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Enzyme Immunoassay for Studying Intracellular Levels of cAMP

cause cAMP can activate a cascade of reactions, the involvement of cAMP greatly amplifies the response to a hormonal stimulus.

Nonradioactive Detection of cAMP

figure 1

Since changes in cAMP levels can result in the activation of proteins known to play key roles in signal transduction cascades, quantitative measurment of cAMP can serve to identify signaling events. Popular methods for determining cAMP levels include radioimmunoassays (RIA). Although these assays are highly sensitive, they require radioisotopes and time-consuming protocols. In contrast, Stratagenes nonradioactive EIA cAMP Kit uses a competitive EIA (figure 1) assay for simple and sensitive measurement of intracellualar cAMP levels. The assay is based on the competition between cAMP present in the sample and a fixed quantity of a cAMP-peroxidase conjugate that binds to rabbit anti-cAMP-specific antibodies.

figure 2

The assay is performed in a microtiter plate, with each well precoated with goat anti-rabbit IgG. Rabbit anti-cAMP antibodies are combined with the sample and cAMP-horseradish peroxidase conjugate (tracer) and applied to the microtiter well. Samples containing cAMP compete with a fixed quantity of tracer for the rabbit anti-cAMP-specific antibodies. All unbound reagents are r
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