Homogeneous immunoassays using rare earth cryptates and time resolved fluorescence: principles and specific...

We have developed a new type of long lived fluorescent tracer, the rare earth cryptates formed by the inclusion of a Eu3+ ion into the intra molecular cavity of a macrobicyclic ligand containing bipyridine units. This tracer is used in a new homogeneous assay format based on spectral and temporal selectivity as well as on an amplification of the cryptate fluorescence. As a consequence of these features, the measurement of the specific signal is totally shielded from media interaction and corrected in real time for sample to sample optical variation. We show the example of a PSA assay where an analytical sensitivity of 0.03 ng/ml is obtained and an AFP assay where measurements in the first minutes of the incubation allow a rapid estimation of the AFP concentration and therefore an immediate dilution of the samples if required. These results illustrate the performance of this new homogeneous method and point out the specific advantages it allows in terms of sensitivity, precision and flexibility. It is therefore particularly well adapted for the assay of analytes like tumor markers where both sensitivity and wide dynamic range are needed.