Rare earth cryptates and homogeneous fluoroimmunoassays with human sera

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).