Evaluation of fluorescence excitation transfer immunoassay for the measurement of plasma cortisol

Fluorescence excitation transfer immunoassay is a form of homogeneous assay suitable for the measurement of both large and small molecules. F6rster in 1948 [1] demonstrated that energy can be transferred through space from a fluorescent donor to an acceptor molecule; immunoassays based on this observation have now been developed. In fluorescence excitation transfer immunoassay, antibody is labelled with acceptor molecule or 'quencher', and the antigen with fluorescer molecules. When the antigen and antibody bind, the fluorescer and quencher molecules are brought into close proximity and the fluorescence decreases. Antigen in the sample competes with the labelled antigen for antibody sites. Thus a high analyte concentration results in a low level of quenching and vice versa [2]. The extent of quenching partly depends on the distance between the quencher and fluorescer [3]; the quenching rapidly decreasing with increasing distance. For this reason antibody rather than antigen is labelled with quencher. If the antibody was labelled with fluorescer, molecules bound at sites distant from the antigen-antibody binding site may not be effectively quenched by labelled antigen. The quencher must absorb at the wavelength of the fluorescence emission; the higher the molar absorptivity the greater the quenching efficiency. For a homogeneous immunoassay the number of quencher molecules coupled to the antibody is limited by the effect on the protein solubility and by any background fluorescence due to the quencher itself [2].