143

Study of GPCR pharmacology using the DiscoverX HitHunter cAMP HS assay on a BMG LABTECH microplate reader

Julie M.-N. Rainard, Stewart E. Mireylees, Mark G. Darlison School of Biomedical and Natural Sciences, Nottingham Trent University 09/2006

G-protein coupled receptors (GPCRs) are cell surface receptors, which represent the most predominant drug targets. Following receptor stimulation, intracellular signaling pathways are activated, which in case of a Gs receptor leads to an increase and in case of a Gi receptor to a decrease of the second messenger cAMP.

 

The HitHunterTM cAMP assay by DiscoverX employs competitive binding of sample cAMP and peptide-labelled cAMP that is able to complement an enzyme acceptor added to the reaction. In the presence of cAMP in the sample, antibody binding sites are occupied leading to free peptide-labelled cAMP that complements the enzyme. The completed and active ß-Galactosidase enzyme converts a substrate under emission of light.

 

Using a cAMP standard curve as well as PC12 cells together with a well-known agonist the PHERAstar® microplate reader proved to be highly sensitive and suitable to read the assay.

go to top