Optimized homogeneous immunoassay based on XeCI-laser excited foirster resonance energy transfer

We have investigated a commercial fluorescence immunoassay (FIA) reader (KRYPTOR-Cezanne), designed for homogeneous immunoassays, which is based on a time-resolved measurement of Forster resonance energy transfer (FRET) from a europium cryptate (Eu-TBP) donor to a crosslinked allophycocyanin (XL665) acceptor. The system uses a nitrogen laser as excitation source and a two-channel PMT detection system with filter-based wavelength separation. The most important factor for FIA to compete with chemiluminescent immunoassay systems is sensitivity. Compared to N₂-laser excitation the limit of detection (LOD) with the XeCl-laser was decreased by a factor of 3 in the same reader system by simply exchanging lasers. Using the so called Gate-Delay-LOD Matrix the optimal delay time and measuring window time (gate) in which the luminescence signal is integrated can be determined . This is an important tool for applying system changes (e.g. excitation wavelength or energy) to different FIA tests