cAMP-Screen Direct™ Cyclic AMP Immunoassay System

The cAMP-Screen Direct™ 96-well Cyclic AMP Immunoassay System enables ultrasensitive determination of cyclic AMP (cAMP) levels in cell lysates. This competitive chemiluminescent immunoassay is formatted with maximum flexibility to permit either manual reagent additions or automated high-throughput processing. The cAMP assay utilizes the highly sensitive chemiluminescent CSPD™ Substrate with Sapphire-II™ Enhancer that is triggered by an enzyme conjugate composed of cAMP bound to alkaline phosphatase (cAMP-AP). The chemiluminescent substrate/enhancer formulation is a ready-to-use reagent that generates a sustained-glow light emission from 30 minutes after addition. This assay is mainly used in secondary screening and pre-clinical research, where sensitivity and no false positives are essential.

• The highest sensitivity of any commercially available cAMP assay
• Hours of read time with little or no degradation of the signal
• A simple protocol with cells grown directly on the assay plate
• Useful for a wide range of receptor activation studies

High Sensitivity and Hours of Steady Glow Time
This chemiluminescent assay is designed to provide the highest sensitivity of any commercially available cAMP assay. As few as 60 femtomoles of cAMP can be detected. The assay has a wide dynamic range, detecting from 0.06 to 6,000 picomoles of cAMP without the need for sample dilution or manipulations such as acetylation. This is especially helpful in cell-based assays when measuring Gs- or Gi-coupled agonist or antagonist stimulation and/or inhibition. Intra-assay precision for duplicate samples is typically 5% or less. Once the substrate/enhancer formulation reaches glow signal, the plate can be read for hours with little or no degradation of the signal. This is useful in screens where several plates are compared with each other. In addition, the assay exhibits exceptionally low cross-reactivity with other adenosine-containing or cyclic nucleotides.

For Receptor Activation Studies
The cAMP-Screen Direct™ system is designed for quantitation of cellular cAMP for functional assays of receptor activation. The assay has been used with established cell lines for functional measurements with endogenous receptors, cell lines with exogenously expressed ligand receptors on the cell surface, primary cell cultures, and tissues in response to treatment with the appropriate ligands. In addition it has been used for receptor characterization, orphan receptor ligand identification, and the characterization of novel chimeric receptors. The assay can be used for high-throughput screens for compounds that stimulate or interfere with these signal transduction pathways.

A Simple Protocol—No Lysate Transfer Required
The assay follows a simple protocol. Cells are seeded into plates, cultured, and treated with test compounds as desired. Cell lysates are prepared in either the presence or absence of culture media. Lysates are incubated with a cAMP-AP conjugate and an anti-cAMP antibody in a coated microplate; the resulting immune complexes are captured in the plate. In samples without cAMP, all of the cAMP-AP conjugate is captured on the coated surface, resulting in a high signal. In the presence of cAMP, the amount of cAMP-AP conjugate captured decreases as a result of competition for binding with unlabeled cAMP, causing a reduced signal; signal reduction is proportional to the amount of cAMP present in the cell lysate. After washing to remove unbound cAMP-AP, the chemiluminescent substrate is added, and the resulting glow signal is measured in a luminometer.

This cAMP-Screen Direct™ system has all of the same benefits as the cAMP-Screen™ system, but also eliminates the need to transfer cell lysates from a tissue culture plate to the precoated microplates because the cells can be grown directly in the microplates. This provides better assay precision with one less transfer step. Many different cell types have been grown successfully on the precoated microplates, which also have a clear bottom to allow monitoring of cells.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.