Clinical Pathology: General Principles, Lab Management, Clinical Chemistry

• In time-resolved fluorescent immunoassays, a lanthanide ion (usually europium [Eu³⁺]) is used. The fluorescence of the Eu³⁺ ion alone is weak and is, therefore, bound to an organic ligand, which significantly enhances its fluorescence.

• When the Eu³⁺ organic complex is excited by light irradiation, the organic ligand absorbs energy and electrons are raised from the ground to a singlet excited state. While in the excited state, these electrons can pass to a triplet state.

• If the ligand is chelated to Eu³⁺, there can be an energy transfer from the triplet excited state of the ligand to the Eu³⁺ ion, which then can move to an excited singlet state and emit fluorescence that is characteristic of Eu³⁺.

• The major advantage of time-resolved immunoassay is its long fluorescence lifetime. In conventional fluorescence assays, the fluorescence lasts about 1 μsec, whereas in time-resolved assays, the fluorescence can be measured at intervals of 400 to 800 μsec. This prolonged fluorescence eliminates the noise created by background fluorescence in biological samples, cuvettes, and solvents. As a result, the sensitivity of the assay is increased.

• Another advantage of time-resolved fluorescent assays is the long Stokes shift. The Stokes shift in some time-resolved assays can be greater than 200 nm. As a result, there is no overlap between the excitation and emission wavelengths. For example, the excitation and emission wavelengths for a Eu³⁺ organic complex are 295 and 612 nm, respectively.