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Demonstration of a Homogeneous Noncompetitive Immunoassay Based on Bioluminescence Resonance Energy Transfer

✍ Scribed by Ryoichi Arai; Hideyuki Nakagawa; Kouhei Tsumoto; Walt Mahoney; Izumi Kumagai; Hiroshi Ueda; Teruyuki Nagamune

Publisher
Elsevier Science
Year
2001
Tongue
English
Weight
77 KB
Volume
289
Category
Article
ISSN
0003-2697

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✦ Synopsis

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 to approximately 10 microg/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.

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