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Fluorescence polarisation immunoassay

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  387

2026-11-05

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Fluorescence polarisation immunoassay

Fluorescence polarisation immunoassay (FPIA) is another example of a homogenous immunoassay, and uses fluorescein-labelled antigen as the tracer molecule (Dandliker et al. 1973). It has been widely used as the basis of assay systems to analyse drugs in urine (Colbert et al. 1985). The fluorescein-labelled antigen rotates rapidly when not bound by an antibody. When bound by the antibody, the rotation is slowed dramatically compared to the unbound molecule. To generate the assay signal, a fluorimeter shines light, at the excitation wavelength for fluorescein, through a vertical polarising filter. Rapidly rotating unbound fluorescein molecules emit light in a different plane from the incident light, whereas the relatively stationary antibody bound fluorescein returns light in a similar plane, which is detected via the polarising filter. Drug added via the sample competes for binding to the antibody with the fluorescein labelled hapten, thereby reducing the amount of fluorescein bound to the antibody, which results in less emitted fluorescence being detected via the polarised filter. The background fluorescence found with many biological samples means it is usual to take a blank reading of the sample and reagents before the addition of the fluorescent tracer to the mixture. This method is the basis of the Abbott ADx system.