The rare earth cryptates are used as long-lived fluorescent labels. They are formed by inclusion of a europium ion in the intramolecular cavity of a macropolycyclic ligand containing bipyridine groups as light absorbers. The use of fluoride ions in the measuring medium allows a total shielding of the label fluorescence. Here, I have established the conditions under which the signal of these cryptates can be amplified through the use of nonradiative energy transfer, and applied these conditions to homogeneous immunoassays by using allophycocyanin as the acceptor. Because only a low proportion of the cryptate label is involved in the transfer amplification, and because allophycocyanin emission occurs between the europium emission lines, time-resolved measurement of europium and allophycocyanin emission allows real-time correction of the optical properties of the assay medium. These features have allowed the development of a rapid homogeneous immunoassay of prolactin that can detect as little as 0.3 microgram/L (3rd International Standard 84/500).

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A homogeneous assay method based on the long-lived fluorescence of rare earth cryptates and amplification by nonradiative energy transfer has been developed for immunoassays. The principles of the assay allow a double discrimination of the emitted signal through spectral and temporal selectivity. The cage structure of the complex, ion pairing around europium, as well as double-wavelength detection, fully shield the assay from perturbations of media. Events based on short-range interactions involving biomolecules are of tremendous importance in many domains of biology, either for analytical purposes or for molecular mechanism studies. Therefore, the principles and the reagents used to devise this homogeneous assay were adapted to various models representative of molecular and cellular processes and were chosen from the signaling pathways involved in cellular communication and expression: epidermal growth factor (EGF) receptor-ligand interaction, EGF receptor kinase activity, Jun/Fos protein-protein interaction, and DNA hybridization. Evaluation of the homogeneous assays yielded results compatible with those from comparison assays and demonstrates the versatility and wide range of applicability of this methodology.

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