Antibody free ELISA-like assay for the detection of transcription factors based on double-stranded DNA thermostability.,Analyst

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Antibody free ELISA-like assay for the detection of transcription factors based on double-stranded DNA thermostability.
Analyst ( IF 3.6 ) Pub Date : 2020-03-27 , DOI: 10.1039/c9an02631b
Yue Sun ₁ , Zhiyan Li , Choiwan Lau , Jianzhong Lu

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Transcription factors (TFs) play critical roles in gene expression regulation and disease development. In this paper, we report an antibody free ELISA-like assay which could be used to analyze transcription factor NF-κB p50 with comparatively low cost and high throughput. This assay is based on the stabilization of a duplex DNA probe by binding with a transcription factor. The double-stranded DNA (dsDNA) probe immobilized on a 96-well plate was too short in length to stabilize its duplex structure at a relatively high temperature and would unwind into a single strand. In the presence of a target TF, the protein bound to a specific TF-binding site and prevented the specific dsDNA probe from being unzipped at the washing temperature, thus holding the chemiluminescence signal. This method could sensitively detect NF-κB p50 with a detection limit of 0.5 nM. The proposed strategy provides a convenient, cheap and high throughput detection method of TFs, which can potentially help the development of drug discovery and disease diagnosis.

中文翻译：

基于双链DNA热稳定性的无抗体ELISA样测定法，用于检测转录因子。

转录因子（TFs）在基因表达调控和疾病发展中起关键作用。在本文中，我们报告了一种无抗体的ELISA样测定法，可用于以相对较低的成本和较高的通量分析转录因子NF-κBp50。该测定基于通过与转录因子结合来稳定双链DNA探针。固定在96孔板上的双链DNA（dsDNA）探针长度太短，无法在相对较高的温度下稳定其双链体结构，因此会解开成单链。在目标TF存在的情况下，蛋白质结合到特定的TF结合位点，并阻止了特定的dsDNA探针在洗涤温度下解开拉链，从而保留了化学发光信号。该方法可以灵敏地检测NF-κBp50，检测限为0。5 nM。提出的策略提供了一种方便，廉价且高通量的TF检测方法，可潜在地帮助开发药物发现和疾病诊断。

更新日期：2020-03-09

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