Antibody free ELISA-like assay for detection of transcription factor based on double-stranded DNA thermostability

Transcription factors (TFs) play critical roles in gene expression regulation and disease development. In this paper，we reported 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 was based on the stabilization of the duplex DNA probe by binding with transcription factor. The double-stranded DNA (dsDNA) probe immobilized on 96-well plate was too short in length to stabilize its duplex structure at relatively high temperature and would unwind into single strand. In the presence of target TF, the protein bound to specific TF-binding site and prevented specific dsDNA probe from being unzipped at 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.