A protein L-based immunodiagnostic approach utilizing time-resolved Förster resonance energy transfer

Satu Hepojoki; Visa Nurmi; Antti Vaheri; Klaus Hedman; Olli Vapalahti; Jussi Hepojoki

doi:10.1371/journal.pone.0106432

A protein L-based immunodiagnostic approach utilizing time-resolved Förster resonance energy transfer

PLoS One. 2014 Sep 2;9(9):e106432. doi: 10.1371/journal.pone.0106432. eCollection 2014.

Authors

Satu Hepojoki¹, Visa Nurmi¹, Antti Vaheri², Klaus Hedman², Olli Vapalahti³, Jussi Hepojoki¹

Affiliations

¹ Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.
² 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.
³ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies, Bacterial / immunology
Antibodies, Monoclonal / immunology
Antigens, Bacterial / immunology
Bacterial Proteins / immunology*
Carbocyanines / metabolism
DNA-Binding Proteins / immunology*
Enzyme-Linked Immunosorbent Assay
Europium / metabolism
Fluorescence Resonance Energy Transfer / methods*
Humans
Immunoassay
Immunoglobulin Fab Fragments / metabolism
Immunoglobulin G / immunology
Immunologic Tests / methods*
Limit of Detection
Protein Binding
Time Factors

Substances

Alexa Fluor 647
Antibodies, Bacterial
Antibodies, Monoclonal
Antigens, Bacterial
Bacterial Proteins
Carbocyanines
DNA-Binding Proteins
Immunoglobulin Fab Fragments
Immunoglobulin G
L-protein, Peptococcus magnus
Europium

Grants and funding

Finnish Funding Agency for Technology and Innovation grant numbers 440071 and 440118. KH received these funds (http://www.tekes.fi/en/tekes/). Kliinisen Kemian tutkimussäätiö (SH), The Foundation for Finnish Inventions (KH), Integrative Life Science doctoral program (SH), and Laboratoriolääketieteen edistämissäätiö (SH) provided smaller funds. Grant numbers do not apply. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.