KR20070103547A - Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof - Google Patents
Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof Download PDFInfo
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- KR20070103547A KR20070103547A KR1020060035230A KR20060035230A KR20070103547A KR 20070103547 A KR20070103547 A KR 20070103547A KR 1020060035230 A KR1020060035230 A KR 1020060035230A KR 20060035230 A KR20060035230 A KR 20060035230A KR 20070103547 A KR20070103547 A KR 20070103547A
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- antigen
- monoclonal antibody
- sars coronavirus
- sars
- gly
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Abstract
Description
도 1은 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체를 생산하는 17개의 하이브리도마 세포 클론의 배양 사진이다.1 is a culture photograph of 17 hybridoma cell clones producing monoclonal antibodies against the nucleocapsid protein of SARS coronavirus.
도 2는 17개의 하이브리도마 세포 배양액에서 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체의 생산유무를 간접 ELISA법으로 조사한 결과이다.Figure 2 shows the results of the production of monoclonal antibodies against SARS coronavirus nucleocapsid protein in 17 hybridoma cell cultures by indirect ELISA.
도 3은 17개의 하이브리도마 세포 배양액에서 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체를 단백질 G 컬럼으로 정제한 양상을 관찰하기 위해 SDS-PAGE를 수행한 결과이다.FIG. 3 shows the results of SDS-PAGE for observing the purification of monoclonal antibodies against SARS coronavirus nucleocapsid protein in a protein G column in 17 hybridoma cell cultures.
도 4는 샌드위치 ELISA법으로 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체가 비변성 항원에 대한 반응성을 분석한 그래프이다.Figure 4 is a graph analyzing the reactivity of monoclonal antibodies against non-denatured antigen of SARS coronavirus nucleocapsid protein by sandwich ELISA method.
도 5는 면역블롯으로 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체가 변성 항원에 대한 반응성 및 선형 항원결정기를 분석한 결과이다.Figure 5 shows the results of analyzing the reactivity and linear epitope of the monoclonal antibody against denatured antigen of the SARS coronavirus nucleocapsid protein in an immunoblot.
도 6은 뉴클레오캡시드 단백질의 아미노산 서열에 근거한 펩타이드를 이용하여 경쟁 ELISA를 통해 얻은 단클론 항체의 항원결정기에 대한 모식도이다.6 is a schematic diagram of epitopes of monoclonal antibodies obtained through competition ELISA using peptides based on amino acid sequences of nucleocapsid proteins.
도 7은 간접면역형광항체법으로 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체가 바이러스가 감염된 세포에서 항원의 검출능을 조사한 결과이다.FIG. 7 shows the results of investigating the ability of the monoclonal antibody to nucleocapsid protein of SARS coronavirus to detect antigen in virus-infected cells by indirect immunofluorescence antibody method.
도 8은 간접면역형광항체법으로 사스 코로나바이러스의 뉴클레오캡시드 단백질에 대한 단클론 항체가 사람코로나바이러스에 대한 교차반응을 조사한 결과이다.FIG. 8 shows the results of cross-reaction of monoclonal antibodies against human coronavirus by the indirect immunofluorescent antibody method against nucleocapsid protein of SARS coronavirus.
기술분야Field of technology
본 발명은 사스 진단에 유용한 단클론 항체에 관한 것이다. 보다 구체적으로, 본 발명은 사스 코로나바이러스 뉴클레오캡시드 (nucleocapsid) 항원으로 면역시킨 마우스 비장 (spleen) 세포와 골수종 세포가 융합된 하이브리도마 (hybridoma) 클론, 하이브리도마로부터 생산되는 단클론 항체 및 이들을 제조하는 방법에 관한 것이다. 또한 본 발명은 사스 코로나바이러스의 단클론항체를 이용한 진단시약 및 진단키트에 관한 것이다.The present invention relates to monoclonal antibodies useful for the diagnosis of SARS. More specifically, the present invention provides monoclonal antibodies produced from hybridoma clones, hybridomas, and fusions of mouse spleen cells and myeloma cells immunized with SARS coronavirus nucleocapsid antigen, It relates to a manufacturing method. The present invention also relates to diagnostic reagents and diagnostic kits using monoclonal antibodies of SARS coronavirus.
종래기술Prior art
사스 (중증급성호흡기증후군, Severe Acute Respiratory Syndrome, SARS)는 2002년 11월부터 중국 광동지역을 중심으로 발생하여 홍콩, 싱가포르, 캐나다 등 전 세계적으로 확산되었던 신종 전염병으로 발열과 기침, 호흡곤란, 폐렴 등의 증상을 보이는 증후군이다. 사스는 전염성과 치사율이 높은 질환으로서 WHO에 따르면 발병 이후 2003년 8월 7일까지 32개국에서 8,422명의 환자가 발생했고, 이중 916명이 사망한 것으로 보고 되었다. 따라서 사스는 감염의 조기 진단이 매우 중요하다. 그러나 사스는 다른 호흡기증상과 임상적으로 유사하여 이들을 구분하는 것은 용이하지 않다. 비록 현재까지 한국 내에서 사스 발병사례가 없고, 전 세계적으로 이 질병의 전파가 통제되고 있지만 재발의 위험과 국내유입 가능성을 간과할 수는 없다. 따라서 사스의 국내유입 여부를 신속히 판정하고, 질병의 빠른 확산을 막기 위해 감염여부를 초기에 진단할 수 있는 효과적이고 신속한 진단법의 개발이 절실히 요구된다. 특히 기타 호흡기바이러스 및 사람 코로나바이러스와의 감별진단이 가능한 방법의 개발이 매우 시급한 실정이다. SARS (Severe Acute Respiratory Syndrome, SARS) is a new pandemic that has spread worldwide in Hong Kong, Singapore and Canada since November 2002. It is a syndrome with such symptoms. SARS is a highly contagious and fatal disease. According to the WHO, 8,422 cases have occurred in 32 countries since August 7, 2003, with 916 deaths reported. Therefore, SARS is very important for early diagnosis of infection. However, SARS is clinically similar to other respiratory symptoms, so it is not easy to distinguish them. Although there are no cases of SARS in Korea to date, and the spread of the disease is controlled globally, the risk of recurrence and the possibility of domestic inflow cannot be overlooked. Therefore, it is urgently needed to develop an effective and rapid diagnosis method that can quickly determine whether SARS enters the country and to diagnose infection early to prevent the spread of disease. In particular, it is very urgent to develop a method for differential diagnosis with other respiratory viruses and human coronaviruses.
사스 병원체는 사스 코로나바이러스임이 밝혀졌고, 바이러스 전체 게놈의 염기 서열 분석이 이루어졌다. 사스 코로나바이러스는 외피를 지닌 양성가닥 (positive stranded) RNA 바이러스로서 전체 게놈이 29,727 뉴클레오타이드로 구성되며, 현재까지 알려진 RNA 바이러스 중에서 가장 큰 게놈을 갖는 바이러스이다. 사스 코로나바이러스의 게놈은 11개의 오픈리딩프레임 (open reading frame, ORF)을 갖고 있으며 23종에 달하는 단백질을 코딩하고 있는 것으로 알려졌다. 사스 코로나바이러스의 주요 구조단백질은 뉴클레오캡시드 (nucleocapsid, N), 스파이크 (spike, S), 멤브레인 (membrane, M), 스몰 엔벨로프 (small envelope, E) 단백질임이 밝혀졌다. 이들 단백질에 대한 염기서열 분석결과, 사스 코로나바이러스가 다른 코로나바이러스와 비교하여 약 40 ~ 50% 정도의 매우 낮은 서열 상동성을 가짐이 밝혀졌다. 코로나바이러스의 항원성에 따른 계통발생학적 분류에 따르면, 사스 코로나바이러스는 I, II 및 III 그룹 중 II 그룹과 유연관계가 높은 것으로 나타났다. SARS pathogens were found to be SARS coronavirus, and sequencing of the entire virus genome was performed. SARS coronavirus is an enveloped, positive stranded RNA virus with a total genome of 29,727 nucleotides and the largest genome known to date. The genome of SARS coronavirus has 11 open reading frames (ORFs) and is known to encode 23 proteins. The major structural proteins of SARS coronavirus have been found to be nucleocapsid (N), spike (S), membrane (membrane, M), small envelope (E) proteins. Sequence analysis of these proteins revealed that SARS coronaviruses had very low sequence homology of about 40-50% compared to other coronaviruses. According to the phylogenetic classification according to the antigenicity of coronavirus, SARS coronavirus was found to have a flexible relationship with group II among the I, II and III groups.
사스 코로나바이러스의 조기 진단을 위해, 검출 용이성, 신속진단 및 바이러스 핵산에 대한 안정성 등을 고려하여 항원검출법이 이용되어 왔다. 대표적인 항원검출법은 간접면역형광항체법 (indirect immunofluorescence assay, indirect IFA)과 항원 검출용 효소면역항체법 (sandwich ELISA) 이다. 그러나 항원 검출용 간접면역형광항체법과 효소면역항체법을 구축하기 위해서는 바이러스 항원에 특이적인 항체가 반드시 필요하다. 종래에는 사스 코로나바이러스 전체 항원에 대한 항혈청을 이용하여 진단하였다. 그러나 항혈청내에는 바이러스배양에 이용된 세포성분에 대한 항체를 포함하고 있기 때문에 사람세포와 위양성을 보일 수 있고, 사스 코로나바이러스는 사람코로나바이러스와 유연관계가 비교적 높기 때문에 전체 항원에 대한 항혈청내에는 사람 코로나바이러스에 대한 항원과 비특이 반응을 보이는 항체가 포함되어 있기 때문에 사람 코로나바이러스와 감별진단이 어렵다. 그러나 단클론 항체는 단일 항원결정기 (epitope)를 인식하기 때문에 특이반응을 보이는 단클론 항체만을 선별할 수 있다. 또한 전체 항원내에는 항체형성에 큰 영향을 미치는 주요항원부위가 존재하는데, 이 항원부위에 대한 단클론 항체의 이용은 항혈청을 이용한 항원진단법에 비해 민감도를 향상시킬 수 있다. 따라서 특이적인 항원에 대한 단클론 항체의 생산 및 이를 이용한 진단제에 대한 개발의 요구가 지속되어 왔다.For early diagnosis of SARS coronavirus, antigen detection has been used in consideration of ease of detection, rapid diagnosis, and stability against viral nucleic acids. Representative antigen detection methods are indirect immunofluorescence assay (indirect IFA) and enzyme detection antibody (sandwich ELISA) for antigen detection. However, in order to construct an indirect immunofluorescent antibody method and an enzyme immune antibody method for antigen detection, an antibody specific for a viral antigen is necessary. Conventionally, antisera against the SARS coronavirus whole antigen have been diagnosed. However, antiserum may show false positives with human cells because it contains antibodies to the cell components used in the culture of virus. Since SARS coronavirus has a high degree of flexibility with human coronavirus, Differentiation with human coronavirus is difficult because it contains antibodies that have a nonspecific reaction with antigens for coronavirus. However, since monoclonal antibodies recognize a single epitope, only monoclonal antibodies that are specific can be selected. In addition, there are major antigenic sites in the whole antigen that have a great effect on antibody formation. The use of monoclonal antibodies against these antigenic sites can improve sensitivity compared to the antiserum-based antigen diagnosis method. Therefore, there has been a continuous need for the production of monoclonal antibodies directed against specific antigens and the development of diagnostic agents using the same.
본 발명자들은 특이적인 항원으로 판정된, 재조합 베큘로바이러스에서 생산 된 사스 코로나바이러스 N 항원 (특허출원:10-2005-0109726)을 면역원으로 이용하여 마우스에 면역시키고, 면역된 마우스의 비장세포와 골수종세포가 융합된 하이브리도마 클론을 제조하고 이로부터 사스 코로나바이러스 N 항원에 대한 단클론항체를 생산함으로써 본 발명을 완성하였다. 본 발명에 따른 단클론 항체는 사스 코로나바이러스 N 항원에 대해 우수한 친화성 (affinity)을 나타내고, 특히 사람 코로나바이러스 혈청에 대하여 교차반응성하지 않았으며, 간접면역형광항체, 효소면역항체법 및 웨스턴 블롯법 등의 다양한 진단법에 이용 가능한 진단용 항체이다. The present inventors immunized mice using SARS coronavirus N antigen (patent application: 10-2005-0109726) produced from recombinant baculovirus determined as a specific antigen as an immunogen, and splenocytes and myeloma of the immunized mouse. The present invention was completed by preparing hybridoma clones in which cells were fused and producing monoclonal antibodies against SARS coronavirus N antigen. The monoclonal antibody according to the present invention shows excellent affinity for SARS coronavirus N antigen, and in particular, has not been cross-reactive with human coronavirus sera, indirect immunofluorescent antibody, enzyme immunoassay method and Western blot method. It is a diagnostic antibody which can be used for various diagnostic methods.
본 발명은 사스 진단을 위해 사스 코로나바이러스 N 항원에 대한 단클론 항체를 생산하는 하이브리도마 클론을 제공하는 것을 목적으로 한다. It is an object of the present invention to provide a hybridoma clone that produces monoclonal antibodies against SARS coronavirus N antigen for SARS diagnosis.
또한 본 발명의 목적은 사스 코로나바이러스 N 항원에 대한 정제된 단클론 항체를 제공하는 것이다.It is also an object of the present invention to provide purified monoclonal antibodies against SARS coronavirus N antigen.
또한 본 발명의 목적은 단클론 항체를 이용한 사스 진단방법을 제공하는 것이다.It is also an object of the present invention to provide a SARS diagnostic method using a monoclonal antibody.
또한 본 발명의 목적은 사스 진단을 위한 진단키트를 제공하는 것이다. It is also an object of the present invention to provide a diagnostic kit for SARS diagnosis.
상기 목적을 달성하기 위하여, 본 발명은 사스 진단을 위해 사스 코로나바이러스 N 항원에 대한 단클론 항체를 생산하는 하이브리도마 클론을 제공한다. 바람직하게는 상기 단클론 항체를 생산하는 하이브리도마 클론은 단클론 항체를 장기적으로 생산할 수 있도록 마우스 비장세포와 골수종세포를 융합시킨 것으로, 제한 희 석법(limited dilution method)에 의해 단일 항원결정기(epitope)에 대한 항체만을 생산하는 세포클론이다. 또한 본 발명은 사스 코로나바이러스의 구조 단백질인 N 항원을 특이적으로 인식하는, 정제된 단클론항체를 제공한다. In order to achieve the above object, the present invention provides a hybridoma clone that produces a monoclonal antibody against SARS coronavirus N antigen for SARS diagnosis. Preferably, the hybridoma clone that produces the monoclonal antibody is a fusion of mouse splenocytes and myeloma cells to produce long-term monoclonal antibodies, and to a single epitope by a limited dilution method. It is a cell clone that produces only antibodies against. The present invention also provides a purified monoclonal antibody that specifically recognizes N antigen, which is a structural protein of SARS coronavirus.
또한 본 발명은 사스 코로나바이러스 N 항원을 면역원으로 마우스에 면역시키고, 면역된 마우스의 비장세포와 골수종세포를 융합하여 하이브리도마 클론을 선별하는 단계를 포함하는 항체 생산방법을 제공한다. 바람직하게는 본 발명의 제조방법은 단백질 G-레진(resin)을 이용한 크로마토그래피에 의한 항체 정제공정을 추가로 포함할 수 있다. In another aspect, the present invention provides an antibody production method comprising the step of immunizing a mouse with SARS coronavirus N antigen to an immunogen, fusing the splenocytes and myeloma cells of the immunized mouse to screen for hybridoma clones. Preferably, the preparation method of the present invention may further include an antibody purification step by chromatography using protein G-resin.
또한, 본 발명은 사스 진단용 키트를 제공한다. 본 발명의 진단키트는 상기 단클론항체, 기질과 반응하여 발색하는 표지체와 축합된 항체 및 발색기질을 포함한다. 본 발명의 진단키트에서 상기 단클론항체는 기판에 흡착된 상태로 제공된다. 상기 기판으로는 PVDF 막, 플레이트 및 슬라이드가 사용될 수 있다. 표지체로는 HRP, FITC (fluorescein isothiocyanate), 발색기질로는 TMB가 바람직하지만 이에 한정되는 것은 아니다. 이렇게 본 발명은 본 발명의 제조방법에 따라 제조된 사스 코로나바이러스 N 항원에 특이적인 단클론 항체를 이용하여 사람에서 사스를 진단하기 위한 진단키트를 제공한다. 진단키트의 최종 검출방법으로는 효소면역항체법 (ELISA), 웨스턴 블롯 또는 IFA가 이용될 수 있다. 바람직하게는 본 발명의 진단키트에 사용되는 검출방법은 효소면역항체법이다. 효소면역항체법을 이용한 키트의 경우, 본 발명의 단클론 항체가 코팅된 플레이트의 각 웰에 사스 의심환자로부터 채취된 검체를 반응시키고, HRP (Horseradish peroxidase)-축합 사스 단클론 항체를 첨가한 후, TMB (3,3',5,5' - tetramethylbenzidine) 기질용액을 각 웰에 처리하여 발색반응을 흡광도로 측정하는 것으로 구성된다. 바람직하게는, 본 발명의 진단 키트는 사람 코로나바이러스로부터 사스의 감별진단을 위한 특이적인 단클론 항체를 이용하며 양성대조군 및 정량적 분석이 가능하도록 본 발명자들에 의해 특허출원된 재조합 N 항원 (한국특허출원:10-2005-0109726)을 추가로 포함할 수 있다.The present invention also provides a SARS diagnostic kit. The diagnostic kit of the present invention includes the monoclonal antibody, an antibody condensed with a label that reacts with the substrate, and a color substrate. In the diagnostic kit of the present invention, the monoclonal antibody is provided as adsorbed on a substrate. PVDF films, plates and slides can be used as the substrate. HRP, fluorescein isothiocyanate (FITC) as a label, and TMB as a chromogenic substrate are preferred, but are not limited thereto. Thus, the present invention provides a diagnostic kit for diagnosing SARS in humans using monoclonal antibodies specific for SARS coronavirus N antigen prepared according to the preparation method of the present invention. Final detection of the diagnostic kit may be performed by enzyme-linked immunosorbent assay (ELISA), Western blot or IFA. Preferably, the detection method used in the diagnostic kit of the present invention is an enzyme immunosorbent method. In the case of the kit using the enzyme-immunized antibody method, a sample collected from suspected SARS was reacted to each well of a plate coated with the monoclonal antibody of the present invention, and after adding HRP (Horseradish peroxidase) -condensed SARS monoclonal antibody, TMB (3,3 ', 5,5'-tetramethylbenzidine) Substrate solution was treated to each well and the color reaction was measured by absorbance. Preferably, the diagnostic kit of the present invention utilizes a specific monoclonal antibody for the differential diagnosis of SARS from human coronavirus and is a recombinant N antigen patented by the present inventors to enable positive control and quantitative analysis (Korean Patent Application 10-2005-0109726).
이와 같이, 본 발명자들은 사스를 보다 더 신속하고 정확하게 진단하기 위해, 사스 코로나바이러스 N 항원에 높은 친화도와 특이도를 갖는 단클론 항체를 제조하였다. 먼저, 정제된 사스 코로나바이러스 N 항원을 면역증강제와 함께 섞어 balb/c 마우스의 복강내에 1차 면역시키고, 2주 간격으로 추가 면역시켰으며 마우스의 비장세포와 골수종세포를 폴리에틸렌글리콜을 이용하여 융합된 하이브리도마 세포를 제조하였다. 다음 HAT(hypoxanthine, aminopteri, thymidine) 배지에서 배양하여 융합되지 않은 마우스 비장세포와 골수종 세포로부터 융합된 하이브리도마 세포를 선별한 후, 제한 희석법에 의해 단일 항원 결정기에 대한 항체만을 생산하는 하이브리도마 세포 클론을 제조하고, 재조합 사스 코로나바이러스 N 항원을 이용한 간접 ELISA에 의해 단클론항체를 생산하는 하이브리도마 클론을 선별하였다. As such, we have prepared monoclonal antibodies with high affinity and specificity for SARS coronavirus N antigen to diagnose SARS more quickly and accurately. First, purified SARS coronavirus N antigen was mixed with an adjuvant for primary immunization of the abdominal cavity of balb / c mice, and further immunized every two weeks. The splenocytes and myeloma cells of the mouse were fused using polyethylene glycol. Hybridoma cells were prepared. Next, hybridoma cells fused from unfused mouse splenocytes and myeloma cells were cultured in HAT (hypoxanthine, aminopteri, thymidine) medium, and then hybridomas producing only antibodies against a single antigenic determinant by restriction dilution. Cell clones were prepared and hybridoma clones producing monoclonal antibodies were selected by indirect ELISA with recombinant SARS coronavirus N antigen.
본 발명에 따른 단클론항체는 사스 코로나바이러스 감염에 대한 진단 시약, 진단 키트 또는 사스의 예방 및 항체치료제의 제조에 유용한 성분으로서 이용될 수 있을 것이다. The monoclonal antibody according to the present invention may be used as a diagnostic reagent for a SARS coronavirus infection, a diagnostic kit, or as a useful component in the preparation of SARS prevention and antibody therapeutics.
본 발명에 사용된 용어 사스 코로나바이러스 N 단백질과 사스 코로나바이러 스 N 항원은 의미상 동일한 표현이다.As used herein, the terms SARS coronavirus N protein and SARS coronavirus N antigen are semantically identical expressions.
이하 실시예에 기초하여 본 발명을 기술한다. 하기 실시예들은 본 발명을 예시하고자 하는 것으로, 본 발명을 제한하고자 하는 것이 아니다. 본 발명에 개시된 모든 문헌은 참조로서 통합된다.The present invention is described based on the following examples. The following examples are intended to illustrate the invention, not to limit the invention. All documents disclosed in the present invention are incorporated by reference.
실시예Example
실시예 1: 사스 코로나바이러스 N 항원을 이용한 면역Example 1 Immunization with SARS Coronavirus N Antigen
본 발명자들에 의해 특허출원된 재조합 사스 코로나바이러스 N 항원 (서열번호: 1) (특허출원:10-2005-0109726)을 프로인트완전애주번트 (Freund's complete adjuvant)와 동량으로 혼합하고, balb/c 마우스(샘타코, 9주령)의 복강 내에 주입하여 1차 면역을 실시하였다. 1차 면역 후 2주 간격으로 정제된 항원과 프로인트불완전애주번트 (Freund's incomplete adjuvant)를 동량으로 혼합하여 2회 추가 면역을 실시한 후 세포융합 3일 전에 항원 성분만 마우스의 꼬리정맥 내로 주입하여 2차 면역 (booster)을 유도하였다. Recombinant SARS coronavirus N antigen (SEQ ID NO: 1) (Patent Application: 10-2005-0109726) patented by the inventors was mixed in the same amount with Freund's complete adjuvant, balb / c Primary immunization was performed by intraperitoneal injection of mice (Sam taco, 9 weeks old). Two additional immunizations were performed by mixing the same amount of purified antigen and Freund's incomplete adjuvant at two weeks interval after the first immunization, followed by two additional immunizations, and injecting only the antigen components into the tail vein of the
실시예 2: 하이브리도마 세포 제조 Example 2: Hybridoma Cell Preparation
면역시킨 마우스의 비장(spleen)을 무균적으로 적출하여 얻어진 비장세포와 골수종세포(myeloma cell)인 SP-2 세포 (메타볼렙사)의 세포융합(cell fusion)은 폴리에틸렌글리콜 1500 (PEG 1500, Boehringer Mannheim)을 이용하여 콜러 (Kohler) 및 밀스테인 (Milstein)의 방법 (참고문헌: Kohler G., and Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497)에 따라 실시하였다. 최종 면역 3~4일 후 마우스 의 비장을 무균적으로 적출하고 26-게이지 (gauge) 주사바늘로 적출한 비장을 세절한 후 혈청이 첨가되지 않은 얼음 냉각 DMEM (Dulbecco's Modified Eagle's medium)에 부유시켰다. 부유액은 50 ㎖ 코니컬 튜브 (cornical tube)에 수집하여 2분간 정치시킨 후 DMEM으로 1,000 rpm에서 10분씩 3회 원심 및 세척하였다. 침전된 림프구를 SP-2 세포와 7:1 내지 10:1 정도의 비율로 혼합하고 37℃로 미리 가온한 DMEM으로 1회 세척한 후 파스퇴르 피펫 (pasteur pipet)을 이용하여 상층액을 완전히 제거하였다. 이 혼합세포에 37℃로 미리 가온한 50% PEG 1500 1 ㎖를 1분에 걸쳐 첨가했다. PEG를 가한 후 2분간 혼합 세포가 들어있는 튜브를 흔들어주며 세포 융합을 유도하였다. 이 튜브에 DMEM 10 ㎖를 천천히 첨가한 후 37℃ 항온수조에서 5분간 정치시켰다. 혼합액을 1,000 rpm에서 10분간 원심분리한 후 상층액을 제거하였다. 침전된 세포는 HAT (0.1mM hypoxanthine, 0.4 μM aminopteri, 16 μM thymidine, SIGMA)와 20% 우태아혈청이 첨가된 DMEM에 1 x 106 세포/㎖의 농도로 부유시켰다. 이 부유액을 96 웰 마이크로플레이트에 100 ㎕씩 분주하고, 37℃, 5% CO2 항온기에서 하룻밤 배양한 다음 배지를 2일 간격으로 첨가해 주며 2주간 배양함으로써 융합된 세포를 선별하였다. 융합 2주 후부터는 HT 보충액 (100 μM hypoxanthine, 16 μM thymidine, SIGMA)과 10% 우태아혈청이 첨가된 DMEM으로 배양하였고, 3주 후부터는 10% 우태아 혈청만 첨가된 DMEM으로 배양하였다 (도 1 참조). Cell fusion between splenocytes obtained by aseptic extraction of spleen from immunized mice and SP-2 cells (metabolepsa), myeloma cells, was performed using polyethylene glycol 1500 (PEG 1500, Boehringer). Method of Kohler and Milstein using Mannheim (Kohler G., and Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity.Nature 256: 495-497) It was carried out according to. Three to four days after the final immunization, the spleens of the mice were aseptically extracted, and the spleens extracted with a 26-gauge needle were cut and suspended in ice-cold Dulbecco's Modified Eagle's medium (serum-free). The suspension was collected in a 50 ml conical tube, allowed to stand for 2 minutes, and centrifuged and washed three times for 10 minutes at 1,000 rpm with DMEM. Precipitated lymphocytes were mixed with SP-2 cells at a ratio of about 7: 1 to 10: 1, washed once with DMEM pre-warmed to 37 ° C, and the supernatant was completely removed using a pasteur pipet. . To this mixed cell was added 1 ml of 50% PEG 1500, previously warmed to 37 ° C., over 1 minute. After adding PEG, the tube containing the mixed cells was shaken for 2 minutes to induce cell fusion. 10 ml of DMEM was slowly added to the tube and allowed to stand for 5 minutes in a 37 ° C constant temperature water bath. The mixed solution was centrifuged at 1,000 rpm for 10 minutes and the supernatant was removed. Precipitated cells were suspended at a concentration of 1 × 10 6 cells / ml in DMEM supplemented with HAT (0.1 mM hypoxanthine, 0.4 μM aminopteri, 16 μM thymidine, SIGMA) and 20% fetal bovine serum. 100 μl of this suspension was dispensed into 96-well microplates, incubated overnight at 37 ° C. in a 5% CO 2 incubator, and then cultured for 2 weeks by adding medium every two days. Two weeks after the fusion, HT supplementation (100 μM hypoxanthine, 16 μM thymidine, SIGMA) and 10% fetal bovine serum were added to DMEM. ).
실시예 3: 하이브리도마 세포의 클로닝Example 3: Cloning of Hybridoma Cells
융합된 세포의 단클론을 확보하기 위해 96 웰 플레이트의 첫 번째 웰에 약 10개의 세포가 들어가도록 세포 부유액을 넣은 후 행으로 2배 단계 희석하고, 이를 다시 열로 2배 단계 희석하는 방법으로 2회 클로닝 하였다. To secure the monoclonal of the fused cells, cell suspension was added so that about 10 cells were placed in the first well of the 96 well plate, followed by two-fold dilution in rows, followed by two-fold dilutions in columns. It was.
실시예 4: 하이브리도마 클론에서 항체생성 조사Example 4: Investigation of Antibody Production in Hybridoma Clones
융합된 세포의 항체 생성 여부는 정제된 N 항원과 융합된 세포 배양액을 이용하여 간접 ELISA를 실시하여 관찰하였다. 정제된 사스 코로나바이러스 N 항원 단백질을 탄산염 완충액 (carbonate buffer) (pH 9.4)으로 10 ㎍/㎖ 농도로 희석한 다음 Maxisorp ELISA 플레이트 (Nunc사)의 각 웰 당 100 ㎕씩 첨가하고, 상온에서 16시간 반응시켜 항원을 코팅하였다. 항원이 코팅된 각 웰에 1% BSA (Bovine Serum Albumin)가 포함된 인산완충용액을 처리하여 37℃에서 1시간 동안 비특이 반응을 차단하였다. 각 웰에 융합된 세포배양액을 100 ㎕씩 첨가하고, 37℃에서 1 시간 반응한 다음 인산완충용액으로 3회 세척하였다. 세척 후 1:1000배 희석한 HRP (Horseradish peroxidase)-축합 항-마우스 IgG+IgA+IgM 항체 (Abcam, 1:2000)를 각 웰 당 100 ㎕씩 첨가하고 37℃에서 1 시간 반응시킨 다음 인산완충용액으로 3회 세척하였다. 세척 후 TMB (3,3',5,5' - tetramethylbenzidine) (입수처: Dade behring사) 기질용액을 각 웰에 100 ㎕씩 첨가하고 암소에서 30분간 반응시켜 발색한 후 1N H2SO4를 처리하여 효소반응을 정지시켰다. 반응 후 ELISA 리더를 이용하여 405 nm에서 흡광도를 측정하였다. 그 결과 사스 코로나바이러스 N 항원과 반응하는 17개의 하이브리도마 클론을 얻었다 (도 2 참조). 이들 중 07-19-11 하이브리도마 세포를 한국세포주연구재단에 수탁번호 KCLRF-BP-00126으로 2005년 12월 28일에 기탁하였다. 선별된 하이브리도마 클론은 10% 우태아 혈청과 5% DMSO (dimethyl sulfoxide)가 함유된 DMEM배지에 현탁시켜 액체질소에 보관하였다. Antibody production of the fused cells was observed by indirect ELISA using a cell culture fused with purified N antigen. Purified SARS coronavirus N antigen protein was diluted to 10 μg / ml in carbonate buffer (pH 9.4), and then 100 μl of each well of Maxisorp ELISA plate (Nunc Co.) was added, followed by 16 hours at room temperature. The reaction was coated with the antigen. Each well coated with antigen was treated with a phosphate buffer solution containing 1% BSA (Bovine Serum Albumin) to block nonspecific reaction at 37 ° C. for 1 hour. 100 μl of the cell culture solution fused to each well was added and reacted at 37 ° C. for 1 hour, and then washed three times with phosphate buffer solution. After washing, 100 μl of HRP (Horseradish peroxidase) -condensed anti-mouse IgG + IgA + IgM antibody (Abcam, 1: 2000) diluted 1: 1000 after washing was added and reacted at 37 ° C. for 1 hour, followed by phosphate buffering. Wash three times with solution. After washing, TMB (3,3 ', 5,5'-tetramethylbenzidine) (available from Dade behring) was added to the wells of 100 µl of each substrate solution and reacted for 30 minutes in the dark to develop 1N H 2 SO 4 . Treatment stopped the enzyme reaction. After the reaction, the absorbance was measured at 405 nm using an ELISA reader. As a result, 17 hybridoma clones were reacted with SARS coronavirus N antigen (see FIG. 2). Of these, 07-19-11 hybridoma cells were deposited with the Korea Cell Line Research Foundation on Dec. 28, 2005 with accession number KCLRF-BP-00126. Selected hybridoma clones were suspended in DMEM medium containing 10% fetal calf serum and 5% DMSO (dimethyl sulfoxide) and stored in liquid nitrogen.
실시예 5: 단클론 항체의 정제Example 5: Purification of Monoclonal Antibodies
항체를 생성하는 융합 세포를 25 cm2 배양 플라스크로 옮겨서 배양한 뒤 제한 희석법(limited dilution method)을 이용하여 클로닝을 실시하고 37℃, 5% CO2 항온기에서 배양하였다. 클로닝된 융합 세포들의 항체 생성 여부를 다시 한번 확인한 후 항체를 생성하는 융합세포를 선별하여 배양하고, 배양 후 얻어진 세포 상층액으로부터 항체를 정제하였다. 항체의 정제는 이뮤노퓨어 (ImmunoPure) (G) IgG 정제 키트 (PIERCE)를 이용하였으며 실험과정은 제조사의 방법에 따라 수행하였다. BCA 단백질 분석 키트 (PIERCE)를 이용하여 정제된 항체량을 측정한 후 정제양상을 SDS-PAGE를 실시하여 확인하였다. 각 하이브리도마 클론으로부터 생산되는 단클론 항체를 단백질 G 컬럼을 이용하여 손쉽게 정제할 수 있었으며 SDS-PAGE로 분석한 결과 정제도 (purity)가 약 90% 이상에, 융합된 세포배양액 1㎖당 약 1mg/㎖ 농도의 정제된 항체를 수득할 수 있었다 (도 3 참조). 레인 M은 단백질표준분자량 표지(standard protein marker) 이고, 레인 1은 단백질 G 레진에서 3번째로 추출된 단클론 항체이고, 레인 2는 단백질 G 레진에서 4번째로 추출된 단클론 항체를 보여준다.The fusion cells producing the antibody were transferred to a 25 cm 2 culture flask, followed by cloning using a limited dilution method, and cultured in a 37 ° C., 5% CO 2 incubator. After confirming the production of the antibodies of the cloned fusion cells once again, the fusion cells producing the antibody were selected and cultured, and the antibody was purified from the cell supernatant obtained after the culture. Purification of the antibody was performed using an ImmunoPure (G) IgG Purification Kit (PIERCE) and the experiment was carried out according to the manufacturer's method. The amount of purified antibody was measured using a BCA protein analysis kit (PIERCE), and the purified form was confirmed by performing SDS-PAGE. Monoclonal antibodies produced from each hybridoma clone could be easily purified using a protein G column, and analyzed by SDS-PAGE, the purity was greater than about 90%, and about 1 mg per ml of fused cell culture solution. Purified antibody at a concentration of / ml could be obtained (see FIG. 3). Lane M is the standard protein marker,
실시예 6: 이소타이핑에 의한 단클론 항체의 선별Example 6: Screening for Monoclonal Antibodies by Isotyping
선별된 17개의 하이브리도마 클론으로부터 정제된 단클론 항체의 이소타입 (isotype)의 결정은 이뮤노퓨어 단클론항체 이소타이핑 키트 (ImmunoPure Monoclonal Antibody Isotyping Kit) II (PIERCE)를 이용하여 공지된 제조사의 방법에 따라 수행되었다. 정제된 항원을 탄산염 완충액 (carbonate buffer) (pH 9.4)로 10 ㎍/㎖ 농도로 희석한 다음 Maxisorp ELISA 플레이트 (Nunc)의 각 웰 당 100 ㎕씩 첨가하고, 상온에서 16시간 반응시켜 항원을 코팅하였다. 항원이 코팅된 각 웰에 1% BSA가 포함된 인산완충용액을 처리하여 37℃에서 1시간 동안 비특이 반응을 차단하였다. 각 단클론 항체를 1% BSA가 포함된 인산완충용액을 이용하여 20 ㎍/㎖ 농도로 희석한 다음 각 웰에 100 ㎕씩 첨가하고, 37℃에서 1 시간 반응한 다음 인산완충용액으로 3회 세척하였다. 50 ㎕의 정상 토끼 혈청 (음성 대조군)과 서브클래스-특이적 항-마우스 Ig을 각 웰에 첨가하고, 37℃에서 1 시간 반응한 다음 인산완충용액으로 3회 세척하였다. 세척 후 1:500배 희석한 알칼라인 탈인산화효소 축합 항-토끼 (Alkaline phosphatase-conjugated anti-rabbit) IgG를 각 웰 당 100 ㎕씩 첨가하고, 37℃에서 1 시간 반응시킨 다음 인산완충용액으로 3회 세척하였다. 세척 후 기질용액(1 mg/㎖, pNPP)을 각 웰에 100 ㎕씩 첨가하고 암소에서 30분간 반응시켜 발색하였다. 반응 후 ELISA 리더를 이용하여 405 nm에서 흡광도를 측정하였다. 이소타입을 결정한 결과, 표 1에 정리된 바와 같이 중쇄 (heavy chain)의 서브클래스에 따라서 분류하면 2개는 IgG1 서브클래스, 3개는 IgG2b 서브클래스, 나머지 12개는 IgM 서브클래스였고, 모든 단클론 항체의 경쇄 (light chain)는 카파 사 슬 (kappa chain)으로 구성되어 있었다. 일반적으로 IgM 항체는 진단제 개발을 위해 이용되는 고상표면이나 표적 (target) 항원 이외의 다른 성분과 비특이적으로 흡착하는 특성을 갖고 있다. 따라서 본 발명을 통해 진단용 단클론 항체로 이용 가능한 항체는 5개의 IgG 서브클래스 항체로 선정하였다. 이후 제반실험에서는 이 항체만을 이용하여 특성분석을 실시하였다. Determination of the isotype of the monoclonal antibody purified from the 17 hybridoma clones selected was carried out in the methods of known manufacturers using ImmunoPure Monoclonal Antibody Isotyping Kit II (PIERCE). Was performed accordingly. Purified antigen was diluted with carbonate buffer (pH 9.4) at a concentration of 10 μg / ml, and then 100 μl of each well of Maxisorp ELISA plate (Nunc) was added and reacted at room temperature for 16 hours to coat the antigen. . Each well coated with antigen was treated with a phosphate buffer solution containing 1% BSA to block nonspecific reaction at 37 ° C. for 1 hour. Each monoclonal antibody was diluted to 20 μg / ml using a phosphate buffer solution containing 1% BSA, and then 100 μl was added to each well, reacted at 37 ° C. for 1 hour, and washed three times with phosphate buffer solution. . 50 μl of normal rabbit serum (negative control) and subclass-specific anti-mouse Ig were added to each well, reacted for 1 hour at 37 ° C. and washed three times with phosphate buffer. After washing, 100 μl of alkaline phosphatase-conjugated anti-rabbit IgG diluted 1: 500 times was added to each well, and reacted at 37 ° C. for 1 hour, followed by 3 times with phosphate buffer solution. Washed. After washing, 100 μl of substrate solution (1 mg / ml, pNPP) was added to each well, and the resultant was reacted for 30 minutes in the dark and developed. After the reaction, the absorbance was measured at 405 nm using an ELISA reader. As a result of determining the isotype, as shown in Table 1, when classified according to the heavy chain subclass, two were IgG 1 subclass, three were IgG 2b subclass, and 12 were IgM subclass. The light chain of all monoclonal antibodies consisted of kappa chains. In general, IgM antibodies have the characteristic of nonspecific adsorption with components other than solid surface or target antigen used for the development of diagnostics. Therefore, antibodies that can be used as diagnostic monoclonal antibodies according to the present invention were selected as five IgG subclass antibodies. In the following experiments, the antibody was characterized using only this antibody.
표 1. 단클론항체 이소타입Table 1. Monoclonal Antibody Isotypes
실시예 7: 단클론 항체의 친화도 분석Example 7: Affinity Analysis of Monoclonal Antibodies
진단용 단클론 항체의 선별을 위한 단클론 항체의 친화도 조사는 정제된 N 항원과 단계희석된 단클론 항체를 이용하여 간접 ELISA법에 의해 실시되었다. 정제된 항원을 탄산염 완충액(pH 9.4)로 10 ㎍/㎖ 농도로 희석한 다음 Maxisorp ELISA 플레이트 (Nunc)의 각 웰 당 100 ㎕씩 첨가하고, 상온에서 16시간 반응시켜 항원을 코팅하였다. 항원이 코팅된 각 웰에 1% BSA가 포함된 인산완충용액을 처리하여 37℃에서 1시간 동안 비특이 반응을 차단하였다. 각 단클론 항체를 1% BSA가 포함된 인산완충용액을 이용하여 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 및 0.3 nM 농도로 희석한 다음 각 웰에 첨가하고, 37℃에서 1 시간 반응시킨 다음 인산완충용액으로 3회 세척하였다. 세척 후 1:2000배 희석한 HRP-축합 항-마우스 IgG+IgA+IgM 항체 (Abcam)를 각 웰에 100 ㎕씩 첨가하고 37℃에서 1 시간 반 응시킨 다음 인산완충용액으로 3회 세척하였다. 세척 후 TMB 기질용액을 각 웰에 100 ㎕씩 첨가하고, 암소에서 30분간 반응시켜 발색한 후 1N H2SO4를 처리하여 효소반응을 정지시켰다. 반응 후 ELISA 리더를 이용하여 405 nm에서 흡광도를 측정하였다. 간접 ELISA를 수행한 결과, 단클론 항체의 농도에 따른 친화도를 보이는 용량-의존적 곡선 (dose-response curve)를 얻을 수 있었다. 이 곡선으로부터 1.00의 흡광도 (OD405)를 보이는 단클론 항체의 농도를 산출한 결과, 21-10-06 단클론 항체는 4.55 nM, 22-05-03 단클론 항체는 1.19 nM, 07-19-11 단클론 항체는 1.45 nM, 07-19-21 단클론 항체는 2.23 nM, 그리고 21-10-11 단클론 항체는 14.54 nM로 나타났다 (표 2 참조). 따라서 21-10-11 단클론 항체를 제외한 나머지 항체는 항원과의 친화도가 뛰어났으며, 향후 진단제 개발을 위해 유용할 것이다. Affinity investigation of monoclonal antibodies for the selection of diagnostic monoclonal antibodies was carried out by indirect ELISA using purified N antigen and step-diluted monoclonal antibodies. Purified antigen was diluted with carbonate buffer (pH 9.4) at a concentration of 10 μg / ml, and then 100 μl was added to each well of Maxisorp ELISA plate (Nunc), and reacted at room temperature for 16 hours to coat the antigen. Each well coated with antigen was treated with a phosphate buffer solution containing 1% BSA to block nonspecific reaction at 37 ° C. for 1 hour. Each monoclonal antibody was diluted to 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.3 nM concentrations using a phosphate buffer solution containing 1% BSA and added to each well, The reaction was carried out at 37 ° C. for 1 hour and then washed three times with phosphate buffer solution. After washing, 100 μl of HRP-condensed anti-mouse IgG + IgA + IgM antibody (Abcam) diluted 1: 2000 times was added to each well, reacted at 37 ° C. for 1 hour, and washed three times with phosphate buffer solution. After washing, 100 µl of the TMB substrate solution was added to each well, followed by reaction for 30 minutes in the dark, followed by development of 1N H 2 SO 4 to stop the enzymatic reaction. After the reaction, the absorbance was measured at 405 nm using an ELISA reader. As a result of indirect ELISA, a dose-response curve showing affinity according to the concentration of monoclonal antibody was obtained. From this curve, the concentration of monoclonal antibody showing an absorbance of 1.00 (OD 405 ) was calculated. As a result, 21-10-06 monoclonal antibody was 4.55 nM, 22-05-03 monoclonal antibody was 1.19 nM, and 07-19-11 monoclonal antibody. 1.45 nM, 07-19-21 monoclonal antibody was 2.23 nM, and 21-10-11 monoclonal antibody was 14.54 nM (see Table 2). Therefore, the other antibodies except 21-10-11 monoclonal antibody had excellent affinity with antigen and may be useful for future diagnostic development.
표 2. 흡광도Table 2. Absorbance
실시예 8: 비변성 N 항원에 대한 단클론 항체의 반응성 분석Example 8: Reactivity Analysis of Monoclonal Antibodies to Undenatured N Antigens
본 발명을 통해 확보된 단클론 항체는 항원진단용 키트의 개발 및 면역정제법 (Immunoprecipitation)등에 이용될 수 있기 때문에 비변성의 N 단백질과의 반응성에 따른 단클론 항체의 특성을 조사할 필요가 있었다. 이에 본 발명에서는 단클론 항체와 비변성 단백질과의 반응성을 확인하기 위해 정제된 단클론 항체와 항원을 이용하여 샌드위치 ELISA를 수행하였다. 우선 정제된 22-05-03 단클론항체에 비 오틴을 표지하기 위해 EZ-Link Sulfo-NHS-LC-Biotinylation kit (PIERCE)를 이용하여 제조사의 방법에 따라 실시하였다. 정제된 단클론 항체 (표지되지 않은 22-05-03 단클론항체)를 탄산염 완충액 (pH 9.4)로 10 ㎍/㎖ 농도로 희석한 다음 Maxisorp ELISA 플레이트 (Nunc)의 각 웰 당 100 ㎕씩 첨가하고, 상온에서 16시간 반응시켜 항체를 코팅하였다. 50 ㎕의 각 단클론 항체(20 ㎍/㎖)와 50 ㎕의 정제된 N 항원 (10㎍/㎖)을 혼합하고, 37℃에서 30분간 반응시킨 후, 항체가 코팅된 웰에 항체-항원 반응액 전량을 첨가하고, 37℃에서 60분간 반응시켰다. 인산완충용액으로 3회 세척하고, 1:2000배 희석한 HRP-축합 사스 단클론항체 (07-19-11 단클론항체)를 각 웰 당 100 ㎕씩 첨가하여 37℃에서 60분간 반응시킨 다음 인산완충용액으로 3회 세척하였다. 세척 후 TMB (3,3',5,5' - tetramethylbenzidine) (입수처: Dade behring사) 기질용액을 각 웰에 100 ㎕씩 첨가하고, 암소에서 30분간 반응시켜 발색한 후 1N H2SO4를 처리하여 효소반응을 정지시켰다. 반응 후 ELISA 리더를 이용하여 405 nm에서 흡광도를 측정하였다. 단클론 항체와 비변성의 N 항원과의 반응성을 분석한 결과, 21-10-06과 21-10-11 단클론 항체에 비해 22-05-03, 07-19-11, 07-19-21 단클론 항체가 비변성 N 항원과 다소 높은 반응성을 보였다 (도 4 참조). 비록 반응도에 차이는 보였지만 5개의 IgG 서브클래스의 단클론항체는 모두 항원진단용 키트의 개발 및 면역정제법에 이용 가능할 것이다.Since the monoclonal antibody obtained through the present invention can be used for the development of an antigen diagnosis kit, immunoprecipitation, etc., it was necessary to investigate the characteristics of the monoclonal antibody according to its reactivity with non-denatured N protein. In the present invention, a sandwich ELISA was performed using the purified monoclonal antibody and antigen to confirm the reactivity between the monoclonal antibody and the non-denatured protein. First, to label biotin on purified 22-05-03 monoclonal antibody, EZ-Link Sulfo-NHS-LC-Biotinylation kit (PIERCE) was used according to the manufacturer's method. Purified monoclonal antibody (unlabeled 22-05-03 monoclonal antibody) was diluted to 10 μg / ml concentration with carbonate buffer (pH 9.4), then 100 μl of each well of Maxisorp ELISA plate (Nunc) was added and room temperature The reaction was coated for 16 hours at. 50 [mu] l of each monoclonal antibody (20 [mu] g / ml) and 50 [mu] l of purified N antigen (10 [mu] g / ml) were mixed and reacted at 37 [deg.] C. for 30 minutes, followed by antibody-antigen reaction solution to the antibody-coated wells. The whole amount was added and reacted at 37 degreeC for 60 minutes. After washing three times with phosphate buffer solution, 100 μl of HRP-condensed SARS monoclonal antibody (07-19-11 monoclonal antibody) diluted 1: 2000 times was added to each well and reacted at 37 ° C. for 60 minutes, followed by phosphate buffer solution. Washed three times. After washing, 100 μl of TMB (3,3 ', 5,5'-tetramethylbenzidine) (obtained by Dade behring) was added to each well, and reacted for 30 minutes in the dark, followed by 1N H 2 SO 4 Was treated to stop the enzymatic reaction. After the reaction, the absorbance was measured at 405 nm using an ELISA reader. As a result of analyzing the reactivity of monoclonal antibody with non-denatured N antigen, 22-05-03, 07-19-11, 07-19-21 monoclonal antibody compared to 21-10-06 and 21-10-11 monoclonal antibody Showed somewhat higher reactivity with undenatured N antigen (see FIG. 4). Although there were differences in responsiveness, all five monoclonal antibodies of the IgG subclass would be available for the development of immunoassay kits and immunoassays.
실시예 9: 변성 N 항원에 대한 단클론 항체의 반응성 분석 및 항원결정기 분석Example 9: Reactivity and epitope analysis of monoclonal antibodies against denatured N antigens
특정 항원에 특이적인 단클론 항체는 면역블롯처럼 변성조건에서 항원을 검출하는 진단제 개발에 이용될 수 있다. 이에 본 발명에서는 정제된 사스 코로나바이러스 N 항원을 환원제인 β-메르캅토에탄올 (mercaptoethanol)로 처리하여 이황화결합을 끊어준 후, SDS-PAGE와 면역블롯을 실시하여 각 단클론 항체의 반응성을 조사하였다. 정제된 N 항원 (1 ㎍/㎖)을 이용하여 라엠리 (Laemmli) 등의 방법 (참고문헌: Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685)에 따라 10% 아크릴아미드겔 (acrylamide gel)에서 SDS-PAGE를 실시하고, 영동이 끝난 후 겔 상의 N 항원은 타우빈 (Towbin) 등 의 방법(참고문헌: Towbin H, Staehelin T, Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354)에 따라 PVDF (Polyvinylidene fluoride) 막으로 이적시켰다. 단백질이 이적된 막은 적정한 크기의 스트립 (strip)으로 절단하고, 3% 탈지유 (skim milk)가 포함된 인산완충용액으로 비특이 반응을 차단하였다. 3% 탈지유가 함유된 인산완충용액에 100 ㎍/㎖ 농도가 되도록 희석한 각 단클론 항체를 각 막 스트립에 처리하여 상온에서 1시간 반응시켰다. 반응 후 인산완충용액으로 3회 세척하고, HRP-축합 항-마우스 IgG+IgA+IgM 항체 (Abcam)를 3% 탈지유가 포함된 인산완충용액으로 1:4,000으로 희석하여 첨가한 후 상온에서 1시간 동안 반응시켰다. 반응 후 인산완충용액으로 3회 세척하고, DAB 기질용액 (Sigma)을 이용하여 발색시켰다. 도 5에서 레인 1은 21-10-06 단클론 항체, 레인 2는 22-05-03 단클론 항체, 레인 3은 07-19-11 단클론 항체, 레인 4는 07-19-21 단클론 항체, 레인 5는 21-10-11 단클론 항체, 레인 P는 양성대조군인 항-His 항체로 처리한 결과를 보여 주고 있는데, 도 5에서 보는 바와 같이 21-10-06, 21-10-11, 22-05-03, 07-19-11, 07-19-21 단클론 항체는 49kDa의 전체크기의 N 항원과 반응하는 것으로 나타났다. 따라서 모든 단클론 항체는 선형의 항원결정기(epitope)와 반응함을 알 수 있으며, 변성된 항원검출을 위한 진단제 개발에 본 발명에서 확보된 단클론 항체는 유용할 것이다. Monoclonal antibodies specific for specific antigens can be used to develop diagnostics that detect antigens under denaturing conditions, such as immunoblots. Therefore, in the present invention, after dissolving disulfide bonds by treating purified SARS coronavirus N antigen with β-mercaptoethanol, a reducing agent, and performing immunoblot with SDS-PAGE, the reactivity of each monoclonal antibody was examined. Laemmli et al. (Ref .: Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-) using purified N antigen (1 μg / ml). SDS-PAGE was performed on a 10% acrylamide gel according to 685), and N-antigen on the gel was taubin (Towbin) et al. (Reference: Towbin H, Staehelin T, Gordon J). (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA 76: 4350-4354) were transferred to PVDF (Polyvinylidene fluoride) membranes. The protein-transferred membrane was cut into strips of appropriate size, and the non-specific reaction was blocked with phosphate buffer solution containing 3% skim milk. Each monoclonal antibody diluted to a concentration of 100 μg / ml in a phosphate buffer solution containing 3% skim milk was treated to each membrane strip and allowed to react at room temperature for 1 hour. After the reaction, the solution was washed three times with phosphate buffer solution, and HRP-condensed anti-mouse IgG + IgA + IgM antibody (Abcam) was added diluted 1: 4,000 with phosphate buffer solution containing 3% skim milk, followed by 1 hour at room temperature. Reacted for a while. After the reaction, the mixture was washed three times with phosphate buffer solution and developed using DAB substrate solution (Sigma). In Figure 5
또한 도 5에서 양성대조군인 항-His 항체와 반응하는 단편(약 25kDa)은 아미노말단 단편이고, 반응하지 않는 단편(약 28kDa)은 카르복시말단 단편이었다. 따라서 22-05-03, 07-19-11, 07-19-21 단클론항체는 사스 코로나바이러스 N 항원의 아미노말단부위와 반응하고, 21-10-06과 21-10-11 단클론 항체는 카르복시 말단부위와 반응함을 알 수 있다. 이를 확인하고, 단클론 항체의 항원결정기를 조사하기 위해 사스 코로나바이러스 N 단백질에 대한 15 내지 20개 길이의 아미노산으로 구성된 펩타이드(Peptron Inc.)를 이용하여 경쟁 ELISA (competition ELISA)를 실시하였다 (표 4 참조). 그 결과, 21-10-06과 21-10-11 단클론 항체는 사스 코로나바이러스 N 단백질의 아미노 말단에서부터 215 ~ 239 아미노산 부위와 반응하고, 22-05-03, 07-19-11, 07-19-21 단클론 항체는 135 ~ 150 아미노산 부위와 반응하였고, 17 ~ 32 아미노산 부위와는 약하게 반응하였다. 따라서 22-05-03, 07-19-11, 07-19-21 단클론항체가 도 5에서의 결과와 같이 전체 사스 코로나바이러스 N 항원의 아미노 말단부위, 특히 135 ~ 150 및 17 ~32 아미노산 부위와 반응하고, 21-10-06과 21-10-11 단클론항체는 카르복시 말단부위, 특히 215 ~ 239 아미노산 부위와 반 응함을 알 수 있다. 따라서 본 발명을 통해 항원결정기가 다른 2 그룹의 단클론 항체를 확보할 수 있었으며 각 그룹의 단클론 항체들은 사스 항원진단제 개발뿐만 아니라 사스 코로나바이러스에 대한 기초연구에서도 유용하게 이용될 수 있다 (도 6 참조). In addition, the fragment (about 25 kDa) reacting with the anti-His antibody which is a positive control in FIG. 5 was an amino terminal fragment, and the fragment which does not react (about 28 kDa) was a carboxy terminal fragment. Thus, 22-05-03, 07-19-11, 07-19-21 monoclonal antibodies reacted with the amino terminus of SARS coronavirus N antigen, and 21-10-06 and 21-10-11 monoclonal antibodies reacted with the carboxy terminus. It can be seen that the reaction. To confirm this, competition ELISA (competition ELISA) was performed using Pepttron Inc. consisting of 15-20 amino acids in length against SARS coronavirus N protein to examine epitopes of monoclonal antibodies (Table 4 Reference). As a result, 21-10-06 and 21-10-11 monoclonal antibodies reacted with 215-239 amino acid sites from the amino terminus of SARS coronavirus N protein, 22-05-03, 07-19-11, 07-19 The -21 monoclonal antibody reacted with 135-150 amino acid sites and weakly with 17-32 amino acid sites. Thus, 22-05-03, 07-19-11, and 07-19-21 monoclonal antibodies may be combined with the amino terminus of the entire SARS coronavirus N antigen, in particular 135-150 and 17-32 amino acid sites, as shown in FIG. It can be seen that the 21-10-06 and 21-10-11 monoclonal antibodies react with the carboxy terminus, particularly the 215-239 amino acid region. Therefore, the present invention was able to secure two groups of monoclonal antibodies with different epitopes, and the monoclonal antibodies of each group can be usefully used in basic research on SARS coronavirus as well as SARS diagnosis (see FIG. 6). ).
표 4. 사스 코로나바이러스 N 단백질의 아미노산 단편Table 4. Amino Acid Fragments of SARS Coronavirus N Protein
실시예 10: 사스 코로나바이러스가 감염된 세포에서 단클론 항체의 반응성 분석Example 10 Reactivity Analysis of Monoclonal Antibodies in SARS Coronavirus Infected Cells
최근 까지는 바이러스 진단을 위해 표준진단법으로 면역형광항체법 (immunofluorescence assay)이 널리 이용되어 왔기 때문에 본 발명에서 확보된 단클론 항체들과 사스 코로나바이러스의 반응성을 조사하기 위해 바이러스가 접종된 베로 (vero) 세포 (ATCC, CCL-81)를 이용하여 간접면역형광항체법을 수행하였다. 사스 코로나바이러스를 단층배양된 베로세포에 접종하고, 세포병변효과가 관찰될 때 세포침전물을 수확하였다. 세포침전물을 인산완충용액으로 3회 세척한 후 FA (fluorescence assay)용 슬라이드에 도말하여 건조시키고, 바이러스를 불활성화하기 위해 UVC (ultra-violet C)를 30분간 처리하였다. FA 슬라이드는 제반실험을 수행하기 전까지 -70 ℃보관하거나 100% 메탄올에서 5분간 고정하였다. 고정된 FA 슬라이드에 각 단클론 항체를 1% BSA가 포함된 인산완충용액을 이용하여 20 ㎍/㎖ 농도로 희석하여 처리하고, 37 ℃에서 30분간 반응시킨 후 인산완충용액을 이용해서 3회 세척하였다. 세척 후 FITC 축합 항-마우스 IgG+IgA+IgM 항체 (Cappel, 20 ㎍/㎖)를 처리하고, 37 ℃에서 30분간 반응시킨 후 인산완충용액을 이용해서 3회 세척하였다. 세척 후 FITC 축합 항-마우스 IgG+IgA+IgM 항체 (Cappel, 20 ㎍/㎖)를 처리하고, 37 ℃에서 30분간 반응시켰다. 반응 후 인산완충용액으로 3회 세척하고, 증류수로 1회 세척하였다. FA 슬라이드를 건조시킨 후 형광현미경하에서 관찰하였다. 도 7에서 보는 바와 같이, 21-10-06을 제외한 22-05-03, 07-19-11, 07-19-21 단클론항체는 모두 사스 코로나바이러스가 감염된 세포에서 N 항원을 검출할 수 있었다. 따라서, 22-05-03, 07-19-11, 07-19-21 단클론항체는 사스 코로나바이러스가 감염된 세포에서 N 항원의 검출용 진단제 개발을 위해 유용할 것이다.Until recently, since immunofluorescence assay has been widely used as a standard diagnostic method for virus diagnosis, vero cells inoculated with virus to investigate the reactivity of monoclonal antibodies and SARS coronavirus obtained in the present invention. (ATCC, CCL-81) was used to perform the indirect immunofluorescent antibody method. SARS coronavirus was inoculated into monolayer cultured Vero cells, and cell precipitates were harvested when the cytopathic effect was observed. The cell precipitate was washed three times with phosphate buffer solution, plated on a slide for FA (fluorescence assay), dried, and treated with UVC (ultra-violet C) for 30 minutes to inactivate the virus. FA slides were stored at −70 ° C. or fixed for 5 minutes in 100% methanol until the experiments were performed. Each monoclonal antibody was treated with a phosphate buffer solution containing 1% BSA on a fixed FA slide at a concentration of 20 μg / ml, and reacted at 37 ° C. for 30 minutes and washed three times with phosphate buffer solution. . After washing, the FITC condensed anti-mouse IgG + IgA + IgM antibody (Cappel, 20 μg / ml) was treated, and reacted at 37 ° C. for 30 minutes, and then washed three times using phosphate buffer solution. After washing, the FITC condensed anti-mouse IgG + IgA + IgM antibody (Cappel, 20 μg / ml) was treated and reacted at 37 ° C. for 30 minutes. After the reaction was washed three times with phosphate buffer solution, and washed once with distilled water. FA slides were dried and observed under a fluorescence microscope. As shown in FIG. 7, 22-05-03, 07-19-11, and 07-19-21 monoclonal antibodies except 21-10-06 were able to detect N antigen in cells infected with SARS coronavirus. Therefore, 22-05-03, 07-19-11, 07-19-21 monoclonal antibodies will be useful for the development of diagnostics for the detection of N antigen in cells infected with SARS coronavirus.
실시예 11: 단클론 항체의 사람 코로나바이러스에 대한 교차반응 조사Example 11: Cross-Reaction Investigation of Monoclonal Antibodies to Human Coronavirus
사스 코로나바이러스의 진단제 개발을 위해서는 비교적 높은 유연관계를 보이는 사람 코로나바이러스와의 감별진단이 매우 중요하다. 이에 본 실시예에서는 사스 코로나바이러스 N 항원에 대한 단클론 항체가 사람 코로나바이러스와 교차반응하는 지를 조사하기 위해 MRC-5 세포 (ATCC, CCL-171)에서 배양된 바이러스를 이 용해 간접면역형광항체법을 수행하였다. 각 사람코로나바이러스 OC43 주(ATCC, VR-1558)를 단층배양된 MRC-5 세포에 접종하고, 세포병변효과가 관찰될 때 세포침전물을 수확하였다. 세포침전물을 인산완충용액으로 3회 세척한 후 FA용 슬라이드에 도말하여 건조시키고, 바이러스를 불활성화하기 위해 UVC를 30분간 처리하였다. FA 슬라이드는 제반실험을 수행하기 전까지 -70℃에서 보관하거나 100% 메탄올에서 5분간 고정하였다. 고정된 FA 슬라이드에 각 단클론 항체, 사람 코로나바이러스 OC43 N 항원에 대한 항체(Chemicon, MAB 9013)를 1:100배 희석하여 처리하고, 37 ℃에서 30분간 반응시킨 후 인산완충용액을 이용해서 3회 세척하였다. 세척 후 FITC 축합 항-마우스 IgG (Abicam, 20 ㎍/㎖)를 처리하고, 37 ℃에서 30분간 반응시켰다. 반응 후 인산완충용액으로 3회 세척하고, 증류수로 1회 세척하였다. FA 슬라이드를 건조시킨 후 형광현미경하에서 관찰하였다. 도 8에서 보는 바와 같이 본 발명의 각 단클론 항체는 사람 코로나바이러스와 교차반응하지 않는 것으로 관찰되었다. 따라서 본 발명을 통해 획득된 단클론 항체는 사스 코로나바이러스의 감염을 감별진단 할 수 있는 특이적인 항체임을 알 수 있었다. Differential diagnosis with human coronavirus, which has a relatively high degree of flexibility, is very important for the development of diagnosis of SARS coronavirus. In this example, an indirect immunofluorescent antibody method using a virus cultured in MRC-5 cells (ATCC, CCL-171) was used to investigate whether monoclonal antibodies against SARS coronavirus N antigen cross-react with human coronavirus. Was performed. Each human coronavirus OC43 strain (ATCC, VR-1558) was inoculated into monolayer cultured MRC-5 cells, and cell precipitates were harvested when the cytopathic effect was observed. The cell precipitate was washed three times with a phosphate buffer solution and then plated on a slide for FA and dried, and treated with UVC for 30 minutes to inactivate the virus. FA slides were stored at −70 ° C. or fixed for 5 minutes in 100% methanol until the experiments were conducted. Each monoclonal antibody and antibody to human coronavirus OC43 N antigen (Chemicon, MAB 9013) were diluted 1: 100 on fixed FA slides, reacted at 37 ° C for 30 minutes, and then washed three times using phosphate buffer solution. Washed. After washing, the FITC condensed anti-mouse IgG (Abicam, 20 µg / ml) was treated and reacted at 37 ° C for 30 minutes. After the reaction was washed three times with phosphate buffer solution, and washed once with distilled water. FA slides were dried and observed under a fluorescence microscope. As shown in FIG. 8, it was observed that each monoclonal antibody of the present invention did not cross react with human coronavirus. Therefore, the monoclonal antibody obtained through the present invention was found to be a specific antibody that can differentially diagnose the infection of SARS coronavirus.
실시예 12: 진단키트Example 12: Diagnostic Kit
본 발명은 본 발명의 단클론항체를 포함하는, 사스 코로나바이러스의 감염을 진단할 수 있는 진단키트를 제공한다. 본 발명의 진단키트는 본 발명의 단클론항체, 기질과 반응하여 발색하는 표지체와 축합된 항체 및 발색기질을 포함한다. 상세하게는, 상기 진단키트는 본 발명의 단클론항체를 탄산염 (pH 9.4)으로 희석하여 최종 농도가 10 ㎍/㎖인 단클론항체 희석액이 코팅된 플레이트, 1:2,000배로 희석 된 HRP-축합 사스 단클론항체, 인산완충용액, TMB 및 1N H2SO4 로 구성된다.The present invention provides a diagnostic kit for diagnosing the infection of SARS coronavirus, comprising the monoclonal antibody of the present invention. The diagnostic kit of the present invention includes a monoclonal antibody of the present invention, an antibody condensed with a label which reacts with a substrate, and a color substrate. Specifically, the diagnostic kit is a monoclonal antibody diluted with carbonate (pH 9.4) and a monoclonal antibody dilution-coated plate having a final concentration of 10 μg / ml, and HRP-condensed SARS monoclonal antibody diluted 1: 2,000 times. , Phosphate buffer solution, TMB and 1N H 2 SO 4 .
본 발명의 진단키트를 이용하여, 사스 의심 환자 (피검자)에 대해 사스 코로나바이러스 감염여부를 진단하는 방법은 다음과 같다. 단클론항체가 코팅된 플레이트에 피검자로부터 채취한 검체 (여기서, 검체는 피검자의 비강세척액, 인후도말액, 대변 또는 혈액이다)를 각 웰 당 100 ㎕ 씩 첨가하고 37 ℃에서 60분 동안 반응시킨 다음 인산완충용액으로 플레이트를 3회 세척한다. 다음으로, HRP-축합 사스 단클론항체를 플레이트의 각 웰 당 100 ㎕ 씩 첨가하고 37 ℃에서 60분 동안 반응시킨 다음 인산완충용액으로 플레이트를 3회 세척한다. 그런 다음, HRP의 기질인 TMB를 플레이트의 각 웰 당 100 ㎕ 씩 첨가하고 암소에서 30분 동안 반응시켜 발색반응을 유도한다. 그런 다음, 1N H2SO4를 플레이트의 각 웰 당 100 ㎕ 씩 첨가하여 효소반응을 정지시킨다. 본 발명의 진단키트를 이용하여 이렇게 처리한 플레이트를 ELISA 리더 (reader)를 사용하여 405 nm에서 흡광도를 측정한다. 측정 결과, 흡광도가 0.2 이상인 경우 양성으로 판정한다.Using the diagnostic kit of the present invention, a method for diagnosing SARS coronavirus infection in a suspected SARS (test subject) is as follows. 100 μl of each sample from the subject (wherein the sample is the nasal wash, throat smear, feces, or blood) of the monoclonal antibody-coated plate was added to each well and reacted at 37 ° C. for 60 minutes, followed by phosphoric acid. Wash the plate three times with buffer. Next, 100 μl of HRP-condensed SARS monoclonal antibody was added to each well of the plate, reacted at 37 ° C. for 60 minutes, and the plate was washed three times with phosphate buffer solution. Then, 100 μl of TMB, a substrate of HRP, is added to each well of the plate and reacted in the dark for 30 minutes to induce a color reaction. Then, 1 N H 2 SO 4 is added to 100 μl of each well of the plate to stop the enzyme reaction. The plate thus treated using the diagnostic kit of the present invention is measured for absorbance at 405 nm using an ELISA reader. As a result of the measurement, when the absorbance is 0.2 or more, it is determined as positive.
또한, 본 발명의 진단키트는 FA 슬라이드, 100% 메탄올, 1% BSA가 포함된 인산완충용액으로 희석하여 최종 농도가 20 ㎍/㎖인 단클론항체, FITC 축합 항-마우스 IgG+IgA+IgM 항체 (20 ㎍/㎖), 인산완충용액 및 증류수로 구성될 수도 있다. 이렇게 구성된 진단키트를 이용하여, 사스 의심 환자 (피검자)에 대해 사스 코로나바이러스 감염여부를 진단하는 방법은 다음과 같다.In addition, the diagnostic kit of the present invention is diluted with a phosphate buffer solution containing FA slide, 100% methanol, 1% BSA, monoclonal antibody having a final concentration of 20 μg / ml, FITC condensed anti-mouse IgG + IgA + IgM antibody ( 20 μg / ml), phosphate buffer solution and distilled water. Using the diagnostic kit configured as described above, the method of diagnosing SARS coronavirus infection in the suspected SARS (test subject) is as follows.
피검자로부터 채취한 검체 100 ㎕를 FA 슬라이드에 도말하여 건조시키고, 바 이러스를 불활성화하기 위해 UVC (ultra-violet C)를 30분 동안 처리한다. 그런 다음, FA 슬라이드를 -70 ℃에 보관하거나 100% 메탄올에 5분 동안 처리하여 고정시킨다. 고정된 FA 슬라이드에 1% BSA가 포함된 인산완충용액으로 희석하여 최종 농도가 20 ㎍/㎖인 단클론항체에 처리하고 37 ℃에서 30분 동안 반응시킨 후 인산완충용액으로 3회 세척한다. 세척 후, FITC 축합 항-마우스 IgG+IgA+IgM 항체 (20 ㎍/㎖)를 처리하고 37 ℃에서 30분 동안 반응시킨다. 반응 후, 인산완충용액으로 3회 세척하고 증류수로 1회 세척한다. 그런 다음, FA 슬라이드를 건조시키고 형광현미경하에서 관찰한다.100 μl of sample from the subject is plated on FA slides and dried, and UVC (ultra-violet C) is treated for 30 minutes to inactivate the virus. The FA slides are then stored and fixed at −70 ° C. or treated in 100% methanol for 5 minutes. The immobilized FA slide was diluted with phosphate buffer solution containing 1% BSA, treated with monoclonal antibody having a final concentration of 20 μg / ml, reacted at 37 ° C. for 30 minutes, and washed three times with phosphate buffer solution. After washing, the FITC condensed anti-mouse IgG + IgA + IgM antibody (20 μg / ml) is treated and reacted at 37 ° C. for 30 minutes. After the reaction, it is washed three times with phosphate buffer solution and once with distilled water. The FA slides are then dried and observed under fluorescence microscopy.
본 발명의 진단 키트는 사람 코로나바이러스로부터 사스의 감별진단을 위한 특이적인 단클론 항체를 이용하며 양성대조군 및 정량적 분석이 가능하도록 본 발명자들에 의해 특허출원된 재조합 N 항원 (한국특허출원:10-2005-0109726) (서열번호: 1)을 추가로 포함할 수 있다.The diagnostic kit of the present invention utilizes a specific monoclonal antibody for differential diagnosis of SARS from human coronavirus and is a recombinant N antigen patented by the present inventors to enable positive control and quantitative analysis (Korean Patent Application: 10-2005 -0109726) (SEQ ID NO: 1) may be further included.
본 발명에 따른 사스 코로나바이러스 N 항원에 대한 17개의 단클론 항체중에 5개의 IgG 서브클래스의 항체는 진단제 개발을 위해 매우 유용한 진단용 항체를 제공하고, 이들 단클론 항체는 하이브리도마 클론 세포의 배양을 통해 지속적으로 생산이 가능할 뿐만 아니라 높은 순도로 정제가 용이하였고, 사람 코로나바이러스 항원에 대해 교차반응하지 않았으며 사스 코로나바이러스 N 항원과 친화도가 매우 뛰어났다. 또한 이들 단클론 항체는 N 항원의 아미노말단과 카르복시말단과 반응하는 두 그룹의 단클론 항체들로 구분할 수 있었다. 특히 21-10-06, 22-05-03, 07-19-11, 07-19-21 단클론 항체는 높은 친화도를 보이면서 간접 ELISA, 면역블롯, 면역 형광항체법(IFA), 샌드위치 ELISA와 같은 다양한 진단제 개발에 이용 가능하였다 (표 5 참조). 따라서 이와 같은 N 단백질에 대한 단클론 항체는 사스 코로나바이러스의 감염에 대한 진단시약 또는 진단키트, 사스 예방 또는 항체치료제의 제조 등에 이용될 수 있다. Among the 17 monoclonal antibodies directed against SARS coronavirus N antigen according to the present invention, five IgG subclass antibodies provide diagnostic antibodies which are very useful for the development of diagnostic agents. Not only was it continuously produced, it was easy to purify with high purity, did not cross-react with human coronavirus antigens, and was highly affinity with SARS coronavirus N antigen. In addition, these monoclonal antibodies could be divided into two groups of monoclonal antibodies that react with the amino and carboxy termini of the N antigen. In particular, 21-10-06, 22-05-03, 07-19-11, and 07-19-21 monoclonal antibodies have high affinity, such as indirect ELISA, immunoblot, immunofluorescent antibody (IFA) and sandwich ELISA. Various diagnostic agents were available (see Table 5). Therefore, such a monoclonal antibody to the N protein can be used for the production of diagnostic reagents or diagnostic kits for SARS coronavirus infection, SARS prevention or antibody therapeutics.
표 5. 하이브리도마 클론으로부터 생산된 단클론항체 검출방법 및 유용성Table 5. Methods and usefulness for detecting monoclonal antibodies produced from hybridoma clones
a: ++ = 강한 양성; + = 양성; - = 음성.a: ++ = strong positive; + = Positive; -= Negative.
이상 살펴본 바와 같이, 본 발명의 하이브리도마 클론에서 생산되는 단클론 항체는 감수성 및 특이성이 우수하여, 사스 코로나바이러스 감염에 대한 진단시약 또는 진단키트, 사스 예방 또는 항체치료제의 제조 등에 매우 유용하게 이용될 수 있다.As described above, the monoclonal antibody produced in the hybridoma clone of the present invention is excellent in sensitivity and specificity, and thus may be very useful for preparing a diagnostic reagent or diagnostic kit for SARS coronavirus infection, prevention of SARS or antibody treatment. Can be.
<110> Korea Center for Disease Control and Prevention <120> Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use thereof <130> IPM-29809 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Nucleocapsid protein of SARS coronavirus <400> 1 Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile 1 5 10 15 Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 20 25 30 Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn 35 40 45 Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 50 55 60 Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly 65 70 75 80 Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg 85 90 95 Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr 100 105 110 Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys 115 120 125 Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 130 135 140 Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu 145 150 155 160 Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly 165 170 175 Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 180 185 190 Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro 195 200 205 Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu 210 215 220 Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln 225 230 235 240 Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser 245 250 255 Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr 260 265 270 Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly 275 280 285 Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln 290 295 300 Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg 305 310 315 320 Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly 325 330 335 Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile 340 345 350 Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu 355 360 365 Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro 370 375 380 Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp 385 390 395 400 Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 405 410 415 Ala Asp Ser Thr Gln Ala 420 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N1 <400> 2 Met Ser Asp Asn Gly Pro Gly Ser Asn Gln Arg Ser Ala Pro Arg Ile 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N17 <400> 3 Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N35 <400> 4 Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> N50 <400> 5 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 1 5 10 15 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N65 <400> 6 Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile 1 5 10 <210> 7 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> N76 <400> 7 Asn Thr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala 1 5 10 15 Thr Arg Arg Val Arg Gly Gly Asp Gly Lys 20 25 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N101 <400> 8 Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N117 <400> 9 Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys Glu Gly Ile Val 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N135 <400> 10 Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg 1 5 10 15 <210> 11 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> N153 <400> 11 Asn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr Thr Leu 1 5 10 15 Pro Lys Gly Phe Tyr Ala Glu Gly Ser 20 25 <210> 12 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N177 <400> 12 Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 1 5 10 15 Gly Asn Ser Arg Asn Ser 20 <210> 13 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N196 <400> 13 Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro Ala Arg Met 1 5 10 15 Ala Ser Gly Gly Gly Glu 20 <210> 14 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> N215 <400> 14 Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln 1 5 10 15 Leu Glu Ser Lys Val Ser Gly Lys Gly 20 25 <210> 15 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> N245 <400> 15 Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg 1 5 10 15 Gln Lys Arg Thr Ala Thr Lys Gln 20 <210> 16 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N258 <400> 16 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr Gln 1 5 10 15 Ala Phe Gly Arg Arg Gly 20 <210> 17 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N355 <400> 17 Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys 1 5 10 15 Lys Asp Lys Lys Lys Lys 20 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> N371 <400> 18 Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro Gln Arg 1 5 10 15 Gln Lys Lys Gln 20 <210> 19 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> N385 <400> 19 Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp 1 5 10 15 Met Asp Asp Phe Ser Arg Gln 20 <210> 20 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N401 <400> 20 Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 1 5 10 15 Ala Asp Ser Thr Gln Ala 20 <110> Korea Center for Disease Control and Prevention <120> Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use <130> IPM-29809 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 422 <212> PRT <213> Artificial Sequence <220> Nucleocapsid protein of SARS coronavirus <400> 1 Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile 1 5 10 15 Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 20 25 30 Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn 35 40 45 Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 50 55 60 Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly 65 70 75 80 Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg 85 90 95 Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr 100 105 110 Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys 115 120 125 Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 130 135 140 Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu 145 150 155 160 Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly 165 170 175 Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 180 185 190 Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro 195 200 205 Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu 210 215 220 Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln 225 230 235 240 Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser 245 250 255 Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr 260 265 270 Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly 275 280 285 Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln 290 295 300 Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg 305 310 315 320 Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly 325 330 335 Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile 340 345 350 Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu 355 360 365 Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro 370 375 380 Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp 385 390 395 400 Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 405 410 415 Ala Asp Ser Thr Gln Ala 420 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N1 <400> 2 Met Ser Asp Asn Gly Pro Gly Ser Asn Gln Arg Ser Ala Pro Arg Ile 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N17 <400> 3 Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N35 <400> 4 Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> N50 <400> 5 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 1 5 10 15 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N65 <400> 6 Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile 1 5 10 <210> 7 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> N76 <400> 7 Asn Thr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala 1 5 10 15 Thr Arg Arg Val Arg Gly Gly Asp Gly Lys 20 25 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N101 <400> 8 Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N117 <400> 9 Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys Glu Gly Ile Val 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> N135 <400> 10 Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg 1 5 10 15 <210> 11 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> N153 <400> 11 Asn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr Thr Leu 1 5 10 15 Pro Lys Gly Phe Tyr Ala Glu Gly Ser 20 25 <210> 12 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N177 <400> 12 Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 1 5 10 15 Gly Asn Ser Arg Asn Ser 20 <210> 13 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N196 <400> 13 Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro Ala Arg Met 1 5 10 15 Ala Ser Gly Gly Gly Glu 20 <210> 14 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> N215 <400> 14 Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln 1 5 10 15 Leu Glu Ser Lys Val Ser Gly Lys Gly 20 25 <210> 15 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> N245 <400> 15 Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg 1 5 10 15 Gln Lys Arg Thr Ala Thr Lys Gln 20 <210> 16 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N258 <400> 16 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr Gln 1 5 10 15 Ala Phe Gly Arg Arg Gly 20 <210> 17 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N355 <400> 17 Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys 1 5 10 15 Lys Asp Lys Lys Lys Lys 20 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> N371 <400> 18 Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro Gln Arg 1 5 10 15 Gln Lys Lys Gln 20 <210> 19 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> N385 <400> 19 Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp 1 5 10 15 Met Asp Asp Phe Ser Arg Gln 20 <210> 20 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> N401 <400> 20 Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 1 5 10 15 Ala Asp Ser Thr Gln Ala 20
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| CN113238048A (en) * | 2021-05-11 | 2021-08-10 | 上海真测生物科技有限公司 | Diagnostic marker and application thereof in distinguishing new coronavirus infection and new coronavirus inactivated vaccination |
| CN113588948A (en) * | 2021-08-03 | 2021-11-02 | 江苏量界生物技术有限公司 | Kit for quantitatively detecting novel coronavirus N protein based on ELISA method |
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| JP2023528876A (en) * | 2020-06-02 | 2023-07-06 | バテル メモリアル インスティチュート | Systems and Methods for Screening for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) (COVID-19) 2019 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113238048A (en) * | 2021-05-11 | 2021-08-10 | 上海真测生物科技有限公司 | Diagnostic marker and application thereof in distinguishing new coronavirus infection and new coronavirus inactivated vaccination |
| CN113238048B (en) * | 2021-05-11 | 2024-03-15 | 抗码(苏州)生物科技有限公司 | Diagnostic markers and their use in differentiating between new coronavirus infection and new coronavirus inactivated vaccination |
| CN113588948A (en) * | 2021-08-03 | 2021-11-02 | 江苏量界生物技术有限公司 | Kit for quantitatively detecting novel coronavirus N protein based on ELISA method |
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