This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor κB (NF-κB). 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-κB binding consensus sequence for capturing activated NF-κB in 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-κB. Finally, the probes protected by NF-κB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-κB without the need for NF-κB 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.

Introduction

Nuclear factor κB (NF-κB) was found to be a transcription factor that specifically bound a 10-bp GGGACTTTCC sequence in the immunoglobulin κ light-chain enhancer of B lymphocytes (1). Further studies revealed that the NF-κB family includes five members [c-Rel, p65 (RelA), RelB, p50 (NF-κB 1), and p52 (NF-κB2)] that share a common Rel homology region (RHR) (2). Now, NF-κB is known as a ubiquitous transcription factor that plays a pivotal role in controlling important cellular processes, ranging from normal cell growth and differentiation to apoptosis and cancer (3,4). In the absence of activation, NF-κB is detained in the cytoplasm by an inhibitory protein, IκB (5). With stimulation, NF-κB is activated and migrates into the nucleus, binds to its target sequence, and regulates expression of target genes. It has been demonstrated that NF-κB is involved in many diseases (6,7); therefore, it is attracting increasing interest as a promising target for drug development (8).

Almost all NF-κB studies require measurement of the level of activated NF-κB; therefore, the development of methods for assaying the DNA binding activity of NF-κB is important. The general method for doing this has been the electrophoretic mobility shift assay (EMSA) (9,10), in which the DNA binding activity of NF-κB is evaluated by measuring the intensity of a retarded band produced by a NF-κB/double-stranded DNA (dsDNA) complex in native polyacrylamide gel electrophoresis (PAGE). This method is still indispensable for NF-κB studies; however, EMSA is time-consuming and laborious. Moreover, the procedure requires the use of radioactivity and is not adaptable to automation and high-throughput analyses. For these reasons, several alternative NF-κB assay methods have been developed, such as an enzyme-linked immunosorbent assay (ELISA)-type assay in which NF-κB is measured by first capturing NF-κB with a dsDNA-coupled plate and then detected using a standard ELISA procedure (11,12,13). The ELISA-type assay provides an easy, nonisotopic, sensitive, quantitative, and high-throughput method for the assay of NF-κB. However, the assay relies on a specific anti-NF-κB antibody, and therefore is challenged by the need to prepare such a high-quality antibody.

We have previously described an alternative NF-κB detection approach that does not require a specific NF-κB antibody (14). In this assay, NF-κB is measured by a liquid-phase exonuclease protection assay with a fluorescence resonance energy transfer (FRET)-dsDNA probe. However, this method is limited by its relatively low-throughput and the high cost of the FRET probe. In this report, we describe a substantial improvement to the exonuclease protection assay. Our new method, called exonuclease-mediated ELISA-like assay (EMEA), provides a cost-effective means to assay NF-κB that is independent of the NF-κB antibody. We show that EMEA is an easy, nonisotopic, sensitive, quantitative, and high-throughput technique for measuring NF-κB; in addition, we provide evidence suggesting that the method should be generally applicable to other transcription factors.

Materials and Methods

Preparation of dsDNA-Coupled Plate

Table 1 lists the oligonucleotides (BIOASIA Biological Technology, Shanghai, China) used to prepare dsDNA-coupled plates for the detection of NF-κB (Table 1, nos. 1–4) and activating protein 1 (AP1) (Table 1, nos. 5 and 6). Two oligonucleotides (Table 1, nos. 1 and 5) were modified with a primary amino group at their 3′ ends so that they could be covalently coupled to the DNA-BIND™ plate (Corning Costar, Cambridge, MA, USA) that was coated with a layer of reactive N-oxysuccinimide esters (referred as NOS groups). The sense oligonucleotide was first dissolved in oligonucleotide binding buffer (50 mM Na₃PO₄, pH 8.5, 1 mM EDTA) at a concentration 0.25 pmol/µL, then 100 µL/well were added to the DNA-BIND plate. The plate was incubated overnight at 4°C and washed three times with maleate buffer (100 mM maleate, 150 mM NaCl, pH 7.5) to remove uncoupled DNA. Then 200 µL/well 3% bovine serum albumin (BSA) dissolved in oligonucleotide binding buffer were added to the plate and incubated for 30 min at 37°C to block unreacted NOS groups. The plate was washed three times with washing buffer (maleate buffer containing 0.05% Tween® 20), then 100 µL/well hybridization solution [5× sodium chloride/sodium citrate (SSC), 1.0% casein, 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate (SDS)] containing 50 nM antisense oligonucleotide were added. The plate was incubated at 65°C for 1 h and washed twice with washing solution (2× SSC, 0.1% SDS) preheated to 65°C, each for 5 min. Finally, the plate was dehumidified and stored in foil bags with desiccant at room temperature. The plate could be kept for 1 year.