title: A protein L-based immunodiagnostic approach utilizing time-resolved Förster resonance energy transfer.
abstract: Chelated lanthanides such as europium (Eu) have uniquely long fluorescence emission half-lives permitting their use in time-resolved fluorescence (TRF) assays. In Förster resonance energy transfer (FRET) a donor fluorophore transfers its emission energy to an acceptor fluorophore if in sufficiently close proximity. The use of time-resolved (TR) FRET minimizes the autofluorescence of molecules present in biological samples. In this report, we describe a homogenous immunoassay prototype utilizing TR-FRET for detection of antibodies in solution. The assay is based on labeled protein L, a bacterial protein that binds to immunoglobulin (Ig) light chain, and labeled antigen, which upon association with the same Ig molecule produce a TR-FRET active complex. We show that the approach is functional and can be utilized for both mono- and polyvalent antigens. We also compare the assay performance to that of another homogenous TR-FRET immunoassay reported earlier. This novel assay may have wide utility in infectious disease point-of-care diagnostics.
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Satu
authors.initials: S
authors.lastname: Hepojoki
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Visa
authors.initials: V
authors.lastname: Nurmi
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland; Helsinki University Central Hospital, Hospital District of Helsinki and Uusimaa, Laboratory Services, HUSLAB, Helsinki, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Vaheri
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland; Helsinki University Central Hospital, Hospital District of Helsinki and Uusimaa, Laboratory Services, HUSLAB, Helsinki, Finland.
authors.forename: Klaus
authors.initials: K
authors.lastname: Hedman
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland; Helsinki University Central Hospital, Hospital District of Helsinki and Uusimaa, Laboratory Services, HUSLAB, Helsinki, Finland; Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Olli
authors.initials: O
authors.lastname: Vapalahti
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Jussi
authors.initials: J
authors.lastname: Hepojoki
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D002232:Carbocyanines / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005063:Europium / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D007159:Immunologic Tests / Q000379:methods*; D057230:Limit of Detection; D011485:Protein Binding; D013997:Time Factors
chemical_list: C569686:Alexa Fluor 647; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002232:Carbocyanines; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; C116593:L-protein, Peptococcus magnus; D005063:Europium