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One-step EOC Biosensors for Bacterial Antigens and Anti-virus Antibodies as Diagnostic Indicators

박테리아 항원 혹은 항 바이러스 항체 탐지용 1단계 EOC형 바이오센서 개발

  • 전진우 (대학원 바이오마이크로시스템기술협동과정 바이오마이크로시스템기술전공)

초록/요약

Early detection of disease symptoms and diagnosis in time prevents serious casualties and reduces mortality rate in humans as well as animals. Rapid and accurate detection of biomarkers indicating diseases by use of simple detection kits always has remained a precedence and concern for detection kit developers and researchers. Therefore, detection kits with a high sensitivity and short detection time are essential for early diagnosis and proper treatment. Enzyme-linked immunosorbent immunoassays (ELISA)-on-a-chip (EOC), developed in our laboratory, is the test device based on the rapid immuno-chromatograpy concept employing horseradish peroxidase (HRP) as a signal generator. Herein, we have developed a rapid diagnostic biosensor by combining one-step EOC technology and immuno-magnetic separation (IMS) for an increased sensitivity, convenience, and reproducibility. This has been applied to early detection of food-contaminating microorganisms (e.g., Salmonella species) and highly contagious foot-and-mouth disease virus. An immunosensor system has been developed for sensitive detection of Salmonella species, food-contaminating microorganisms, by using the EOC technology. To produce immuno-reagents to the targets, antisera were raised using rabbits immunized with the two different heat-killed microbes, Salmonella typhimurium and Salmonella choleraesuis that belong to serogroups B and C1, respectively. Since these contain the most common O-antigen of lipopolysaccharides (LPS) offering major antibody-combining site, LPS B was used as the ligand in immuno-affinity purification of the mixed antisera on the gel column. Such purified polyclonal antibody was employed in the development of immunosensor as the both capture and detection binders where, in case of the detection, the binder was labeled with an enzyme, HRP. Such newly developed biosensor was able to detect a minimum of 1×104 Salmonella typhimurium CFU/mL., 1×105 Salmonella enteritidis CFU/mL, and 1×105 Salmonella choleraesuis CFU/mL, effectively. Further a standard screening test is proposed, for the detection of the food poisoning microbes in real samples (e.g., fish intestines as matrix), by introducing the combined IMS-EOC technology and selenite selective culture method. When the proposed standard test protocol was applied to the tests, approximately 100-fold increased sensitivity in the responses was achieved, i.e., the detection of 1 CFU Salmonella species/10 g food sample within 6 hours for Salmonella typhimurium, Salmonella enteritidis, and Salmonella choleraesuis. These results indicated that the detection protocol can early detect about 90% of the Salmonella serogroups. In case of FMD disease detection, we first employed the recombinant viral antigen as the both capture and detection, after the HRP labeling, for the analysis of the antibodies to the virus in sample. Such method resolves the problems of the current ELISA where a secondary antibody was used, only allowing for one animal species detection. Thus, this would allow a universal diagnosis for the samples from different animal species. Secondly, an automatic signal generation EOC device containing built-in enzyme substrate tank was constructed to attain an improved accuracy and reproducibility. As a result, a rapid determination about the antibody existence in the specimen with only a 10 mL sample was achieved within 20 minutes. It is notable that the same one-step EOC technology was also applied to the detection of the Salmonella species above. In conclusions, the one-step EOC devices developed for the different analytes in this study were able to improve convenience, accuracy, and reproducibility. Since they can measure antigens as well as antibodies as diagnostic indicators, the utilities would be broad in terms of the analytical sectors such as biodefense, environmental monitoring, and clinical diagnosis in addition to public health maintenance and veterinary. In the near future, we plan to develop an EOC biosensor with a long-term stability and miniatured transducer by enhancing the enzyme stability and introducing an image sensor, respectively.

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목차

제1장.서론
1.1. 연구의 필요성 1
1.2. 연구배경 3
1.2.1. 분석대상 질병의 병리 및 진단 3
1.2.1.1. Salmonella 증의 병리 및 진단법 3
1.2.1.2. 구제역의 병리 및 진단법 5
1.2.2. 면역분석기술 연구개발 배경 8
1.2.3. ELISA-on-a-Chip 바이오센서의 개념 9
1.3. 연구의 목적 12
제2장.재료 및 방법 14
2.1. 재료 14
2.2. 전처리 및 항체 정제 15
2.2.1. 항혈청 생산 16
2.2.2. 전처리를 통한 교차반응성 제거 16
2.2.3. 전처리 효과 확인 16
2.2.4. Ammonium sulfate 침전을 통한 IgG 정제 17
2.2.5. Protein G 면역친화크로마토그래피 17
2.2.6. LPS를 고정상으로 사용하는 column 제작 18
2.2.7. LPS 면역친화크로마토그래피 18
2.2.8. 항체 특성 확인 19
2.3. 신호발생 중합체 제조 20
2.3.1. 탐지항체-HRP 중합체 제조 20
2.3.2. 탐지항원-HRP 중합체 제조 21
2.4. 면역분석시스템 제작 22
2.4.1. 플라스틱칩제조 22
2.4.2. 면역스트립제조 23
2.4.2.1. Salmonella 탐지용 면역스트립 23
2.4.2.2. 구제역바이러스 항체 탐지용 면역스트립 24
2.4.3. 기질 저장탱크 제조 24
2.4.4. ELISA-on-a-Chip 바이오센서 조립 25
2.5. 면역분석시스템 성능확인 25
2.5.1. 농도응답곡선 작성 26
2.5.2. 교차반응성 확인 27
2.6. 면역자성비드 제조 및 농축비 확인 27
2.6.1. 면역자성비드제조 27
2.6.2. 자성농축 비 확인 28
2.7. 표준 현장 진단용 검사법 29
제3장.결과 및 토론 31
3.1. One-step ELISA 도입 31
3.2. Salmonella 탐지용 바이오센서 개발 34
3.2.1. 다중 특이 항혈청 특성화 34
3.2.1.1. 혈청 혼합비에 따른 최적화 34
3.2.1.2. 특이 항체 정제 및 반응성 확인 36
3.2.2. 면역분석시스템 성능평가 39
3.2.2.1. 면역분석시스템 구축 39
3.2.2.2. 농도응답곡선 작성 40
3.2.2.3. 분석특이성 평가 45
3.2.3. 자성농축 도입을 통한 고감도 바이오센서 개발 47
3.2.4. 실제 시료를 통한 바이오센서 성능평가 49
3.2.4.1. 선택배지에 따른 균배양 조건 확인 50
3.2.4.2. 시간별 실제시료 검사결과 비교 52
3.3. 구제역바이러스 항체 탐지용 바이오센서 개발 55
3.3.1. 범용진단용 신호발생 중합체 제조 55
3.3.1.1. 유전자 재조합 구제역 바이러스 항원 특성화 분석 56
3.3.1.2. 항원-HRP 중합체 제조 58
3.3.2. 면역분석시스템 구축 및 성능평가 59
제4장.결론 62
참고 문헌 66

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