HitHunter® cGMP Assay

Functional, Homogeneous Assay for Quantifying cGMP in Cells

Cyclic guanosine 3’,5’-monophosphate (cGMP) is an important second messenger molecule that plays a crucial role in cell-signaling pathways involving the regulation of smooth muscle relaxation, ion channel activity, intracellular calcium levels, and gene expression. cGMP is synthesized by the enzyme guanylate cyclase from GTP and serves as a signal transducer by activating various downstream effector proteins. In drug discovery and development, cGMP is often used as the readout to screen molecules that modulate phosphodiesterase (PDE) activity. The dysregulation of cGMP signaling has been linked to various diseases, including hypertension, heart failure, and erectile dysfunction.

Eurofins DiscoverX® provides an easy-to-use, functional HitHunter cGMP cell-based assay for accurately detecting cellular cGMP levels. This homogeneous, competitive immunoassay assay is scalable and excludes the need for wash steps, optimization, or specialized equipment.

Product Highlights

  • Assay Ready Kits – Complete, ultra-convenient kits for cGMP detection with optimized reagents and a chemiluminescent read-out to minimize false positives from auto-fluorescence
  • Homogeneous, High Throughput Amendable – Simple, rapid assays with scalable from 96-wells through 3456-well plate formats and high-throughput capability for screening small molecule and biologic therapeutics
  • Multiple Applications – Detect cellular cGMP in any cell line and investigate complex signaling networks involving cGMP and its downstream effectors

HitHunter cGMP Assay Principle

Measure cellular cGMP directly in cells. The HitHunter cGMP assay is a cell-based competitive immunoassay amenable for high-throughput screening. The assay utilizes the Enzyme Fragment Complementation (EFC) technology, where a ß-galactosidase (ß-gal) enzyme donor (ED) fragment is conjugated with cGMP. Free cGMP from cell lysate competes for antibody binding against labeled ED-cGMP conjugate. With low levels of cellular cGMP, most of the ED-cGMP binds to the anti-cGMP Ab, making the ED-cGMP unable to complement with the enzyme acceptor (EA) fragment of the ß-gal enzyme. With high levels of cellular cGMP, the anti-cGMP antibody becomes saturated, allowing the ED-cGMP complex to complement with the EA fragment and form an active ß-gal enzyme. The active enzyme then hydrolyzes a substrate to produce a chemiluminescent signal directly proportional to the amount of cGMP in the cell lysate.

The HitHunter cGMP assay is a cell-based functional immunoassay with a chemiluminescent readout. The easy-to-use assay is robust and available as a complete kit, containing all the reagents and plates you need for measuring the levels of cGMP in cells. After plating and stimulation of cells with a small molecule or biologic, the user simply adds the HitHunter cGMP assay reagents to the cell following the homogeneous, simple protocol provided and then reads the signal on any standard luminometer. The flexible assay system has been designed to be high throughput and scalable using 96-well through 3456-well plate formats.

Quantify cGMP in Cells

Quantify cGMP in Cells

The HitHunter cGMP assay can be used to quantify cGMP in cell lysates.  Atrial natriuretic peptide (ANP), a stimulator of guanylate cyclase, was added in a dose-dependent manner to PathHunter NPR1 (Natriuretic Peptide Receptor 1) cells (Cat. No. 93-0804C2). Activation of the cells led to increased cGMP production, quantified using the HitHunter cGMP assay. EC50 of 1.9 nM and signal-to-background of 4.3.