Detection of NFκB Activity in Primary CLL Cells Using a Sensitive Oligo-Based Chemiluminescent ELISA.

Abstract

Abstract 4709Contemporary research on cellular signaling in oncogenesishas undergone a shift of focus from qualitative measurements of single signaling pathways to high-throughput quantitation of comprehensive signaling networks. Notably, NFkB is a family of transcription factors involved in immune and inflammatory responses, developmental processes and especially in cellular growth and apoptosis, which are central in oncogenesis: NFkB signaling is deregulated in a number of hematologic malignancies like Hodgkin's lymphoma or chronic lymphocytic leukemia. We have established a chemiluminescent oligonucleotide-based ELISA (co-ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFkB components (i.e. RelA, p50, p52, RelB, and c-Rel). In addition, it can make use of whole extract and does not require cumbersome nuclear/cytosolic fractionation, saving time and resources. Co-ELISA has a 3.5- to 43-fold higher signal-over-noise ratio than currently available assays, while the standard error (SEM) is 3-to 6-fold lower. Co-ELISA is suited for the assessment of NFkB activity in primary tumor cells or in high-throughput experimentation i.e. functional evaluation of novel compounds. The method is faster than EMSA, not restricted to transfectable cells as is the case for luciferase reporter assays, making it applicable to primary tumor cells, and ten times more cost-efficient than commercially available ELISA assays. Co-ELISA is a quantitative, sensitive, fast, and cost-efficient measurement of the activity of all DNA-binding NFkB proteins, also in primary tumor cells. Disclosures:No relevant conflicts of interest to declare.