fluoroimmunoassay

A fluoroimmunoassay is an immunoassay that uses fluorescent labels to detect and quantify an antigen or antibody. The assay relies on specific antigen–antibody interactions, with a fluorescent signal produced by a labeled molecule when excited by light at a suitable wavelength. The emitted fluorescence is measured and is proportional to the amount of target present.

Common formats include direct and indirect fluoroimmunoassays and, within those, competitive and sandwich designs. In direct assays, a labeled antibody binds to the target; in indirect assays, an unlabeled antigen or antibody is detected with a labeled secondary reagent. In competitive assays, labeled antigen competes with the sample antigen for binding sites, giving a signal inversely related to analyte concentration. In sandwich assays, a capture antibody binds the antigen and a labeled detection antibody completes the complex, with signal proportional to analyte concentration.

Fluoroimmunoassays can be homogeneous (no separation of bound and free reagents) or heterogeneous (requiring washing steps to separate bound from free labels). Fluorophores used include organic dyes and more specialized tracers such as europium or other lanthanide chelates, which enable time-resolved fluorescence (TRFIA) to reduce background and improve sensitivity.

Detection is performed with a fluorometer or fluorescence plate reader, often in microplate format. Advantages include high sensitivity and a broad dynamic range and the potential for multiplexing with different emission wavelengths. Limitations include autofluorescence from samples, photobleaching of fluorophores, and the need for specialized instrumentation.

Applications span clinical diagnostics (such as infectious disease and hormone assays), therapeutic drug monitoring, environmental and food testing, and various research settings, where fluorescence provides sensitive, rapid readouts of target concentrations.