A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

Transcription factors (TFs) are nuclear proteins that bind to the elements upstream of promoter genes, thereby regulating gene expression [1]. Thus, aberrant activity of TFs (such as oncogenic transcription factors) may lead to a broad range of dysfunctions of human and animal cells, causing diseases such as cancers [2,3]. Therefore, TFs are potential cellular bioindicators for medical diagnosis and targets for drug development [4]. Examples of such important TFs are nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor-1 (HIF-1).To analyze the potential activation of transcription factors in tumors, it is necessary to develop a simple method to measure the DNA binding capacity of target proteins. Although Western blotting is a good method to detect the content of specific proteins, it can only provide information regarding the total number of the target TFs. Western blotting cannot be used to distinguish between active or inactive TFs [5]. The activity of transcription factors are not always correlated with the TF amounts present in the cells; only the active TFs bound to the transcription factor binding site represent instances of gene expression [6]. Electrophoresis mobility shift assay (EMSA) is the current method used to detect the activity of TFs. Essentially, dsDNA probes containing the TF binding sequences are labeled with the [32P]-radioisotope, and the activity of TFs is determined after electrophoresis based on radioactivity levels. In addition, non-radioactive EMSA has been developed, but it is limited by high background and long detection time [5]. Although EMSA can be used to measure the activity of TFs, the devices required and steps of the EMSA procedure do not provide possibilities to develop a high-throughput assay. Although modified enzyme-linked immunosorbent assay (ELISA) assays were developed for measuring the activity of transcription factors in nuclear proteins [7], the procedures for extracting nuclear proteins hindered the application