Quantitative Chemiluminescent Immunoassay for NF‐κB–DNA Binding Activity

Abstract

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous redox-sensitive transcription factor involved in the pro-inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF-kappaB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. The conventional method for detection of NF-kappaB -DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time-consuming and non-quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF-kappaB DNA-binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF-kappaB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3-5h post sample preparation, (2) Greater sensitivity: 10pg NF-kappaB/well, (3) Quantitative: linear range: 10-1000pg NF-kappaB; r2 = 0.999 (4) High throughput adaptability: 96-well plate format can analyze up to 40 samples in duplicate, (5) SAFETY: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF-KB which is translocated into the nucleus and total (bound + unbound) NF-kappaB present in the cytosol/cell.

A graphene oxide (GO)-based molecular beacon for DNA-binding transcription factor detection

“…To date, a variety of methods have been explored for DNAbinding transcription factor detection. These involve the electrophoretic mobility shift assay (EMSA), 7 DNA footprinting, 8 enzyme-linked immunosorbent assay (ELISA) 9 and electrochemical techniques. 10 However, the aforementioned methods are generally tedious, laborious and expensive, which hinder them from more extensive applications.…”

Fok I cleavage–inhibition strategy for the specific and accurate detection of transcription factors

“…The conventional methods for transcription factor detection are electrophoretic mobility shift assay (EMSA) [11], DNA footprinting assay [12], Western blotting assay [13] and enzyme-linked immunosorbent assay (ELISA) [14]. These methods require radioisotope labels and/or specific antibodies against each target protein, which limits their extensive applications.…”

Determination of theophylline concentration in serum by chemiluminescent immunoassay

“…It is necessary to establish a new excellent method. In recent years, chemiluminescent immunoassay (CLIA) has been widely applied to the detection of drug concentration (Feng et al, 2005;Gupta et al, 2005;Shukla and Krag, 2005;Zamora et al, 1999). A study showed that CLIA detected chloramphenicol successfully (Lin et al, 2005).…”