학술연구용역사업최종결과보고서 뇌척수액내프리온검출진단법개발연구 Development of PrPsc(rogue prion) diagnosis in CSF(Cerebrospinal fluid) 주관연구기관 : ( 주 ) 피플바이오 질병관리본부
뇌척수액내 프리온 검출진단법 개발연구 2 0 1 0 질병관리본부
목 차 Ⅰ. 연구개발결과요약문 ( 한글 ) 뇌척수액내프리온검출진단법개발연구 ( 영문 ) Development of PrPsc(rogue prion) diagnosis in CSF(Cerebrospinal fluid) Ⅱ. 학술연구용역사업연구결과 제1장최종연구개발목표제2장최종연구개발내용및방법제3장최종연구개발결과제4장연구결과고찰및결론제5장연구성과제6장참고문헌제7장첨부서류
과제명 뇌척수액내프리온검출진단법개발연구 중심단어 뇌척수액, 프리온 주관연구기관 ( 주 ) 피플바이오주관연구책임자강성민 연구기간 2009. 5. 20-2010. 5. 19 현재까지환자가검물 ( 혈액, 뇌척수액등 ) 을이용하여프리온을검출할수있는실험실확인진단방법이개발되어있지않으며, CJD를진단하는데사용되고있는뇌척수액의 14-3-3 protein 검출은다른뇌질환환자에서도발현되는지표로특이도가떨어진다. 그러므로뇌척수액등의가검물에서프리온을검출할수있는실험실확인진단방법개발이시급하다. 이에사람검체에서프리온검출가능성이높은뇌척수액을이용하여동물혈액진단용으로개발되어있는 MDS (Multimer Detection System) 방법을적용하여진단법을개발하고자한다. MDS 기술은항원의같은부위에반응하는항체들을포획 (capturing) 과검출 (detection) 항체로동시에사용하여모노머와멀티머를구분할수있는새로운개념의진단시스템으로모노머 ( 단량체 ) 상태에서는질병을유발하지않으나, 멀티머 ( 다량체 ) 상태에서는질병을유발하는단백질에대하여 MDS는그단백질이모노머인지멀티머인지를판별함으로써질병의감염여부를판단한다. 뇌척수액 (CSF, Cerebrospinal fluid) 을이용한 CJD 진단기술개발중 1차년도에는뇌척수액진단키트용포획및진단항체조사를통해총 13종의인간프리온단백질에반응성이있다고알려진상업용항체를확보하였고프리온단백질과뇌척수액샘플을이용하여 MDS antibody set(3e7/t2hrp) 을선발하였다. 뇌척수액을이용한 MDS 진단기술확립을위해선발된 MDS antibody set을 non-cjd CSF에적용하였을때비특이반응이발생하였고이를낮추기위해 protease를사용하여비특이반응을줄일수있었다. 2차년도에는이조건을바탕으로최적화를진행할예정이다. 또한, 뇌척수액안정성유지를위한샘플 protocol 확립과 non-cjd CSF 샘플확보및확보방안을논의중에있다. 본연구를통해개발된기술은 CJD 진단을보다정확하게 cross-check 할수있는보강 (confirmatory) 또는진단기술로활용할수있으며, 감염성이높은것으로알려져있는뇌척수액을이용한진단법개발로 CJD 진단의정확도를제고할수있고 CJD 조기발견을통한환자관리및 2차감염을사전에차단하는데적용될것으로기대된다. - 1 -
Title of Project Key Words Development of PrPsc(rogue prion) diagnosis in CSF(Cerebrospinal fluid) CSF, Prion Institute PeopleBio Project Leader Sungmin Kang Project Period 2009. 5. 20-2010. 5. 19 The diagnostic method of detecting rogue prion from bodily fluids(blood, CSF etc.) is not developed and detection of 14-3-3 protein from CSF in use of diagnosis for CJD lacks specificity because it's used as indexes in other brain disease as well. This indicates the necessity for the development of laboratorial diagnostic method of detecting rogue prion from bodily fluids. Hereby, we are developing the diagnostic method of detecting rogue prion from human CSF by using MDS(Multimer Detection System) technology which was developed for blood screening test for scrapie in sheep. Multimer Detection System (MDS) is a platform technology, which can differentiate only multimers from monomer by creating competition between capturing and detection antibodies for the overlapping epitope. MDS discriminates infectiousness of disease by detecting only multimeric feature of rogue prion protein(prpsc) in contrast to monomeric form of cellular prion protein. During the first year of development of CJD diagnostic technology using CSF, we assured total of 13 commercialized antibody which is known to be respond to human prion protein through search of capturing and detecting antibody which can be applied in CSF sample and selected MDS antibody set(3e7/t2hrp) by applying to recombinant human prion protein and CSF samples. While MDS antibody set was applied to non-cjd CSF, non-specific binding occurred and we were able to decrease non-specific binding by using protease. We will make progress to optimization based on these condition in the second year, also we are in discussion of securing non-cjd CSF samples and establishing protocol for sample preparation. The developed technology from this project can be utilized as diagnostic technology for CJD or confirmatory to improve accuracy of diagnosis. We hope it will bring more effective and systematic patient care by early diagnosis of CJD and isolate the second infection. - 2 -
학술연구용역사업연구결과 동물세포햄스터쥐양사슴소사람 잠복기 20-30 일 60 일 120 일 18-36 개월 18-36 개월 3-5 년 7-40 년 - 3 -
1.2 목표달성도 - 4 -
1.3 국내 외기술개발현황 - 5 -
제2장최종연구개발내용및방법 2.1 멀티머검출시스템 (Multimer Detection System, MDS) - 6 -
2.2 연구개발내용및방법 - 7 -
제3장최종연구개발결과 3.1 뇌척수액진단키트용포획및진단항체조사및선발 - 8 -
Detection antibodies 1E4HRP T2HRP 3E7HRP recombinant human PrP 반응성 X O Capturing antibodies recombinant human PrP 반응성 1E4 group 6D11 ICSM35 3C10 1E4 MA1-750 T2 group 3E7 SAF53 SAF54 SAF61 SAF70 1E2 O O ICSM 18 3.2 Candidate MDS antibody set 의비특이반응확인 - 9 -
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3.3 뇌척수액안정성유지를위한샘플 protocol 확립 - 11 -
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70000 100n 60000 1ng/ 50000 40000 30000 20000-14 -
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제 4 장연구결과고찰및결론 2 차년도추진체계 - 21 -
제 5 장연구성과 5.1 활용성과 과제명뇌척수액내프리온검출진단법개발연구 (2 차년도 ) 과제책임자강성민 / 피플바이오 / 생물학 번호논문제목저자명저널명집 ( 권 ) 페이지 Impact factor 1 국내 / 국외 SCI여부 번호발표제목발표형태발표자학회명연월일발표지국내 / 국제 1 Detection of total prion concentrations in CSF from confirmed and possible CJD patients in Korea. Poster Kim SY, An SS, et al Neuroprion 2009-9-22 Porto Carras, Greece 국제 번호출원 / 등록 1 특허명출원 ( 등록 ) 인출원 ( 등록 ) 국출원 ( 등록 ) 번호 IPC 분류 국내 ( 질병관리본부, 국립수의과학검역원 ) 와국외 ( 일본국립동물위생연구소, 프랑스혈액원 ) 의연구기관과의형성된연구개발네트워크를이용하여국내변종 CJD 환자감시체계를강화하며, 국내변종 CJD 환자발생시효과적이고신속한대책마련및대응을할수있을것으로사료됨 뇌척수액과같은임상검체를이용하여개발된기술은크로이츠펠트야콥병조기혈액진단스크리닝시스템 개발에적용할수있어 CJD 의예방및관리, 안정성을확보하는데본연구를활용할수있을것으로사료됨 해당사항없음 해당사항없음 - 22 -
5.2 활용계획 제 6 장기타중요변경사항 제 7 장참고문헌 - 23 -
제 8 장첨부서류 - 24 -