Fig. (5) cAMP assay principle. In this competitive assay, an anti-cAMP antibody is labeled with a cryptate donor and cAMP is labeled with acceptor d2. Once both of them are place together, the binding of antibody to cAMP brings donor and acceptor into proximity range. Upon the excitation of donor at 337 nm, the energy is transferred from donor to acceptor. Donor and acceptor generate emission at 620 nm and 665 nm, respectively. In the cAMP assay, the level of free cAMP generated by the signal cascade can be measured by competing with the cAMP d2 for antibody binding. IP-One assay from HTRF has the similar principle.