Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer

We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA), The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V-H)-Renilla luciferase (Rluc) and an antibody light-chain fragment (V-L)-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 similar to 10 mug/ml antigen hen egg lysozyme, Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration, (C) 2001 Academic Press.