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HitHunter® cGMP Assay Platform

HitHunter cGMP is an in-vitro based, competitive immunoassay whereby free cGMP from the sample competes for antibody binding against labeled ED (a small peptide fragment of β-galactosidase) conjugated to cGMP. Unbound ED-cGMP is free to complement with EA (an inactive β-gal EFC enzyme), forming active β-galactosidase enzyme, which hydrolyzes substrate. The amount of luminescent signal is directly proportional to free cGMP.

Measuring cellular cGMP with EFC chemiluminescent detection



HitHunter cGMP is an in-vitro based competitive immunoassay. Free cGMP from cell lysates compete for antibody binding against labeled ED-cGMP conjugate, a small peptide fragment of β-galactosidase (β-gal). Unbound ED-cGMP is free to complement with EA, an inactive β-gal EFC enzyme, to form active enzyme, which subsequently hydrolyzes luminescent substrate. The amount of luminescent signal produced is directly proportional to the amount of free cGMP in the cell lysate

cGMP Analysis After Cell Stimulation & Inhibition

HitHunter cGMP Standard Curve

   

cGMP PDE V Inhibition

cGMP-standard-curve   cGMP-PDE-V-Inhibition-curve-data
A representative standard curve is shown in the graph.  HitHunter cGMP has been designed to exhibit robust and reliable assay performance as defined by the following parameters: EC50 < 20nM; Z'factor > 0.75 [click graph to enlarge].
 
The HitHunter cGMP assay can be used to identify PDE inhibitors.  12.5 U/well PDE V enzyme was used in this assay, dose dependent inhibition of the enzyme was observed.  IBMX yielded an EC50 of 1.62 uM [click graph to enlarge].

HitHunter cGMP Platform Features & Benefits

  • Directly measure cGMP in cell-based assays
  • Ideal for cGMP-dependent phosphodiesterases
  • Determine activity of guanylate cyclase
  • Directly measure cGMP from cells in suspension
  • Chemiluminescent detection - minimizes false positives from autofluorescence
  • HTS-friendly assay with high Z' (0.75)
  • Scalable assay adaptable to 1536 applications
  • Robust assay with large assay windows