CAMP, fluorescent assays

YTX has also been shown to reduce cAMP levels in exposed cells through enhanced phosphodiesterase activity. This mechanism has been used as the basis for another functional assay for YTXs. Phosphodiesterase and anthranloyl-cAMP (fluorescent) were incubated with YTX or sheUflsh extracts and the decrease in EL measured. The rate of cAMP hydrolysis was shown to be linearly correlated to YTX concentration (0.5-10 pM). Two shellfish extracts gave the same results... 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Prystay, L., Gagne, A., Kasila, P., Yeh, L.A., and Banks, P., Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMP, /. Biomol. Screen., 6, 75, 2001. 

Wandelt et al. [87] have reported the application of a FILA for nucleotide monitoring. The assays were based on the fluorescence quenching of 3-diphenyl-6-vinyl-1 //-pyrazole 3.4-/ quinoline (PAQ), by guanosine 30,50-cyclic monophosphate (cGMP), adenosine 30,50-cyclic monophosphate (cAMP), and cytidine 30,50-cyclic monophosphate (cCMP). Although no analytical characterization of the polymers was reported, the authors carried out a rebinding study to ascertain the applicability of the MIPs for such application. 

Surface-modified nanobeads, beads where the chelates have shorter distance to the respective acceptor-labeled reactant, are also used to create proximity in TR-FRET assays such as cAMP immtmoassay and assays of kinase activities by the Molecular Devices IMAP system. The kinase assays apply fluorescent peptides as substrates, which after phosphorylation are collected to beads by complex formation between phosphor and the trivalent metalhc ions on the beads. Fluorescent terbium chelates, substituted with a phosphor group, provide thereafter the energy-donating groups to create sensitized signal on bead surfaces [37]. 

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