High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay.
Contemporary research on cellular signaling has undergone a shift of focus from qualitative measurements of single signaling pathways to high-throughput quantitation of comprehensive signaling networks. Notably, nuclear factor-kappaB (NFkappaB) is a family of transcription factors involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis and is deregulated in a number of disease states. We have established a chemiluminescent oligonucleotide-based enzyme-linked immunosorbent assay (co-ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFkappaB 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, whereas the percent relative standard deviation is 3- to 6-fold lower. Furthermore, the novel method is faster than electrophoretic mobility shift assay, not restricted to transfectable cells as is the case for luciferase reporter assays and 10 times more cost efficient than commercially available ELISA assays. Co-ELISA is a sensitive, fast and cost-efficient quantitation method for all DNA-binding NFkappaB proteins that can be used in high-throughput experimentation.
Bhattacharya
Nupur
N
Department of Internal Medicine III, University Hospital Ulm, Ulm, Germany.
Sarno
Antonio
A
Idler
Irina S
IS
Führer
Marita
M
Zenz
Thorsten
T
Döhner
Hartmut
H
Stilgenbauer
Stephan
S
Mertens
Daniel
D
D000970:Antineoplastic Agents / Q000494:pharmacology; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D023201:CD40 Ligand / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000378:metabolism*; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000970:Antineoplastic Agents; D001897:Boronic Acids; D016328:NF-kappa B; D009841:Oligonucleotides; D011719:Pyrazines; D014409:Tumor Necrosis Factor-alpha; D023201:CD40 Ligand; D000069286:Bortezomib