Cellular Cyclic AMP Quantitation

Cyclic AMP (cAMP) is a key second messenger in intracellular signal transduction across a variety of biological pathways in many different organisms. The Applied Biosystems® cAMP-Screen® and cAMP-Screen Direct® chemiluminescent immunoassay systems were designed for rapid and convenient quantitation of cellular cAMP in functional assays of receptor activation, with both adherent and nonadherent cell types.

Flexible Applications

The cAMP-Screen® and cAMP-Screen Direct® assays are used with established cell lines for functional measurements of endogenous receptors (Figure 1), with cell lines expressing exogenous ligand receptors, and with primary cell cultures to investigate their response to treatment with the appropriate ligands. The cAMP-Screen® assay has also been used for cAMP quantitation from tissues. In addition, the assays can be used for high-throughput screening assays for compounds which stimulate or inhibit cAMP production via interference with receptor function or signal transduction pathways. 

The cAMP-Screen® and cAMP-Screen Direct® assay systems have been used with a wide range of cell lines containing endogenous receptors, including epithelial, neuronal, endocrine, pancreatic, and tumor-derived cell lines, as well as stably- and transiently-transfected CHO and HEK cells exogenously expressing many different GPCRs, neurotransmitter, adenosine, and hormone receptors. Primary cell types include epithelial, neuronal, adipose, osteoblast, and dendritic cells as well as platelets, hepatocytes, myoblasts, fibroblasts, and T cells. Mouse and Drosophila brain tissues have also been used with these assay systems.

Effect of forskolin and an antagonist on cAMP levelsEffect of forskolin and an antagonist on cAMP levels 
Figure 1. Effect of forskolin and an antagonist on cAMP levels. Duplicate cAMP-Screen® assays were performed after using the indicated concentration of forskolin to induce cAMP production in SK-N-MC cells in the absence or presence of a forskolin antagonist (human NPY). Measurements were performed on a microplate luminometer.

Rapid and Simple Detection Method

The cAMP-Screen® and cAMP-Screen Direct® competitive immunoassay systems are formatted for maximum flexibility to permit either manual assay or automated high-throughput screening with 96- or 384-well microplates. The system utilizes CSPD® substrate, a high-sensitivity chemiluminescent alkaline phosphatase (AP) substrate with Sapphire-II™ luminescence enhancer. This ready-to-use substrate/enhancer reagent generates a light signal that is measured 30 minutes after addition, with glow emission lasting several hours. 

With the cAMP-Screen Direct® system, cells are grown and lysed directly in the assay plate. For cells requiring specialized growth surfaces, we recommend using the cAMP-Screen® system after culturing cells in appropriate surface-modified culture plates (Figure 2). Lysates are incubated with a cAMP-AP conjugate and an anti-cAMP antibody in the secondary antibody–coated assay plate; the resulting immune complexes are captured in the plate. The captured immune complexes are washed to remove unbound cAMP-AP, chemiluminescent substrate/enhancer is added, and the resulting signal is measured in a luminometer. A low level of cAMP yields minimal competition with the cAMP-AP conjugate for binding to anti-cAMP antibody, resulting in a high chemiluminescence signal (Figure 3). Increased levels of cAMP compete with and thus reduce the amount of cAMP-AP conjugate in the captured immune complexes. Standard curves display an inverse correlation between signal intensity and free cAMP concentration and allow quantitation of the amount of cAMP.

Overview of cAMP-Screen® and cAMP-Screen Direct® systems 
Figure 2. Overview of cAMP-Screen® and cAMP-Screen Direct® systems. Cells are cultured, treated, and lysed in tissue culture plates. (A) In the cAMP-Screen® system, cell lysates are then transferred to a solid white assay plate precoated with secondary antibody. (B) In the cAMP-Screen Direct® system, cells are assayed directly in the culture plate; clear-bottom wells permit visual inspection of cells prior to start of the assay.
Sensitivity and dynamic range of the cAMP-Screen Direct® system 
Figure 3. Sensitivity and dynamic range of the cAMP-Screen Direct® system.
HEK 293 cells were plated at the indicated densities and grown in cAMP-Screen Direct® 96-well assay plates, followed by duplicate cAMP-Screen Direct® assays in the presence of cAMP standards. Sensitivity of the assay to exogenously added cAMP is unchanged following growth of cells in the assay plate; signal intensity differences result from basal intracellular levels of cAMP. Signal was measured with the TR717™ microplate luminometer.

High Sensitivity and Wide Dynamic Range

The cAMP-Screen® and cAMP-Screen Direct® systems enable ultrasensitive determination of cAMP levels in cell lysates: less than 0.2 pmol/well cAMP in the 384-well format. The dynamic range covers from less than 0.06 to greater than 6000 pmol/well cAMP in the 96-well format, and from less than 0.2 to greater than 200 pmol/well cAMP in the 384-well format, without sample dilution or modification. In addition, the assay exhibits very low cross-reactivity with other adenosine-containing or cyclic nucleotides (Table 1).

Table 1. Cross-reactivity of the cAMP-Screen® system with related nucleotides. Percent cross-reactivity is the ratio of the cAMP concentration to the cross-reactive nucleotide concentration that provides the same assay signal intensity.
Nucleotide
Cross-reactivity (%)
cAMP
100
AMP
0.15
ATP
0.15
cIMP
0.15
cTMP
0.06
ADP
0.03
cGMP
0.02
cUMP
0.012

Take Advantage of Maximum Flexibility

The cAMP-Screen® and cAMP-Screen Direct® Chemiluminescent Immunoassay Systems enable rapid and convenient quantitation of cellular cAMP. Formatted for maximum flexibility, the cAMP-Screen assays are available in either 96-well or 384-well formats, and in sizes that are ideal for evaluation, research and development, or primary and secondary screening. Learn More About cAMP-Screen® and cAMP-Screen Direct® Chemiluminescent Immunoassay Systems.

PLEASE NOTE: These products will not be available through Invitrogen website until November 7th in 2011. Please visit the Applied Biosystems web site for ordering information.

 

Product Quantity Cat. No.
cAMP-Screen® 96-Well Immunoassay System192 assays (2 plates)4412182
960 assays (10 plates)4412183
cAMP-Screen® 384-Well Immunoassay System768 assays (2 plates)T1501
19,200 assays (50 plates)T1504
cAMP-Screen Direct® 96-Well Immunoassay System192 assays (2 plates)4412186
960 assays (10 plates)4412187
cAMP-Screen Direct® 384-Well Immunoassay System768 assays (2 plates)T1506
19,200 assays (50 plates)T1508

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