The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

• DOI: 10.1111/php.12556
• Publication Date: 2015-12-09T18:30:30Z
• Authors: Daria V. Smirnova; Jeanne V. Samsonova; Natalia N. Ugarova
• Keywords: Immunoassay; 0301 basic medicine; 03 medical and health sciences; Luminescence; Energy Transfer; Luciferases, Firefly; Coloring Agents; Progesterone

AbstractThe 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 λmax.em = 590 nm (RedLuc) and the “green” mutant with λmax.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.