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Enzyme Immunoassay for Studying Intracellular Levels of cAMP

Sensitive, nonradioactive cyclic-AMP detection kit and adenylate cyclase (AC) toxin

Danny Q. Hoang
Stratagene Cloning Systems, Inc.

Xinping Yang and Timothy G. Kingan, Ph.D.
American Qualex

Stratagenes EIA cAMP Kit is a highly sensitive and specific antibody-based assay kit for nonradioactive, quantitative measurement of intracellular levels of cAMP. The assay is based on the competition between cAMP present in the sample and a fixed quantity of a cAMP-horseradish peroxidase conjugate for a limited number of binding sites on cAMP-specific antibodies. When the amounts of antibody and peroxidase-labeled cAMP are fixed, the amount of peroxidase-labeled cAMP that binds to the antibody is inversely proportional to the concentration of added, unlabeled cAMP in the sample. In this enzyme immunoassay (EIA) format, each microtiter plate is precoated with a secondary antibody, goat anti-rabbit IgG, which binds to a fixed amount of rabbit anti-cAMP. The amount of peroxidase-labeled cAMP bound to the antibody is determined by the addition of a colorimetric substrate, and the resulting color is measured by reading the spectrophotometric absorbance at 450 nm. The sensitivity of the EIA cAMP Kit is in the nanomolar range, and sensitivity is increased to the picomolar range with acetylated cAMP.

Cyclic adenosine monophosphate (cAMP) plays a key role as an intracellular second messenger for transduction events that follow a number of extracellular signals. Typically, the binding of a hormone or neuromodulator to its receptor is followed by the activation of a G protein, which, in turn, activates the effector adenylyl cyclase evoking the production of cAMP. The activation of a protein kinase by cAMP results in the phosphorylation of substrate proteins, which include enzymes, ion channels and transcription regulators. Be
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