A new microsphere-based immunoassay for measuring the activity of transcription factors

Abstract

There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

Figure 1

Experimental design of a microsphere-based immunoassay for measuring the activity of transcription factors (MIA-TF). a Flowchart of MIA-TF. The biotin-labeled dsDNA probes were made to react with TFs in a nuclear extract of the cells. Subsequently, NeutrAvidin microspheres were added into the reactions. After incubation, the mixtures were probed with anti-transcription factor antibody and detected with a fluorescent secondary antibody by flow cytometry. b Dot plot (SSC vs. FSC) of the MIA-TF method. The DNA-bound TFs were detected by an immunoassay using a transcription factor-specific antibody and then analyzed using flow cytometry. Microspheres not incubated with nuclear extract were used as negative controls (left) to gate the range of effective signals (R1). The microspheres incubated with the nuclear extracts increased the fluorescence values in the range of effective signals (right). bio-DNA biotin-labeled dsDNA, TF transcription factor.

Figure 2

The MIA-TF method was used to detect the activity of transcription factors. a The MIA-TF method was used to detect the changes in NF-κB expression in 500 ng of nuclear extract. b The MIA-TF method detected the changes of HIF-1 expression in 500 ng of nuclear extract. Relative total mean = Events_R1 × Mean_R1/Events_R2 × Mean_R2. The results are expressed as mean ± SD. *p < 0.05, n = 6. NBS biotin-labeled dsDNA containing five NF-κB binding site, cNBS non-biotin-labeled NBS, HBS biotin-labeled dsDNA containing seven HIF binding sites, cHBS non-biotin-labeled HBS, VP16 non-biotin-labeled dsDNA containing no HBS and NBS.

Figure 3

Sensitivity of MIA-TF detection. a The MIA-TF method was used to detect different contents of recombinant human NF-κB p50 transcription factors. b The MIA-TF method was used to detect NF-κB activity in different contents of nuclear extract. c The MIA-TF method was used to detect HIF-1 activity in different contents of nuclear extract. The results are expressed as mean ± SD. *p < 0.05 and, compared with the control group (column II), n = 6. NBS biotin-labeled dsDNA containing five times NF-κB-binding site, HBS biotin-labeled dsDNA containing seven times HIF-binding site.

Figure 4

MIA-TF detected the activity of transcription factors in cells after drug treatment. a After treatment with 25 μM hydroquinone for 16 h, the MIA-TF method was used to detect the changes of NF-κB expression in HeLa cells. b After treatment with 100 μM desferrioxamine (DFO) for 16 h, the MIA-TF method was also used to detect the changes of HIF-1 expression in HeLa cells. The results are expressed as mean ± SD. *p < 0.05, n = 6. NBS biotin-labeled dsDNA containing five times NF-κB-binding sites, HBS biotin-labeled dsDNA containing seven HIF-binding sites.

Figure 5

Modified-MIA-TF directly measured the TF activity in 96-well plates. The activity of NF-κB was analyzed using a modified-MIA-TF system. After seeding 3,500 HeLa cells/well in 96-well plates, cells were treated with 25 μM hydroquinone for 16 h. The cells were lysed and the activity of transcription factors in cell lysates in 96-well plates was measured. The results are expressed as mean ± SD. *p < 0.05, n = 6. NBS biotin-labeled dsDNA containing five times NF-κB-binding site.