fluoroimmunoassays

Fluoroimmunoassays (FIA) are immunoassays that detect analytes using fluorescence as the readout. They rely on antigen–antibody interactions in which either the antigen or antibody is labeled with a fluorescent probe, or a fluorescent tracer bound to the complex allows quantitation. The emitted fluorescence is proportional to the amount of target present in the sample.

Two common formats are competitive and sandwich assays. In competitive FIA, labeled antigen competes with unlabeled analyte for binding to a specific antibody; the signal is inversely proportional to the analyte concentration. In sandwich FIA, a capture antibody binds the analyte, and a labeled detection antibody provides a direct signal proportional to the analyte amount. Detection often uses fluorophores such as FITC, Alexa dyes, or lanthanide chelates in time-resolved fluorescence to improve signal-to-noise. Time-resolved fluorescence immunoassays (TR-FIA) use long-lived emitters like europium to reduce background.

Modern FIA platforms include plate-based assays in microplates and flow-based fluorescence immunoassays. Instrumentation includes fluorescence plate readers and flow cytometers, enabling multiplexing by using multiple fluorophores with distinct emission spectra.

Applications span clinical diagnostics, therapeutic drug monitoring, plant and veterinary tests, infectious disease screening, and environmental analysis. Advantages include high sensitivity, wide dynamic range, fast turnaround, and multiplexing capability; limitations include autofluorescence interference, photobleaching, the need for specialized detectors, and potential cross-reactivity or matrix effects.