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Terbium and Rhodamine as Labels in a Homogeneous Time-resolved Fluorometric Energy Transfer Assay of the ß Subunit of Human Chorionic Gonadotropin in Serum

^a Author for correspondence. Fax 358-2-2678357; e-mail kaj.blomberg{at}wallac.fi

Background: Fluorescence resonance energy transfer (FRET) is a powerful tool in analytical chemistry. The aim of the present work was to use FRET to design a homogeneous immunoassay.

Methods: We used a highly fluorescent terbium (Tb³⁺) chelate (donor) and the organic fluorochrome rhodamine (acceptor) combined with time-resolved detection of the acceptor emission in homogeneous assay format for the measurement of the ß subunit of human chorionic gonadotropin (ßhCG) in serum. We used two antibodies labeled with Tb³⁺ and rhodamine, respectively, recognizing different epitopes on ßhCG. The close proximity between the labels in the immunocomplex permitted energy transfer between the pulse-excited Tb³⁺ donor (decay time >1 ms) and the acceptor rhodamine (decay time of 3.0 ns). The prolonged emission of donor-excited acceptor (energy transfer) was measured after the short-lived background and acceptor emissions had decayed. The emission of donor-excited rhodamine was measured at a wavelength of where the emission of unbound donor is minimal.

Results: The energy transfer signal was directly proportional to the ßhCG concentration in the sample. The limit of detection was 0.43 µg/L, and the assay was linear up to 200 µg/L. Total assay imprecision in the range 10–185 µg/L was between 7.5% and 2.8%.

Conclusions: Although less sensitive than heterogeneous, dissociation-enhanced europium-based separation assays, the presented assay format has advantages such as speed and simplicity, which make the assay format ideal for assays requiring a high throughput.© 1999 American Association for Clinical Chemistry

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