建立新型微粒子免疫分析法以測定轉錄因子活性

Abstract

轉錄因子在基因調控方面佔相當重要的地位，藉由偵測細胞內轉錄因子的活化程度，可以判斷細胞的生理狀況、癌化程度或檢視藥物造成的各種反應。傳統上已有許多方式用以偵測轉錄因子活化程度，例如EMSA、ELISA、報導基因活性分析等，然而各方法仍有其缺失之處，例如背景值過高或是靈敏度差等，因此發展出一個新方法以避免舊有之缺點可使轉錄因子活性分析研究進展更加快速。 我們參考流式細胞儀可偵測微粒子所帶螢光的方法，發展出新型微粒子免疫偵測法（MIA-TF），利用avidin與biotin的鍵結，使結合有目標轉錄因子的核酸片段接合上微粒子，後續利用免疫法標定螢光於目標轉錄因子上，最後使用流式細胞儀偵測。於此研究中使用HIF-1α及NF-κB作為系統之所偵測之目標轉錄因子，實驗結果顯示MIA-TF可測得細胞中目標轉錄因子活性並可準確測得細胞在藥物作用下其目標轉錄因子之活性改變，此外ELISA及報導基因活性分析與MIA-TF相比較；此方法之最大特點為可不經萃取核蛋白而直接偵測到細胞內轉錄因子之活性，可更加簡便且快速的完成分析，其靈敏度高於傳統ELISA一百倍，並可發展為多目標偵測之高生產力系統，未來可大量應用至醫療診測、細胞生理研究及藥物篩選平台。由各種方面看來，MIA-TF為一個相當有前瞻性的轉錄因子活性偵測方法。

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.