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The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone
Author(s) -
Smirnova Daria V.,
Samsonova Jeanne V.,
Ugarova Natalia N.
Publication year - 2015
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/php.12556
Subject(s) - luciferase , immunoassay , bioluminescence , conjugate , chemistry , polyclonal antibodies , alexa fluor , homogeneous , antigen , mutant , acceptor , antibody , microbiology and biotechnology , chromatography , fluorescence , biochemistry , transfection , biology , physics , mathematical analysis , genetics , mathematics , quantum mechanics , immunology , gene , thermodynamics , condensed matter physics
The sensitive BRET system for the homogeneous immunoassay of a low‐molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)—the “red” mutant with λ max.em = 590 nm (RedLuc) and the “green” mutant with λ max.em = 550 nm (GreenLuc)—were tested as the donors. The water‐soluble Alexa Fluor 610× ( AF ) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody ( AF –Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL −1 .