급성의 열성 전염병을 유발하여 높은 이환율과 폐사율을 나타내는 바이러스성 질병이다.(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, one of the most common viral infectious diseases in dogs, is a viral disease with high morbidity and mortality caused by highly infectious acute or subacute recessive diseases (Bush Mitchell, Montali RJ). , Brownstein D, James AE, Appel MJG.Vaccine-induced canine distemper in lesser panda.JAVMA 1976; 169: 959-960)
개 디스템퍼 바이러스는 파라믹소바이러스(Paramyxovirus)과의 모르빌리바이러스(Morbillivirus)속에 속한다.(Pringle CR. Paramyxoviridae, Classification and nomenclature of viruses. Arch Virol 1992;242-246)Canine distemper virus belongs to the genus Morbillivirus with Paramyxovirus (Pringle CR. Paramyxoviridae, Classification and nomenclature of viruses. Arch Virol 1992; 242-246)
개 디스템퍼 바이러스는 (-) 단일가닥 RNA로서 크기는 약 16,000 bp이다.(Bernard NF, David NK, Peter MH. Fields virology. 3rd ed. Lippincott Raven Publishers, Philadelphia : 1996;1177-1204)Canine distemper virus is a negative single-stranded RNA, approximately 16,000 bp in size (Bernard NF, David NK, Peter MH. Fields virology. 3rd ed. Lippincott Raven Publishers, Philadelphia: 1996; 1177-1204).
바이러스의 모양은 구형 또는 필라멘트형태로 100 내지 700 nm의 크기를 나타내며, 뉴클레오캡시드와 관련된 3개의 뉴클레오캡시드(nucleocapsid: NP) 단백질, 포스포캡시드 단백질, 포스포 단백질 및 거대 단백질(large(L) protein)이 있다. 또한 막과 관련된 기질단백질(matrix(M) protein), 융합단백질(fusion(F) protein) 및 부착단백질(attachment(H) protein)으로 구성되어 있다.(Appel MJCT. Canine distemper virus, In : Virus infections of carnivors. Elsevier Science Publisher, Amsterdam : 1987;1:133-159)The shape of the virus is 100 to 700 nm in spherical or filamentous form, with three nucleocapsid (NP) proteins, phosphocapsid proteins, phosphoproteins and large proteins (L) (L) associated with nucleocapsids. ) protein). It is also composed of membrane-related matrix proteins (matrix (M) protein, fusion (F) protein) and attachment protein (attachment (H) protein). (Appel MJCT.Canine distemper virus, In: Virus infections of carnivors.Elsevier Science Publisher, Amsterdam: 1987; 1: 133-159)
개 디스템퍼 바이러스는 주로 호흡기를 통하여 감염되며, 감염된 바이러스는 폐의 대식세포에 탐식된 후 임파절로 이동하여 임파절 괴사를 일으킨다. 그 결과로 면역저하를 유발하고, 탈수초성뇌염(demyelinating encephalitis)으로 인한 간질성 경련(epileptiform convulsion)을 유발한다.(Cornwell 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, 112:54-59)Canine distemper virus is mainly infected through the respiratory tract, and the infected virus is phagocytosed into the macrophages of the lungs and then migrates to the lymph nodes causing lymph node necrosis. The result is immunodeficiency and epileptiform convulsions due to demyelinating encephalitis (Cornwell 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, 112: 54-59)
개 디스템퍼는 감염 초기에는 항체가가 낮지만 모체이행항체를 보유한 개와예방접종을 받은 개에게서는 높은 중화항체가를 나타낸다.(Appel MJG, Gillespie JH. Canine distemper virus. Virol Monogr 1972; 11:1-94) 따라서, 중화항체를 검사하여 감염여부를 진단하는 것은 정확하지 않다.(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)Canine distemper has low antibody titer early in infection but high neutralizing antibody titer in dogs vaccinated with maternally-transfected dogs (Appel MJG, Gillespie JH. Canine distemper virus. Virol Monogr 1972; 11: 1-94 Therefore, it is not accurate to test for neutralizing antibodies to diagnose 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)
개 디스템퍼의 확실한 진단을 위하여는 바이러스를 직접 분리하거나 바이러스 항원에 대한 신속하고 높은 특이성을 갖는 방법이 필요한 현실이나, 바이러스를 분리하기 위하여는 수일에서 수주까지의 장시간이 소요되어 실질적인 진단방법으로 적용할 수 없다.In order to reliably diagnose dog distemper, it is necessary to isolate virus directly or have rapid and high specificity for virus antigen, but it takes a long time from several days to several weeks to isolate the virus. Can't.
최근에 RT-PCR(reverse transcription-polymerase chain reaction) 방법에 의하여 진단하는 방법을 대학 동물병원 등에서 시행하고 있지만 시료의 전처리가 필요하고 최소 수시간 소요되는 단점이 있어 일반 동물병원에서는 적용하기 어려운 문제가 있어 보다 신속하면서 정확한 진단법이 요구된다.Recently, the method of diagnosing by reverse transcription-polymerase chain reaction (RT-PCR) has been performed in university animal hospitals, but it is difficult to apply in general animal hospitals because it requires the pretreatment of samples and takes at least several hours. More rapid and accurate diagnostics are required.
본 발명은 개 디스템퍼 바이러스에 대한 모노클로날 항체를 생산할 수 있는 하이브리도마를 제공하는 것을 목적으로 한다.An object of the present invention is to provide a hybridoma capable of producing monoclonal antibodies against canine distemper virus.
또한 본 발명은 개 디스템퍼 바이러스에 대한 모노클로날 항체를 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a monoclonal antibody against canine distemper virus.
또한 본 발명은 모노클로날 항체를 이용한 개 디스템퍼 바이러스의 진단방법을 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a method for diagnosing canine distemper virus using a monoclonal antibody.
또한 본 발명은 신속, 간편하게 개 디스템퍼 바이러스를 진단할 수 있는 진단키트를 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a diagnostic kit that can quickly and easily diagnose the dog distemper virus.
상기 목적을 달성하기 위하여 본 발명은 항원 개 디스템퍼 바이러스(Canine distemper virus)에 대한 모노클로날 항체를 제공한다.In order to achieve the above object, the present invention provides a monoclonal antibody against the antigen canine distemper virus.
또한 본 발명은In addition, the present invention
(a) 개 디스템퍼 바이러스 대한 모노클로날 항체;(a) monoclonal antibodies against canine distemper virus;
(b) 모노클로날 항체를 포함하는 반응기; 및(b) a reactor comprising a monoclonal antibody; And
(c) 항원-항체반응을 확인하기 위한 검출 수단;(c) detection means for confirming the antigen-antibody response;
을 포함하는 개 디스템퍼 바이러스 진단키트를 제공한다.It provides a dog distemper virus diagnostic kit comprising a.
또한 본 발명은 동물의 혈청에 대하여 상기의 모노클로날 항체로 분석하여 이루어지는 개 디스템퍼 바이러스의 진단방법을 제공한다.The present invention also provides a method for diagnosing a dog distemper virus, which is obtained by analyzing the serum of an animal with the above monoclonal antibody.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 디스템퍼로 진단된 동물의 분변을 채취하여 디스템퍼 바이러스를 분리하였고, 상기 바이러스에 대한 모노클로날 항체를 제조하였다.We isolated the distemper virus by collecting feces from animals diagnosed with distemper, and prepared monoclonal antibodies against the virus.
모노클로날 항체를 분비하는 하이브리도마의 제조는 케일러 및 밀슈타인 등의 방법(Nature, 256, p495∼497, 1975) 및 그에 준하는 방법에 따라 실시할 수 있다. 즉 면역화된 포유동물로부터 취득되는 비장, 임파절, 골수 또는 편도세포와 마우스, 래트,모르못, 햄스터, 토끼 또는 사람 등의 포유동물에 유래한골수종(myeloma) 세포를 융합시킴으로써 제조한다. 바람직하기는 비장에 함유된 항체 생성 세포와 마우스, 래트 또는 사람에 유래한 자기 항체 생성능이 없는 골수종 세포를 세포 융합시킴으로써 제조한다.The production of hybridomas that secrete monoclonal antibodies can be carried out according to methods such as Kale and Milstein (Nature, 256, p495-497, 1975) and the corresponding methods. In other words, it is prepared by fusing spleen, lymph node, bone marrow or tonsil cells obtained from an immunized mammal with myeloma cells derived from mammals such as mice, rats, morphs, hamsters, rabbits or humans. Preferably, the antibody-producing cells contained in the spleen are prepared by cell fusion of myeloma cells having no ability to produce autoantibodies derived from mice, rats or humans.
모노클로날 항체를 생성하는 하이브리도마의 스크리닝은 하이브리도마를 배양하고, 증식이 보인 웰의 배양 상징액의 항원반응성을 측정하여 실시할 수 있다.Screening of hybridomas that produce monoclonal antibodies can be carried out by culturing the hybridomas and measuring the antigenic reactivity of the culture supernatant of the wells showing growth.
하이브리도마로부터 모노클로날 항체의 제조는 하이브리도마를 인비트로(in vitro)로 배양하여 제조된 모노클로날 항체를 분리하거나, 생체내주입하여 분리할 수 있다. 바람직하기로는 마우스, 래트, 햄스터 또는 토끼 등의 복수에 삽입하고 복수로부터 분리, 정제하는 것이다.Production of monoclonal antibodies from hybridomas can be performed by separating the monoclonal antibodies prepared by culturing the hybridomas in vitro, or by in vivo injection. Preferably, it inserts into the ascites of a mouse, a rat, a hamster, a rabbit, etc., and isolates and purifies from the ascites.
모노클로날 항체의 분리 및 정제는 배양 상징액 또는 복수를 이온교환 크로마토그래피(DEAE 또는 DE52 등), 항 면역글로불린 칼럼 또는 프로테인 A컬럼 등의 친화성 크로마토그래피를 이용하여 실시할 수가 있다.Isolation and purification of monoclonal antibodies can be carried out using affinity chromatography such as ion supernatant chromatography (DEAE or DE52), anti-immunoglobulin column or protein A column, or the culture supernatant or ascites.
또한 본 발명은 디스템퍼 모노클로날 항체를 이용한 개 디스템퍼 진단방법을 제공한다. 진단방법은 모노클로날 항체를 동물의 혈청에 반응시켜 반응성을 확인하는 것이다. 이때 반응성 유무의 검출은 항원-항체반응을 확인할 수 있는 통상의 방법으로 실시가능하다. 바람직하기로는 효소면역정량법(EIA), 형광면역정량법(FIA), 발광면역정량법(LIA) 또는 방사능면역정량법(RIA)가 있으며, 이에 한정되는 것은 아니다.In another aspect, the present invention provides a dog distemper diagnostic method using a distemper monoclonal antibody. The diagnostic method is to check the reactivity by reacting the monoclonal antibody to the serum of the animal. At this time, the detection of the presence or absence of reactivity can be carried out by a conventional method capable of confirming the antigen-antibody reaction. Preferably, enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA) or radioimmunoassay (RIA) is not limited thereto.
또한 본 발명은 디스템퍼 진단키트를 제공한다.The present invention also provides a distemper diagnostic kit.
상기 진단키트는 디스템퍼 모노클로날 항체, 반응기, 검출수단 및 반응액을포함하며, 이외 통상적인 도구 또는 완충액을 더욱 포함할 수 있다. 검출수단은 효소면역정량법(EIA), 형광면역정량법(FIA), 발광면역정량법(LIA) 또는 방사능면역정량법(RIA)이 있을 수 있으며, 이에 한정되지 않는다.The diagnostic kit includes a distemper monoclonal antibody, a reactor, a detection means, and a reaction solution, and may further include other conventional tools or buffers. The detection means may include, but is not limited to, enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), or radioimmunoassay (RIA).
바람직한 디스템퍼 진단키트는 디스템퍼 모노클로날 항체를 포함하는ELISA 키트 또는 스트립 키트이다. ELISA 키트는 디스템퍼 모노클로날 항체가 코팅된 반응기, 세척완충액, 검출수단을 포함한다. 검출수단의 예로 바이오틴 또는 퍼옥시다아제를 포함하는 이차항체 및 발색기질이 있다. 스트립 키트는 스트립에 디스템퍼 모노클로날 항체를 흡수 또는 코팅시키고, 여기에 면역반응을 검출하기 위한 검출수단을 반응시키거나, 스트립에 검출수단을 포함하는 디스템퍼 모노클로날 항체를 흡수 또는 코팅시켜 제조할 수 있다.Preferred distemper diagnostic kits are ELISA kits or strip kits comprising distemper monoclonal antibodies. The ELISA kit includes a reactor coated with distemper monoclonal antibodies, wash buffer, and detection means. Examples of detection means include secondary antibodies and color substrates, including biotin or peroxidase. The strip kit may be prepared by absorbing or coating a distemper monoclonal antibody on a strip, reacting a detection means therewith for detecting an immune response, or absorbing or coating a distemper monoclonal antibody comprising a detection means on the strip. Can be.
본 발명에서는 디스템퍼 바이러스 모노클로날 항체와 금입자를 각각 제조한 후 항체-금입자 결합체를 제조하였다. 스트립의 대조군 검사선은 대조군(goat anti-mouse IgG)으로 코팅하고, 검사선은 디스템퍼 바이러스 모노클로날 항체로 코팅하였다. 또한 항체-금입자(콜로이달 골드)를 막 스트립에 부착시킨 후 상기 막 스트립을 스트립에 부착하였다.In the present invention, a distemper virus monoclonal antibody and a gold particle were prepared, respectively, and then an antibody-gold particle conjugate was prepared. The control test line of the strips was coated with a control (goat anti-mouse IgG) and the test line was coated with a distemper virus monoclonal antibody. The antibody-gold particles (colloidal gold) were also attached to the membrane strips followed by attachment of the membrane strips to the strips.
상기 스트립을 시료에 반응시키면, 시료내 디스템퍼 바이러스 항원은 디스템퍼 바이러스 모노클로날 항체-금입자와 결합되어 확산에 의해 스트립을 따라 올라가다가 검사선상의 디스템퍼 바이러스 모노클로날 항체와 결합함으로써 발색선을 나타내게 된다.When the strip is reacted with the sample, the distemper virus antigen in the sample binds to the distemper virus monoclonal antibody-gold particle and rises along the strip by diffusion and binds to the distemper virus monoclonal antibody on the test line to give a color line. do.
본 발명의 디스템퍼 바이러스 진단키트를 사용하여 디스템퍼 바이러스 감염유무를 확인한 결과, RT-PCR방법에서 개 디스템퍼 바이러스 양성으로 확인된 시료는 진단키트에서도 모두 양성으로 판정되었으며, RT-PCR에서 음성인 시료는 음성으로 판정되어 특이도, 민감도가 100 %임을 확인할 수 있었다.As a result of confirming the presence of distemper virus infection using the distemper virus diagnosis kit of the present invention, all the samples identified as dog distemper virus positive by the RT-PCR method were determined as positive in the diagnostic kit, and the samples negative in the RT-PCR were negative. It was determined that the specificity, sensitivity was 100%.
이하 본 발명의 실시예를 기재한다. 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명이 하기 실시예에 한정되는 것은 아니다.Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.
실시예 1: 디스템퍼 모노클로날 항체의 생산Example 1: Production of Distemper Monoclonal Antibodies
1.분변 재료1. Fecal material
충북대학교 동물병원에서 디스템퍼로 진단한 개의 분변에서 총 10개의 분변을 수집하여 디스템퍼 바이러스를 분리하였다.Distemper virus was isolated by collecting 10 feces from dog feces diagnosed with distemper at Chungbuk National University Animal Hospital.
분변재료는 EMEM(Eagles minimum essential medium)에 10 %(w/v)가 되도록 섞어서 5,000 rpm에서 30분간 원심분리하여 상층액을 20 ℃에 보관하여 사용하였다.Fecal material was mixed to 10% (w / v) in EMEM (Eagles minimum essential medium) and centrifuged at 5,000 rpm for 30 minutes to store the supernatant at 20 ℃.
2.모노클로날 항체의 생산2. Production of Monoclonal Antibodies
분리된 바이러스를 배양 정제하여 불완전 프레운즈 보조체(incomplete Freunds Adjuvant)와 1:1로 혼합한 다음 Balb/c 마우스에 바이러스 항원량이 5 ㎍/마리 되도록 복강내에 접종하고 2주후에 동량을 접종하였다. 마지막 접종후 2주째에 3차 접종을 시행하고 3차 접종 4일후 마우스의 비장세포를 채취하였다.The isolated virus was cultured, purified, mixed 1: 1 with incomplete Freunds Adjuvant, and then inoculated to Balb / c mice intraperitoneally so that the virus antigen amount was 5 μg / ml, and 2 weeks later, the same amount was inoculated. The third dose was given 2 weeks after the last inoculation, and splenocytes of mice were collected 4 days after the third dose.
비장세포의 수를 측정하여 106개 되도록 하여 sp2/0 세포 105개와 혼합한 후 1000 g에서 10분간 원심분리하여 상층을 제거하고, 37 ℃의 폴리에틸렌 글리콜 50%용액 2 mL를 원심침전에 점적투여하였다. 여기에 무혈청 배지를 20 mL 첨가하고, 1000 g에서 10분간 원심분리하여 상층을 제거하였다. 침전물을 무혈청 배지로 부유하여 5-10개의 세포가 되도록 하여 96 웰 플레이트에 옮기고 HAT배지를 각 웰에 채웠다. CO2항온기에서 세포를 배양하면서 세포융합여부를 관찰하고 세포가 융합되면 HT배지로 배지를 교체하였다. 융합세포가 증식되면 HT배지에 세포를 옮겨 다시 증식시키는 방법으로 반복하면서 모노클로날 항체를 발현하는 클론을 얻었다. 클론의 일부는 196 ℃ 질소 탱크에 보관하고 일부는 다시 배양하여 세포 부유액을 만들었다.The number of splenocytes was measured to make 10 6, mixed with 10 5 sp2 / 0 cells, centrifuged at 1000 g for 10 minutes to remove the upper layer, and 2 mL of a polyethylene glycol 50% solution at 37 ° C. Administered. 20 mL of serum-free medium was added thereto, and the upper layer was removed by centrifugation at 1000 g for 10 minutes. The precipitate was suspended in serum-free medium to give 5-10 cells, transferred to a 96 well plate and filled with HAT medium in each well. Cell fusion was observed while culturing the cells in a CO 2 incubator, and when the cells were fused, the medium was replaced with HT medium. When the fusion cells were propagated, the cells were transferred to HT medium to be reproliferated, thereby obtaining a clone expressing the monoclonal antibody. Some of the clones were stored in a 196 ° C. nitrogen tank and some were recultured to make cell suspensions.
세포 부유액을 마우스의 복강에 접종(마리당 106개)하고 일주일 후 복수를 채취하여 친화성 크로마토그라피(affinity chromatography)와 이온교환 크로마토그라피(ion exchange chromatography)법을 응용한 일반적인 모노클로날 항체의 정제방법으로 정제하였다.Cell suspensions were inoculated into the abdominal cavity of mice (10 6 per horse), and a week later, ascites was collected and purification of general monoclonal antibodies using affinity chromatography and ion exchange chromatography was performed. Purification by the method.
실시예 2: 진단키트 제조Example 2: Diagnostic Kit Preparation
1. 금입자 제조1. Gold particle manufacturing
끓는 증류수에 1 % 구연산나트륨(HauCl4)을 1.5 v/v%, 3 v/v% 되도록 첨가하여 반응시킨 후 4 % 구연산나트륨 용액을 0.2-0.3 v/v% 되도록 첨가후 상온 냉각하였고 전자현미경으로 관찰하였다.After adding 1% sodium citrate (HauCl 4 ) to 1.5 v / v% and 3 v / v% in boiling distilled water, 4% sodium citrate solution was added to 0.2-0.3 v / v% and cooled to room temperature. Observed by.
2.모노클로날 항체-금입자의 결합체 제조2. Preparation of conjugates of monoclonal antibody-gold particles
40 nm 크기의 금입자에 모노클로날 항체를 1-10 ㎍/mL 되도록 반응시킨 후소혈청 알부민을 2 %을 포함하는 폴리에틸렌 글리콜 20,000을 최종 0.2 %로 혼합한 후 20,000 g, 1시간 원심분리하여 침전을 얻었다.After monoclonal antibody was reacted to 1-10 ㎍ / mL of gold particles of 40 nm size, polyethylene glycol 20,000 containing 2% of bovine serum albumin was mixed with 0.2% of final concentration, and then precipitated by centrifugation for 20,000 g for 1 hour. Got.
3.진단키트의 제조3. Manufacture of diagnostic kit
실시예 1의 모노클로날 항체-금입자 결합체를 포함하는 용액을 막 스트립(membrane strip)에 0.1-1 ㎕ 흡수시키고, 테스트 스트립에 검사라인과 대조군라인을 그었다. 검사라인에는 항 디스템퍼 바이러스 모노클로날 항체를 0.1-1㎕로, 대조군 라인에는 염소 항-마우스 IgG를 0.1-1㎕ 되도록 흡수시켰다. 막 스트립과 테스트 스트립을 샘플을 받아들일 수 있는 패드에 접착하여 진단시약의 형태를 갖추었다.The solution containing the monoclonal antibody-gold particle conjugate of Example 1 was absorbed into the membrane strip by 0.1-1 μl, and test and control lines were drawn on the test strip. The test line was absorbed with 0.1-1 μl of anti-temper virus monoclonal antibody and the control line with 0.1-1 μl of goat anti-mouse IgG. Membrane strips and test strips were bonded to pads that could accept samples to form diagnostic reagents.
실시예 3: 개 디스템퍼 바이러스의 검출Example 3: Detection of Canine Distemper Virus
개 디스템퍼 바이러스의 농도가 1 ㎍/100㎕ - 0.1 ng/100㎕ 되도록 샘플을 제조하여 실시예 2의 신속진단키트에 100 ㎕씩 반응시켰다.Samples were prepared such that the concentration of the dog distemper virus was 1 μg / 100 μl-0.1 ng / 100 μl, and 100 μl was reacted to the rapid diagnosis kit of Example 2.
1 ㎍-10 ng의 농도에서는 샘플을 반응시킨지 1분 후부터 검사라인에 양성결과를 눈으로 관찰할 수 있었고, 1 ng에서는 5-10분 후 알아 볼 수 있는 수준이었으나 1 ng이하에서는 관찰이 불가능하여 검출한계는 1 ng이었다.At 1 ㎍-10 ng, positive results were observed in the test line 1 minute after the sample was reacted, and at 1 ng, it was recognizable after 5-10 minutes, but not below 1 ng. The detection limit was 1 ng.
실시예 4: 개 디스템퍼 바이러스의 진단키트의 정확도 측정Example 4 Measurement of Accuracy of Diagnostic Kit of Canine Distemper Virus
서울, 충청지역의 디스템퍼 환축 50마리를 대상으로 RT-PCR법과 실시예 1의 진단키트를 이용한 검사를 실시하였다.50 distemper cyclists in Chungcheong, Seoul, were tested using the RT-PCR method and the diagnostic kit of Example 1.
RT-PCR 양성으로 나타난 샘플은 신속진단시약에서도 모두 양성으로 판독되어 민감도 100 %이었고, 같은 지역에서의 디스템퍼 음성인 애완견의 혈청 50개에서는신속진단시약에서도 모두 음성으로 판독되어 특이도가 100 %였다.RT-PCR-positive samples were all read positive for the rapid diagnostic reagents and were 100% sensitive, and 50 sera of distemper-negative dogs in the same region were all negative for the rapid diagnostic reagents and 100% specificity. .
상기에 언급한 바와 같이, 애완견에서 흔히 발병하는 디스템퍼 바이러스의 항원검사용 신속진단시약은 검출한계, 민감도 및 특이도가 우수하였다. 따라서 본 발명은 개 디스템퍼 바이러스 신속진단시약으로서 사용할 수 있다.As mentioned above, rapid diagnostic reagents for antigen testing of distemper virus, which commonly occurs in dogs, were excellent in detection limit, sensitivity and specificity. Therefore, the present invention can be used as a dog distemper virus rapid diagnostic reagent.
Claims (4)
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| KR1020010069271A KR20020066171A (en) | 2001-11-07 | 2001-11-07 | Monoclonal antibody for canine distemper virus and test kit for canine distemper virus using the same |
| PCT/KR2002/002069 WO2003040186A1 (en) | 2001-11-07 | 2002-11-06 | Monoclonal antibody against canine distemper virus and method of diagnosis of distemper |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020010069271A KR20020066171A (en) | 2001-11-07 | 2001-11-07 | Monoclonal antibody for canine distemper virus and test kit for canine distemper virus using the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20020066171A true KR20020066171A (en) | 2002-08-14 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020010069271A KR20020066171A (en) | 2001-11-07 | 2001-11-07 | Monoclonal antibody for canine distemper virus and test kit for canine distemper virus using the same |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20020066171A (en) |
| WO (1) | WO2003040186A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100593286B1 (en) * | 2004-04-14 | 2006-06-26 | (주)에니젠 | Kit for detecting canine distemper virus antibody using immunochromatography |
| KR20150080277A (en) * | 2013-12-31 | 2015-07-09 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Apparatus for detecting Canine distemper virus comprising quartz crystal microbalance biosensor and detecting method using thereof |
| CN115094043A (en) * | 2022-08-02 | 2022-09-23 | 长春西诺生物科技有限公司 | Hybridoma cell strain for canine coronavirus and canine parvovirus, monoclonal antibody and application |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090061524A1 (en) * | 2005-04-15 | 2009-03-05 | Judith Rishpon | Enzyme-Channeling Based Electrochemical Biosensors |
| CN102618503B (en) * | 2012-03-26 | 2013-06-12 | 江苏省农业科学院 | Hybrid tumor cell strain 1D7 capable of secreting high neutralizing activity canine distemper virus monoclonal antibody |
| CN103992988B (en) * | 2014-04-11 | 2016-08-24 | 吉林特研生物技术有限责任公司 | A kind of monoclonal antibody of the anti-canine distemper disease viral N proteins of hybridoma cell strain and generation thereof |
| CN111856015B (en) * | 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) | Duck plague virus antibody detection kit and application thereof |
| CN112876561B (en) * | 2021-01-14 | 2021-11-16 | 中国农业科学院特产研究所 | Antibody pair for detecting canine distemper virus and application thereof |
| CN116462754B (en) * | 2023-06-12 | 2023-09-12 | 北京纳百生物科技有限公司 | Monoclonal antibody for identifying N protein of canine distemper virus, detection reagent and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60130600A (en) * | 1983-12-16 | 1985-07-12 | Nippon Zenyaku Kogyo Kk | Anti-canine-distemper monoclonal antibody and assay therewith |
-
2001
- 2001-11-07 KR KR1020010069271A patent/KR20020066171A/en not_active Application Discontinuation
-
2002
- 2002-11-06 WO PCT/KR2002/002069 patent/WO2003040186A1/en not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60130600A (en) * | 1983-12-16 | 1985-07-12 | Nippon Zenyaku Kogyo Kk | Anti-canine-distemper monoclonal antibody and assay therewith |
Non-Patent Citations (3)
| Title |
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| J Gen Virol. 1985 Mar;66 ( Pt 3):443-56, Orvell C * |
| J Gen Virol. 1991 Nov;72 ( Pt 11):2827-30, Hirayama N * |
| J Wildl Dis. 1994 Jan;30(1):95-8, Lopez-Pena M * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100593286B1 (en) * | 2004-04-14 | 2006-06-26 | (주)에니젠 | Kit for detecting canine distemper virus antibody using immunochromatography |
| KR20150080277A (en) * | 2013-12-31 | 2015-07-09 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Apparatus for detecting Canine distemper virus comprising quartz crystal microbalance biosensor and detecting method using thereof |
| CN115094043A (en) * | 2022-08-02 | 2022-09-23 | 长春西诺生物科技有限公司 | Hybridoma cell strain for canine coronavirus and canine parvovirus, monoclonal antibody and application |
| CN115094043B (en) * | 2022-08-02 | 2023-08-25 | 长春西诺生物科技有限公司 | Hybridoma cell strain for canine coronavirus and canine parvovirus, monoclonal antibody and application |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2003040186A1 (en) | 2003-05-15 |
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