Direct cAMP EIA Kit from Enzo Life Sciences

The role of 3'-5'-cyclic adenosine monophosphate (cAMP) as a second messenger in many biological processes has made intracellular cAMP measurement of basic importance in understanding intracellular regulatory pathways. A number of hormones activate adenylate cyclase, which converts ATP into cAMP. The Direct cAMP EIA kit from Enzo Life Sciences is useful in the quantitative determination of cAMP from tissue samples or cells grown in culture medium. It provides everything you need (except the instruments!) for cAMP determination in a 96-well format. The kit also provides a sheet with 96 grids resembling a microplate where you can write what sample is in each well.

Samples are first mixed with 0.1 M HCl to stop endogenous phosphodiesterase activity and to permit cell lysis. A cAMP covalently attached to an alkaline phosphatase molecule is added to standards and samples, then a polyclonal antibody raised against cAMP binds, in a competitive manner, to the cAMP in the standard or sample or to the cAMP coniugate-alkaline phosphatase conjugate. After incubation at room temperature and a washing step to remove the excess reagents, the substrate p-nitrophenyl phosphate is added. Finally, the reaction of alkaline phosphatase with its substrate is stopped by adding trisodium phosphate and the yellow color generated read at 405 nm.
The net optical density for standards and samples, obtained by subtracting the standard zero optical density from each measurement, can be used to calculate the cAMP concentration since it is inversely proportional to the concentration of cAMP.

This kit is suitable to measure low amounts of cAMP (typically under 5 pmol/mL) since it includes an acetylating reagent (e.g. acetic anhydride to be dissolved in triethylamine). The acetylated version is more sensitive because antiserum has greater affinity for the acetylated form of the cAMP than for the cyclic nucleotide itself. In the acetylated version, the highest value of the standard is 20 pmol/mL, the lowest value of the standard curve is 0.078 pmol/mL, and the sensitivity is 0.037 pmol/mL. If acetylation is not performed, the assay can be used for high amounts of cAMP (with standards from 200 to 0.78 pmol/mL) with a sensitivity of 0.39 pmol/mL. (Sensitivity is defined as the amount of cAMP needed to reduce the zero dose binding by two standard deviations.)

The optimal preparation of reagents is fundamental to achieve best results. It is critical to follow basic good laboratory practice: mix all samples and reagents thoroughly before use, fill wells to the top and completely drain each time, and always change and pre-rinse pipet tips for each reagent. Furthermore, standards and samples must be processed within 60 or 30 minutes from their preparation in the non-acetylated and acetylated version, respectively. The whole procedure is a little bit time-consuming; it takes at least 4 hours.

I successfully used this kit to evaluate the cAMP variation in the adrenal cell response to neuropeptide in vitro stimulation. Overall, the Direct cAMP EIA kit provides an effective (but not inexpensive) method to study the cAMP variation in tissues and cell culture.