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

Smirnova, D.V.; Samsonova, J.V.; Ugarova, N.N.

Авторы: Smirnova Daria V., Samsonova Jeanne V., Ugarova Natalia N.
Журнал: Photochemistry and Photobiology
Том: 92
Номер: 1
Год издания: 2016
Издательство: American Society for Photobiology
Местоположение издательства: United States
Первая страница: 158
Последняя страница: 165
DOI: 10.1111/php.12556
Аннотация: The high-performance 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 (RLuc) and the “green” mutant with λmax.em = 550 nm (GLuc) – were tested as the donors. The water-soluble Alexa Fluor 610х (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 GLuc and RLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal anti-progesterone antibody (AF–Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated an efficient BRET. 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.
Добавил в систему: Угарова Наталья Николаевна

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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 Progesteroneстатья

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