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Immunoassay of Theophylline by fluorescence polarisation


Abstract
Fluorescence immunoassay is applied to the detection of theophylline, a drug used for myocardial stimulation. Interference from other sample components is limited by use of polarization measurements, which discriminate between bound and unbound fluorophores. The detection system includes a T-format sampling module mounting two emission spectrometers and programmable polarizers. A calibration curve is generated and the technique demonstrated by running test samples in serum.


Introduction
Immunoassay methods are based on competitive-binding reactions between labeled and unlabeled ligand for receptor sites on a specific antibody.1 Measurement of some physical property of the label permits the construction of a calibration curve of that property versus concentration of the unlabeled ligand. The selectivity of the antibodies for a specific antigen allows the analysis of the ligand (analyte) of interest in the presence of similar substances.

To date, the most popular immunoassay technique has been radioimmunoassay (RIA) because of its high sensitivity. There are a number of problems, however, associated with RIA.2 A license is required to handle radioactive material, waste disposal is complicated, and reagents have short shelf-lives. Consequently, other techniques have been developed to supplant RIA. One of the most promising, generally applicable approaches, is fluorescence immunoassay (FIA). With FIA, the label is a small fluorescent molecule (the probe), which is bound covalently to the analyte. The intensity of fluorescence produced by this probe is related to the ligand concentration, to produce the calibration curve.

Ideally, a fluorescent label should satisfy certain criteria:
Chemical
The labeled ligand should be water-soluble to match physiological conditi
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