GPCRs transmit information via two major signaling pathways: Gs and Gi receptors change cAMP levels whereas Gq-coupled GPCRs induce a cytoplasmic Ca2+ release by inositol triphosphate (IP3).
The IP-One and cAMP HTplex™ assay is a competitive immunoassay that uses two antibodies labelled with Lumi4-TbTM (anti-cAMP Cryptate and anti-IP1 Cryptate as donors) and two acceptors (cAMP-green dye and IP1-red dye). In the inactivate state, a high TR-FRET signal is seen for both the red and green emissions. As cAMP or IP1 (degradation product of IP3) is produced upon GPCR activation, the tracer green-cAMP or the tracer red-IP1 will be uncoupled from the Tb-cryptate antibody, thereby causing a decrease in TR-FRET signal.
Reading the HTplex™ assay on a microplate reader from BMG LABTECH, the dose response effect of vasopressin on CHO-V2R cells was measured. At lower concentration an initial cAMP response was observed and at higher concentrations a latent IP1 accumulation was determined.