Development of virus nanoparticles for sensitive antibody detection via genetic and unnatural amino acid modification of the coat proteins
Development of virus nanoparticles for sensitive antibody detection via genetic and unnatural amino acid modification of the coat proteins
- 주제(키워드) immuno pcr , phage display , antigen detection
- 발행기관 아주대학교
- 지도교수 유태현
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021654
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The virus particle has attractive features as materials, for example, homogenous size, specific modifications of particle surface via genetic and chemical manipulations, and self-amplification via its own genetic information. These properties make researchers try to develop biomaterials, sensor and scaffolds based on phage. Detection by immuno-PCR exhibits high sensitivity, but it usually requires a complex manufacturing process especially for protein-DNA conjugates. We anticipated that a genetically modified M13 bacteriophage can be used as a detection molecule in immuno-PCR. In this study, we have developed a novel bacteriophage particle which displays Z domain, an IgG binding protein, at the pIII coat proteins. The virus particle has not only a template for PCR but also a binding capability to IgG; thus the new sensing reagent can be used for immune-PCR directly by itself. The cTnI myocardial infarction marker is chosen for the demonstration of an immuno-PCR assay method using the modified bacteriophage. An initially developed immuno-PCR method showed a limit of detection as 0.092 ng cTnI/mL, which is about 4-fold lower than an ELISA method. In order to improve the platform, we are applying the site-specific modification strategies using unnatural amino acids by incorporating the analogues into the pIII and/or pVIII coat proteins. For an example, we are developing a simple method to conjugate IgG to the Z domain-displayed phage particle via a light-mediated reaction.
more목차
1. Introduction 1
1.1. ELISA, the antibody based detection method 1
1.2. Immuno-PCR 3
1.3. Incorporation of unnatural amino acids into proteins 5
1.4. Phage display technology 6
2. Materials and Methods 12
2.1. Construction of plasmids 12
2.2. Preparation of bacteriophage particles 13
2.3. Determination of phage titer 15
2.4. Copper-free azide-dibenzocyclooctyl cycloaddition reaction 15
2.5. Photocrosslinking protein conjugation using benzophenone 15
2.6. Western blot 16
2.7. ELISA 16
2.8. Immuno-PCR 17
2.9. Real-time PCR 18
3. Results and discussions 20
3.1. Preparation of Z domain displaying bacteriophage particles 20
3.2. Detection of phage by PCR 24
3.3. Binding analysis of phage using ELISA 26
3.4. Optimization of phage-mediated IPCR 28
3.4.1. Blocking reagent 28
3.4.2. Phage concentration 30
3.4.3. Antibody pairs 32
3.4.4. Coating antibody 34
3.4.5. Primary antibody 34
3.5. Comparison of IPCR with standard ELISA for cTnI detection 36
4. Conclusions 40
5. References 42
6. Abstract in Korean 45
7. Acknowledgement 46
