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.