Technologies & Platforms / Enzyme Fragment Complementation Technology / HitHunter EFC Biochemical Assay Platform / Competitive Immunoassays / cAMP Assay Platform

HitHunter® cAMP Assay Platform

Our HitHunter® cAMP assays are competitive immunoassays in which cAMP is labeled with β-galactosidase (β-gal) enzyme donor (ED).  This cAMP-ED conjugate and cAMP released from the testing cells compete for binding to an anti-cAMP antibody.  In the absence of the cellular cAMP, most of the cAMP-ED conjugate binds to the cAMP antibody, making them unable to complement with the enzyme acceptor (EA).  In the presence of cellular cAMP, the cAMP-ED is sequestered from the antibody by the cellular cAMP, so that it complements with EA to form active β-gal enzyme.  β-gal activity can be detected by DiscoveRx detection reagents using a chemiluminescent readout.

HitHunter® cAMP Assay Principle

cAMP Assay employing EFC using HitHunter
Free cAMP from cell lysates competes for antibody binding against labeled cAMP (ED-cAMP conjugate). Unbound ED-cAMP is free to complement EA to form active enzyme by EFC, which subsequently hydrolyzes substrate to produce signal. A positive signal generated is directly proportional to the amount of free cAMP bound by the binding protein.

Identification of Potent Agonists & Antagonists

 CXCR3

MC4R  
cAMP Hunter CXCR3 cell line (panel 1) was profiled against commercially available chemokines. The compounds differ in potencies as indicated by cAMP levels detected using HitHunter cAMP XS+. The cAMP Hunter MC4R Gs receptor (panel 2) was profiled against three agonists and the results reproduced the published rank order binding potencies (MT II > NDP-MSH > aMSH) [click graph to enlarge].

Features & Benefits

  • Works on any luminometer or multi-format reader
  • Unmatched performance for Gi-coupled receptors
  • Directly measure cAMP in cell-based assays- adherent & suspension mode
  • Ideal for cAMP-dependent phosphodiesterases
  • Chemiluminescent detection - minimizes false positives from autofluorescence