Fluorescent immunoassays are simply a variation of colorimetric ELISA. An enzyme converts a substrate to a reaction product that fluoresces when excited by light of a particular wavelength. The relative fluorescence units (emitted photons of light) that are detected are typically proportional to the amount of analyte being measured.
In comparison to the colorimetric ELISA, fluorescent immunoassays are only slightly more sensitive. However, they widen the dynamic range of the assay by allowing very high readings to be accurately measured as opposed to the 2.0 to 4.0 OD limit imposed on colorimetric assays.
Theory: Fluorescence results from a process that occurs when certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores, fluorochromes, or fluorescent dyes absorb light. The absorption of light by a population of these molecules raises their energy level to a brief excited state. As they decay from this excited state, they emit fluorescent light. The process responsible for fluorescence is illustrated by a simple electronic state diagram.
①Excitation ②Vibrational relaxation ③ Emission
Properties: high sensitivity (more than 1000 times sensitive than the light absorption method); easy to use; efficient; safe; no radiation; but have background fluorescence interference (microplate autofluorescence) and interference of the excitation light to the emitted light.