A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays.

The limitation of current dissociative fluorescence enhancement techniques is that the lanthanide chelate structures used as molecular probes are not stable enough in one-step assays with high concentrations of complexones or metal ions in the reaction mixture since these substances interfere with lanthanide chelate conjugated to the detector molecule. Lanthanide chelates of diethylenetriaminepentaacetic acid (DTPA) are extremely stable, and we used EuDTPA derivatives conjugated to antibodies as tracers in one-step immunoassays containing high concentrations of complexones or metal ions. Enhancement solutions based on different ß-diketones were developed and tested for their fluorescence-enhancing capability in immunoassays with EuDTPA-labelled antibodies. Characteristics tested were fluorescence intensity, analytical sensitivity, kinetics of complex formation and signal stability. Formation of fluorescent complexes is fast (5 min) in the presented enhancement solution with EuDTPA probes withstanding strong complexones (ethylenediaminetetra acetate (EDTA) up to 100 mM) or metal ions (up to 200 µM) in the reaction mixture, the signal is intensive, stable for 4 h and the analytical sensitivity with Eu is 40 fmol/L, Tb 130 fmol/L, Sm 2.1 pmol/L and Dy 8.5 pmol/L. With the improved fluorescence enhancement technique, EDTA and citrate plasma samples as well as samples containing relatively high concentrations of metal ions can be analysed using a one-step immunoassay format also at elevated temperatures. It facilitates four-plexing, is based on one chelate structure for detector molecule labelling and is suitable for immunoassays due to the wide dynamic range and the analytical sensitivity.

Novel detection strategies for drug discovery.

The Human Genome Project is expected to increase the number of potential drug targets from the current figure of 500 to approximately 3,000-4,000. This increased number of targets, and increasing knowledge of signaling-pathway networks and their complexities, sets new demands for efficiency on HTS assay technologies. Assessment of the total efficacy of a given drug candidate requires not only the classical assays, but also a wide variety of assays related to signaling cascades and cellular functions. Discrete functional assays traditionally involved Ca(2+) flux, kinases and cAMP, but today extend to the whole signaling network, from ligand binding to expression. This review discusses emerging novel non-radioisotopic assays, such as ligand-stimulated GTP-binding, the inositol triphosphate assay, cellular receptor trafficking, and protein-protein interactions.