Abstract
Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.
Highlights
Forster resonance energy transfer (FRET) is a process in which a donor molecule in excited state transfers its excitation energy through dipole-dipole coupling [1] to an acceptor fluorophore, when the two are brought into close proximity [2,3]
In this report we describe a novel time-resolved FRET (TR-FRET) based approach for detection of antibodies from biological fluids using a solutionphase homogenous assay and solid-phase heterogenous assays
We demonstrated with donor- and acceptor-labeled SA that addition of a specific antibody to a mixture of antigens each carrying one of the two labels induces the formation of immune complexes producing a TR-FRET signal promptly after combining the reagents
Summary
Forster resonance energy transfer (FRET) is a process in which a donor molecule in excited state transfers its excitation energy through dipole-dipole coupling [1] to an acceptor fluorophore, when the two are brought into close proximity (typically less than 10 nm) [2,3]. FRET between chromophores is characterized by Forster radius (R0), the distance at which the efficiency of FRET is 50% [5]. This phenomenon has been applied, among others, to study of protein-protein interactions, antigen-antibody binding, ligand-receptor interactions, DNA hybridization and DNA-protein binding [2,6,7,8,9,10,11]. Time-resolved fluorometry (TRF) relies on the long fluorescence emission half-life of lanthanides, rare earth elements such as europium (Eu) and terbium (Tb) [12,13,14,15,16]. In TR-FRET, chelated or cryptic lanthanides are typically used as donors for acceptors such as Alexa FluorsTM or cyanine dyes (e.g. Cy5)
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