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 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.
Arai
R
R
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Nakagawa
H
H
Tsumoto
K
K
Mahoney
W
W
Kumagai
I
I
Ueda
H
H
Nagamune
T
T
D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers; D004735:Energy Transfer; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria