Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay)

The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antib ody HyHEL-10 variable region was monitored with fluorescence resonance ener gy transfer (FRET) between fluorolabeled heavy chain (VH) and light chain ( V-L) fragments. The VH and V-L fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored . When excited at 490 nm, significant decrease in the fluorescence at 520 n m and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1-100 mu g/mL. The assay, named open sandwich fluoro-immunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use o f appropriate antibodies, it is thought to be a quick and inexpensive alter native to the conventional laborious and/or expensive immunoassays.