Develope a new microsphere-based immunoassay for measuring the activities of transcription factors

• Main Authors: Yun-Ju Lai, 賴韻如
• Other Authors: Kuang-Wen Liao
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/93874270799892520444

碩士 === 國立交通大學 === 生物科技系所 === 95 === Transcription factors play pivotal roles in regulation of gene expression. By measuring the DNA-binding activities of transcription factors, we know the physiology of cells, cancerization or the response to drug treatment. There have been several traditional methods for analyzing activities of transcription factors, such as EMSA、ELISA、reporter gene activity assay. But all of these methods have its own disadvantages like high background or low sensitivity. For the purpose of developing a new method to avoid those disadvantages, we develop a new microsphere-based immunoassay to measure the activities of transcription factors（MIA-TF）. For MIA-TF, NeutrAvidin-labeled microspheres have been used as solid phase to capture the biotin-labeled double-strand DNA. The DNA-bound transcription factors are detected by immunoassay using transcription factor-specific antibody, then analysis by flow cytometry. We use HIF-1α and NF-κB individually as target transcription factors. MIA-TF can detect the difference in activities of transcription factors in cells after drug treatment. We compared MIA-TF with ELISA and reporter gene activity assay in sensitivity and optimize time. The distinguishing feature of MIA-TF is that we can work without the purification of nuclear protein so lead to high-throughput assay. When compared to ELISA method, MIA-TF shows a 100-fold higher sensitivity. Furthermore, MIA-TF can extend to multiplex targets analysis. The development of MIA-TF would make a further progress in clinical analysis and drug screening system. In all respects, MIA-TF is a high potential methods to detect activities of transcription factors.