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

Abstract

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. 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 beta subunit of human chorionic gonadotropin (betahCG) in serum. We used two antibodies labeled with Tb3+ and rhodamine, respectively, recognizing different epitopes on betahCG. 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. The energy transfer signal was directly proportional to the betahCG concentration in the sample. The limit of detection was 0.43 microgram/L, and the assay was linear up to 200 microgram/L. Total assay imprecision in the range 10-185 microgram/L was between 7.5% and 2.8%. 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.