Assay Principle:

The DetectX® Cyclic AMP Direct ELISA Kit provides a highly sensitive and efficient method for quantitatively measuring Cyclic AMP (cAMP) in various samples. These sample types include cell lysates, urine, plasma, saliva, tissue, and tissue culture media. This competitive ELISA has a run time of 2.5 hours. It is multi-format and capable of detecting both acetylated and non-acetylated cAMP across different sample types. Read the complete kit insert before running the cAMP ELISA assay. A Cyclic AMP standard is included to establish an accurate standard curve.

Protocol Summary:

• Introduce standards or diluted samples into the provided transparent microtiter plate coated with donkey anti-sheep IgG antibody.
• Add Cyclic AMP peroxidase conjugate and Cyclic AMP sheep antibody to initiate the immunological reaction.
• Incubate the mixture for 2 hours at room temperature with shaking. The reaction varies inversely with the Cyclic AMP concentration in the sample.
• After incubation, wash away excess conjugate. Then add the TMB substrate and incubate 30 minutes. TMB reacts with the bound conjugate to produce a measurable signal.
• Use a plate reader to detect the signal at 450nm and calculate Cyclic AMP concentration based on the standard curve.

Background:

Sutherland and Rall discovered Adenosine-3’, 5’-cyclic monophosphate, or cyclic AMP (cAMP), in 1957. It is an important secondary messenger and an essential intracellular regulator. cAMP plays a critical role in mediating the activity of various hormones, including epinephrine, glucagon, and ACTH. It is produced by the activation of adenylate cyclase, which is stimulated by hormones such as glucagon and adrenaline, and by G protein activation. The liver’s adenylate cyclase has a stronger response to glucagon, while muscle adenylate cyclase responds more robustly to adrenaline.

The enzyme phosphodiesterase catalyzes the decomposition of cAMP into AMP. The Human Metabolome Database reflects Cyclic AMP’s broad role in cellular signaling and regulation, listing 166 metabolic enzymes that convert cAMP. The DetectX® Cyclic AMP Direct ELISA Kit is an important resource for researchers studying cellular signaling pathways, hormonal regulation, and metabolic processes.