title: Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.
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.
authors.affiliation: Department of Virology, Infection Biology Research Program, Haartman Institute, University of Helsinki, Helsinki, Finland. satu.saraheimo@helsinki.fi
authors.forename: Satu
authors.initials: S
authors.lastname: Saraheimo
authors.forename: Jussi
authors.initials: J
authors.lastname: Hepojoki
authors.forename: Visa
authors.initials: V
authors.lastname: Nurmi
authors.forename: Anne
authors.initials: A
authors.lastname: Lahtinen
authors.forename: Ilkka
authors.initials: I
authors.lastname: Hemmilä
authors.forename: Antti
authors.initials: A
authors.lastname: Vaheri
authors.forename: Olli
authors.initials: O
authors.lastname: Vapalahti
authors.forename: Klaus
authors.initials: K
authors.lastname: Hedman
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D057230:Limit of Detection; D012698:Serologic Tests / Q000379:methods*; D019809:Streptavidin / Q000276:immunology; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D019809:Streptavidin