Development of human procollagen type I c-terminal peptides detection sandwich ELISA kit
Development of human procollagen type I c-terminal peptides detection sandwich ELISA kit
- 주제(키워드) Collagen , Sandwich ELISA kit
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000025690
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Procollagen type I carboxy-terminal peptide (PICP) is the heterotrimeric molecule which is derived from type I collagen, an important extracellular matrix (ECM) protein in our body. There are a few methods to purify the PICP, which described only about isolating the native PICP from the fibroblast culture medium, not the recombinant protein. However, these steps are time-consuming and laborious due to complicated purification procedures, resulting in low yield. Here I report a novel method to express in mammalian cells recombinant human heterotrimeric PICP. The designed recombinant PICP not only improved expression level, but also simplified purification process. In addition, I selected scFv antibodies by screening phage-display library against purified recombinant PICP, and also obtained anti-PICP polyclonal antibody by immunizing with recombinant PICP. Finally, I developed a sandwich PICP ELISA kit which can precisely determine serum level of the native PICP from the fibroblast. The expression level of PICP indicate how many type I collagen was synthesized. Therefore, quantification of the existing PICP through ELISA system can be used for diagnosis of tissue regeneration or testing cosmetics. I hope that developed sandwich ELISA kit also can be used as useful tool for determining changes of collagen type I expression level.
more목차
1. Introduction 1
1.1 Collagen 1
1.2 Procollagen type I c-terminal peptide (PICP) 4
2. Experiment materials and methods 7
2.1 Reagents and Cell lines 7
2.2 Design and purification of PICP 7
2.3 Validation of purified recombinant PICP 11
2.4 Production of anti-PICP antibodies 12
2.5 Purification and validation of antibodies 13
2.6 Establishment and application of sandwich ELISA 18
3. Results 20
3.1 Purification and validation of recombinant PICP 20
3.2 Screening and selection of PICP antibodies using recombinant PICP 25
3.3 Biochemical characterization of anti-PICP antibodies 28
3.4 Development and application of PICP sandwich ELISA system 31
4. Discussion 37
5. References 40
국문요약 42
