Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors:: measurement of activated NF-κB

This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA)for detecting the DNA binding activity of nuclear factor kappa B (NF-kappa B). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT contained a NF-kappa B binding consensus sequence for capturing activated NF-kappa B ill analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-kappa B. Finally, the probes protected by NF-kappa B were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-kappa B without the need for NF-kappa B antibodies. EMEA may provide a general approach for assays of DNA, sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.