title: Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol.
abstract: We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.
authors.affiliation: Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. katri.kuningas@utu.fi
authors.forename: Katri
authors.initials: K
authors.lastname: Kuningas
authors.forename: Telle
authors.initials: T
authors.lastname: Ukonaho
authors.forename: Henna
authors.initials: H
authors.lastname: Päkkilä
authors.forename: Terhi
authors.initials: T
authors.lastname: Rantanen
authors.forename: Jaana
authors.initials: J
authors.lastname: Rosenberg
authors.forename: Timo
authors.initials: T
authors.lastname: Lövgren
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D004958:Estradiol / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics
chemical_list: D004958:Estradiol