Adenosine 3’:5’-cyclic monophosphate (cyclic AMP, cAMP) is one of the most important secondary messengers involved in signal transduction, gene regulation, steroid hormone biosynthesis and intracellular signaling. Its regulatory role extends to the nervous system, cell growth and differentiation, immune mechanisms and general metabolism. cAMP production is stimulated by compounds (hormones, drugs, neuromodulators) binding to target cells through cell membrane receptors. These binding events activate G-proteins which in turn activate the enzyme adenylate cyclase. Adenylate cyclase converts ATP to cAMP. cAMP-dependent protein kinases can then be activated resulting in phosphorylation of substrate proteins. Cyclic AMP levels may aid in understanding the mechanisms of action of a variety of hormones and biologically active substances.
This is a competitive ELISA for the quantitative analysis of cAMP levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the cAMP in the sample for a limited number of binding sites.
First, the sample or standard solution is added to the microplate. Next, the diluted enzyme conjugate is added and the mixture is shaken and incubated at room temperature for one hour. During the incubation, competition for binding sites is taking place. The plate is then washed to remove all unbound material. The bound enzyme conjugate is detected by the addition of substrate which generates an optimal color after 30 minutes. Quantitative test results may be obtained by measuring absorbance readings with a microplate reader with a 450nm or 650nm filter and then comparing the readings of the samples against the readings of the standards. The extent of color development is inversely proportional to the amount of cAMP in the sample or standard. For example, the absence of cAMP in the sample will result in a bright blue color, where as the presence of Cyclic AMP will result in decreased or no color development.