Quantitative Chemiluminescent Immunoassay for NF‐κB–DNA Binding Activity

Abstract

Nuclear factor‐κB (NF‐κB) is a ubiquitous redox‐sensitive transcription factor involved in the pro‐inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF‐κB 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‐κB ‐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‐κB 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‐κB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3–5 h post sample preparation, (2) Greater sensitivity: 10 pg NF‐κB/well, (3) Quantitative: linear range: 10–1000 pg NF‐κB; 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‐κB which is translocated into the nucleus and total (bound + unbound) NF‐κB present in the cytosol/cell.

Keywords:

• NF‐κB
• Oxidative stress
• Quantitative
• Chemiluminescent immunoassay
• Assay
• Cell extracts
• Human lymphocytes
• Rat liver

Acknowledgment

Supported, in part, by National Cancer Institute. Grant#: R43 CA91658–01.